Perspectives of Stem Cells

Henning Ulrich
Editor

Perspectives
of Stem Cells
From Tools for Studying
Mechanisms of Neuronal
Differentiation towards Therapy

123
Editor
Prof. Henning Ulrich
Universidade de Sao Paulo
Instituto de Quimica
Departamento de Bioquimica
Av. Prof. Lineu Prestes 748
Sao Paulo-SP
Brazil
henning@iq.usp.br

ISBN 978-90-481-3374-1 e-ISBN 978-90-481-3375-8

DOI 10.1007/978-90-481-3375-8
Springer Dordrecht Heidelberg London New York
Library of Congress Control Number: 2009940404

Springer Science+Business Media B.V. 2010

No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by
any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written
permission from the Publisher, with the exception of any material supplied specifically for the purpose of
being entered and executed on a computer system, for exclusive use by the purchaser of the work.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Stem cells are fascinating cell types. They can replicate themselves forever while
retaining the potential to generate progeny with specific functions. Because of these
special properties, stem cells have been subjects of intensive investigation, from
understanding basic mechanisms underlying tissue generation, to modeling human
diseases, to application for cell replacement therapy. Stem cells come in different
forms. For example, mouse embryonic stem cells can general all cell types in a body,
either in a dish or when put back into mouse embryos. On the other hand, neural
stem cells in the adult brain generate neurons and glia cells that contribute to the
brains plasticity. Rapid progress has been made in the stem cell field with discover-
ies published in a record speed. A quick Pubmed search has returned 2789 hits for
embryonic stem cells and 815 hits for adult neural stem cells/neurogenesis in the
year 2008 alone. It remains a taunting task for all who are interested in stem cells to
keep up with rapidly accumulating literatures. The Perspectives of Stem Cells by a
truly international team of experts provides a timely and invaluable highlight of the
stem cell field gearing toward future therapeutic applications in the nervous system.
Stem cells with neural potentials have attracted a lot of attention because of their
promise for cell replacement therapy, ranging from degenerative neurological disor-
ders to spinal cord injuries. Before such potentials to be realized, however, we need
to understand the basic biology of these stem cells. For example, understanding how
stem cell behaviors are controlled by intrinsic and extrinsic factors will help to direct
stem cells into a specific fate while avoiding undesired tumorigenesis. Equally impor-
tant, we need to understand adult nervous system milieu where substitute neurons
need to integrate into proper circuitry for maximal recovery. In this regard, the recent
discovery of functional neurogenesis in discrete regions of the adult mammalian
brain, including humans, has a major impact on regenerative medicine. Not only
there exist residual adult neural stem cells as endogenous cellular sources for neu-
rogenesis, the adult nervous system itself exhibits surprising plasticity. They provide
proper signals to support normal neurogenesis and, furthermore, additional signals
upon injuries to activate neural stem cells and guide their neuronal progeny to the
right location. The dogma, In the adult centers, the nerve paths are something fixed,
ended, and immutable. Everything may die, nothing may be regenerated, is now long
gone.
The Prospective covers a broad spectrum of a fast evolving field of stem cells:
from different model systems and stem cell types, to their cell biology and molecu-
lar signaling mechanisms; from overview of protocols for directed neuronal subtype
differentiation from embryonic stem cells, and even the latest induced pluripotent

v
vi Preface

stem cells, to specific considerations for the therapeutic application. The historical
view of neurogenesis since the time of Cajal was a delight retreat; the discussion of
retrotransposons in generating neuronal diversity through neurogenesis was fascinat-
ing. The Prospective provides a much needed overview of the state of the art in the
field and a rich resource of updated information. More importantly, it sets up a stage
for flourishing of new ideas in stem cell biology and for fostering novel therapeutic
applications in the nervous system for years to come.

Clarksville, Maryland Hongjun Song

August 2009
Editor Preface

The field of stem cell biology is geared towards translation into clinical practice
through in vitro tissue production and regeneration therapy. The discovery of neu-
rogenesis in selected areas of the adult brain has revolutionized neuroscience. This
discovery has overturned the central assumption that no new neurons were origi-
nated in the brain after birth, and provided the basis for understanding the molecular
mechanisms of neural differentiation. Several in vitro models have been developed
to investigate signalling pathway of neurogenesis regulation and cell fate specifica-
tion. Massive propagation of embryonic cells into just the right type of phenotype
of a neural progenitor cell or strategies for mobilizing endogenous neural stem or
progenitor cells provide replacement therapies for brain injury resulting from stroke
or neurodegenerative diseases. Such strategies are getting more important, since the
development of ex vivo cultures of stem cells allows collection of multipotent cells
from patients, their differentiation and transplantation into diseased areas.
This book includes a chapter on mechanisms of neural induction in early embryos
as well as a detailed discussion of changes in paradigms in view of the discov-
ery of adult neurogenesis. Neuronal differentiation is detailed using the olfactory
epithelium, one of the tissues bearing neurogenesis along life. In addition to chapters
on therapeutic applicability of embryonic, very small-embryonic like, mesenchymal
stem and neural progenitor cells, this book covers signalling mechanisms guiding
induction to differentiation and selective achievement of specific phenotypes. Cell
diversification of the neuronal system is explained using the example of neural crest
cell differentiation. Alterations in genetic material, such as loss of chromosomes
and retrotransposition, are discussed as possible mechanisms for cell diversification.
Furthermore, fundamental aspects of stem cell biology and neurogenesis, such as the
importance of proliferation induction, programmed cell death, as well as the func-
tion of glia in differentiation of stem cells and development of neuronal circuits, are
also highlighted. The participation of cytoskeletal elements in cell polarization as pre-
requisites of asymmetric division and differentiation induction is discussed. Further
topics include the analysis of extracellular signals, such as neurotrophic factors and
neurotransmitters, their receptor molecules and the propagation of these signals by
intracellular signal transduction leading to activation of selective gene expression dur-
ing differentiation. Rhythmic gene expression for activation and inhibition of Notch
signalling is discussed as a mechanism for regulating the progress of neurogenesis. In
vitro cultures of embryonic, mesenchymal and neural stem cells as well as mobiliza-
tion of endogenous stem and precursor cells for brain repair and replacement therapy
in neurological disorders are important issues of this book.

vii
viii Editor Preface

Each chapter provides an invaluable resource for information on the most current
advances in the field and possible therapeutic applications, with discussions of con-
troversial issues and areas of emerging importance. By providing an up-to-date and
critical view of the state of Science, we hope that this book shall be a base for excit-
ing scientific ideas regarding functions and therapeutic applications of stem cells in
the adult brain. The book is directed to neuroscientists, physicians, students and all
who are engaged and interested in the exciting and rapidly expanding field of modern
neuroscience and stem cell biology.

So Paulo Henning Ulrich

June 2009
Contents

1 Neural Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Karla Loureiro Almeida, Jos Abreu, and C. Y. Irene Yan
2 Neurogenesis: A Change of Paradigms . . . . . . . . . . . . . . . . 11
Luiz E. Mello and Beatriz M. Longo
3 Neurogenesis in the Olfactory Epithelium . . . . . . . . . . . . . . . 35
Bettina Malnic and Lucia Armelin-Correa
4 Cell Diversification During Neural Crest Ontogeny:
The Neural Crest Stem Cells . . . . . . . . . . . . . . . . . . . . . . 47
Elisabeth Dupin, Giordano W. Calloni, and Nicole M. Le
Douarin
5 Intermediate Filament Expression in Mouse Embryonic
Stem Cells and Early Embryos . . . . . . . . . . . . . . . . . . . . . 59
Zhigang Xue, Vivaldo Moura-Neto, Araksya Izmiryan,
Sheila Cristina de Souza Martins, Jean Christophe Larcher,
Denise Paulin, and Zhenlin Li
6 Aneuploidy in Embryonic Stem Cells . . . . . . . . . . . . . . . . . 73
Rafaela C. Sartore, Priscila B. Campos,
Michael J. McConnell, and Stevens K. Rehen
7 Retrotransposition and Neuronal Diversity . . . . . . . . . . . . . . 87
Maria C. N. Marchetto, Fred H. Gage, and Alysson R. Muotri
8 Directing Differentiation of Embryonic Stem Cells
into Distinct Neuronal Subtypes . . . . . . . . . . . . . . . . . . . . 97
Noelle Ammon, Nathaniel Hartman, and Laura Grabel
9 Neurotransmitters as Main Players in the Neural
Differentiation and Fate Determination Game . . . . . . . . . . . . 115
Katia K. Yuahasi, Katia N. Gomes, Marcelo Campos, Arthur
A. Nery, Ariane Nunes-Alves, Cleber A. Trujillo, and Henning Ulrich
10 Rhythmic Expression of Notch Signaling in Neural
Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Hiromi Shimojo, Toshiyuki Ohtsuka, and Ryoichiro Kageyama

ix
x Contents

11 Neuron-Astroglial Interactions in Cell Fate Commitment

in the Central Nervous System . . . . . . . . . . . . . . . . . . . . . 145
Joice Stipursky, Tnia Cristina Leite de Sampaio e Spohr,
Luciana Ferreira Romo, and Flvia Carvalho Alcantara Gomes
12 The Origin of Microglia and the Development of the Brain . . . . . 171
Flavia R. S. Lima, Anna Carolina C. da Fonseca,
Giselle P. Faria, Luiz Gustavo F. Dubois, Trcia R. Alves,
Jane Faria, and Vivaldo Moura Neto
13 Tissue Biology of Proliferation and Cell Death Among
Retinal Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . 191
Rafael Linden, Rodrigo A.P. Martins, Mariana S. Silveira,
Helena L. Borges, Alfred Sholl-Franco, Lucianne
Fragel-Madeira, and Ana Carolina Dudenhoeffer-Carneiro
14 Potential Application of Very Small Embryonic Like
(VSEL) Stem Cells in Neural Regeneration . . . . . . . . . . . . . . 231
Mariusz Z. Ratajczak, Ewa Zuba-Surma, Magda Kucia,
Przemyslaw Nowacki, and Bogdan Machalinski
15 Embryonic Stem Cell Transplantation for the Treatment
of Parkinsons Disease . . . . . . . . . . . . . . . . . . . . . . . . . 245
Asuka Morizane and Jun Takahashi
16 Functional Multipotency of Neural Stem Cells
and Its Therapeutic Implications . . . . . . . . . . . . . . . . . . . 255
Yang D. Teng, Serdar Kabatas, Jianxue Li,
Dustin R. Wakeman, Evan Y. Snyder, and Richard L. Sidman
17 Dual Roles of Mesenchymal Stem Cells in Spinal Cord
Injury: Cell Replacement Therapy and as a Model System
to Understand Axonal Repair . . . . . . . . . . . . . . . . . . . . . 271
Cecile King, Shyam Patel, Treena Livingston Arinzeh,
and Pranela Rameshwar
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Contributors

Jos Abreu Cellular and Developmental Biology Program, Institute of Biomedical

Sciences, Universidade Federal do Rio de Janeiro, Centro de Cincias da Sade
bloco F, Cidade Universitria, Rio de Janeiro, RJ 21949-590, Brazil,
garciajr@anato.ufrj.br
Karla Loureiro Almeida Departamento de Morfologia, Centro de Cincias da
Sade, Universidade Federal do Esprito Santo - Vitria/ES - Brazil,
karlaloureiro25@hotmail.com
Ariane Nunes-Alves Departamento de Bioqumica, Instituto de Qumica,
Universidade de So Paulo, So Paulo, Brazil, anunesalves@usp.br
Trcia R. Alves Programa de Anatomia, Instituto de Cincias Biomdicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
terciabio@yahoo.com.br
Noelle Ammon Hall-Atwater Laboratories, Biology Department, Wesleyan
University, Middletown, CT, USA, nammon@wesleyan.edu
Lucia Armelin-Correa Departamento de Bioqumica, Instituto de Qumica,
Universidade de So Paulo, So Paulo, Brazil, armelin@iq.usp.br
Helena L. Borges Instituto de Cincias Biomdicas, Universidade Federal do Rio
de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil, hborges@anato.ufrj.br
Giordano W. Calloni CNRS UPR2197 Laboratoire Dveloppement, Evolution et
Plasticit du Systme Nerveux, Institut de Neurobiologie Alfred Fessard, 91198
Gif-sur-Yvette, France; Departamento de Biologia Celular, Embriologia e Gentica,
Centro de Cincias Biolgicas, Universidade Federal de Santa Catarina,
Florianpolis, Brazil, giordano@ccb.ufsc.br
Priscila B. Campos Instituto de Cincias Biomdicas, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, Brazil, brittopris@anato.ufrj.br
Marcelo Campos Departamento de Bioqumica, Instituto de Qumica,
Universidade de So Paulo, So Paulo, Brazil, camposm@iq.usp.br
Anna Carolina C. da Fonseca Programa de Anatomia, Instituto de Cincias
Biomdicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
annafonseca@anato.ufrj.br
Luiz Gustavo F. Dubois Programa de Anatomia, Instituto de Cincias Biomdicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, dubois@anato.ufrj.br

xi
xii Contributors

Ana Carolina Dudenhoeffer-Carneiro Instituto de Biofsica, Universidade

Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil,
anacarol@biof.ufrj.br
Elisabeth Dupin CNRS UPR2197 Laboratoire Dveloppement, Evolution et
Plasticit du Systme Nerveux, Institut de Neurobiologie Alfred Fessard, 91198
Gif-sur-Yvette, France, dupin@inaf.cnrs-gif.fr
Giselle P. Faria Programa de Anatomia, Instituto de Cincias Biomdicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
giselle.faria@gmail.com
Jane Faria Programa de Anatomia, Instituto de Cincias Biomdicas, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brazil, jane@anato.ufrj.br
Lucianne Fragel-Madeira Instituto de Biofsica, Universidade Federal do Rio de
Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil, lufragel@biof.ufrj.br
Fred H. Gage Laboratory of Genetics, The Salk Institute for Biological Studies, La
Jolla, CA, USA, gage@salk.edu
Katia N. Gomes Departamento de Bioqumica, Instituto de Qumica, Universidade
de So Paulo, So Paulo, Brazil, knevesgomes@yahoo.com.br
Flvia Carvalho Alcantara Gomes Laboratrio de Neurobiologia Celular,
Programa de Biologia Celular e do Desenvolvimento, Instituto de Cincias
Biomdicas, Centro de Cincias da Sade, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil, fgomes@anato.ufrj.br
Laura Grabel Hall-Atwater Laboratories, Biology Department, Wesleyan
University, Middletown, CT, USA, lgrabel@wesleyan.edu
Nathaniel Hartman Hall-Atwater Laboratories, Biology Department, Wesleyan
University, Middletown, CT, USA, nhartman@wesleyan.edu
Araksya Izmiryan UPMC Univ Paris 6, UMR 7079, Paris, France,
araksya.izmiryan@inserm.fr
Serdar Kabatas Department of Neurosurgery, Baskent University Istanbul
Hospital, Istanbul, Turkey, kabatasserdar@hotmail.com
Ryoichiro Kageyama Institute for Virus Research, Kyoto University, Kyoto, Japan;
Japan Science and Technology Agency, CREST, Kyoto, Japan,
rkageyam@virus.kyoto-u.ac.jp
Cecile King UMDNJ-New Jersey Medical School, Department of Medicine,
Newark, NJ, USA, kingc4@umdnj.edu
Magda Kucia Stem Cell Institute at James Graham Brown Cancer Center,
University of Louisville, Louisville, KY, USA, mjkuci01@louisville.edu
Jean Christophe Larcher Laboratoire de Biologie du Dveloppement, Paris,
France, jclarche@snv.jussieu.fr
Nicole M. Le Douarin CNRS UPR2197 Laboratoire Dveloppement, Evolution et
Plasticit du Systme Nerveux, Institut de Neurobiologie Alfred Fessard, 91198
Gif-sur-Yvette, France, nicole.ledouarin@academie-sciences.fr
Contributors xiii

Jianxue Li Department of Neurology, Beth Israel Deaconess Medical Center,

Harvard Medical School, Boston, MA, USA, jli7@caregroup.harvard.edu
Zhenlin Li UPMC Univ Paris 6, UMR 7079, Paris, France, zhenlin.li@upmc.fr
Flavia R.S. Lima Programa de Anatomia, Instituto de Cincias Biomdicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, flima@anato.ufrj.br
Rafael Linden Instituto de Biofsica, Universidade Federal do Rio de Janeiro,
Cidade Universitaria, Rio de Janeiro, Brazil, rlinden@biof.ufrj.br
Treena Livingston Arinzeh New Jersey Institute of Technology, Department of
Biomedical Engineering, Newark, NJ, USA, arinzeh@adm.njit.edu
Beatriz M. Longo Departamento de Fisiologia, Universidade Federal de So Paulo,
So Paulo, Brazil, beatriz.longo@unifesp.br
Bogdan Machalinski Department of Physiopathology Pomeranian Medical
University, Szczecin, Poland, machalin@pam.szczecin.pl
Bettina Malnic Departamento de Bioqumica, Instituto de Qumica, Universidade
de So Paulo, So Paulo, Brazil, bmalnic@iq.usp.br
Maria C. N. Marchetto Laboratory of Genetics, The Salk Institute for Biological
Studies, La Jolla, CA, USA, marchetto@salk.edu
Sheila Cristina de Souza Martins Instituto de Cincias Biomdicas-Universidade
Federal de Rio de Janeiro, Rio de Janeiro, Brazil, sheila@anato.ufrj.br
Rodrigo A.P. Martins Instituto de Biofsica, Universidade Federal do Rio de
Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil, Rodrigo.Martins@biof.ufrj.br
Michael J. McConnell Crick-Jacobs Center for Theoretical and Computational
Biology, Salk Institute for Biological Studies, La Jolla, California, USA,
mikemc@salk.edu
Luiz E. Mello Departamento de Fisiologia, Universidade Federal de So Paulo, So
Paulo, Brazil, lemello@unifesp.br
Asuka Morizane Department of Biological Repair, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto, Japan, asuka.morizane@frontier.kyoto-u.ac.jp
Vivaldo Moura-Neto Instituto de Cincias Biomdicas-Universidade Federal de
Rio de Janeiro, Rio de Janeiro, Brazil, vivaldo@anato.ufrj.br
Alysson Muotri University of California at San Diego, School of Medicine,
Department of Pediatrics/Rady Childrens Hospital San Diego and Department of
Pediatrics/Cellular and Molecular Medicine, La Jolla, CA, USA, muotri@ucsd.edu
Arthur A. Nery Departamento de Bioqumica, Instituto de Qumica, Universidade
de So Paulo, So Paulo, Brazil, arthur.nery@gmail.com
Przemyslaw Nowacki Department of Physiopathology Pomeranian Medical
University, Szczecin, Poland, rektor@szczecin.pl
Toshiyuki Ohtsuka Institute for Virus Research, Kyoto University, Kyoto, Japan;
Japan Science and Technology Agency, CREST, Kyoto, Japan,
tohtsuka@virus.kyoto-u.ac.jp
xiv Contributors

Shyam Patel UMDNJ-New Jersey Medical School, Department of Medicine,

Newark, NJ, USA, patel120@umdnj.edu
Denise Paulin UPMC Univ Paris 6, UMR 7079, Paris, France,
denise.paulin@upmc.fr
Pranela Rameshwar UMDNJ-New Jersey Medical School, Department of
Medicine, Newark, NJ, USA, rameshwa@umdnj.edu
Mariusz Z. Ratajczak Stem Cell Institute at James Graham Brown Cancer Center,
University of Louisville, Louisville, KY, USA; Department of Physiopathology
Pomeranian Medical University, Szczecin, Poland, mzrata01@louisville.edu
Stevens K. Rehen Instituto de Cincias Biomdicas, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, Brazil, srehen@anato.ufrj.br
Luciana Ferreira Romo Laboratrio de Neurobiologia Celular, Programa de
Biologia Celular e do Desenvolvimento, Instituto de Cincias Biomdicas, Centro de
Cincias da Sade, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
romao@anato.ufrj.br
Rafaela C. Sartore Instituto de Cincias Biomdicas, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, Brazil, fa_sartore@yahoo.com.br
Hiromi Shimojo Institute for Virus Research, Kyoto University, Kyoto, Japan;
Japan Science and Technology Agency, CREST, Kyoto, Japan,
hshimojo@virus.kyoto-u.ac.jp
Alfred Sholl-Franco Instituto de Biofsica, Universidade Federal do Rio de
Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil, asholl@biof.ufrj.br
Richard L. Sidman Department of Neurology, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, MA, USA,
Richard_Sidman@hms.harvard.edu
Mariana S. Silveira Instituto de Biofsica, Universidade Federal do Rio de Janeiro,
Cidade Universitaria, Rio de Janeiro, Brazil, silveira@biof.ufrj.br
Evan Y. Snyder Stem Cell and Regeneration Program, The Burnham Institute, La
Jolla, CA, USA, esnyder@burnham.org
Tnia Cristina Leite de Sampaio e Spohr Laboratrio de Neurobiologia Celular,
Programa de Biologia Celular e do Desenvolvimento, Instituto de Cincias
Biomdicas, Centro de Cincias da Sade, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil, spohr@anato.ufrj.br
Joice Stipursky Laboratrio de Neurobiologia Celular, Programa de Biologia
Celular e do Desenvolvimento, Instituto de Cincias Biomdicas, Centro de Cincias
da Sade, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
joice@anato.ufrj.br
Jun Takahashi Department of Biological Repair, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto, Japan, jbtaka@frontier.kyoto-u.ac.jp
Contributors xv

C.Y. Irene Yan Department of Cell and Developmental Biology, Institute of

Biomedical Sciences, Universidade de So Paulo, Av. Prof Lineu Prestes 1524, So
Paulo, SP 05508-900, Brazil, ireneyan@usp.br
Yang D. Teng Department of Neurosurgery, Harvard Medical School, Brigham and
Womens Hospital, and Childrens Hospital Boston, Boston, MA, USA; Department
of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding
Rehabilitation Hospital, Boston, MA, USA; Division of SCI Research, Veterans
Affairs Boston Healthcare System, Boston, MA, USA, yang_teng@hms.harvard.edu
Cleber A. Trujillo Departamento de Bioqumica, Instituto de Qumica,
Universidade de So Paulo, So Paulo, Brazil, clebertrujillo@yahoo.com.br
Henning Ulrich Departamento de Bioqumica, Instituto de Qumica, Universidade
de So Paulo, So Paulo, Brazil, henning@iq.usp.br
Dustin R. Wakeman Stem Cell and Regeneration Program, The Burnham Institute,
La Jolla, CA, USA; University of California at San Diego: Graduate Program in
Biomedical Sciences, La Jolla, CA 92093, USA, dwakeman@burnham.org
Zhigang Xue UPMC Univ Paris 6, UMR 7079, Paris, France,
zhigang.xue@upmc.fr
Katia K. Yuahasi Programa de Ps-graduao em Neurologia, Departamento de
Neurologia e Neurocirurgia, Universidade Federal de So Paulo, So Paulo, Brazil,
yuahasi@usp.br
Ewa Zuba-Surma Stem Cell Institute at James Graham Brown Cancer Center,
University of Louisville, Louisville, KY, USA, ewa.zuba-surma@uj.edu.pl
Chapter 1

Neural Induction

Karla Loureiro Almeida, Jos Abreu, and C. Y. Irene Yan

Contents layer of complexity to the interpretation of the results

1.1 Introduction . . . . . . . . . . . . . . . . . 1
1.2 Neural Induction in the Xenopus Embryo
The Early Experiments . . . . . . . . . . . 2
1.3 Neural Default Model . . . . . . . . . . . . 3
1.4 BMP and the Neural Inducers . . . . . . . . 4
1.5 Challenges to the Neural Default Model . . . . 4
1.6 Neural Induction and the Avian Node . . . . . 4
1.7 Epiblast The Responsive Tissue . . . . . . . 5
1.8 Inhibition of BMP in the Avian Context . . . . 6
1.9 FGF Signaling and Neural Induction . . . . . 7
References . . . . . . . . . . . . . . . . . . . . 8
Abstract Neural induction, i.e. definition of the
neural domain from the ectoderm, is a fundamental
topic that has fascinated developmental biologists for
years. The concept was first proposed by Spemman
and Mangold after their classic experiment in the
amphibian Xenopus laevis where transplantation of
the embryos dorsal blastopore lip induced a com-
plete neural axis from the acceptor embryos ectoderm.
Since then, much effort has been applied into iden-
tifying the signals that bias the ectoderm into neural
fate and the resulting picture clearly indicates that
neural induction is a multi-step process that requires
the interplay of various pathways. A major part of
our current understanding of neural induction orig-
inates from the original amphibian model Xenopus
laevis. Recently, the chick embryo has added another
C.Y.I. Yan ()
Department of Cell and Developmental Biology, Universidade
de So Paulo, Av Prof. Lineu Prestes, 1524, So Paulo, SP,
05508-900, Brazil
e-mail: ireneyan@usp.br
obtained from the amphibian model. Here, we will
focus on the landmark experiments that address the
earliest step of neural induction in these two models.
Specifically, we will discuss the Neural Default model
that was generated from experiments in the amphib-
ian embryo to explain the choice between epidermal
and neural precursor fate and the modifications on this
model based on conclusions derived from the chick
embryo.
Keywords BMP signaling Ectoderm FGF Neural
induction Smad Xenopus Chick
Abbreviations
BMP bone morphogenetic protein
TGF- transforming growth factor
FGF fibroblast growth factor
MAPK mitogen activated protein kinase
1.1 Introduction
The induction of neural tissue is a fundamental ques-
tion that has fascinated developmental biologists since
the classic experiment by Spemmann and Mangold.
In 1924, based on their results from grafting experi-
ments performed in amphibian embryos, the authors
proposed for the first time the concept of neural induc-
tion. At the time, it was known that the blastopore
lip initial involution site during gastrulation marked
the dorsal region of the embryo, and that the future
neural plate arose from the dorsal ectoderm the ven-
tral ectoderm forms mainly epidermal tissue. Spemann

H. Ulrich (ed.), Perspectives of Stem Cells, 1

DOI 10.1007/978-90-481-3375-8_1, Springer Science+Business Media B.V. 2010
2 K.L. Almeida et al.
and Mangold transplanted the blastopore lip of donor
embryos to the ventral region of host embryos in gas-
trula stage. The host embryos went on to develop
a second, ventral neuraxis and anterior nervous sys-
tem. More strikingly, the duplicate nervous system
was fully composed of host tissue, while the trans-
plant gave rise to a second notochord (dorsal meso-
derm) underlying it. This result suggested strongly that
the grafted tissues determinative influences on its
surroundings converted the surrounding ventral ecto-
derm into the second nervous system (Spemann and
Mangold, 1924). The authors named the dorsal blasto-
pore lip the Organizer, and hypothesized that during
normal development this region determined the choice
of a neural fate for the dorsal ectoderm. They also
proposed that the effect of the Organizer on the respon-
sive ectoderm necessarily would involve cell-to-cell
communication.
In the ensuing years, much effort has been applied
for identifying the exact signals that emanate from the
Organizer and activate the signaling pathways that bias
the ectoderm into neural fate in vertebrates. The result-
ing picture, derived from data obtained by various
groups, indicates that neural induction is a multi-step
process. The amphibian model, Xenopus laevis, has
continued to be of major importance to our understand-
ing of neural induction due to the ease of experimental
readout of neural induction in ectoderm explants. In
recent years, the chick embryo has added another
layer of complexity to the interpretation of the results
obtained from the amphibian model. In the following
sections we will present the major results derived from
both model systems and the model that is emerging
from those results. For the purposes of this chapter, we
will focus our discussion on the earliest step of neural
induction, which is the choice between epidermal and
neural precursor fate.
1.2 Neural Induction in the Xenopus
Embryo The Early Experiments
In the decade of 19902000, the search for the
Organizers neural inducing factors intensified and was
mainly performed in the Xenopus embryo. Based on
the characteristic of the Organizer, it was agreed that
a bona fide candidate for direct neural inducer had to
fulfill certain criteria: it should cause axis duplication
in whole embryos, it should be expressed in the dor-
sal blastopore (Organizer) region and elimination of its
activity should interfere with normal neural develop-
ment. The experimental paradigm used to screen for
candidate neural inducers was based on the fact that,
by definition, induction involves a signaling source
and a responsive target. Based on Spemmans exper-
iment, the endogenous source of neural inducers is
the Organizer and the responsive tissue the ectoderm.
Thus, ectoderm explants assays were used as an initial
screen for candidates. Ectoderm explants (also known
as animal caps) are cultured from a piece of ecto-
derm excised from the animal pole of late blastulas,
the lower part of which constitutes the blastocoele
roof (Fig. 1.1a). At this stage, the ectoderm is not
yet committed to an epidermal or neural fate and
responds to growth factors in the media or overex-
pression of relevant mRNAs by adopting different cell
fates, which are verified through the expression of
marker genes. When cultured as an intact tissue in
saline solution, ectoderm explants express genes char-
acteristic of epidermal tissue (Kintner and Melton,
1987). However, if the explant is co-cultured with a
dorsal blastopore lip, neural markers are expressed
instead (Kintner and Melton, 1987). Thus, a genes
neural-inducing activity is identified if there is upregu-
lation of the expression of neural markers and decrease
in the expression of epidermal genes. Importantly,
because the Organizer is part of the dorsal meso-
derm, genes that increased neural marker expression
but also induced mesoderm markers, were not con-
sidered direct neural inducers, as their effect could
be indirect, through additional factors secreted by the
mesoderm.
The first molecule to fulfill all of the above-
mentioned criteria for direct neural induction was
Noggin, a secreted polypeptide first identified by
Smith and Harland (1992) in the Xenopus. Afterwards,
Follistatin (Hemmati-Brivanlou et al., 1994) and
Chordin (Sasai et al., 1994), were also isolated from
Xenopus embryos on the basis of their neuraliz-
ing activity. All of these factors fulfilled the above-
mentioned conditions, including expression at the
Organizer. At the time, these molecules were thought
to act by directly stimulating neural fate, albeit through
an as yet unidentified mechanism.
1 Neural Induction 3

Fig. 1.1 Epidermal default

model versus neural default
Model. (a) In the epidermal
default model the normal fate
of an ectodermic tissue would
be epidermal, unless this
ectoderm is stimulated by
external factors (such as those
provided by addition of the
dorsal blastopore lip). (b) In
the neural default model,
the intact ectoderm secretes
anti-neural/pro-epidermal
factors. Induction of neural
fate occurs either by
co-culture with the dorsal
blastopore lip, which secretes
neuralizing factors or
dissociation of the ectoderm.
In the former, neuralizing
factors counteract the effect of
endogenous pro-epidermal
factors. In the latter case,
dissociation of the ectoderm
dilutes these factors and
generates neural fate.
Addition of ectoderm extract
restores epidermal fate

1.3 Neural Default Model of neural fate (Godsave and Slack, 1989; Grunz and
Tacke, 1989; Sato and Sargent, 1989). Thus, it was
proposed that the ectoderm has neural default fate,
Insight on the mode of action of these molecules came
which is revealed in the absence of exogenous sig-
from a second series of experiments that explored
naling (reviewed by Muoz-Sanjun and Brivanlou,
the effect of cell dissociation on ectoderm cell fate.
2002).
When ectoderm explants are dissociated into individ-
The addition of concentrated ectodermal super-
ual cells and cultured as such for a set period of time,
natant to dissociated cell cultures prevented the expres-
they express neural markers, instead of epidermal ones
sion of neural markers after ectodermal dissociation
(Fig. 1.1b). Remarkably, this occurs in the absence of
(Grunz and Tacke, 1990). Thereafter, candidate pro-
the dorsal blastopore lip and without the addition of
teins for the role of epidermal factor were added
exogenous factors (Godsave and Slack, 1989; Grunz
onto dissociated cultures and tested for their ability to
and Tacke, 1989; Sato and Sargent, 1989; Wilson et al.,
restore epidermal fate while suppressing neuralization.
1997). These data led to the hypothesis that neural-
These screens identified Bone Morphogenetic Protein
ization is the default fate for ectodermal cells, and
4 (BMP4), a member of the Transforming Growth
that the cellcell interactions that occur in an intact
Factor beta (TGF-) superfamily as a potent epidermal
ectodermic tissue somehow inhibit this developmen-
inducer. When BMP4 is added to a culture of cells dis-
tal path, resulting in an epidermal fate (Fig. 1.1b).
sociated from the ectoderm it induces the expression of
Once the tissue is dissociated, these epidermal fac-
epidermal markers (Wilson and Hemmati-Brivanlou,
tors are sufficiently diluted so as to allow development
1995). Moreover, the expression pattern of BMPs in
4 K.L. Almeida et al.
the Xenopus gastrula is consistent with the role of
epidermal factor: BMP4 is found throughout the
ectoderm prior to gastrulation but, afterwards it is
excluded from the neural plate (Fainsod et al., 1994;
Hemmati-Brivanlou and Thomsen, 1995). Finally,
inhibition of BMP signaling in ectodermal cells with
dominant-negative receptors or antisense BMP4 RNA
neuralizes ectodermal cells (Sasai et al., 1995). This
last set of data was consistent with the model that
inhibition of endogenous BMP signaling, through
dilution, directs dissociated ectodermal cells towards
neural fate.
1.4 BMP and the Neural Inducers
The discovery of the neuralization-suppressing effect
of BMP4 suggested a new hypothesis for the mode
of action of the direct neuralizers (Noggin, Chordin
and Follistatin), that is through the inhibition of BMP4
action. Further experiments showed that, indeed,
Noggin and Chordin directly bind to BMP4 protein and
interfere with its ligation to its receptor (Zimmerman
et al., 1996; Piccolo et al., 1996). Follistatin also
binds to BMPs and, while still allowing ligation to its
receptor, forms a trimeric complex that inhibits sig-
naling (Nakamura et al., 1990; Fainsod et al., 1997;
Iemura et al., 1998). Interestingly, molecular studies
have shown that different from Noggin and Follistatin
the inhibitory activity of Chordin on BMP resides in
specific cysteine-rich (CR) domains and is phylogenet-
ically conserved (Abreu et al., 2002).
The model that emerged was one in which the deci-
sion on the neural or epidermal fate of the ectoderm
depends on the level of BMP signaling. When BMP
signaling is decreased, either through dilution in disso-
ciated cultures or inhibition by neural inducers, ecto-
derm will progress towards a neural fate. Conversely,
when BMP signaling prevails, the ectoderm will form
epidermis.
This model is consistent with the conditions occur-
ring during normal Xenopus development: On the ven-
tral ectoderm of the gastrulating embryo, which is dia-
metrically opposite to the Organizer and which devel-
ops into the epidermis, high levels of BMP are detected
(Jones et al., 1996; Reem-Kalma et al., 1995). In con-
trast, the dorsal ectoderm, where neurulation occurs, is
in close proximity to the Organizer, which is the source
of BMP-inhibiting neural inducers. Accordingly, it
has relatively low levels of BMP signaling. Likewise,
this model explains the double-neural axis phenotype
in Spemann and Mangolds original Organizer graft
experiment: the grafting of an additional Organizer in
the ventral region provided a source of neural inducers
that inhibited BMP signaling in that region, allow-
ing the ventral ectodermal cells to follow their default
neural fate.
1.5 Challenges to the Neural Default
Model
The model of neural induction based on the sim-
ple inhibition of BMP signaling by its antagonists
expressed at the Organizer has been challenged, how-
ever, by results which suggest that neural induction
is a more complex process, involving additional fac-
tors. One of these might be Fibroblast Growth Factor
(FGF; Kengaku and Okamoto, 1993). FGF treatment
increases expression of neural markers and decreases
that of epidermal markers, (Kengaku and Okamoto,
1993, 1995; Lamb and Harland, 1995; Uzgare et al.,
1998). Furthermore, dominant-negative FGF receptor
inhibits the neuralizing effects of ectoderm dissocia-
tion and of noggin overexpression in whole embryos
(Hongo et al., 1999; Launay et al., 1996). Together,
these data suggested that FGF might also be nec-
essary to promote neural induction. This was just
the beginning of a series of questions regarding the
sufficiency of BMP inhibition in the neural induc-
tion model, which was primarily based on amphibian
embryos. The strongest evidence against the neural
default model of BMP inhibition, however, came from
experiments conducted in chick embryos.
1.6 Neural Induction and the Avian Node
Unlike the Xenopus embryos, whose development is
completely external, the avian embryo initiates its
development in the oviduct (reviewed in Wittler and
Kessel, 2004). The initial cleavage cycles that occur
there generate a flat blastoderm disc overlying the yolk.
When the egg is laid, the avian embryo is a translu-
cent disc composed of an epithelial monolayer the
1 Neural Induction
epiblast , which is subdivided into a central area pel-
lucida and an yolk-rich, extra-embryonic area opaca.
The circumference where the pellucida and the opaca
meet is known as the Marginal Zone. After a few hours,
a half-moon-shaped thickened region appears at the
Marginal Zone. This structure is known as Kohlers
sickle and is the morphological landmark for the pos-
terior end of the embryo and the site for initiation of
gastrulation. At the stage of its appearance, the epiblast
cells migrate posteriorly in a bilaterally symmetric
movement and anteriorly at the midline, forming the
primitive streak through which epiblast cell ingress and
form the definitive endoderm and mesoderm (Hatada
and Stern, 1994; Voiculescu et al., 2007; Joubin and
Stern, 1999 ). When sickle cells and the central epiblast
cells meet at the anteriormost edge of the primi-
tive streak, they form a thickened structure known as
Hensens Node, or simply the node (Fig. 1.2; Lawson
and Schoenwolf, 2001; Bachvarova et al., 1998 ). As
gastrulation continues, the primitive streak continues
Fig. 1.2 The expression pattern of BMP, Chordin and FGF during different stages of early chick development. The first row
represents a simplified dorsal view of the pre-gastrula and gastrulating embryo
5
expanding anteriorly and bisects the embryo into left
and right regions (Fig. 1.2).
The node is considered the avian homologue of the
amphibian dorsal blastopore lip. Its neural inductive
abilities and gene expression pattern are reminiscent
of the Organizer: transplantation of the node to the
extraembryonic area opaca induces a secondary neu-
raxis (Waddington, 1932; Storey et al., 1992), with
minimal participation of donor node cells (Storey
et al., 1992). Furthermore, the node expresses the avian
homologues of Goosecoid (Izpisua-belmonte et al.,
1993), Goosecoid-like gene (Gsx, Lemaire et al., 1997)
and Chordin (Streit et al., 1998), which are found in the
Xenopus Organizer.
1.7 Epiblast The Responsive Tissue
The induction and patterning of the avian nervous
system is a stepwise process that can be subdivided
6 K.L. Almeida et al.
into the ability of the epiblast to respond to neu-
ralizing signals (competence), the progressive sta-
bilization of this response (specification) and the
subsequent patterning of the neural region in its
diverse axis. The initial experiments by Waddington
(1932, 1993) showed that the avian blastulas epi-
blast layer is competent to respond to neuralizing
signals derived from the node. Indeed, fate mapping
experiments show that neural structures arise from
a widespread region of the epiblast prior to gas-
trulation (Hatada and Stern, 1994; Garca-Martnez
et al., 1993). Waddingtons conclusions were fur-
ther refined by Storey et al. who transplanted ectopic
nodes to progressively older host embryos and deter-
mined that the epiblast can generate a full antero-
posterior neural axis up to early gastrula stages (Storey
et al., 1992; Streit et al., 1997). Thereafter, the
epiblast cannot be induced to form anterior neural
structures.
The precise stage at which the epiblast first demon-
strates that it is competent to follow neural fate has
been progressively pushed back as more molecular
markers have become available. For instance, the early
neural marker Sox3 and late marker Sox2 have been
used as standard indicators of chick neural specifi-
cation (Rex et al., 1997; Streit et al., 2000, 1997;
Uchikawa et al., 2003). Sox3 is detected throughout
the epiblast before neural induction in pre-gastrula
embryos and becomes restricted to the future neu-
roectoderm as development progresses. Sox2 is first
detected around the time when neural induction is
believed to occur and its expression is limited to
the neuroectoderm (Rex et al., 1997; Muhr et al.,
1999).
Accordingly, immediately prior to gastrulation, the
potential of different regions of the epiblast differ.
Cultures of explants derived from central epiblast gen-
erated Sox2 and Sox3-positive cells whereas cultures
derived from explants removed from regions closer
to the marginal zone did not. Rather, these periph-
eral explants express genes indicative of epidermal
fate (Fig. 1.2, Wilson et al., 2000). Thus, by fol-
lowing the expression of Sox3 and Sox2 in cultured
epiblast explants, the earliest stage in which epiblast is
compartmentalized into neural and epidermal domains
was identified to be immediately prior to egg-laying
(Wilson et al., 2000). At this stage, neural fate is
restricted to the central epiblast and epidermal fate to
the peripheral epiblast.
1.8 Inhibition of BMP in the Avian
Context
The search for avian neural inducers that compartmen-
talize the epiblast into neural or epidermal fate was
initially based on a parallelism between the inductive
abilities of Hensens Node and Spemanns Organizer.
In support of this idea was the expression pattern of
BMPs and its inhibitors in late Primitive Streak stages:
Prior to egg-laying, BMP is present throughout the
epiblast but, when neuro-epidermal compartmentaliza-
tion occurs, it becomes excluded from the prospective
neural tissue (Wilson et al., 2000; Streit et al., 1998,
Watanabe and Le Douarin, 1996; Streit et al., 1998).
Likewise, the TGF-beta inhibitors chordin and noggin,
which are expressed anterior to Kohlers Sickle prior to
gastrulation, are found at the anterior tip of the prim-
itive streak in early gastrulas and are restricted to the
notochord and the node in late gastrulas (Streit et al.,
1998; Streit and Stern, 1999; Connolly et al., 1997).
Altogether, these data suggested that in chick, simi-
lar to Xenopus, BMP and its inhibitors are present in
complementary regions and that definition of a BMP-
activity-free neural domain plays a crucial role in
neural induction.
However, contrary to the results obtained in
amphibian embryos, application of ectopic chordin
onto early gastrula embryos cannot induce neural fate
in non-neural ectoderm (Streit et al., 1998). Moreover,
it would be expected, from the results in the frog
model, that exposure to ectopic BMP would con-
vert the presumptive neural domain into epidermal.
Surprisingly, application of BMP onto early gastru-
las neural domains does not inhibit Sox3 or Sox2
expression (Streit et al., 1998). Inhibition of BMP
signaling through overexpression of Smad6 or dom-
inant negative BMP receptor is also not sufficient
for neural induction (Linker and Stern, 2004). These
results, together with the findings that central epiblast
is specified as neural prior to egg-laying (see previ-
ous section), indicated that at early gastrula stages
the neuro-ectodermal regions are already specified and
that the search for the initial neuralizing step should
include earlier developmental stages.
Thus, Wilson and collaborators investigated the
identity of the signals that compartmentalized the cen-
tral and peripheral epiblast into their respective neural
and epidermal fates in pre-gastrula embryos. At this
1 Neural Induction
stage, the central epiblast is still susceptible to BMP
and will respond to its presence by converting from
neural to epidermal fate (Streit et al., 1998; Wilson
et al. 2000). Thus, in early chick epiblasts, the Xenopus
neural induction model holds true, in that BMP sig-
naling confers an epidermal bias and that its absence
is necessary for neural fate. The dynamics of BMP
expression at this stage is consistent with its role as the
endogenous epidermalizing signal BMP is downreg-
ulated in central epiblast and maintained in peripheral
epiblast (Streit et al., 1998; Wilson et al., 2000). This
plasticity ends with the onset of gastrulation (HH4)
(Fig. 1.2; Wilson et al., 2000). The neural domains
progressive resistance to BMP reflects the gradual
commitment to neural fate that occurs during normal
embryonic development.
1.9 FGF Signaling and Neural Induction
The question that remains is: what is the identity of
the endogenous factor(s) that inhibit BMP signaling in
the pre-gastrula central epiblast? Contrary to expecta-
tions, BMP signaling cannot be directly antagonized
by secreted BMP-inhibitors in pre-gastrula embryo.
Although Chordin is expressed at the gastrulas node,
neither Chordin, Noggin, Follistatin or Caronte were
detected in central or peripheral epiblast in pre-gastrula
embryos (Levin, 1998; Wilson et al., 2000). Moreover,
these inhibitors cannot induce neural markers by them-
selves (Streit et al., 1998, 2000). In other words, an
alternative signaling mechanism must maintain the
central epiblast BMP-free for the initial step in neural
induction to occur.
The answer came from a series of elegant experi-
ments that provided strong evidence that FGF meets all
the requirements for a role as an endogenous inhibitor
of BMP in avian blastulas. Firstly, FGF3 is expressed
in pre-gastrula central epiblast (Mahmood et al., 1995;
Wilson et al., 2000, 2001). Furthermore, exogenously
applied FGF can induce the expression of early neural
markers (Streit et al., 2000). Blockade of endogenous
FGF signaling inhibits expression of Sox3. Inhibition
of FGF signaling blocks neuralization and induction
of ectopic neural plate by a grafted organizer (Streit
et al., 2000). Lastly, the FGF pathway is required
for downregulation of BMP levels in the central epi-
blast, and absence of FGF signaling in the central
7
epiblast can be compensated for by the addition of
BMP inhibitors (Streit et al., 2000; Wilson et al., 2000,
2001). Together, these data suggest that FGF is a puta-
tive early neural inducer that acts by counteracting
BMP signaling in the central epiblast.
These results agree with the previously mentioned
effects of FGF on Xenopus embryos. However, at the
time that those reports appeared, FGF was considered
mainly a posteriorizing signal that acted secondarily on
the neural domain generated by inhibition of BMP sig-
naling. In light of the compelling data obtained from
chick embryos, the role of FGF as a primary neural-
izing signal was revisited in the amphibian embryo as
well. This reassessment was done with ex vivo ectoder-
mal explants and in vivo analysis of ventral ectoderm
fate in whole embryos. The results derived from in
vivo experiments differed somewhat from the clas-
sical ex vivo experiments. While overexpression of
truncated TGF-beta receptor was sufficient to induce
Sox2 expression in amphibian ectodermal explants
(Wilson and Hemmati-Brivanlou, 1995), it did not
induce a similar response in whole embryo ventral
ectoderm (Linker and Stern, 2004; Delaune et al.,
2005). In this experimental paradigm, ectopic expres-
sion of neural markers was achieved when there was
concomitant inhibition of BMP and stimulation of FGF
signaling (Linker and Stern, 2004). Moreover, in the
absence of FGF signaling, the ectoderm cannot be neu-
ralized by inhibition of BMP (Delaune et al., 2005).
These results strongly suggest that, similar to the
avian embryo, neuralization in the amphibian embryo
requires interaction of the FGF and BMP pathways.
The interaction between both pathways has been
mapped to Smad1, a downstream nuclear effector of
the BMP pathway. Smad1 nuclear translocation and
transcriptional activity are increased when it is phos-
phorylated at the carboxy-terminal upon activation of
the BMP receptor serine/threonine kinase (Massagu
and Chen, 2000). This activity is required for BMP-
induced epidermal fate (Wilson et al., 1997; Nakayama
et al., 1998). In contrast, when Smad1 is phospho-
rylated by MAPK in the central linker region, both
nuclear translocation and transcription are inhibited
(Kretzschmar et al., 1997).
FGF signals through receptor tyrosine kinases that
ultimately activate MAPK, which in turn phospho-
rylates Smad1 (Pera et al., 2003). Underscoring the
importance of the MAPK pathway during Xenopus
neural development, MAPK activity is required for
8 K.L. Almeida et al.
Fig. 1.3 Neural and
epidermal fate are
determined by Smad1
activity, which in turn is
regulated by
phosphorylation of its
serine/threonine residues.
FGF-induced phosphorylation
of the linker region retains
Smad1 in the cytoplasm and
results in neural fate, whereas
BMP-induced N-
phosphorylation of the
carboxy terminal promotes
translocation of Smad1 to the
nucleus and results in
epidermal fate
neural induction by FGF and cell dissociation in ecto-
derm explants (Uzgare et al., 1998; Kuroda et al.,
2005). Thus, Smad1 integrates signals from the FGF
and BMP pathway. Its activity results from the oppos-
ing effects between FGF-induced linker region phos-
phorylation versus the BMP-driven phosphorylation of
the carboxy-region. Consistent with this idea, overex-
pression of a MAPK-kinase insensitive Smad1 inhib-
ited neural development in whole embryos, whereas
mutation of both MAPK and BMP-sensitive regions
resulted in very mild phenotype (Pera et al., 2003).
Thus, the final model that emerges places Smad1
in the centre of the choice between neural and epi-
dermal fate. In the presence of high levels of BMP
signaling, Smad1 is phosphorylated in the carboxy
terminal, which activates its nuclear activity and cul-
minates in epidermal fate. This epidermalizing effect
can be counteracted by FGF, which phosphorylates the
Smad1 linker, inhibits its nuclear functions, resulting
in adoption of neural fate (Fig. 1.3).
Although this model accounts for most of the results
in the field, there are some points that must be consid-
ered: firstly, besides FGF there are other growth factors
that can activate MAPK activity, which raises the pos-
sibility that additional secreted proteins can modulate
neural induction (Linker and Stern, 2004). Second,
MAPK has other target proteins, amongst them Smad2
and Smad3, components of another TGF-beta pathway.
Therefore, it is possible that FGF modulates additional
pathways for its neuralizing effect. Indeed, there is
evidence that suppression of both Smad1 and Smad2
activity are necessary for neural induction in ventral
ectoderm (Chang and Harland, 2007). Furthermore,
Chordin
Noggin
FGF
BMP
MAPK
P P
MH1 LINKER MH2 -C
NEURAL EPIDERMAL
the FGF pathway itself is modulated by other signals
that are present during acquisition of neural compe-
tence. For instance, in the chick embryo, the Wnt
pathway suppresses FGF signaling in the lateral epi-
blast (Wilson et al., 2001). Lastly, as mentioned above,
cell fate induction occurs in a continuous and progres-
sive fashion. Therefore, the response of a target tissue
to neuralizing or epidermalizing signals depends on its
differentiation state at the time of exposure. An exam-
ple of this is neuralization through BMP inhibition in
Xenopus embryos. The response to BMP inhibition
is lost prior to the onset of gastrulation (Wawersik
et al., 2005). Likewise, neural induction in Xenopus
embryos is most sensitive to removal of FGF signaling
during mid-blastula transition (Delaune et al., 2005).
Although these results are still under discussion (de
Almeida et al., 2008) and the exact period when each
identified player is required for normal progression of
neural development is still unclear, it is the general
consensus that the plasticity of the ectoderm decreases
with time due to stabilization of cell fate (Streit et al.,
1998; Wawersik et al., 2005; Linker and Stern, 2004;
reviewed in Stern, 2005).
In conclusion, since the molecular identification
of direct neural inducers the development field has
proposed and refined models for the signaling that
underlies the choice between epidermal and neural fate
from the ectoderm. Even though the current model
does not account for all the complexity that occurs in
this process, the speed with which new findings are col-
lected and incorporated into the most recent hypothesis
has increased, and a more comprehensive panorama
should emerge in the next few years.
1 Neural Induction
References
Abreu JG, Coffinier C, Larrain J, Oelgeschlager M, De Robertis
EM (2002) Chordin-like CR domains and the regulation
of evolutionarily conserved extracellular signaling systems.
Gene 287:3947.
Bachvarova RF, Skromne I, Stern CD (1998) Induction of primi-
tive streak and Hensens node by the posterior marginal zone
in the early chick embryo. Development 125:35213534.
Chang C, Harland RM (2007) Neural induction requires contin-
ued suppression of both Smad1 and Smad2 signals during
gastrulation. Development 134:38613872.
Connolly DJ, Patel K, Cooke J (1997) Chick noggin is expressed
in the organizer and neural plate during axial develop-
ment, but offers no evidence of involvement in primary axis
formation. Int J Dev Biol 41:389396.
de Almeida I, Rolo A, Batut J, Hill C, Stern CD, Linker C (2008)
Unexpected activities of Smad7 in Xenopus mesodermal and
neural induction. Mech Dev 125:421431.
Delaune E, Lemaire P, Kodjabachian L (2005) Neural induction
in Xenopus requires early FGF signaling in addition to BMP
inhibition. Development 132:299310.
Fainsod A, Deissler K, Yelin R, Marom K, Epstein M, Pillemer
G, Steinbeisser H, Blum M (1997) The dorsalizing and neu-
ral inducing gene follistatin is an antagonist of BMP-4. Mech
Dev 63:3950.
Fainsod A, Steinbeisser H, De Robertis EM (1994) On the
function of BMP-4 in patterning the marginal zone of the
Xenopus embryo. EMBO J 13:50155025.
Garcia-Martinez V, Alvarez IS, Schoenwolf GC (1993)
Locations of the ectodermal and nonectodermal subdivisions
of the epiblast at stages 3 and 4 of avian gastrulation and
neurulation. J Exp Zool 267:431446.
Godsave SF, Slack JM (1989) Clonal analysis of mesoderm
induction in Xenopus laevis. Dev Biol 134:486490.
Grunz H, Tacke L (1989) Neural differentiation of Xenopus
laevis ectoderm takes place after disaggregation and
delayed reaggregation without inducer. Cell Differ Dev 28:
211217.
Grunz H, Tacke L (1990) Extracellular matrix components pre-
vent neural differentiation of disaggregated Xenopus ecto-
derm cells. Cell Differ Dev 32:117123.
Hatada Y, Stern CD (1994) A fate map of the epiblast of the early
chick embryo. Development 120:28792889.
Hemmati-Brivanlou A, Kelly OG, Melton DA (1994) Follistatin,
an antagonist of activin, is expressed in the Spemann
organizer and displays direct neuralizing activity. Cell 77:
283295.
Hemmati-Brivanlou A, Thomsen GH (1995) Ventral mesoder-
mal patterning in Xenopus embryos: expression patterns and
activities of BMP-2 and BMP-4. Dev Genet 17:7889.
Hongo I, Kengaku M, Okamoto H (1999) FGF signaling and
the anterior neural induction in Xenopus. Dev Biol 216:
561581.
Iemura S, Yamamoto TS, Takagi C, Uchiyama H, Natsume
T, Shimasaki S, Sugino H, Ueno N (1998) Direct binding
of follistatin to a complex of bone-morphogenetic protein
and its receptor inhibits ventral and epidermal cell fates
in early Xenopus embryo. Proc Natl Acad Sci USA 95:
93379342.
9
Izpisua-Belmonte JC, De Robertis EM, Storey KG, Stern CD
(1993) The homeobox gene goosecoid and the origin of orga-
nizer cells in the early chick blastoderm. Cell 74:645659.
Jones CM, Dale L, Hogan BL, Wright CV, Smith JC (1996)
Bone morphogenetic protein-4 (BMP-4) acts during gas-
trula stages to cause ventralization of Xenopus embryos.
Development 122:15451554.
Joubin K, Stern CD (1999) Molecular interactions continu-
ously define the organizer during the cell movements of
gastrulation. Cell 98:559571.
Kengaku M, Okamoto H (1993) Basic fibroblast growth fac-
tor induces differentiation of neural tube and neural crest
lineages of cultured ectoderm cells from Xenopus gastrula.
Development 119:10671078.
Kengaku M, Okamoto H (1995) bFGF as a possible morphogen
for the anteroposterior axis of the cental nervous system in
Xenopus. Development 121:31213130.
Kintner CR, Melton DA (1987) Expression of Xenopus N-CAM
RNA in ectoderm is an early response to neural induction.
Development 99:311325.
Kretzschmar M, Doody J, Massague J (1997) Opposing BMP
and EGF signalling pathways converge on the TGF-beta
family mediator Smad1. Nature 389:618622.
Kuroda H, Fuentealba L, Ikeda A, Reversade B, De Robertis EM
(2005) Default neural induction: neuralization of dissociated
Xenopus cells is mediated by Ras/MAPK activation. Genes
Dev 19:10221027.
Lamb TM, Harland RM (1995) Fibroblast growth factor is a
direct neural inducer, which combined with noggin gen-
erates anterior-posterior neural pattern. Development 121:
36273636.
Launay C, Fromentoux V, Shi DL, Boucaut JC (1996) A trun-
cated FGF receptor blocks neural induction by endogenous
Xenopus inducers. Development 122:869880.
Lawson A, Schoenwolf GC (2001) Cell populations and mor-
phogenetic movements underlying formation of the avian
primitive streak and organizer. Genesis 29:188195.
Lemaire L, Roeser T, Izpisua-Belmonte JC, Kessel M (1997)
Segregating expression domains of two goosecoid genes dur-
ing the transition from gastrulation to neurulation in chick
embryos. Development 124:14431452.
Levin M (1998) The roles of activin and follistatin signaling in
chick gastrulation. Int J Dev Biol 42:553559.
Linker C, Stern CD (2004) Neural induction requires BMP inhi-
bition only as a late step, and involves signals other than FGF
and Wnt antagonists. Development 131:56715681.
Mahmood R, Kiefer P, Guthrie S, Dickson C, Mason I (1995)
Multiple roles for FGF-3 during cranial neural development
in the chicken. Development 121:13991410.
Massague J, Chen YG (2000) Controlling TGF-beta signaling.
Genes Dev 14:627644.
Muhr J, Graziano E, Wilson S, Jessell TM, Edlund T (1999)
Convergent inductive signals specify midbrain, hindbrain,
and spinal cord identity in gastrula stage chick embryos.
Neuron 23:689702.
Muoz-Sanjuan I, Brivanlou AH (2002) Neural induction, the
default model and embryonic stem cells. Nat Rev Neurosci
3:271280.
Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H
(1990) Activin-binding protein from rat ovary is follistatin.
Science 247:836838.
10
Nakayama T, Gardner H, Berg LK, Christian JL (1998) Smad6
functions as an intracellular antagonist of some TGF-beta
family members during Xenopus embryogenesis. Genes
Cells 3:387394.
Pera E, Ikeda A, Eivers E, de Robertis EM (2003) Integration of
IGF, FGF and anti-BMP signals via Smad1 phosphorylation
in neural induction. Genes Dev 17:30233028.
Piccolo S, Sasai Y, Lu B, De Robertis EM (1996) Dorsoventral
patterning in Xenopus: inhibition of ventral signals by direct
binding of chordin to BMP-4. Cell 86:589598.
Reem-Kalma Y, Lamb T, Frank D (1995) Competition between
noggin and bone morphogenetic protein 4 activities may reg-
ulate dorsalization during Xenopus development. Proc Natl
Acad Sci USA 92:1214112145.
Rex M, Orme A, Uwanogho D, Tointon K, Wigmore PM, Sharpe
PT, Scotting PJ (1997) Dynamic expression of chicken Sox2
and Sox3 genes in ectoderm induced to form neural tissue.
Dev Dyn 209:323332.
Sasai Y, Lu B, Steinbeisser H, Geissert D, Gont LK, De Robertis
EM (1994) Xenopus chordin: a novel dorsalizing factor
activated by orgaizerspecific homeobox genes. Cell 79:
779790.
Sasai Y, Lu B, Steinbeisser H, de Robertis EM (1995) Regulation
of neural induction by the Chd and Bmp-4 antagonistic
patterning signals in Xenopus. Nature 376:333336.
Sato SM, Sargent TD (1989) Development of neural induc-
ing capacity in dissociated Xenopus embryos. Dev Biol
134:263266.
Smith WC, Harland RM (1992) Expression cloning of noggin, a
new dorsalizing factor localized to the Spemann organizer in
Xenopus embryos. Cell 70:829840.
Spemann H, Mangold H (1924) Induction of embryonic primor-
dia by implantation of organizers from a different species.
Rouxs Arch Entw Mech 100:599638. Re-published in Int J
Dev Biol 45:1338.
Stern CD (2005) Neural induction: old problem, new findings,
yet more questions. Development 132:20072021.
Storey KG, Crossley JM, De Robertis EM, Norris WE, Stern
CD (1992) Neural induction and regionalisation in the chick
embryo. Development 114:729741.
Streit A, Berliner AJ, Papanayotou C, Sirulnik A, Stern CD
(2000) Initiation of neural induction by FGF signalling
before gastrulation. Nature 406:7478.
Streit A, Lee KJ, Woo I, Roberts C, Jessell TM, Stern CD
(1998) Chordin regulates primitive streak development and
the stability of induced neural cells, but is not sufficient
for neural induction in the chick embryo. Development 125:
507519.
K.L. Almeida et al.
Streit A, Sockanathan S, Perez L, Rex M, Scotting PJ, Sharpe
PT, Lovell-Badge R, Stern CD (1997) Preventing the loss
of competence for neural induction: HGF/SF, L5 and Sox-2.
Development 124:11911202.
Streit A, Stern CD (1999) Establishment and maintenance of the
border of the neural plate in the chick: involvement of FGF
and BMP activity. Mech Dev 82:5166.
Uchikawa M, Ishida Y, Takemoto T, Kamachi Y, Kondoh
H (2003) Functional analysis of chicken Sox2 enhancers
highlights an array of diverse regulatory elements that are
conserved in mammals. Dev Cell 4:509519.
Uzgare AR, Uzman JA, El-Hodiri HM, Sater AK (1998)
Mitogen-activated protein kinase and neural specification in
Xenopus. Proc Natl Acad Sci USA 95:1483314838.
Voiculescu O, Bertocchini F, Wolpert L, Keller RE, Stern
CD (2007) The amniote primitive streak is defined by
epithelial cell intercalation before gastrulation. Nature 449:
10491052.
Waddington CH (1932) Experiments on the development of
chick and duck embryos cultivated in vitro. Philos Trans R
Soc Lond B Biol Sci 221:179230.
Waddington CH (1933) Induction by the primitive streak and its
derivatives in the chick. J Exp Biol 10:3848.
Watanabe Y, Le Douarin NM (1996) A role for BMP-4 in the
development of subcutaneous cartilage. Mech Dev 57:6978.
Wawersik S, Evola C, Whitman M (2005) Conditional BMP
inhibition in Xenopus reveals stage-specific roles for BMPs
in neural and neural crest induction. Dev Biol 277:
425442.
Wilson SI, Graziano E, Harland R, Jessell TM, Edlund T (2000)
An early requirement for FGF signalling in the acquisition of
neural cell fate in the chick embryo. Curr Biol 10:421429.
Wilson PA, Hemmati-Brivanlou A (1995) Induction of epi-
dermis and inhibition of neural fate by Bmp-4. Nature
376:331333.
Wilson PA, Lagna G, Suzuki A, Hemmati-Brivanlou A
(1997) Concentration-dependent patterning of the Xenopus
ectoderm by BMP4 and its signal transducer Smad1.
Development 124:31773184.
Wilson SI, Rydstrom A, Trimborn T, Willert K, Nusse R, Jessell
TM, Edlund T (2001) The status of Wnt signalling regu-
lates neural and epidermal fates in the chick embryo. Nature
411:325330.
Wittler L, Kessel M (2004) The acquisition of neural fate in the
chick. Mech Dev 121:10311042.
Zimmerman LB, De Jesus-Escobar JM, Harland RM (1996) The
Spemann organizer signal noggin binds and inactivates bone
morphogenetic protein 4. Cell 86:599606.
Chapter 2

Neurogenesis: A Change of Paradigms

Luiz E. Mello and Beatriz M. Longo

Contents under physiological and pathological conditions from

the self repair and medical perspective. We discuss fac-
2.1 Historical Overview . . . . . . . . . . . . . 12 tors that influence adult neurogenesis and stem cell
2.2 Neurogenesis and Neurogenic Regions . . . . 14 behavior and their potential therapeutic implications.
2.3 Cell Death and Neurogenesis . . . . . . . . . 17
2.4 Neurogenesis and Inflammation . . . . . . . 20 Keywords Adult neurogenesis Cell death
2.5 Stem Cell Therapies for CNS Disorders . . . . 23
2.6 Concluding Remarks . . . . . . . . . . . . 25 Epilepsy Inflammation Stem cell
References . . . . . . . . . . . . . . . . . . . . 26
Abbreviations

Abstract Neurogenesis and neural stem cells in the AD Alzheimers disease

adult brain are relatively recent discoveries in neu- BDNF brain-derived neurotrophic factor
roscience. Since Cajals statement in 1928 it was bFGF basically fibroblast growth factor
conceived that in the adult brain no new neurons BrdU bromodeoxyuridine
were formed. However, along the history of neuro- CXCR4 chemokine receptor 4
science new findings and observations did not fit to the DG dentate gyrus
established paradigm and novel concepts were formed. EGF epidermal growth factor
Thereafter, neurogenesis, a process of generation of EGFP enhanced green fluorescent protein
new neurons was proved to occur in the adult brain. GABA -aminobutyric acid
Later, the existence of stem cells made adult neuroge- GFAP glial fibrillary acidic protein
nesis more plausible, and thus dividing cells leading GFP green fluorescent protein
GDNF glial-derived neurotrophic factor
to new neurons in the adult brains became widely
HD Huntingtons disease
accepted. These two concepts, adult neurogenesis and
hNSC human neural stem cell(s)
neural stem cells, together overturned the so-called
IFN- interferon-gamma
no-new-neurons dogma and shifted the old paradigm.
IGF-1 insulin-like growth factor-1
Currently, adult neurogenesis is largely accepted by IL-1 interleukin-1 alpha
the scientific community, and is a classic example of IL-1 interleukin-1 beta
a long-held scientific theory being brought down. Here IL-6 interleukin-6
we review the most recent findings in neurogenesis and LPS lipopolysaccharide
neural stem cell and their implication in brain function NeuN neuronal nuclear antigen
NPC neuroprogenitor cell(s)
NPY neuropeptide Y
PD Parkinsons disease
L.E. Mello ()
Departamento de Fisiologia, Universidade Federal de So Pilo pilocarpine
Paulo, So Paulo, Brazil POMC pro-opiomelanocortin
e-mail: lemello@unifesp.br RMS rostral migratory stream

H. Ulrich (ed.), Perspectives of Stem Cells, 11

DOI 10.1007/978-90-481-3375-8_2, Springer Science+Business Media B.V. 2010
12
SDF1 stromal cell-derived factor-1
SE status epilepticus
SGZ subgranular zone
SRMS spontaneous recurrent motor seizures
SVZ subventricular zone
TLE temporal lobe epilepsy
TNF- tumor necrosis factor-alpha
2.1 Historical Overview
Thomas Kuhn, in his book The Structure of Scientific
Revolutions (1962) stated that a scientific paradigm is a
set of practices that define a scientific discipline during
a particular period of time. Kuhn described a paradigm
shift as a change in basic assumptions within the rul-
ing theory of science, and wrote Successive transition
from one paradigm to another via revolution is the
usual developmental pattern of mature science.
By the end of the nineteenth century, the great
anatomist Ramon y Cajal, together with other main fig-
ures of that time, Koelliker and His, proposed that the
structure of the adult mammalian CNS remains fixed
and constant (Gross, 2000).
In his 1928 paper, Cajal wrote: Once development
was ended, the fonts of growth and regeneration of
the axons and dendrites dried up irrevocably. In the
adult centers, the nerve paths are something fixed and
immutable: everything may die, nothing may be regen-
erated. (Ramon y Cajal, 1928). This suggestion was
well accepted and since then, it was thus created a
paradigm that in the adult brain no new neurons were
formed.
However, along the history of neuroscience new
findings and observations did not fit to the estab-
lished paradigm and new concepts were formed.
Neurogenesis and neural stem cells in the adult brain
are relatively recent discoveries in neuroscience. The
former is a process of generation of new neurons in
the CNS of adult brain (Kempermann, 2006), and the
latter refers to the presence of undifferentiated cells in
the adult brain that show the properties of undergoing
unlimited self-renewal and the potency to generate at
least two different cell types (McKay, 1997; van der
Kooy and Weiss, 2000; Weissman et al., 2001). Adult
neurogenesis originates from undifferentiated (precur-
sor) cells in the adult brain. In this logic, these two con-
cepts, adult neurogenesis and neural stem cell, together
L.E. Mello and B.M. Longo
overturned the so-called no-new-neurons dogma and
shifted the old paradigm.
With respect to Cajals studies in 1928, with the
technology available at that time, he could not distin-
guish newly formed neurons from neurons that were
already there since birth in adult brain. Cajal never
found evidence whether neurons could divide, and he
was right. Even today it remains established (at least
for the moment) that neurons do not divide.
The first evidence that the adult brain contained
dividing and proliferating cells with neural phenotypes
was reported by Joseph Altman in the 1960s (Altman,
1962, 1963; Altman and Das, 1965). Although histori-
cally the first suggestion of the existence of dividing
cells in the adult CNS was raised by Hamilton (see
Trujillo et al., 2009 for review), no credits were given
to him, and the nonexistence of new postnatal neurons
became a central dogma in neuroscience for almost
a century. Autoradiography with 3 H-thymidine con-
tributed for the advances in this area. 3 H-thymidine
incorporates into the DNA during the S phase of
the cellular cycle, thus enabling cell division to be
visualized by autoradiography, and demonstrating that
progenitor cells divide and their progeny shows neural
phenotypes. Altman combined intracranial injection of
tritiated thymidine with brain lesions in rats, and found
labeled neurons and neuroblasts suggesting the possi-
bility of proliferation of cells and differentiation onto
neurons in adult rats. In his paper he wrote: It is
commonly stated that in higher vertebrates neogene-
sis of nerve cells is restricted to the early stages of
embryonic development. This belief is based on the
observation that neurons with mitotic figures are absent
in the central nervous system of higher vertebrates.
However, this does not definitely rule out the neogene-
sis of neurons in the adult, for new neurons might arise
from nondifferentiated precursors, such as ependymal
cells. . . . If the general observation is valid that mitotic
figures are absent in the brain of adult mammals,
these findings might suggest that the labeled neurons
were formed from undifferentiated cells which divided
mitotically during the period at which the adminis-
tered thymidine-H3 was available. The presence of
labeled neuroblasts, mostly in fiber tracts, would sup-
port such a process of neurogenesis (Altman, 1962).
Altman was very careful in interpreting his findings
and was aware of the false impression of neuronal
labeling (Nottebohm, 1985). At that time, the concept
of stem cell in the adult brain did not yet exist, causing
2 Neurogenesis: A Change of Paradigms
adult neurogenesis to remain an issue that was hard to
sustain.
Fifteen years later, a series of electron microscopy
studies by Michael Kaplan demonstrated the neuronal
nature of the cells labeled with tritiated thymidine in
the hippocampus, the olfactory bulb, and the visual
cortex (Kaplan and Hinds, 1977). In these labeled cells
they identified somatic synapses and small neurites
which are typical of neural cells (Kaplan, 1981, 1985;
Kaplan and Bell, 1984).
Although well documented in these previous stud-
ies, neurogenesis could not be confirmed in rhesus
monkeys. Pasko Rakic in 1984, did not find radiola-
beled neurons in the adult brain of rhesus monkeys,
and concluded that all neurons of the rhesus monkey
brain are generated during prenatal and early postna-
tal life (Eckenhoff and Rakic, 1984). Based on this,
Rakic speculated that, differently of rodents, a stable
population of neurons in primates may be important
for the continuity of learning and memory over a life-
time (Eckenhoff and Rakic, 1988). In 1999, however,
also Rakic published evidence for neurogenesis in the
dentate gyrus of adult primates (Kornack and Rakic,
1999).
In 1981, Fernando Nottebohm published an elegant
paper named A brain for all seasons: cyclical anatom-
ical changes in song control nuclei of the canary
brain (Nottebohm, 1981), followed by a series of
studies on adult neurogenesis in songbirds (Goldman
and Nottebohm, 1983; Paton and Nottebohm, 1984;
Nottebohm, 1985; Burd and Nottebohm, 1985;
Nottebohm, 1996). Interestingly, Nottebohms studies
on the canary system of singing demonstrated that the
production of new neurons was related to the song
learning. These contributions corroborated with the
idea that new functional neurons with a clear behav-
ioral function could be generated in the adult brain in
the learning context.
During the 1990s, with the advances of new
techniques, a considerable number of experiments
demonstrated the presence of newborn neurons in the
adult mammal brain. Confirmation of adult neuro-
genesis came with important studies combining the
3 H-thymidine labeling method with cell type-specific
markers for immunohistochemical identification of the
phenotype of newly generated cells (Cameron et al.,
1993; Okano et al., 1993; Seki and Arai, 1993). The
other important contribution in this area was the use
of the immunohistochemicaly detectable thymidine
13
synthetic analog bromodeoxyuridine (BrdU). BrdU is
a halopyrimidine used therapeutically as antiviral and
antineoplastic agent (Begg et al., 2000; Freese et al.,
1994). BrdU labeling was developed as an alterna-
tive approach for determining the proliferative index
of tumors (Hoshino et al., 1989; Struikmans et al.,
1997), and was introduced for studying cell prolif-
eration in the developing brain by Nowakowski and
colleagues (1989). George Kuhn, working with Fred
Gage (1996), replaced tritiated thymidine by BrdU and
double-labeled cells to investigate the newly born cells
in the adult hippocampus.
Two areas of the adult mammalian brain are con-
sidered as neurogenic regions, the subgranular zone
of hippocampal dentate gyrus and subventricular zone
(see details about these neurogenic regions in the
next section). With these techniques (3 H-thymidine
and BrdU), several laboratories confirmed the pres-
ence of new neurons in these regions of the adult
brain. They reported adult neurogenesis to be modu-
lated by a broad range of physiological, experimental
and pathophysiological conditions, in both rodents and
primates.
Factors that increase neurogenesis include dietary
restriction (Lee et al., 2000), enriched environments
(Barnea and Nottebohm, 1994; Kempermann et al.,
1998; Nilsson et al., 1999; van Praag et al., 1999),
hippocampal-dependent learning-tasks (Gould et al.,
1999a; Lemaire et al., 2000), and voluntary physical
activity (van Praag et al., 1999; Kronenberg et al.,
2003). However, other studies showed that cell pro-
liferation decreases with age (Altman and Das, 1965;
Seki and Arai, 1995; Kempermann et al., 1998) and
can also be reduced by stressful experiences (Gould
et al., 1997, 1998; Tanapat et al., 2001) related to
glucocorticoid levels, which are released in response
to stress, suggesting that stress-induced inhibition of
cell proliferation is mediated by glucocorticoids (see
Gould and Gross, 2002, for an overview). There is also
some evidence that the decrease in the number of new
neurons affects performance in some hippocampal-
dependent learning-tasks (Shors et al., 2001, 2002) or
the acquisition in the water maze task (Kempermann
and Gage, 2002). Reduced neurogenesis might con-
tribute to major depression (Santarelli et al., 2003;
Kempermann and Kronenberg, 2003).
Similar studies showed that various trophic factors,
neurotransmitters and drugs modulate neurogenesis
in the dentate gyrus and subventricular zone (Craig
14
et al., 1996; Kuhn et al., 1997; Brezun and Daszuta,
1999; Malberg et al., 2000; Jin et al., 2006). Using
immunohistochemical detection of markers of the cell
cycle or BrdU incorporation, it has been demonstrated
that neurogenesis increased in the subventricular zone
of Huntingtons disease (HD), and in the hippocam-
pus of Alzheimers disease (AD) brains (Curtis et al.,
2003; Jin et al., 2004; Tattersfield et al., 2004). Newly
formed neurons are also increasingly found in the hip-
pocampus and subventricular zone of epileptic animals
(Parent et al., 1997), in strokes (Liu et al., 1998) and
traumatic brain injuries (Dash et al., 2001).
By the end of 1990s, some authors started to sug-
gest that adult neurogenesis could also occur in brain
areas other than hippocampus and subventricular zone
(Shankle et al., 1999; Gould et al., 1999b; Zhao et al.,
2003), although others, including Rakic (2002), have
questioned the scientific evidence of these findings.
Neurogenesis had also been described in the dentate
gyrus of human aged subjects (Ericksson et al., 1998).
Newborn neuronal cells immunopositive for BrdU and
neuronal nuclear antigen (NeuN), a marker of mature
neurons, were identified in the human adult dentate
gyrus of postmortem tissue samples obtained from
cancer patients who had been administered with the
drug as part of their treatment. In 2002, van Praag
and coworkers used retrovirus labeled with the green
fluorescent protein (GFP) to visualize newly gener-
ated cells in living tissue slices and were thus able to
confirm that newborn neurons in the adult rodent hip-
pocampus become functional granule cells (van Praag
et al., 2002). The occurrence of neurogenesis in adult
rodents was also confirmed by BrdU and retroviral
labeling in other studies (Seki and Arai, 1993; Corotto
et al., 1993; Kuhn et al., 1996; Yamada et al., 2004).
Neural stem cells in turn were characterized in the
adult brain by Brent Reynolds and Sam Weiss, by
Perry Barlett and colleagues and by Fred Gage and col-
leagues in the early 1990s (Reynolds and Weiss, 1992;
Richards et al., 1992; Kilpatrick et al., 1993; Kilpatrick
and Barlett, 1995; Palmer et al., 1995; Ray et al.,
1995). Weiss and Reynolds observed the formation of
spheres in cell cultures from adult mice striatum grown
in suspension (Reynolds and Weiss, 1992). The authors
called these structures neurospheres, and suggested
they derived from the adult brain cell population able
to divide dynamically. The potential of self-renewal
and the differentiation of stem cells could be observed
in neurospheres in vitro, which in turn, reflected the
L.E. Mello and B.M. Longo
potential of generating neurospheres in the presence of
growth factors.
The existence of stem cells, thus, made adult neuro-
genesis more plausible and thus dividing cells leading
to new neurons in the adult brains became widely
accepted. Finally, adult neurogenesis has been largely
accepted by the scientific community, and is a classic
example of a long-held scientific theory being brought
down.
2.2 Neurogenesis and Neurogenic
Regions
Adult neurogenesis and stem cells are firmly asso-
ciated to one another. To better understand the phe-
nomena of neurogenesis in the adult brain, the stem
cell concept should be considered. A stem cell is an
undifferentiated (unspecialized) cell that has two main
properties: the ability to divide (self-renewal) infinitely
(or seemingly so), and the potential to give rise to
specialized cell types (multipotency). Conceptually,
the immediate progeny of stem cells are the pro-
genitor cells that in turn generate differentiated cells
(Kempermann, 2006). To ensure self-renewal, stem
cells undergo two types of cell division: symmetric
division, that gives rise to two identical daughter cells
with the same stem cell properties, and asymmet-
ric division (Fig. 2.1a), that produces only one stem
cell and a progenitor cell with limited self-renewal
potential. Progenitors can go through several rounds
of cell of asymmetric division leading to progenitor
cell and a specialized cell (Fig. 2.1b) before terminally
differentiating into a mature cell.
The adult stem cell is an undifferentiated cell that is
found in a differentiated (specialized) tissue, which can
renew itself for a lifetime. Adult stem cells are asso-
ciated with most tissues of the body probably as part
of a tissue/cell renewal mechanism. Sources of adult
stem cells have been found in almost all areas of the
body including bone marrow, blood stream, cornea and
retina of the eye, the dental pulp of the tooth, liver,
skin, gastrointestinal tract, and the pancreas. Indeed, it
seems that the discovery of more sources of adult stem
cells in the body is only a question of time.
Traditionally, adult stem cells are primarily multi-
potent, meaning that they appear to be more limited
in their differentiation potential and to be committed
2 Neurogenesis: A Change of Paradigms
Fig. 2.1 True neural stem
cells provide slow occurring
unlimited self-replication in
asymmetric divisions that
produces progenitor cells
with limited self-renewal
potential and another stem
cell (a). Neural progenitor
cells are already committed to
a neural lineage
(compromised cells) leading
to specialized cells terminally
differentiating into mature
cells
to their tissue of origin, as compared to embryonic
stem cells, which are able to give rise to all cell types
of the body. This view was dropped into question
by a number of studies that seemed to suggest that
stem or progenitor cells from one organ could cross
limits of tissue specificity and generate differentiated
cells in other organs, implying that adult stem cells
have a wider potential for differentiation (transdiffer-
entiation or plasticity) than previously assumed. For
example, studies have shown the generation of blood
cells from neural stem cells and of neurons and glia
from hematopoietic stem cells (Brazelton et al., 2000;
Mezey et al., 2000). Moreover, Purkinje neurons origi-
nated from transplanted bone marrow cells were found
in the cerebellum one year after bone marrow trans-
plantation (Priller et al., 2001; Weimann et al., 2003).
Likewise, in the brains of female patients who had
received a bone marrow graft from male donors, brain
autopsy results revealed neurons carrying the Y chro-
mosome (Mezey et al., 2000; Cogle et al., 2004). In
our hands, cells originating from the bone marrow have
the capacity to migrate proliferate and populate brain
regions in epileptic chimeric animals (Longo et al.,
2005). However, in all of these studies the experimen-
tally observed plasticity of adult stem cells occurs in
a very low frequency in terms of numbers of cells,
15
with even a smaller number assuming neuronal char-
acteristics suggesting the presence of a specific genetic
program of these cells of non-neural origin. In view
of that their progeny and, as a consequence, their
cellular identity is somewhat limited (Avots et al.,
2002).
Stem cells can be found in the adult brain. These
neural stem cells (NCS) are self-renewing, multipotent
cells that generate the main phenotypes of the nervous
system, including neurons and glial cells (astrocytes,
oligodendrocytes) (McKay, 1997; van der Kooy and
Weiss, 2000; Weissman et al., 2001).
Adult neurogenesis is defined as the process of gen-
erating new neurons in the adult brain, after fetal and
early postnatal development has ceased. The process
of adult neurogenesis comprises many steps, from the
division of a stem cell, called precursor cell, to the
full integration of the new neurons into the working
brain. It involves decisions at the precursor level, such
as whether symmetric or asymmetric divisions occur
before become a neuron.
A brain region that can generate neurons is called
neurogenic region, a term that implies the pres-
ence of immature precursor cells and more importantly
a microenviroment that is permissive for neuroge-
nesis to occur. New neurons are continually born
16 L.E. Mello and B.M. Longo

throughout adulthood in predominantly two regions of
the mammalian brain:
The subventricular zone (SVZ), where the new
cells migrate to the olfactory bulb via the rostral
migratory stream (Fig. 2.2a,b).
The subgranular zone (SGZ), part of the dentate
gyrus of the hippocampal complex, where new
granule cells are produced (Fig. 2.2c,d).
For these two regions several types of progenitor
cells were identified.
In the adult SVZ, new neurons are continuously
produced suggesting that neural stem cells persist
within this germinal layer (Alvarez-Buylla and Garca-
Verdugo, 2002). The SVZ is defined as a one- or two-
cell body wide zone below the ependyma, lining the
lateral ventricles, adjacent to striatum (Kempermann,
2006). In the adult olfactory system, the population
of precursor cells is found in the SVZ in the tempo-
ral walls of the lateral ventricles. Progenies migrate
over long distances along the rostral migratory stream
to the olfactory bulb, where they differentiate into
interneurons in the granule and periglomerular lay-
ers. The olfactory system also comprises the olfactory
epithelium, which is part of the peripheral nervous sys-
tem and in which massive adult neurogenesis occurs.
The SVZ, also known as ependymal region, con-
tains at least four different cell types defined by their
morphology, ultrastructure, and molecular markers.
Glial fibrillary acidic protein (GFAP)-expressing astro-
cytic cells act as the primary stem cells in the adult
SVZ (Doetsch et al., 1997). The authors of this work
named these astrocytes as B cells. Another type, a
transiently amplifying progenitor cell (C cell), origi-
nates from B cells and, in turn, give rise to immature
neurons or neuroblasts (A cells). A cells are migra-
tory cells passing through the rostral migratory stream
(RMS) and reaching the olfactory bulb, where they dif-
ferentiate into interneurons. Type C cells are highly
proliferative precursors and form clusters next to the
chains of migrating A cells. The SVZ is largely sepa-
rated from the ventricle cavity by a layer of ependymal
cells (type E cells). B cells interact closely with E cells
and occasionally contact the ventricle lumen. Ongoing
replication of B and C cells suggests that one or both
of these cell types could be involved in the generation
of the new neurons, the A cells (Garca-Verdugo et al.,
1998; Lois et al., 1996; Doetsch and Alvarez-Buylla,
1996). Because A cells, themselves, divide (Lois and
Alvarez-Buylla, 1994; Menezes et al., 1995; Thomas
et al., 1996), it was also formally possible that they
simply generate more A cells. As many cell types in
the subventricular zone incorporate 3 H-thymidine in

Fig. 2.2 The two

neurogenic regions in the
brain, where the presence of
immature precursor cells
and a microenviroment
permissive allow for
neurogenesis.
Immunofluorescence for
BrdU stained cells in (a) and
(b) subventricular zone
(SVZ), where the new cells
migrate to the olfactory bulb
via the rostral migratory
stream; and in (c) and (d) the
subgranular zone (SGZ), part
of the dentate gyrus of the
hippocampal complex, where
new granule cells are
produced
2 Neurogenesis: A Change of Paradigms
adult mice, it is believed that more than one cell type
is dividing in this region. There is some controversy,
however, related to the identity of the stem cells that
could be E cells (Johansson et al., 1999) or type B
astrocytes (Doetsch et al., 1999).
For several decades, evidence identifying the hip-
pocampus as a region with a large degree of structural
plasticity has been accumulated. Investigations were
initially focused on exploring the organization of the
hippocampal formation, proposing that the hippocam-
pus is constantly changing under normal conditions.
According to these studies, the hippocampal forma-
tion is a dynamic area whose synapses and dendrites
are undergoing continuous rearrangement (see Gould,
2007).
In the adult hippocampus, neural precursor cells
reside in a narrow band of tissue in the border zone
between the granule cell layer and the hilus, the sub-
granular zone (SGZ). SGZ is defined as a layer about
three cell nuclei wide, including the basal cell band of
the granule cell layer and a two nucleus-wide zone in
the hilus (Kempermann, 2006). The progeny of these
continuously dividing cells migrate varying distances
into the granule cell layer where they differentiate
(Altman and Das, 1965; Cameron et al., 1993; Kuhn
et al., 1996; Palmer et al., 2000). They then extend
their dendrites, as all other granule cells do, into the
molecular layer and send axons along the mossy fiber
tract to the CA3, forming together with the hilus the
projection area of granule cells.
As in the SVZ, the putative stem cells of the
adult SGZ reveal a characteristic morphology resem-
bling radial glia and astrocytic properties (Seri et al.,
2001). Seri and colleagues demonstrated that BrdU
labeled astrocytes after antimytotic treatment in the
hippocampus generated neurons, astrocytes and oligo-
dendrocytes. Thus, astrocytes from the SGZ were able
to generate the three main types of cells in the brain,
exhibiting characteristics of multipotentiality of stem
cell.
Seaberg and Van der Kooy (2002), however, ques-
tioned whether the hippocampus could indeed contain
stem cells. They speculated that the detection of stem
cell in the primary precursor cell cultures derived from
the hippocampus could in fact be due to stem cell
contamination from the ventricular wall. It may also
be possible that the discrepancy resulted from differ-
ences in cell culture and isolation protocols, leading
to the enrichment of different cell population. The in
17
vivo neurogenesis observed in the adult dentate gyrus
is thus currently considered as derived from a neu-
ronal progenitor population and not from true stem cell
population.
2.3 Cell Death and Neurogenesis
Similarly to developmental neurogenesis, adult neuro-
genesis is also followed by significant apoptotic cell
death. During adult neurogenesis, neuronal progenitor
cells present in neurogenic areas divide and migrate to
their appropriate destination, while differentiating into
their mature phenotypes and connecting to their target
cells. These new neurons need support from neighbor-
ing glial cells and nutrients from blood to survive and
become part of the working brain. Importantly, as a
vital condition, they need to make connections with
other neurons. This process also includes migration
and differentiation of the progeny, and proliferation
processes (dendrites and axons establishing connec-
tions and forming synapses), and finally the presence
of sodium currents and the ability to generate action
potentials. Without these connections, the new neu-
rons shrink and die. In fact, many of these newborn
cells die shortly after their birth (being not much differ-
ent from the process taking place during development,
except for the massive scale of the latter), while the
surviving cells become functionally integrated into the
surrounding brain tissue.
It has been suggested that adult neurogenesis reca-
pitulates events occurring during embryonic develop-
ment (Kintner, 2002). In this view, newly produced
neurons in adult brain may die for the same reasons as
embryonic neurons do during development, resulting
from loss of trophic support and lack of connections.
Neurons that are not integrated in the neuronal network
are not functional and thus are removed by apopto-
sis. Since the generation and integration of these new
neurons takes time, it seems reasonable and a good
strategy to have some of them in stock all the time. In
case of necessity they can be integrated fast. If they
are not anymore needed, cells die and are replaced
by new ones (Kempermann et al., 2004). Although
in the adult brain the large amount of the dying cells
are immature cells, the mature neurons generated dur-
ing development also undergo apoptosis (Buss et al.,
2006; Kempermann, 2006). Coincidently, of all adult
18
brain regions the neurogenic zones show the high-
est incidence of apoptotic cell death, being about a
hundred times higher than in the rest of the brain
(Blaschke et al., 1996; Biebl et al., 2000), suggesting
that these two processes, apoptosis and neurogene-
sis are strongly linked. Moreover, in these neuro-
genic zones the location of apoptotic profiles coincides
exactly with the area where dividing progenitor cells
reside, as observed in cells double-immunostained for
apoptotic profiles and immature neuronal cells (Kuhn
et al., 2005). In the rodent brain, it was demonstrated
that approximately 70% of newly produced DG cells
and approximately 50% of olfactory bulb cells undergo
apoptosis within 30 days of their birth (Biebl et al.,
2000; Kempermann et al., 1997).
Different views have been proposed for the rela-
tionship between these two events (adult neurogenesis
and apoptosis). In fact there are at least two possi-
bilities for this interconnection. One is that already
mentioned whereby apoptosis allows the selection of
cells generated in excess (neurogenesis followed by
apoptosis). The other is the condition in which apopto-
sis is followed by neurogenesis as a sort of replacement
strategy. Magavi and colleagues (2000) showed that
degeneration-induced apoptosis in layer VI of the ante-
rior cortex induces proliferation of endogenous neural
precursors. These precursors showed the ability of dif-
ferentiation into neurons from the damaged region, and
these new neurons were able to reestablish appropri-
ate cortical-thalamic connections. As proposed by the
authors, the induction of highly selective apoptotic cell
death in individual cells could trigger neurogenesis.
A number of different combinations of both strategies
could take place given that apoptosis and neurogenesis
may in addition to the above represent different strate-
gies adapt to changing conditions. Furthermore it must
be considered that no process occurs in isolation and
one may affect the other.
The fact that neurogenesis might ensue after nat-
urally occurring or more obvious and widespread
lesional phenomena has clear implications for cell
replacement therapies. Nowhere has this been devel-
oped as much and as long as in treatment of
Parkinsons disease (PD) by Sweden scientists
(Lindvall et al., 1990; Bjrklund and Lindvall, 2000).
Yet the pioneering studies on the subject came from the
efforts by Madrazo in Mexico, who showed improve-
ment in PD symptoms after transplanting adrenal and
fetal substantia nigra cell extracts into the striatum of
L.E. Mello and B.M. Longo
patients with PD (Madrazo et al., 1987, 1988). The
studies by these and various other groups indicated that
transplanted cells from a number of different sources
can replace dopaminergic neurons damaged in PD,
thereby restoring and regenerating brain function.
In the hippocampus, the link between neurogen-
esis and cell death has been well documented (see
Gould in Andersen, 2007) as observed in studies
with adrenalectomy (Gould et al., 1991; Cameron
and Gould, 1994), mechanical or excitotoxic lesions
(Gould and Tanapat, 1997), ischemia and seizures
(Parent et al., 1997; Scott et al., 2000; Kee et al.,
2001; Kokaia and Lindvall, 2003; Scharfman, 2004),
all inducing cell death and proliferation of granule
cell precursors. Hastings and Gould (2003) suggested
that cell proliferation is enhanced with neuronal dam-
age by two alternative mechanisms: dying neurons
can release molecules that stimulate proliferation of
precursor cells; or neurons in the intact brain release
molecules that suppress cell proliferation. This inhi-
bition is removed after damage. This hypothesis is
reinforced by other lines of arguments based on the
balance between the birth and death of granule cells
(Gould et al., 1991), by the fact that proliferation in
the SVZ is upregulated under inflammatory condi-
tions (Calza et al., 1998), and by the observation that
the neurogenic structures do not grow in size. There
is however evidence for the contrary indicating that
between 3 and 12 months of age (a life period dur-
ing which a mouse is considered an adult) the mouse
dentate granule cell layer incorporates some additional
300,000 new neurons (Bayer, 1982; Bayer et al., 1982;
Kaplan and Hinds, 1977) and that young adult rats
have 9,400 dividing cells in the dentate proliferating
with a cell cycle time of 25 h (Cameron and Mckay,
2001). Therefore the balance between loss and incor-
poration of new neurons is not always maintained both
in physiological and pathological conditions.
Indeed, it has been shown that hippocampi of
patients with epilepsy show remarkable structural
changes of the dentate gyrus that have been initially
labeled granule cell dispersion and more recently tec-
tonic layer displacements (Houser, 1990; Sloviter
et al., 2004). This issue itself has been the subject of
a certain degree of debate regarding whether seizures
are cause or consequence of the cell derangement and
cell proliferation or the reverse (or both). The fact that
adult rodents (Mello et al., 1992; Gray and Sundstrom,
1998; Rougier et al., 2005) and adult primates (our own
2 Neurogenesis: A Change of Paradigms
unpublished observations) subject to status epilepticus
(SE) subsequently develop these altered hippocampal
profiles speaks for the possibility that these may also
be a consequence of seizures. It may thus be that the
adequate balance between loss and birth of neurons
in the hippocampus is not only valid for all species
but in addition is perturbed in neurological conditions
and that the excess of neurons contributes to disease
progression.
A good example of compensatory neurogenesis
replacing older neurons by new ones, renewing neu-
ral circuits, is the seasonal programmed cell death and
neurogenesis of high vocal center neurons in songbirds
(Nottebohm, 2004). As pondered by Nottebohm, there
is little evidence that adult neurogenesis contributes
to neuronal turnover in neurogenic regions. It seems
unlikely that animals in nature should have time to
recover from brain dysfunction by means of neuroge-
nesis process. Nottebohm suggested that neurogenesis
acts as source of neuronal replacement to keep neu-
ronal circuits functionally young (Nottebohm, 2002).
Accordingly, Kempermann proposed the hypothesis
that brain development never ends and that plasticity
can be taken as continued development (Kempermann
et al., 2004). In the hippocampus, new neurons are
added to the existing networks and to the dentate gyrus
throughout life.
Epilepsy, of all the neurological disorders, is the
only condition where there is evidence that more neu-
rogenesis may act as to increase rather than merely
replace dying neurons. Indeed, dentate granule cell
dispersion shown in humans (Houser, 1990; Houser
et al., 1990), initially suggested as a predisposing
developmental event, was later shown to be induced
in adult rodents by status epilepticus (SE) (Mello
et al., 1992) and to result from increased proliferation
rate (Parent et al., 1997). The relative contribution of
this event to the ensuing mossy fiber sprouting that
later develops in the rodent models of SE was subse-
quently discarded given that X-ray irradiated rodents,
in which dentate neurogenesis is impaired, still have
robust mossy fiber sprouting (Parent et al., 1999). The
issue of whether SE-induced neurogenesis is finely
adjusted as a compensatory phenomenon or generates
an excess of cells has been addressed in several stud-
ies (Mohapel et al., 2004; Covolan and Mello, 2000;
Zhao and Overstreet-Wadiche, 2008). Indeed, in the
kindling model of seizures where cell loss is mini-
mal, and may even be non existent in some resistant
19
areas such as the dentate gyrus, there is a sixfold
increase in proliferation rate (Bengzon et al., 1997)
and thus neurogenesis has been shown to be induced
independent of cell death (Ferland et al., 2002). In
the kainate-induced SE mouse model, neurogenesis
largely exceeds dentate cell loss as evidenced by the
marked increase in the area occupied by the dentate
cell layer (Suzuki et al., 1995; Gray and Sundstrom,
1998; Bouilleret et al., 1999). Similarly, a comparative
study using both the kainate and pilocarpine mod-
els of SE, Covolan and Mello (2000) suggested that
increased proliferation in seizures and epilepsy is a
function of stimulating available progenitors by means
of seizures without providing too much excitation and
ending up killing such cells. Therefore, similarly to
a host of naturally occurring conditions that stim-
ulate neural proliferation and neurogenesis, seizures
and epilepsy constitute powerful means to stimulate
this phenomenon (Fig. 2.3). To this end, experimen-
talmodels of epilepsy became widely used as a strategy
for studying the genes and relevant molecules that
regulate adult neural proliferation and neurogenesis
(Elliott et al., 2003; Altar et al., 2004; Hagihara et al.,
2005; Pirtilla et al., 2005; Wang and Baraban, 2007;
Choi et al., 2008 Steiner et al., 2008). Using pro-
opiomelanocortin-enhanced green fluorescent protein
(POMC-EGFP) transgenic mice to identify newborn
granule cells, the group of Linda Overstreet Wadiche
found that the seizure activity not only increased the
number of newborn granule cells, but it also dramati-
cally altered their dendritic morphology (see Zhao and
Overstreet-Wadiche, 2008). Labeling mitotic cells with
BrdU 2 days before SE and quantifying 2 weeks later,
Fig. 2.3 Schematic view of neural proliferation and cell loss
in epilepsy as a function of seizure intensity. The dashed line
represents the resultant outcome (net effect) of proliferation and
cell loss
20
they also suggested that preexisting newborn granule
cells as well as seizure-induced granule cells undergo
abnormal dendrite development during epileptogene-
sis. The authors concluded that seizures accelerated
the dendritic development of newborn granule cells
altering the wiring of individual cells into the existing
circuit.
Not all developmentally triggered protein expres-
sion is associated with seizure-induced proliferation
and migration process in the dentate gyrus (Mello
and Mendez-Otero, 1996), and it has even been sug-
gested that granule cell dispersion is not accompanied
by enhanced neurogenesis in temporal lobe epilepsy
(TLE) patients (Fahrner et al., 2007). It has been shown
in animal models of TLE that migration is disturbed,
leading to ectopic granule cells near the CA3 region
(Scharfman et al., 2000). Thus, careful understanding
of how much of the seizure-induced neurogenic pro-
cesses recapitulate ontogeny is important for the poten-
tial of using electrical stimulation (or even seizures) as
part of therapeutic strategies.
It has been considered that epileptic seizures repre-
sent one of the most severe stimulatory stresses that the
brain is exposed to, and that generalized status epilep-
ticus represents a very severe form of seizure (Kulkarni
and George, 1995). The participation of the newly born
cells in network reorganization and in the chronicity
of epileptic seizure is still unclear. The presence of
newly born cells (in normotypic and ectopic locations)
is systematically observed in the different models of
epilepsy. The mechanisms by which seizure activity
stimulates neurogenesis are still unknown. It remains
unclear to what degree the neurogenic response to
seizures is in attempt at regeneration or part of the
pathology itself.
2.4 Neurogenesis and Inflammation
Adult neurogenesis can be profoundly modulated
by several environmental stimuli, trophic factors,
cytokines, drug treatments, and various physio- and
pathological conditions. It is well established that
specific growth or neurotrophic factors influence neu-
ral precursor proliferation in the adult hippocampus
and SVZ. This group of molecules includes basically
fibroblast growth factor (bFGF), insulin-like growth
factor-1 (IGF-1), epidermal growth factor (EGF),
L.E. Mello and B.M. Longo
neuropeptide Y (NPY) and brain-derived neurotrophic
factor (BDNF) (Kuhn et al., 1997; berg et al., 2003;
Benraiss et al., 2001; Pencea et al., 2001; Scharfman
et al., 2002; Hansel et al., 2001; Howell et al., 2003).
Neurotransmitters, hormones, and psychotropic agents
have been also shown to influence neurogenesis in the
adult brain (see Parent, 2003 for review).
An interesting aspect is the correlation between
neurogenesis and inflammation caused by insults like
ischemia, trauma and seizures (Makowiak et al.,
2004; Bernardino et al., 2005; Vezzani, 2005; Ekdahl
et al., 2009). There is a growing body of evidence
indicating that inflammation in the CNS can either pro-
mote or inhibit adult neurogenesis (Simard and Rivest,
2004; Ekdahl et al., 2009; Bernardino et al., 2005).
A possible molecular explanation for these findings is
that the nervous and immune systems are integrated
in an intense crosstalk (Kerschensteiner et al., 2009).
Both systems produce a range of overlapping factors
that modulate cell growth and differentiation, such as
cytokines and chemokines released by the immune sys-
tem and neurotrophic and growth factors by the CNS,
although neither cytokines nor neurotrophic factors are
completely exclusive to either system (Kerschensteiner
et al., 2003). Expression in the nervous system has
now been described for cytokines of many families
including interleukins, interferons and members of the
tumor necrosis (TNF) family (Neumann et al., 1997;
Knoblach et al., 1999; Krumbholz et al., 2005; Liu
et al., 2007).
The immune-competent cells of the CNS are the
microglia, also considered as part of a very elabo-
rate innate immune system and one of the cell types
that constitute the stem cell niche (Simard and Rivest,
2004; Kempermann, 2006). Microglia are recruited
by many pathological stimuli in the defense of the
neural parenchyma against infection, inflammation,
ischemia, trauma, brain tumors and neurodegenera-
tion. They contribute to the immune response by acting
as antigen presenting cells, cleaning, unwanted debris
by phagocytosis, as well as secreting factors associ-
ated with inflammation, such as cytokines and other
signaling molecules. Over the last few years the role
of microglia in adult neurogenesis in the intact and
injured brain has been discussed. A growing body of
evidence shows that microglia, depending on their state
of activation, can be either detrimental or supportive
for neurogenesis acting on different steps in the for-
mation, maturation and functional integration of the
2 Neurogenesis: A Change of Paradigms
new neurons (see Ekdhal et al., 2009; for review). To
elucidate their neurotoxic and supportive role in adult
neurogenesis, it is necessary first to understand the
microglial function in different activation profiles and
their associated cytokine. An interesting view is that
microglia activation, as an indicator of inflammation, is
not pro- or antineurogenic per se but the net outcome is
dependent on the balance between secreted molecules
with pro- and anti-inflammatory action (Ekdhal et al.,
2009).
Cytokines IL-1, IL-1, IL-6, IFN- and TNF-
in the CNS have been shown to have a suppres-
sive effect over neurogenesis that depends on the
degree of activation of the brain resident microglia
on inflammation (Vallieres et al., 2002; Ekdahl et al.,
2003; Monje et al., 2003; Ben-Hur et al., 2003;
Wong et al., 2004; Heldmann et al., 2005; Cacci
et al., 2005; Iosif et al., 2006; Koo and Duman,
2008) (Table 2.1). Vallieres and colleagues have shown
that interleukin-6 (IL-6) when expressed over the
long term in astroglia of young adult transgenic mice
is able to decrease dentate gyrus neurogenesis by
63% (Vallieres et al., 2002). Other studies demon-
strated that inflammation caused by lipopolysaccha-
ride (LPS), known to induce several inflammatory
responses, inhibits neurogenesis in the hippocampus
(Ekdahl et al., 2003; Monje et al., 2003). Neurogenesis
can be restored by indomethacin, a common nons-
teroidal anti-inflammatory drug (Monje et al., 2003).
Monje and colleagues concluded that cell death under
pathological conditions can be associated with an
antineurogenic inflammatory response. Interestingly,
Wong and colleagues have shown that adult neural
stem cells are able to respond to the inflammatory
cytokines interferon-gamma (IFN-) and tumor necro-
sis factor-alpha (TNF-), with significant effects on
neural stem cell function. While TNF- was toxic
to proliferating neural stem cells, it did not appear
to affect differentiation. IFN-, on the other hand,
inhibited neural stem cell proliferation and promoted
neuronal differentiation and neurite outgrowth (Wong
et al., 2004). Other studies also showed the inhibitory
effect on neurogenesis by IL-6, as well as by other
inflammation-related cytokine products such as IFN-
, interleukin-1beta (IL-1), and TNF- (Heldmann et
al., 2005; Cacci et al., 2005; Iosif et al., 2006; Ben-Hur
et al., 2003; Koo and Duman, 2008).
Interestingly, recent experimental evidence indi-
cates that inflammation or microglia under certain
21
circumstances can be beneficial and supportive of the
different steps in adult neurogenesis and gliogenesis
(Selmaj et al., 1990; Wu et al., 2000; Arnett et al.,
2001; Butovsky et al., 2006; Battista et al., 2006)
(see Table 2.1). By the use of TNF--receptor defi-
cient mice, Arnett and colleagues found that the lack
of TNF- leads to a delay in remyelination, which is
associated with a reduction in the pool of proliferating
oligodendrocyte progenitors followed by a reduction in
the number of mature oligodendrocytes (Arnett et al.,
2001). Moreover, TNF- has been shown to promote
astrocyte proliferation (Selmaj et al., 1990).
A beneficial role of microglia in adult neurogen-
esis is also supported by in vitro studies on neural
stem cells co-cultured with microglia or grown in con-
ditioned media from microglia (Aarum et al., 2003;
Morgan et al., 2004; Walton et al., 2006; Nakanishi
et al., 2007). Microglia and microglia-conditioned
medium rescued the in vitro formation of neurob-
lasts from SVZ neural stem cells, which otherwise
was lost during continued culture (Aarum et al., 2003;
Walton et al., 2006). In a recent paper, Cacci and
colleagues (Cacci et al., 2008), showed that pro-
inflammatory cytokines interleukin (IL-1, IL-1 beta,
IL-6), and TNF- were strongly reduced after chronic
stimulation of microglia, as compared to acute stim-
ulation. The authors suggested that, in a chronically
altered environment, persistently activated microglia
can display protective functions that favor rather
than hinder brain repair processes. They suggested
that acutely activated microglia, or their conditioned
medium, reduced neural progenitor cell survival, pre-
vented neuronal differentiation and strongly increased
glial differentiation, likely through the release of proin-
flammatory cytokines, whereas chronically activated
microglia were permissive to neuronal differentiation
and cell survival, and still supported glial differenti-
ation. Recently, Ziv and colleagues showed that hip-
pocampal neurogenesis could be restored in mice by
the transfer of CNS-reactive T cells (Ziv et al., 2006).
These regulatory T-lymphocytes were also necessary
for the completion of spatial learning and memory
tasks and, notably, for BDNF expression in the den-
tate gyrus. Another relevant molecule associated to
microglial activation under conditions of inflamma-
tory reaction is stromal derived cell factor (SDF1)
that induces microglia migration by inducing CXC
chemokine receptor 4 (CXCR4) expression (Wang
et al., 2008). It was furthermore shown that glutamate
22 L.E. Mello and B.M. Longo

Table 2.1 Examples of studies showing the effect of cytokines and other factors on neurogenesis and gliogenesis
Effect on
Cytokine/factor neurogenesis/gliogenesis Animal model Study
IL-6 Decrease SE (pilo) in transgenic mice Vallieres et al. (2002)
expressing IL-6
IL-1 Decrease LPS Monje et al. (2003)
IL-1 /IL-6 Decrease SE and LPS Ekdall et al. (2003)
IL-1 Decrease Stress and NPC culture Koo and Duman
(2008)
TNF-/IFN- Decrease NPC culture Ben-Hur et al. (2003)
TNF- Decrease Middle cerebral artery Heldmann et al. (2005)
occlusion
TNF- Decrease Hippocampal progenitor cell Cacci et al. (2005)
culture
TNF- Decrease TNF-R1/R2-deficient Iosif et al. (2006)
mice/SE
TNF-/ Decrease Neural stem cell (SVZ) Wong et al. (2004)
IFN- Increase proliferation of culture
BIII-tub+ cells
TNF-/IL-6 Proliferation of astrocyte Cell culture Selmaj et al. (1990)
TNF- Increase Exogenous TNF- Wu et al. (2000)
administration in SVZ/VZ
TNF- Oligodendrocyte TNF-deficient Arnett et al. (2001)
progenitor proliferation mice/cuprizone
IL-4/ Increase NPC co-cultured with IL-4, Butovsky et al. (2006)
IFN- oligodendrogenesis IFN-, LPS
Increase neurogenesis
TGF- Increase Adrenalectomy Battista et al. (2006)
IGF-1 Increase Status Epilepticus Choi et al. (2008)
IGF-1 Increase Igf-1 / mice Beck et al. (1995)
IGF-1 Increase Overexpressed Igf OKusky et al. (2000)
IGF-1 Increase transgenic mice Trejo et al. (2001)
IGF-1 Increase Runner rats and IGF-1 Aberg et al. (2003)
infusion
Hippocampal NPC culture
treated with IGF-1
Microglia Increase Microglia culture from Aarum et al. (2003)
embrionic cortical tissue
Microglia Increase Microglial conditioned Morgan et al. (2004)
medium on cerebellar
cultures
Microglia Increase Neural stem cell adherent Walton et al. (2006)
culture

also increased the CXCR4-mediated T cell chemotac-
tic migration in the direction of the localization of the
key chemokine stromal cell-derived factor-1 (SDF-1)
(Ganor et al., 2003). SDF1-mediated CXCR4 signal-
ing was also shown to influence rat and human cortical
neural progenitor cells (Peng et al., 2004) and to regu-
late the migration of dentate granule cells (Bagri et al.,
2002) placing it at a pivotal step as a candidate for
the trophic effects of seizures over neurogenesis and
associated phenomena.
In epilepsy studies, the presence of immunologi-
cal dysfunctions after seizures has been extensively
demonstrated (Bhatt et al., 2003; Cook and Persinger,
1999; Vezzani, 2005). Inflammatory cells have been
shown to infiltrate the brain parenchyma within
limbic structures after lithium-pilocarpine-induced
seizures (Cook and Persinger, 1999). Moreover, after
amygdala-induced kindling seizures bone marrow pro-
genitor cells were described to proliferate in vitro, sug-
gesting alterations in immunological functions after
2 Neurogenesis: A Change of Paradigms
Fig. 2.4 Expression of
GFP+ /BrdU+ double stained
cells in the hippocampus of
epileptic animals at 24 h, 7
and 24 days after status
epilepticus of chimeric
animals transplanted with
GFP+ bone marrow cells
(kindly provided by S.
Romariz)
chronic seizures (Bhatt et al., 2003). The continuous
traffic of inflammatory cells into the cerebral tissue is
accelerated after injury (Schilling et al., 2003; Vallieres
and Sawchenko, 2003), and is involved in cytokine
response to seizures (Voutsinos-Porche et al., 2004;
Bernardino et al., 2005; Vezzani, 2005). Our own stud-
ies, using chimeric animals transplanted with bone
marrow cells, characterized the temporal pattern of
migration and distribution of bone marrow cells after
epileptic seizures (Fig. 2.4). The majority of these
cells could be labeled with BrdU as well as expressed
microglial markers meaning that the bone marrow
cells proliferated within the brain parenchyma. Our
evidence is consistent with the hypothesis that these
cells participate in inflammatory reaction that follows
epileptic seizures (Romariz et al., 2008).
As speculated by Bernardino and colleagues, neu-
rogenesis that occurs in TLE is under the control
of two opposite driving forces. On one hand, acute
seizures induce differentiation of new neurons. On
the other hand, chronic TLE, inflammation and IL-6
release contribute to inhibit neurogenesis (Bernardino
et al., 2005). The authors suggested that pathological
alterations that occurs in chronic TLE can be influ-
enced by the inflammatory response, and the functional
role of cytokines in inflammation can shift from ben-
eficial to detrimental depending on the type of cells
23
producing and releasing the cytokines, the functional
state of neurons, local cytokine concentration and
period of tissue exposure, the presence of specific
receptors on target cells, and the presence or absence
of modulators of cytokine activity. Indeed, a sus-
tained expression of specific cytokines has been asso-
ciated to chronic TLE. Many authors argued that the
cytokines, up-regulated following seizures, may act
through specific receptors in both protective (Albensi,
2001; Hamano et al., 2002) and toxic ways (Vezzani
et al., 1999; Eriksson et al., 2000; Vezzani et al., 2000)
to control the pattern of neuronal loss and replacement
associated with seizures. The beneficial or detrimental
roles of the innate immune response in the epilep-
tic tissue and its positive or negative modulation on
seizure-induced neurogenesis still needs to be clarified
(Vezzani, 2005).
2.5 Stem Cell Therapies for CNS
Disorders
The CNS, differently from other organs, has a limited
capacity of self repair and self regeneration. Obviously,
these mechanisms are not sufficient for a complete
recovery facing the intense damage to the CNS in some
24
cases. From a clinical perspective, it is a consensus
that there is an incredible need for strategies and thera-
pies developed for preventing or reversing neurological
disturbances. Over the last few years, the therapeutic
potential of stem cells has been suggested as a pos-
sible treatment in various pathological conditions of
the CNS. A growing body of evidence shows that stem
cells from different sources, when transplanted into the
brain, can migrate to the damaged tissue improving
neurological function (Azizi et al., 1998; Kopen et al.,
1999; Lee et al., 2003), and even differentiate into
glial (Wernig et al., 2008) and neuronal cells (Wernig
et al., 2008; Mezey, 2000)!!! Here we address some
of the questions regarding the strategies of therapies
based on stem cell to treat neurological diseases. In
general, stem cell therapy in the CNS can be divided
in two strategies, the transplantation of precursors or
stem cells, and the stimulation of endogenous neural
progenitor cells. The transplanted cell strategy is based
on the replacement potential expecting that these cells
could migrate, differentiate and integrate, and may be
transplanted locally or administered intravenously. The
stimulation of endogenous neural progenitor or stem
cells locally would represent a strategy to promote
regeneration of injured brain (Taupin, 2008).
Neuroprotection can be also provided by the
paracrine effect of stem cell in the lesion site by releas-
ing cytokine and trophic factors, irrespective of the
chosen strategy, whether transplantation or stimula-
tion of progenitor cells (Ruschenschmidt et al., 2005;
Gttinger et al., 2005; Zhao et al., 2004; Borlongan
et al., 2004). Besides triggering differentiation and sur-
vival of transplanted cells by autocrine and paracrine
mechanisms, genetically modified neural stem cells
secreting trophic and differentiation-inducing factors
can also recruit endogenous neural progenitor cells for
tissue repair (Trujillo et al., 2009). Endogenous cells
that proliferate and migrate to the lesioned areas pos-
sibly exert paracrine effects, rescuing damaged cells in
the brain or stimulating neural stem cells and neuro-
genesis. Obviously for the contribution of these stem
cells or progenitors from other tissues to brain repair
makes it hard to unambiguously demonstrate the con-
tribution of true neural stem or progenitor cells. Recent
papers have shown that the paracrine action of these
foreign migrated cells into the brain from transplanted
bone marrow (Zhao et al., 2004), umbilical cord blood
(Borlongan et al., 2004), and embryonic stem cells
(Gttinger et al., 2005) can have positive effect on
L.E. Mello and B.M. Longo
injured areas in the brain. Indeed, freshly injected bone
marrow cells might even be able to block seizures.
In our hands, transplantation of precursor cells only
30 min before a convulsive stimulation in mice and
rats, altered seizure susceptibility, making it more dif-
ficult for a seizure to be triggered in these animals.
Given the narrow temporal window between the trans-
plant and seizure induction in those experiments, it is
likely that this protection is due to a paracrine action
of these recently injected marrow cells (Ferrazoli et al.,
2008).
This paracrine effect opens the possibility of
neuroregenerative therapies, but important points
should be emphasized before proposing a therapy
based on stem cell transplantation. As suggested by
Pluchino and Martino (2005), it is important to be
aware that relevant issues concerning the transplan-
tation strategies, number of cells, timing and route
of administration should be elucidated before any
therapeutic application.
Not less important, questions about the best source
of stem cell to be transplanted and whether trans-
planted cells are functional and integrated to the neu-
ronal circuitry should be also considered. Obviously,
the degree of neural damage and finally, the exper-
imental model elected for therapeutic studies should
be adequate to answer these questions. In this regard,
embryonic stem cells, neural stem cells and bone
marrow cells may constitute the ideal candidates for
transplantation therapy in many neurological diseases
in both clinical and experimental studies (Bossolasco
et al., 2005; Cogle et al., 2004; Chu et al., 2003, 2004;
Silani et al., 2004; Zhang et al., 2004). Hypothetically,
the ideal precursor cell for CNS therapy is of autolo-
gous origin and multipotent in terms of its capacity to
give rise to the three cell lineages comprising the CNS
(neurons, astrocytes and oligodendrocytes), as well as
their restricted capacity to differentiate into other cell
types and their lack of tumorigenic potential (Ziv and
Schwartz, 2008).
One of the criticisms on stem cell therapy and
transplantation is based on the suggestion that these
strategies are less efficacious in chronic neurological
diseases, in neurodegenerative processes, as compared
to the acute period, during disease onset, indicating
a direct correlation with the immune system response
(Glezer et al., 2007). However, recent findings in stud-
ies with Alzheimers disease revealed positive results
in this chronic neurodegenerative disease. The group
2 Neurogenesis: A Change of Paradigms
of Serge Rivest from Laval University of Quebec
have shown that newly differentiated blood-derived
microglia are able to remove -amyloid plaque and
consequently decelerate the progress of Alzheimers
disease, whereas microglia cells resident in CNS are
inefficient in phagocyting the amyloid deposit (Simard
et al., 2006; Napoli and Neumann, 2009). These
authors suggested that therapeutic strategies aiming
to improve microglia recruitment from bone marrow
could potentially lead to a new powerful tool for the
elimination of toxic senile plaques.
In Parkinson disease, as already mentioned here,
much of the present studies about transplantation have
been developed by Bjrklund, Lindvall and their col-
leagues in Sweden (Lindvall et al., 1990; Bjrklund
and Lindvall, 2000). These studies indicated that these
transplanted cells can replace dopaminergic neurons
damaged in PD and restore and regenerate brain func-
tion. Replacement therapies for neurodevelopmental
and neurodegenerative diseases have revealed success
when stem cells were placed into a microenviron-
ment favoring differentiation and survival of newborn
cells. Such environmental conditions can be generated
by transplantation of genetically engineered neural
stem cells, which produce and secrete growth factors
and neurotransmitters with neuroprotective and trophic
actions (Trujillo et al., 2009). In PD, an alternative
method for mechanical pumps delivering glial-derived
neurotrophic factor (GDNF) can be the use of bio-
logical minipumps, such as genetically modified neu-
rospheres. Neurospheres modified to produce GDNF
can be transplanted into the brain for delivery of the
trophic factor. Using an inducible viral system, such as
that described by Capowski and coworkers (2007), it
is possible to control the amounts of GDNF delivery
as well as to stop the therapy at any time. This type of
cell therapy associated with gene therapy is still under
evaluation in animal models (Trujillo et al., 2009).
Concerning the functionality of transplanted stem
cells in neurological diseases, electrophysiological
recordings after transplantation in a model of PD
have shown that grafted neurons were functionally
integrated in the host brain (Wernig et al., 2008;
lvares-Dolado et al., 2006). Morphological analy-
sis of the recorded cells confirmed their differenti-
ation into dopaminergic (Wernig et al., 2008) and
GABAergic neurons (lvares-Dolado et al., 2006).
In the beginning of 1990s two different groups had
published interesting papers about grafting embryonic
25
brain regions into the hippocampus (Bortolotto et al.,
1990; Buzski et al., 1991) and correlated to epilep-
tic activity. Buzski and coworkers had concluded
that hippocampal grafts into the intact brain induce
epileptic patterns. On the other hand, Bortolotto and
colleagues showed that noradrenergic neurons pre-
pared from the locus coeruleus region from E13 or
E14 and injected bilaterally into the hippocampus of
epileptic rats suppressed spontaneous recurrent epilep-
tic seizures in intra-hipocampal grafted rats.
More recently, studies on epilepsy models dealt with
embryonic (Gttinger et al., 2005; Ruschenschmidt
et al., 2005) and human fetal neural stem cells (Chu
et al., 2004) to treat seizures or to study their behav-
ior in epileptic condition. Grafting of human fetal
neural stem cells into rat brains one day after sta-
tus epilepticus induction led to marked suppression of
spontaneous recurrent motor seizures (SRMS). Indeed,
approximately one month after SE onset, 87% of
the untreated animals showed spontaneous recurrence
of seizure activity in contrast to only 13% of the
hNSC-treated animals. Phenotypic analysis indicated
that whereas only a small fraction of the injected
hNSC expressed markers of mature neurons and
yet transplanted cells co-expressed phenotypic mark-
ers of interneurons (GABA, parvalbumin), suggesting
that hNSC can differentiate into GABA-synthesizing
cells after transplantation into the lesioned hippocam-
pus. The authors hypothesize these new GABA-
synthesizing cells may decrease neuronal excitability
and thus suppress spontaneous seizures.
2.6 Concluding Remarks
It is clear that functionally diverse roles expected
of cellular replacement cannot be fulfilled by the
transplantation of any single cell type; rather, spe-
cialized cells or tittered mixtures of cells may be
important in replacement strategies. As claimed by
Kempermann (2006), neural stem cells have some-
times been greeted as the last missing link in an
otherwise obviously coherent picture of what would
constitute successful neuroregeneration. As with the
discovery of precursor cells and adult neurogenesis,
everything should fall into place and neuroregener-
ation become suddenly possible. However, the adult
brain still regenerates poorly. It does so despite the
26
presence of neural stem cells and not, as was previ-
ously thought, because of their absence. To this end,
circumventing this limitation by means foreign to the
brain is not only a difficult task but might in addition
produce mixed results.
If stem cells are mobilized in the case of danger,
than we are facing another type of defense system!
These cells are reacting somehow similar to immuno-
competent cells, which are responsible for the so called
body immune surveillance. Maybe the stem cells,
besides their task to generate all other kind of differen-
tiated cells, are not working on surveillance but rather
are mobilized by specific biochemical signals emitted
by the injured organ in order to regenerate it. Their
tropism for such an organ justifies this hypothesis of
such a signal. This could be universal or specific to
the tissue or more likely specific to the given brain
structure or cell population within a brain structure.
We conclude with Karl Popper statement in his 1934
book The Logic of Scientific Discovery (1959, first
English edition): Science is not a system of certain, or
well-established, statements; nor is it a system which
steadily advances towards a state of finality. Our sci-
ence is not knowledge (episteme): it can never claim to
have attained truth, or even a substitute for it, such as
probability.
Acknowledgments We gratefully acknowledge Dr Aurel
Popescu for suggestions and fertile discussion on immunoin-
flammation, Simone Romariz for the preparation of immunoflu-
orescence assays and images, and Thomas Perlaky for the
assistance with all the references. This work was supported by
FAPESP and CNPq.
References
Aarum J, Sandberg K, Haeberlein SL, Persson MA (2003)
Migration and differentiation of neural precursor cells can be
directed by microglia. Proc Natl Acad Sci USA 100:15983
15988.
Aberg MA, Aberg ND, Palmer TD, Alborn AM, Carlsson-
Skwirut C, Bang P, Rosengren LE, Olsson T, Gage FH,
Eriksson PS (2003) IGF-I has a direct proliferative effect
in adult hippocampal progenitor cells. Mol Cell Neurosci
24:2340.
Albensi BC (2001) Potential roles for tumor necrosis factor and
nuclear factor-kappaB in seizure activity. J Neurosci Res
66:151154.
Altar CA, Laeng P, Jurata LW, Brockman JA, Lemire A,
Bullard J, Bukhman YV, Young TA, Charles V, Palfreyman
L.E. Mello and B.M. Longo
MG (2004) Electroconvulsive seizures regulate gene expres-
sion of distinct neurotrophic signaling pathways. J Neurosci
24:26672677.
Altman J (1962) Are new neurons formed in the brains of adult
mammals? Science 135:11271128.
Altman J (1963) Differences in the utilization of tritiated
leucine by single neurones in normal and exercised rats:
an autoradiographic investigation with microdensitometry.
Nature 199:777780.
Altman J, Das GD (1965) Autoradiographic and histological evi-
dence of postnatal hippocampal neurogenesis in rats. J Comp
Neurol 124:319336.
Alvarez-Buylla A, Garcia-Verdugo J (2002) Neurogenesis in
adult subventricular zone. J Neurosci 22:629634.
Alvarez-Dolado M, Calcagnotto ME, Karkar KM, Southwell
DG, Jones-Davis DM, Estrada RC, Rubenstein JL, Alvarez-
Buylla A, Baraban SC (2006) Cortical inhibition modified by
embryonic neural precursors grafted into the postnatal brain.
J Neurosci 26:73807389.
Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK,
Ting JP (2001) TNF alpha promotes proliferation of oligo-
dendrocyte progenitors and remyelination. Nat Neurosci
4:11161122.
Avots A, Harder F, Schmittwolf C, Petrovic S, Mller AM
(2002) Plasticity of hematopoietic stem cells and cellular
memory. Immunol Rev 187:921.
Azizi SA, Stokes D, Augelli BJ, DiGirolamo C, Prockop DJ
(1998) Engraftment and migration of human bone mar-
row stromal cells implanted in the brains of albino rats
similarities to astrocyte grafts. Proc Natl Acad Sci USA
95:39083913.
Bagri A, Gurney T, He X, Zou YR, Littman DR, Tessier-Lavigne
M, Pleasure SJ (2002) The chemokine SDF1 regulates migra-
tion of dentate granule cells. Development 129:42494260.
Barnea A, Nottebohm F (1994) Seasonal recruitment of hip-
pocampal neurons in adult free-ranging black-capped chick-
adees. Proc Natl Acad Sci USA 91:1121711221.
Battista D, Ferrari CC, Gage FH, Pitossi FJ (2006) Neurogenic
niche modulation by activated microglia: transforming
growth factor beta increases neurogenesis in the adult dentate
gyrus. Eur J Neurosci 23:8393.
Bayer S (1982) Changes in the total number of dentate gran-
ule cells in juvenile and adult rats: A correlated volumetric
and 3H-thymidine autoradiographic study. Exp Brain Res
46:315323.
Bayer S, Yackel JW, Puri PS (1982) Neurons in the rat dentate
gyrus granular layer substantially increase during juvenile
and adult life. Science 216:890892.
Beck D, Gross N, Beretta-Brognara C (1995) Effects of stem cell
factor and other bone marrow-derived growth factors on the
expression of adhesion molecules and proliferation of human
neuroblastoma cells. Eur J Cancer 31A:467470.
Begg AC, Hofland I, Van Der Pavert I, Van Der Schueren B,
Austermans K (2000) Use of thymidine analogues to indicate
vascular perfusion in tumours. Br J Cancer 83:899905.
Ben-Hur T, Ben-Menachem O, Furer V, Einstein O, Mizrachi-
Kol R, Grigoriadis N (2003) Effects of proinflammatory
cytokines on the growth, fate, and motility of multipotential
neural precursor cells. Mol Cell Neurosci 24:623631.
Bengzon J, Kokaia Z, Elmr E, Nanobashvili A, Kokaia M,
Lindvall O (1997) Apoptosis and proliferation of dentate
2 Neurogenesis: A Change of Paradigms
gyrus neurons after single and intermittent limbic seizures.
Proc Natl Acad Sci USA 94:1043210437.
Benraiss A, Chmielnicki E, Lerner K, Roh D, Goldman
SA (2001) Adenoviral brain-derived neurotrophic factor
induces both neostriatal and olfactory neuronal recruitment
from endogenous progenitor cells in the adult forebrain. J
Neurosci 21:67186731.
Bernardino L, Ferreira R, Cristovao AJ, Sales F, Malva
JO (2005) Inflammation and neurogenesis in temporal
lobe epilepsy. Curr Drug Targets CNS Neurol Disord 4:
349360.
Bhatt R, Rameshwar P, Goldstein K, Siegel A (2003) Effects
of kindled seizures upon hematopoiesis in rats. Epilepsy Res
54:209219.
Biebl M, Cooper C, Winkler J, Kuhn HG (2000) Analysis of neu-
rogenesis and programmed cell death reveals a self-renewing
capacity in the adult rat brain. Neurosci Lett 291:1720.
Bjrklund A, Lindvall O (2000) Cell replacement therapies for
central nervous system disorders. Nat Neurosci 3:537544.
Blaschke AJ, Staley K, Chun J (1996) Widespread programmed
cell death in proliferative and postmitotic regions of the fetal
cerebral cortex. Development 122:11651174.
Borlongan CV, Hadman M, Sanberg CD, Sanberg PR (2004)
Central nervous system entry of peripherally injected umbil-
ical cord blood cells is not required for neuroprotection in
stroke. Stroke 35:23852389.
Bortolotto ZA, Calderazzo L, Cavalheiro EA (1990) Some evi-
dence that intrahippocampal grafting of noradrenergic neu-
rons suppresses spontaneous seizures in epileptic rats. Braz J
Med Biol Res 23:12671269.
Bossolasco P, Cova L, Calzarossa C, Rimoldi SG, Borsotti C,
Deliliers GL, Silani V, Soligo D, Polli E (2005) Neuro-glial
differentiation of human bone marrow stem cells in vitro.
Exp Neurol 193:312325.
Bouilleret V, Ridoux V, Depaulis A, Marescaux C, Nehlig A,
Le Gal La Salle G (1999) Recurrent seizures and hippocam-
pal sclerosis following intrahippocampal kainate injection
in adult mice: electroencephalography, histopathology and
synaptic reorganization similar to mesial temporal lobe
epilepsy. Neuroscience 89:717729.
Brazelton TR, Rossi FM, Keshet GI, Blau HM (2000) From
marrow to brain: expression of neuronal phenotypes in adult
mice. Science 290:17751779.
Brezun JM, Daszuta A (1999) Depletion in serotonin decreases
neurogenesis in the dentate gyrus and the subventricular zone
of adult rats. Neuroscience 89:9991002.
Burd GD, Nottebohm F (1985) Ultrastructural characterization
of synaptic terminals formed on newly generated neurons in
a song control nucleus of the adult canary forebrain. J Comp
Neurol 240:143152.
Buss RR, Sun W, Oppenheim RW (2006) Adaptive roles of
programmed cell death during nervous system development.
Annu Rev Neurosci 29:135.
Butovsky O, Ziv Y, Schwartz A, Landa G, Talpalar AE, Pluchino
S, Martino G, Schwartz M (2006) Microglia activated by
IL-4 or IFN-gamma differentially induce neurogenesis and
oligodendrogenesis from adult stem/progenitor cells. Mol
Cell Neurosci 31:149160.
Buzski G, Masliah E, Chen LS, Horvth Z, Terry R, Gage
FH (1991) Hippocampal grafts into the intact brain induce
epileptic patterns. Brain Res 554:12.
27
Cacci E, Ajmone-Cat MA, Anelli T, Biagioni S, Minghetti
L (2008) In vitro neuronal and glial differentiation from
embryonic or adult neural precursor cells are differently
affected by chronic or acute activation of microglia. Glia
56:412425.
Cacci E, Claasen JH, Kokaia Z (2005) Microglia-derived tumor
necrosis factor-alpha exaggerates death of newborn hip-
pocampal progenitor cells in vitro. J Neurosci 80:789797.
Calza L, Giardino L, Pozza M, Bettelli C, Micera A, Aloe L
(1998) Proliferation and phenotype regulation in the subven-
tricular zone during experimental allergic encephalomyelitis:
in vivo evidence of a role for nerve growth factor. Proc Natl
Acad Sci USA 95:32093214.
Cameron HA, Gould E (1994) Adult neurogenesis is regu-
lated by adrenal steroids in the dentate gyrus. Neuroscience
61:203209.
Cameron HA, Mckay R (2001) Adult neurogenesis produces a
large pool of new granule cells in the dentate gyrus. J Comp
Neurol 435:406417.
Cameron HA, Wooley CS, McEwen BS, Gould E (1993)
Differentiation of newly born neurons and glia in the dentate
gyrus if the adult rat. Neuroscience 56:337344.
Capowski EE, Schneider BL, Ebert AD, Seehus CR, Szulc J,
Zufferey R, Aebischer P, Svendsen CN (2007) Lentiviral
vector-mediated genetic modification of human neural pro-
genitor cells for ex vivo gene therapy. J Neurosci Methods
163:338349.
Choi YS, Cho HY, Hoyt KR, Naegele JR, Obrietan K (2008)
IGF-1 receptor-mediated ERK/MAPK signaling couples sta-
tus epilepticus to progenitor cell proliferation in the subgran-
ular layer of the dentate gyrus. Glia 56:791800.
Chu K, Kim M, Jeong SW, Kim SU, Yoon BW (2003) Human
neural stem cells can migrate, differentiate, and integrate
after intravenous transplantation in adult rats with transient
forebrain ischemia. Neurosci Lett 343:129133.
Chu K, Kim M, Jung KH, Jeon D, Lee ST, Kim J, Jeong SW,
Kim SU, Lee SK, Shin HS, Roh JK (2004) Human neu-
ral stem cell transplantation reduces spontaneous recurrent
seizures following pilocarpine-induced status epilepticus in
adult rats. Brain Res 1023:213221.
Cogle CR, Yachnis AT, Laywell ED, Zander DS, Wingard JR,
Steindler DA, Scott EW (2004) Bone marrow transdifferen-
tiation in brain after transplantation: a retrospective study.
Lancet 363:14321437.
Cook LL, Persinger MA (1999) Infiltration of lymphocytes in
the limbic brain following stimulation of subclinical cellular
immunity and low dosages of lithium and a cholinergic agent.
Toxicol Lett 109:7785.
Corotto FS, Henegar JA, Maruniak JA (1993) Neurogenesis per-
sists in the subependymal layer of the adult mouse brain.
Neurosci Lett 149:111114.
Covolan L, Mello LE (2000) Temporal profile of neuronal
injury following pilocarpine or kainic acid-induced status
epilepticus. Epilepsy Res 39:133152.
Craig CG, Tropepe V, Morshead CM, Reynolds BA, Weiss S,
van der Kooy D (1996) In vivo growth factor expansion of
endogenous subependymal neural precursor cell populations
in the adult mouse brain. J Neurosci 16:26492658.
Curtis MA, Connor B, Faull RL (2003) Neurogenesis in the
diseased adult human brain new therapeutic strategies for
neurodegenerative diseases. Cell Cycle 2:428430.
28
Dash PK, Mach SA, Moore AN (2001) Enhanced neurogenesis
in the rodent hippocampus following traumatic brain injury.
J Neurosci Res 63:313319.
Doetsch F, Alvarez-Buylla A (1996) Network of tangential path-
ways for neuronal migration in the adult mammalian brain.
Proc Natl Acad Sci USA 93:1489514900.
Doetsch F, Caille I, Lim DA (1999) Subventricular zone astro-
cytes are neural stem cells in the adult mammalian brain. Cell
97:120.
Doetsch F, Garca-Verdugo JM, Alvarez-Buylla A (1997)
Cellular composition and three-dimensional organization of
the subventricular germinal zone in the adult mammalian
brain. J Neurosci 17:50465061.
Eckenhoff MF, Rakic P (1984) Radial organization of the
hippocampal dentate gyrus: a Golgi, ultrastructural, and
immunocytochemical analysis in the developing rhesus mon-
key. J Comp Neurol 223:121.
Eckenhoff MF, Rakic P (1988) Nature and fate of proliferative
cells in the hippocampal dentate gyrus during the life span of
the rhesus monkey. J Neurosci 8:27292747.
Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O (2003)
Inflammation is detrimental for neurogenesis in adult brain.
Proc Natl Acad Sci USA 100:1363213637.
Ekdahl CT, Kokaia Z, Lindvall O (2009) Brain inflammation and
adult neurogenesis: the dual role of microglia. Neuroscience
158:10211029.
Elliott RC, Miles MF, Lowenstein DH (2003) Overlapping
microarray profiles of dentate gyrus gene expression dur-
ing development- and epilepsy-associated neurogenesis and
axon outgrowth. J Neurosci 23:22182227.
Eriksson PS, Perfilieva E, Bjrk-Eriksson T, Alborn AM,
Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in
the adult human hippocampus. Nat Med 4:13131317.
Eriksson C, Zou LP, Ahlenius S, Winblad B, Schultzberg
M (2000) Inhibition of kainic acid induced expression
of interleukin-1 beta and interleukin-1 receptor antagonist
mRNA in the rat brain by NMDA receptor antagonists. Brain
Res Mol Brain Res 85:103113.
Fahrner A, Kann G, Flubacher A, Heinrich C, Freiman TM,
Zentner J, Frotscher M, Haas CA (2007) Granule cell dis-
persion is not accompanied by enhanced neurogenesis in
temporal lobe epilepsy patients. Exp Neurol 203:320332.
Ferland RJ, Gross RA, Applegate CD (2002) Increased mitotic
activity in the dentate gyrus of the hippocampus of adult
C57bl/6j mice exposed to the flurothyl kindling model of
epileptogenesis. Neuroscience 115:669683.
Ferrazoli E, Senedese A, Romariz S, Bahia L, Santos RR,
Blanco M, Mello LE, Longo B (2008) Avaliao curso-
temporal do efeito protetor do transplante de clulas de
medula ssea em um modelo agudo de crise convulsiva.
J Epil Clin Neurophysiol 14:5455.
Freese A, ORourke D, Judy K, OConnor MJ (1994) The appli-
cation of 5-bromodeoxyuridine in the management of CNS
tumors. J Neurooncol 20:8195.
Ganor Y, Besser M, Ben-Zakay N, Unger T, Levite M (2003)
Human T cells express a functional ionotropic glutamate
receptor GluR3, and glutamate by itself triggers integrin-
mediated adhesion to laminin and fibronectin and chemotac-
tic migration. J Immunol 170:43624372.
Garca-Verdugo JM, Doetsch F, Wichterle H, Lim D, Alvarez-
Buylla A (1998) Architecture and cell types of the adult
L.E. Mello and B.M. Longo
subventricular zone: in search of the stem cells. J Neurobiol
36:234248.
Glezer I, Simard AR, Rivest S (2007) Neuroprotective role of
the innate immune system by microglia. Neuroscience 147:
867883.
Goldman SA, Nottebohm F (1983) Neuronal production, migra-
tion, and differentiation in a vocal control nucleus of the adult
female canary brain. Proc Natl Acad Sci USA 80:23902394.
Gould E (2007) Structural plasticity. In: The Hippocampus Book
(Andersen P, ed), pp. 321335. Oxford University Press,
Oxford.
Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ (1999a)
Learning enhances adult neurogenesis in the hippocampal
formation. Nat Neurosci 2:260265.
Gould E, Gross CG (2002) Neurogenesis in adult mammals:
some progress and problems. J Neurosci 22:619623.
Gould E, McEwen BS, Tanapat P, Galea LA, Fuchs E (1997)
Neurogenesis in the dentate gyrus of the adult tree shrew
is regulated by psychosocial stress and NMDA receptor
activation. J Neurosci 17:24922498.
Gould E, Reeves AJ, Graziano MS, Gross CG (1999b)
Neurogenesis in the neocortex of adult primates. Science
286:548552.
Gould E, Tanapat P (1997) Lesion-induced proliferation of
neuronal progenitors in the dentate gyrus of the adult rat.
Neuroscience 80:427436.
Gould E, Tanapat P, McEwen BS, Flugge G, Fuchs E (1998)
Proliferation of granule cell precursors in the dentate gyrus
of adult of monkeys is diminished by stress. Proc Natl Acad
Sci USA 95:31683171.
Gould E, Woolley C, McEwen BS (1991) Adrenal steroids
regulate postnatal development of the rat dentate gyrus. I.
Effects of glucocorticoids on cell death. J Comp Neurol
313:479485.
Gray W, Sundstrom LE (1998) Kainic acid increases the prolif-
eration of granule cell progenitors in the dentate gyrus of the
adult rat. Brain Res 790:5259.
Gross CG (2000) Neurogenesis in the adult brain: death of a
dogma. Nature Rev Neurosci 1:6773.
Gttinger M, Fedele D, Koch P, Padrun V, Pralong WF, Brustle
O, Boison D (2005) Suppression of kindled seizures by
paracrine adenosine release from stem cell-derived brain
implants. Epilepsia 46:11621169.
Hagihara H, Hara M, Tsunekawa K, Nakagawa Y, Sawada
M, Nakano K (2005) Tonic-clonic seizures induce divi-
sion of neuronal progenitor cells with concomitant changes
in expression of neurotrophic factors in the brain of
pilocarpine-treated mice. Brain Res Mol Brain Res 139:
258266.
Hamano H, Noguchi M, Fukui H, Issiki A, Watanabe Y (2002)
Regulation of brain cell environment on neuronal protection:
role of TNFalpha in glia cells. Life Sci 72:565574.
Hansel DE, Eipper BA, Ronnett GV (2001) Neuropeptide
Y functions as a neuroproliferative factor. Nature 410:
940944.
Hastings NB, Gould E (2003) Neurons inhibit neurogenesis. Nat
Med 9:264266.
Heldmann U, Thored P, Claasen JH, Arvidsson A, Kokaia Z,
Lindvall O (2005) TNF-alpha antibody infusion impairs sur-
vival of stroke-generated neuroblasts in adult rat brain. Exp
Neurol 196:204208.
2 Neurogenesis: A Change of Paradigms
Hoshino T, Prados M, Wilson CB, Cho KG, Lee KS, Davis RL
(1989) Prognostic implications of the bromodeoxyuridine
labeling index of human gliomas. J Neurosurg 71:335341.
Houser C (1990) Granule cell dispersion in the dentate gyrus
of humans with temporal lobe epilepsy. Brain Res 535:
195204.
Houser CR, Miyashiro JE, Swartz BE, Walsh GO, Rich JR,
Delgado-Escueta AV (1990) Altered patterns of dynor-
phin immunoreactivity suggest mossy fiber reorganization in
human hippocampal epilepsy. J Neurosci 10:267282.
Howell OW, Scharfman HE, Herzog H, Sundstrom LE, Beck-
Sickinger A, Gray WP (2003) Neuropeptide Y is neuro-
proliferative for post-natal hippocampal precursor cells. J
Neurochem 86:646659.
Iosif RE, Ekdahl CT, Ahlenius H, Pronk CJ, Bonde S, Kokaia Z,
Jacobsen SE, Lindvall O (2006) Tumor necrosis factor recep-
tor 1 is a negative regulator of progenitor proliferation in
adult hippocampal neurogenesis. J Neurosci 26:97039712.
Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC,
Greenberg DA (2004) Increased hippocampal neurogene-
sis in Alzheimers disease. Proc Natl Acad Sci USA 101:
343347.
Jin K, Wang X, Xie L, Mao XO, Zhu W, Wang Y, Shen J, Mao
Y, Banwait S, Greenberg DA (2006) Evidence for stroke-
induced neurogenesis in the human brain. Proc Natl Acad
Sci USA 103:1319813202.
Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U,
Frisn J (1999) Identification of a neural stem cell in the adult
mammalian central nervous system. Cell 96:2534.
Kaplan MS (1981) Neurogenesis in the 3-month-old rat visual
cortex. J Comp Neurol 195:323338.
Kaplan MS (1985) Formation and turnover of neurons in young
and senescent animals: an electron microscopic and morpho-
metric analysis. Ann NY Acad Sci 457:173192.
Kaplan MS, Bell DH (1984) Mitotic neuroblasts in the 9-day-
old and 11-month-old rodent hippocampus. J Neurosci 4:
14291441.
Kaplan MS, Hinds J (1977) Neurogenesis in the adult rat: elec-
tron microscopic analysis of light radioautographs. Science
197:10921094.
Kee NJ, Preston E, Wojtowicz JM (2001) Enhanced neurogene-
sis after transient global ischemia in the dentate gyrus of the
rat. Exp Brain Res 136:313320.
Kempermann G (2006) Adult Neurogenesis: Stem Cell and
Neuronal Development in the Adult Brain. Oxford University
Press, Oxford.
Kempermann G, Gage FH (2002) Genetic determinants of adult
hippocampal neurogenesis correlate with acquisition, but
not probe trial performance, in the water maze task. Eur
J Neurosci 16:129136.
Kempermann G, Jessberger S, Steiner B, Kronenberg G (2004)
Milestones of neuronal development in the adult hippocam-
pus. Trends Neurosci 27:447452.
Kempermann G, Kronenberg G (2003) Depressed new neurons
adult hippocampal neurogenesis and a cellular plasticity
hypothesis of major depression. Biol Psychiatry 54:499503.
Kempermann G, Kuhn H, Gage FH (1997) More hippocampal
neurons in adult mice living in an enriched environment.
Nature 386:493495.
Kempermann G, Kuhn HG, Gage FH (1998) Experience-
induced neurogenesis in the senescent dentate gyrus. J
Neurosci 18:32063212.
29
Kerschensteiner M, Meinl E, Hohlfeld R (2009) Neuro-
immune crosstalk in CNS diseases. Neuroscience 158:
11221132.
Kerschensteiner M, Stadelmann C, Dechant G, Wekerle H,
Hohlfeld R (2003) Neurotrophic cross-talk between the ner-
vous and immune systems: implications for neurological
diseases. Ann Neurol 53:292304.
Kilpatrick TJ, Bartlett PF (1995) Cloned multipotential precur-
sors from the mouse cerebrum require FGF-2, whereas glial
restricted precursors are stimulated with either FGF-2 or
EGF. J Neurosci 15:36533661.
Kilpatrick TJ, Talman PS, Bartlett PF (1993) The differentiation
and survival of murine neurons in vitro is promoted by sol-
uble factors produced by an astrocytic cell line. J Neurosci
Res 35:147161.
Kintner C (2002) Neurogenesis in embryos and in adult neural
stem cells. J Neurosci 22:639643.
Knoblach SM, Fan L, Faden AI (1999) Early neuronal
expression of tumor necrosis factor-alpha after experimen-
tal brain injury contributes to neurological impairment.
J Neuroimmunol 95:115125.
Kokaia Z, Lindvall O (2003) Neurogenesis after ischaemic brain
insults. Curr Opin Neurobiol 13:127132.
Komack DR, Rakic P (1999) Continuation of neurogenesis in the
hippocampus of the adult macaque monkey. Proc Natl Acad
Sci USA 96:57685773.
Koo JW, Duman RS (2008) IL-1beta is an essential mediator of
the antineurogenic and anhedonic effects of stress. Proc Natl
Acad Sci USA 105:751756.
Kopen GC, Prockop DJ, Phinney DG (1999) Marrow stro-
mal cells migrate throughout forebrain and cerebellum,
and they differentiate into astrocytes after injection into
neonatal mouse brains. Proc Natl Acad Sci USA 96:
1071110716.
Kronenberg G, Reuter K, Steiner B, Brandt MD, Jessberger
S, Yamaguchi M, Kempermann G (2003) Subpopulations
of proliferating cells of the adult hippocampus respond dif-
ferently to physiologic neurogenic stimuli. J Comp Neurol
467:455463.
Krumbholz M, Theil D, Derfuss T, Rosenwald A, Schrader F,
Monoranu CM, Kalled SL, Hess DM, Serafini B, Aloisi F,
Wekerle H, Hohlfeld R, Meinl E (2005) BAFF is produced
by astrocytes and up-regulated in multiple sclerosis lesions
and primary central nervous system lymphoma. J Exp Med
201:195200.
Kuhn T (1962) The Structure of Scientific Revolutions.
University of Chicago Press, Chicago, IL.
Kuhn HG, Biebl M, Wilhelm D, Li M, Friedlander RM, Winkler
J (2005) Increased generation of granule cells in adult Bcl-2-
overexpressing mice: a role for cell death during continued
hippocampal neurogenesis. J Neurosci 22:19071915.
Kuhn HG, Dickison-Anson H, Gage FH (1996) Neurogenesis
in the dentate gyrus of the adult rat: age-related decrease
of neuronal progenitor proliferation. J Neurosci 16:
20272033.
Kuhn HG, Winkler J, Kempermann G, Thal LJ, Gage FH (1997)
Epidermal growth factor and fibroblast growth factor-2 have
different effects on neural progenitors in the adult rat brain. J
Neurosci 17:58205829.
Kulkarni SK, George B (1995) Lithium-pilocarpine neurotoxi-
city: a potential model of status epilepticus. Methods Find
Exp Clin Pharmacol 17:551567.
30
Lee J, Duan W, Long JM, Ingram DK, Mattson MP (2000)
Dietary restriction increases the number of newly generated
neural cells, and induces BDNF expression, in the dentate
gyrus of rats. J Mol Neurosci 15:99108.
Lee J, Kuroda S, Shichinohe H, Ikeda J, Seki T, Hida K, Tada M,
Sawada K, Iwasaki Y (2003) Migration and differentiation of
nuclear fluorescence-labeled bone marrow stromal cells after
transplantation into cerebral infarct and spinal cord injury in
mice. Neuropathology 23:169180.
Lemaire V, Koehl M, Le Moal M, Abrous DN (2000) Prenatal
stress produces learning deficits associated with an inhibi-
tion of neurogenesis in the hippocampus. Proc Natl Acad Sci
USA 97:1102311027.
Lindvall O, Rehncrona S, Brundin P, Gustavii B, Astedt B,
Widner H, Lindholm T, Bjrklund A, Leenders KL, Rothwell
JC (1990) Neural transplantation in Parkinsons disease: the
Swedish experience. Prog Brain Res 82:729734.
Liu Z, Fan Y, Won SJ, Neumann M, Hu D, Zhou L, Weinstein
PR, Liu J (2007) Chronic treatment with minocycline pre-
serves adult new neurons and reduces functional impairment
after focal cerebral ischemia. Stroke 38:146152.
Liu J, Solway K, Messing RO, Sharp FR (1998) Increased neu-
rogenesis in the dentate gyrus after transient global ischemia
in gerbils. J Neurosci 18:77687778.
Lois C, Alvarez-Buylla A (1994) Long-distance neuronal migra-
tion in the adult mammalian brain. Science 264:11451148.
Lois C, Garca-Verdugo JM, Alvarez-Buylla A (1996) Chain
migration of neuronal precursors. Science 271:978981.
Longo B, Blanco M, Senra J, Brustolim D, Bahia L, Soares M,
Mello LE, dos Santos RR (2005) Migration of bone mar-
row stem cell into the brain after status epilepticus or acute
seizures. Epilepsia 46:360361 Suppl. 366.
Madrazo I, Drucker-Colin R, Daz V, Martnez-Mata J, Torres C,
Becerril JJ (1987) Open microsurgical autograft of adrenal
medulla to the right caudate nucleus in two patients with
intractable Parkinsons disease. N Engl J Med 316:831834.
Madrazo I, Leon V, Torres C, Aguilera MC, Varela G, Alvarez F,
Fraga A, Drucker-Coln R, Ostrosky F, Skurovich M (1988)
Transplantation of fetal substantia nigra and adrenal medulla
to the caudate nucleus in two patients with Parkinsons
disease. N Engl J Med 319:370371.
Magavi SS, Leavitt B, Macklis JD (2000) Induction of neuroge-
nesis in the neocortex of adult mice. Nature 405:951955.
Malberg JE, Eisch AJ, Nestler EJ, Duman RS (2000) Chronic
antidepressant treatment increases neurogenesis in adult rat
hippocampus. J Neurosci 20:91049110.
Makowiak M, Chocyk A, Markowicz-Kula K, Wedzony K
(2004) Neurogenesis in the adult brain. Pol J Pharmacol
56:673687.
McKay R (1997) Stem cells in the central nervous system.
Science 276:6671.
Mello LE, Cavalheiro E, Tan AM, Pretorius JK, Babb TL, Finch
DM (1992) Granule cell dispersion in relation to mossy fiber
sprouting, hippocampal cell loss, silent period and seizure
frequency in the pilocarpine model of epilepsy. Epilepsy Res
Suppl 9:5159.
Mello LE, Mendez-Otero R (1996) Expression of 9-O-acetylated
gangliosides in the rat hippocampus. Neurosci Lett 213:
1720.
Menezes JR, Smith CM, Nelson KC, Luskin MB (1995) The
division of neuronal progenitor cells during migration in
L.E. Mello and B.M. Longo
the neonatal mammalian forebrain. Mol Cell Neurosci 6:
496508.
Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR
(2000) Turning blood into brain: cells bearing neuronal
antigens generated in vivo from bone marrow. Science
290:17791782.
Mohapel P, Ekdahl C, Lindvall O (2004) Status epilepti-
cus severity influences the long-term outcome of neu-
rogenesis in the adult dentate gyrus. Neurobiol Dis 15:
196205.
Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade
restores adult hippocampal neurogenesis. Science 302:1760
1765.
Morgan SC, Taylor DL, Pocock JM (2004) Microglia release
activators of neuronal proliferation mediated by activation of
mitogen-activated protein kinase, phosphatidylinositol-
3-kinase/Akt and delta-Notch signalling cascades.
J Neurochem 90:89101.
Nakanishi M, Niidome T, Matsuda S, Akaike A, Kihara T,
Sugimoto H (2007) Microglia-derived interleukin-6 and
leukaemia inhibitory factor promote astrocytic differentia-
tion of neural stem/progenitor cells. Eur J Neurosci 25:
649658.
Napoli I, Neumann H (2009) Microglial clearance function in
health and disease. Neuroscience 158:10301038.
Neumann H, Schmidt H, Wilharm E, Behrens L, Wekerle H
(1997) Interferon gamma gene expression in sensory neu-
rons: evidence for autocrine gene regulation. J Exp Med
186:20232031.
Nilsson SK, Dooner MS, Weier HU, Frenkel B, Lian JB, Stein
GS, Quesenberry PJ (1999) Cells capable of bone production
engraft from whole bone marrow transplants in nonablated
mice. J Exp Med 189:729734.
Nottebohm F (1981) A brain for all seasons: cyclical anatomical
changes in song control nuclei of the canary brain. Science
214:13681370.
Nottebohm F (1985) Neuronal replacement in adulthood. Ann
NY Acad Sci 457:143161.
Nottebohm F (1996) The King Solomon lectures in neuroethol-
ogy. A white canary on Mount Acropolis. J Comp Physiol A
179:149156.
Nottebohm F (2002) Neuronal replacement in the adult brain.
Brain Res Bull 57:737749.
Nottebohm F (2004) The road we travelled: discovery, choreog-
raphy, and significance of brain replaceable neurons. Ann N
Y Acad Sci 1016:628658.
Nowakowski RS, Lewin SB, Miller MW (1989)
Bromodeoxyuridine immunohistochemical determina-
tion of the legths of the cell cycle and the DNA-synthetic
phase for an anatomically defined population. J Neurocytol
18:311318.
Okano HJ, Pfaff DW, Gibbs RB (1993) RB and Cdc2 expression
in brain: correlations with 3H-thymidine incorporation and
neurogenesis. J Neurosci 13:29302938.
OKusky JR, Ye P, DErcole AJ (2000) Insulin-like growth
factor-I promotes neurogenesis and synaptogenesis in the
hippocampal dentate gyrus during postnatal development. J
Neurosci 20:84358442.
Palmer TD, Ray J, Gage FH (1995) FGF-2-responsive neuronal
progenitors reside in proliferative and quiescent regions of
the adult rodent brain. Mol Cell Neurosci 6:474486.
2 Neurogenesis: A Change of Paradigms
Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche
for adult hippocampal neurogenesis. J Comp Neurol 425:
479494.
Parent J (2003) Injury-induced neurogenesis in the adult mam-
malian brain. Neuroscientist 9:261272.
Parent JM, Tada E, Fike JR, Lowenstein DH (1999) Inhibition of
dentate granule cell neurogenesis with brain irradiation does
not prevent seizure-induced mossy fiber synaptic reorganiza-
tion in the rat. J Neurosci 19:45084519.
Parent JM, Yu TW, Leibowitz RT, Gerschwind DH, Sloviter RS,
Lowenstein DH (1997) Dentate granule cell neurogenesis
is increased by seizures and contributes to aberrant net-
work reorganization in the adult rat hippocampus. J Neurosci
17:37273738.
Paton JA, Nottebohm FN (1984) Neurons generated in the
adult brain are recruited into functional circuits. Science
225:10461048.
Pencea V, Bingaman KD, Wiegand SJ, Luskin MB (2001)
Infusion of brain-derived neurotrophic factor into the lat-
eral ventricle of the adult rat leads to new neurons in the
parenchyma of the striatum, septum, thalamus, and hypotha-
lamus. J Neurosci 21:67066717.
Peng H, Huang Y, Rose J, Erichsen D, Herek S, Fujii N,
Tamamura H, Zheng J (2004) Stromal cell-derived factor 1-
mediated CXCR4 signaling in rat and human cortical neural
progenitor cells. J Neurosci Res 76:3550.
Pirttil TJ, Manninen A, Jutila L, Nissinen J, Klviinen R,
Vapalahti M, Immonen A, Paljrvi L, Karkola K, Alafuzoff I,
Mervaala E, Pitknen A (2005) Cystatin C expression is asso-
ciated with granule cell dispersion in epilepsy. Ann Neurol
58:211223.
Pluchino S, Martino G (2005) The therapeutic use of stem cells
for myelin repair in autoimmune demyelinating disorders. J
Neurol Sci 233:117119.
Popper K (2002) The Logic of Scientific Discovery. Routledge
Classics Routledge, London.
Priller J, Flugel A, Wehner T, Boentert M, Haas CA, Prinz
M, Fernandez-Klett F, Prass K, Bechmann I, de Boer BA,
Frotscher M, Kreutzberg GW, Persons DA, Dirnagl U (2001)
Targeting gene-modified hematopoietic cells to the central
nervous system: use of green fluorescent protein uncovers
microglial engraftment. Nat Med 7:13561361.
Rakic P (2002) Neurogenesis in adult primates. Prog Brain Res
138:314.
Ramn y Cajal S (1928) Degeneration and Regeneration of the
Nervous System. Oxford University Press, Oxford.
Ray J, Raymon HK, Gage FH (1995) Generation and cul-
turing of precursor cells and neuroblasts from embryonic
and adult central nervous system. Methods Enzymol 254:
2037.
Reynolds B, Weiss S (1992) Generation of neurons and astro-
cytes from isolated cells of the adult mammalian central
nervous system. Science 255:17071710.
Richards LJ, Kilpatrick TJ, Bartlett PF (1992) De novo genera-
tion of neuronal cells from the adult mouse brain. Proc Natl
Acad Sci USA 89:85918595.
Romariz S, Blanco M, Senra J, Ferrazoli E, Soares M, Santos
RR, Mello LE, Longo B (2008) Avaliao da migrao
e proliferao no hipocampo de camundongos epilpticos
transplantados com clulas de medula ssea GFP. J Epil Clin
Neurophysiol 14:6263.
31
Rougier A, Arthaud S, Zombre N, La Salle L (2005) Patterns of
dentate granule cell responses to perforant path stimulation
in epileptic mice with granule cell dispersion. Epilepsy Res
63:119129.
Ruschenschmidt C, Koch PG, Brustle O, Beck H (2005)
Functional properties of ES cell-derived neurons engrafted
into the hippocampus of adult normal and chronically epilep-
tic rats. Epilepsia 46:174183 Suppl 5.
Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S,
Weisstaub N, Lee J, Duman R, Arancio O, Belzung C, Hen
R (2003) Requirement of hippocampal neurogenesis for the
behavioral effects of antidepressants. Science 301:805809.
Scharfman HE (2004) Functional implications of seizure-
induced neurogenesis. Adv Exp Med Biol 548:192212.
Scharfman HE, Goodman JH, Sollas AL (2000) Granule-like
neurons at the hilar/CA3 border after status epilepticus and
their synchrony with area CA3 pyramidal cells: functional
implications of seizure-induced neurogenesis. J Neurosci
20:61446158.
Scharfman HE, Sollas AL, Smith KL, Jackson MB,
Goodman JH (2002) Structural and functional asym-
metry in the normal and epileptic rat dentate gyrus. J Comp
Neurol 454:424439.
Schilling M, Besselmann M, Leonhard C, Mueller M,
Ringelstein EB, Kiefer R (2003) Microglial activation pre-
cedes and predominates over macrophage infiltration in tran-
sient focal cerebral ischemia: a study in green fluorescent
protein transgenic bone marrow chimeric mice. Exp Neurol
183:2533.
Scott BW, Wojtowicz JM, Burnham WM (2000) Neurogenesis
in the dentate gyrus of the rat following electroconvulsive
shock seizures. Exp Neurol 165:231236.
Seaberg RM, Van der Kooy D (2002) Adult rodent neurogenic
regions: the ventricular subependyma contains neural stem
cells, but the dentate gyrus contains restricted progenitors.
J Neurosci 22:17841793.
Seki T, Arai Y (1993) Highly polysialylated neural cell adhe-
sion molecule (NCAM-H) is expressed by newly generated
granule cells in the dentate gyrus of the adult rat. J Neurosci
13:23512358.
Seki T, Arai Y (1995) Age-related production of new granule
cells in the adult dentate gyrus. Neuroreport 6:24792482.
Selmaj KW, Farooq M, Norton WT, Raine CS, Brosnan CF
(1990) Proliferation of astrocytes in vitro in response to
cytokines. A primary role for tumor necrosis factor. J
Immunol 144:129135.
Seri B, Garca-Verdugo JM, McEwen BS, Alvarez-Buylla A
(2001) Astrocytes give rise to new neurons in the adult
mammalian hippocampus. J Neurosci 21:71537160.
Shankle WR, Rafii MS, Landing BH, Fallon JH (1999)
Approximate doubling of numbers of neurons in postnatal
human cerebral cortex and in 35 specific cytoarchitectural
areas from birth to 72 months. Pediatr Dev Pathol 2:244259.
Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E
(2001) Neurogenesis in the adult is involved in the formation
of trace memories. Nature 410:372376.
Silani V, Cova L, Corbo M, Ciammola A, Polli E (2004)
Stem-cell therapy for amyotrophic lateral sclerosis. Lancet
364:200202.
Simard AR, Rivest S (2004) Role of inflammation in the neuro-
biology of stem cells. Neuroreport 15:23052310.
32
Simard AR, Soulet D, Gowing G, Julien JP, Rivest S (2006)
Bone marrow-derived microglia play a critical role in
restricting senile plaque formation in Alzheimers disease.
Neuron 49:489502.
Sloviter RS, Kudrimoti HS, Laxer KD, Barbaro NM, Chan
S, Hirsch LJ, Goodman RR, Pedley TA (2004) Tectonic
hippocampal malformations in patients with temporal lobe
epilepsy. Epilepsy Res 59:123153.
Steiner B, Zurbog S, Hrster H, Fabel K, Kempermann G (2008)
Differential 24 h responsiveness of Prox1-expressing pre-
cursor cells in adult hippocampal neurogenesis to physical
activity, environmental enrichment, and kainic acid-induced
seizures. Neuroscience 154:521529.
Struikmans H, Rutgers DH, Jansen GH, Tulleken CA, van der
Tweel I, Battermann JJ (1997) S-phase fraction, 5-bromo-2 -
deoxy-uridine labelling index, duration of S-phase, potential
doubling time, and DNA index in benign and malignant brain
tumors. Radiat Oncol Investig 5:170179.
Suzuki F, Junier MP, Guilhem D, Srensen JC, Onteniente
B (1995) Morphogenetic effect of kainate on adult hip-
pocampal neurons associated with a prolonged expres-
sion of brain-derived neurotrophic factor. Neuroscience 64:
665674.
Tanapat P, Hastings NB, Rydel TA, Galea LA, Gould E (2001)
Exposure to fox odor inhibits cell proliferation in the hip-
pocampus of adult rats via an adrenal hormone-dependent
mechanism. J Comp Neurol 437:496504.
Tattersfield AS, Croon RJ, Liu YW, Kells AP, Faull RL, Connor
B (2004) Neurogenesis in the striatum of the quinolinic
acid lesion model of Huntingtons disease. Neuroscience
127:319332.
Taupin P (2008) Adult neurogenesis, neuroinflammation ther-
apeutic potential of adult neural stem cells. Int J Med Sci
5:127132.
Thomas LB, Gates MA, Steindler DA (1996) Young neurons
from the adult subependymal zone proliferate and migrate
along an astrocyte, extracellular matrix-rich pathway. Glia
17:114.
Trejo JL, Carro E, Torres-Aleman I (2001) Circulating insulin-
like growth factor I mediates exercise-induced increases in
the number of new neurons in the adult hippocampus. J
Neurosci 21:16281634.
Trujillo CA, Schwindt TT, Martins AH, Alves JM, Mello LE,
Ulrich H (2009) Novel perspectives of neural stem cell differ-
entiation: from neurotransmitters to therapeutics. Cytometry
A 75:3853.
Vallieres L, Sawchenko PE (2003) Bone marrow-derived cells
that populate the adult mouse brain preserve their hematopoi-
etic identity. J Neurosci 23:51975207.
Vallires L, Campbell II, Gage FH, Sawchenko PE (2002)
Reduced hippocampal neurogenesis in adult transgenic
mice with chronic astrocytic production of interleukin-6. J
Neurosci 22:468492.
Van Praag H, Kempermann G, Gage FH (1999) Running
increases cell proliferation and neurogenesis in the adult
mouse dentate gyrus. Nat Neurosci 2:266270.
Van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD,
Gage FH (2002) Functional neurogenesis in the adult hip-
pocampus. Nature 415:10301034.
Van der Kooy D, Weiss S (2000) Why stem cells? Science
287:14391441.
L.E. Mello and B.M. Longo
Vezzani A (2005) Inflammation and epilepsy. Epilepsy Curr 5:
16.
Vezzani A, Conti M, De Luigi A, Ravizza T, Moneta
D, Marchesi F, De Simoni MG (1999) Interleukin-1beta
immunoreactivity and microglia are enhanced in the rat
hippocampus by focal kainate application: functional evi-
dence for enhancement of electrographic seizures. J Neurosci
19:50545065.
Vezzani A, Moneta D, Conti M, Richichi C, Ravizza T, De Luigi
A, De Simoni MG, Sperk G, Andell-Jonsson S, Lundkvist J,
Iverfeldt K, Bartfai T (2000) Powerful anticonvulsant action
of IL-1 receptor antagonist on intracerebral injection and
astrocytic overexpression in mice. Proc Natl Acad Sci USA
21:1153411539.
Voutsinos-Porche B, Koning E, Kaplan H, Ferrandon A,
Guenounou M, Nehlig A, Motte J (2004) Temporal patterns
of the cerebral inflammatory response in the rat lithium-
pilocarpine model of temporal lobe epilepsy. Neurobiol Dis
17:385402.
Walton NM, Sutter BM, Laywell ED, Levkoff LH, Kearns
SM, Marshall GP 2nd, Scheffler B, Steindler DA (2006)
Microglia instruct subventricular zone neurogenesis. Glia
54:815825.
Wang Y, Baraban SC (2007) Granule cell dispersion and aberrant
neurogenesis in the adult hippocampus of an LIS1 mutant
mouse. Dev Neurosci 29:9198.
Wang Y, Deng Y, Zhou GQ (2008) SDF-1alpha/CXCR4-
mediated migration of systemically transplanted bone mar-
row stromal cells towards ischemic brain lesion in a rat
model. Brain Res 1195:104112.
Weimann JM, Charlton CA, Brazelton TR, Hackman RC, Blau
HM (2003) Contribution of transplanted bone marrow cells
to Purkinje neurons in human adult brains. Proc Natl Acad
Sci USA 100:20882093.
Weissman IL, Anderson DJ, Gage F (2001) Stem and pro-
genitor cells: origins, phenotypes, lineage commitments,
and transdifferentiations. Annu Rev Cell Dev Biol 17:
387403.
Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner
F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch
R (2008) Neurons derived from reprogrammed fibroblasts
functionally integrate into the fetal brain and improve symp-
toms of rats with Parkinsons disease. Proc Natl Acad Sci
USA 105:58565861.
Wong G, Goldshmit Y, Turnley AM (2004) Interferon-gamma
but not TNF alpha promotes neuronal differentiation and
neurite outgrowth of murine adult neural stem cells. Exp
Neurol 187:171177.
Wu JP, Kuo JS, Liu YL, Tzeng SF (2000) Tumor necrosis factor-
alpha modulates the proliferation of neural progenitors in the
subventricular/ventricular zone of adult rat brain. Neurosci
Lett 292:203206.
Yamada M, Onodera M, Mizuno Y, Mochizuki H (2004)
Neurogenesis in olfactory bulb identified by retroviral
labeling in normal and 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-treated adult mice. Neuroscience
124:173181.
Zhang J, Li Y, Chen J, Yang M, Katakowski M, Lu M, Chopp M
(2004) Expression of insulin-like growth factor 1 and recep-
tor in ischemic rats treated with human marrow stromal cells.
Brain Res 1030:1927.
2 Neurogenesis: A Change of Paradigms
Zhao M, Momma S, Delfani K, Carln M, Cassidy RM,
Johansson CB, Brismar H, Shupliakov O, Frisn J, Janson
M (2003) Evidence for neurogenesis in the adult mammalian
substantia nigra. Proc Natl Acad USA 100:79257930.
Zhao CS, Overstreet-Wadiche L (2008) Integration of adult gen-
erated neurons during epileptogenesis. Epilepsia 49(Suppl
5):312.
Zhao LX, Zhang J, Cao F, Meng L, Wang DM, Li YH, Nan X,
Jiao WC, Zheng M, Xu XH, Pei XT (2004) Modification
of the brain-derived neurotrophic factor gene: a portal
33
to transform mesenchymal stem cells into advantageous
engineering cells for neuroregeneration and neuroprotection.
Exp Neurol 190:396406.
Ziv Y, Ron N, Butovsky O, Landa G, Sudai E, Greenberg
N, Cohen H, Kipnis J, Schwartz M (2006) Immune cells
contribute to the maintenance of neurogenesis and spatial
learning abilities in adulthood. Nat Neurosci 9:268275.
Ziv Y, Schwartz M (2008) Orchestrating brain-cell renewal: the
role of immune cells in adult neurogenesis in health and
disease. Trends Mol Med 14:471478.
Chapter 3

Neurogenesis in the Olfactory Epithelium

Bettina Malnic and Lucia Armelin-Correa

Contents neurodegenerative diseases or to induce regeneration

of injured axons.
3.1 Organization of the Mammalian Olfactory
System . . . . . . . . . . . . . . . . . . . 35 Keywords Globose basal cells Horizontal basal
3.2 The Olfactory Epithelium . . . . . . . . . . 36 cells Olfactory ensheating cells Olfactory
3.3 Neurogenesis in the Olfactory Epithelium . . . 38 epithelium Olfactory sensory neurons
3.4 The Olfactory Ensheating Cells . . . . . . . . 41
References . . . . . . . . . . . . . . . . . . . . 42
Abbreviations

GAP-43 growth associated protein of 43-kDa molec-

Abstract The olfactory sensory neurons in the olfac- ular mass
tory epithelium generally live for only about 3060 BG Bowmans glands
days. In addition, they are constantly killed by environ- GBC globose basal cell(s)
mental insults such as pathogens and toxic substances HBC horizontal basal cell(s)
and therefore need to be replaced throughout adult OE olfactory epithelium
life. This unique capability of neuronal regeneration is OEC olfactory ensheating cell(s)
due to the presence of olfactory stem cells localized OMP olfactory marker protein
in the basal layer of the olfactory epithelium which ONF olfactory nerve fibroblast(s)
proliferate and differentiate into new olfactory sen- OSN olfactory sensory neuron(s)
sory neurons. The newly generated neurons are able OST olfactory sulfotransferase
to extend their axons to the olfactory bulb, where NCAM neural cell adhesion molecule
they form synapses with the mitral cells. The olfac- Sus supporting cells
tory ensheating cells, which are specialized olfactory
glial cells, enclose the newly formed axons and help to
direct them through the cribriform plate to the olfac- 3.1 Organization of the Mammalian
tory bulb. Because the olfactory epithelium is readily
Olfactory System
accessible from its location in the nasal cavity, it has
been considered to be a potential source of basal stem
cells and olfactory ensheating cells which could be Mammals can discriminate a vast number of diverse
used in therapeutic applications for the treatment of odorants (Buck, 2000, 2004). Odorants, which are usu-
ally small organic and volatile molecules, are initially
detected by olfactory sensory neurons, located in a spe-
cialized olfactory epithelium lining the nasal cavity.
Each olfactory sensory neuron extends a single den-
B. Malnic ()
drite towards the apical region of the epithelium. The
Departamento de Bioqumica, Instituto de Qumica,
Universidade de So Paulo, So Paulo, Brazil tip of the dendrite carries cilia which are embedded in
e-mail: bmalnic@iq.usp.br the mucus layer of the epithelium, and are therefore

H. Ulrich (ed.), Perspectives of Stem Cells, 35

DOI 10.1007/978-90-481-3375-8_3, Springer Science+Business Media B.V. 2010
36
in direct contact with the environment. Each neuron
also projects an axon to the olfactory bulb, where it
synapses with the dendrites of mitral cells, in spherical
structures called glomeruli. From the olfactory bulb,
sensory information is transmitted to various higher
brain regions via the axons of mitral cells, that project
to the olfactory cortex and other regions.
Odorants enter the nasal cavity and bind to odorant
receptors that are localized in the cilia of the olfactory
sensory neurons (Buck and Axel, 1991). The odor-
ant receptors belong to the super-family of G-protein
coupled receptors and are extremely diverse in their
amino acid sequences, consistent with the ability to
recognize a large variety of odorants. The odorant
receptor genes constitute the largest gene families in
mammalian genomes, with around 1,000 functional
receptors in rodents (Young et al., 2002; Godfrey et al.,
2004; Zhang et al., 2004; Niimura and Nei, 2005)
and 400 functional receptors in humans (Glusman
et al., 2001; Niimura and Nei, 2003; Malnic et al.,
2004).
A series of in situ hybridization experiments using
odorant receptors as molecular probes determined
how sensory information is organized in the olfactory
epithelium and olfactory bulb. These experiments sug-
gested that each olfactory sensory neuron expresses
only one out of the 1,000 odorant receptor genes
(Ressler et al., 1993; Vassar et al., 1993). This was later
verified by examining gene expression in single neu-
rons using single cell RT-PCR (Malnic et al., 1999).
The presence of small amounts of odorant receptor
mRNAs in the axon terminals of the olfactory neu-
rons allowed for a visualization of the glomeruli that
receive input from olfactory neurons expressing differ-
ent odorant receptors. The results indicated that neu-
rons expressing one same given odorant receptor type
converge into one or few glomeruli at two specific sites
in the olfactory bulb (Ressler et al., 1994; Vassar et al.,
1994). Since each mitral cell in the bulb is connected
to a single glomerulus, it receives incoming signals
from only one type of odorant receptor. These results
were confirmed by experiments that used molecular-
genetic techniques to produce transgenic mice were the
olfactory sensory neurons expressing one same type
of odorant receptor co-express tau-lacZ in their axons,
allowing for the visualization of their axonal projec-
tion to the olfactory bulb (Mombaerts et al., 1996).
Interestingly, glomeruli that receive input from each
odorant receptor type show approximately the same
B. Malnic and L. Armelin-Correa
locations in different individuals. These results indi-
cate that the information provided by different odorant
receptors in the nose is organized into a stereotyped
sensory map in the olfactory bulb.
It is not known yet how the sensory information pro-
vided by the different odorant receptors is organized
beyond the olfactory bulb. That is, it is not known how
mitral cells that innervate one given glomerulus con-
nect to the olfactory cortex. Genetic tracing techniques
(Horowitz et al., 1999; Boehm et al., 2005), where one
transneuronal tracer is co-expressed with one given
type of odorant receptor should allow for the visual-
ization of the cortical neurons that receive input from
a particular odorant receptor. Analysis of the neuronal
circuits of different odorant receptors should contribute
to the understanding of how odorant perceptions are
generated by the brain.
3.2 The Olfactory Epithelium
The olfactory epithelium (OE) is found on the nasal
septum and on a series of turbinates in the posterior
region of the nasal cavity (Fig. 3.1a). The OE can be
divided into three major layers: an apical layer, which
contains the supporting cells, a medium layer, which
contains the mature and immature olfactory sensory
neurons, and a basal cell layer, which contains the
horizontal and globose basal cells (Fig. 3.1b).
The supporting cells make up 1518% of the olfac-
tory cells in the OE (Carr et al., 1991). They are
non-neuronal glial-like cells that extend processes
from the surface to the basal lamina of the epithe-
lium. The supporting cells are capped by microvilli
and contain abundant endoplasmic reticulum, but their
functions are not entirely clear. They express mul-
tiple cytochrome P450s, and other biotransformation
enzymes, that are involved in metabolizing foreign
compounds, suggesting a role in detoxifying toxic sub-
stances present in the air, to which the OE is exposed
(Yu et al., 2005). The supporting cells also phagocytose
dead olfactory sensory neurons (OSN) and probably
provide support to the OSN. There is also a second
type of supporting cells, denominated microvillar cells,
which lack the abundant endoplasmic reticulum of
supporting cells (Carr et al., 1991).
Mature OSN are bipolar cells that extend a single
ciliated dendrite to the epithelial surface and project
3 Neurogenesis in the Olfactory Epithelium
Fig. 3.1 The olfactory epithelium. (a) Schematic diagram of
a coronal section through the mouse nasal cavity showing the
septum and the turbinates. (b) Representation of the olfactory
epithelium which is populated by supporting cells (Sus), mature
and immature olfactory sensory neurons (OSNm and OSNim,
respectively), two types of globose basal cells (GBC), the imme-
diate neuronal precursor and the progenitor cells (GBCinp and
GBCprog, respectively), the horizontal basal cells (HBC), the
olfactory ensheating cells (OEC) and the Bowmans gland cells
(BG). Some of the cell specific markers are shown in paren-
thesis: OMP (olfactory marker protein), OST (olfactory sul-
fotransferase), GAP-43 (growth associated protein of 43-kDa
molecular weight), Ngn1 (neurogenin-1), Mash1 (mammalian
achaete-scute homolog 1), K5/K14 (keratin 5 and keratin 14)
a single unmyelinated axon to the olfactory bulb of
the brain. The ciliated dendrites are the primary sites
of odorant transduction. OSN can be identified by
their expression of different proteins. For example,
both mature and immature OSN express the neural cell
adhesion molecule (NCAM) (Key and Akeson, 1990),
but only the mature OSN express the olfactory marker
protein (OMP) (Margolis, 1972) (Fig. 3.1b). OMP is a
cytoplasmic protein that is abundantly and exclusively
expressed in the mature olfactory sensory neurons of
37
both, the olfactory epithelium and the vomeronasal
epithelium (Berghard et al., 1996). The expression of
the olfactory specific adenyl cyclase III (ACIII), and
other proteins involved in odorant signal transduc-
tion, also characterizes mature OSN (Menco et al.,
1992). Immature OSN also express growth associ-
ated protein of 43-kDa molecular weight (GAP-43)
(Verhaagen et al., 1989). The axons of the mature OSN
and their associated (glial) ensheating cells form bun-
dles that run anterior to posterior in the lamina propria
of the epithelium and project to the olfactory bulb,
where they synapse with the mitral cells, forming the
glomeruli.
The basal cells of the olfactory epithelium can
be subdivided into two different cell types, based
on cell morphology (Graziadei and Monti-Graziadei,
1979) and on the expression of specific cell mark-
ers (Holbrook et al., 1995). The horizontal basal cells
(HBC) are flat cells, with elongated nuclei, that stain
darkly with toluidine blue and proliferate at a low rate
(Holbrook et al., 1995). They lie deepest in the olfac-
tory epithelium, in a single-cell layer that is in direct
contact with the basal lamina (Holbrook et al., 1995).
The horizontal cells are the only cells in the olfactory
epithelium that express cytokeratins 5 and 14 (K5 and
K14) (Calof and Chikaraishi, 1989; Holbrook et al.,
1995).
The globose basal cells (GBC) have round nuclei
and show light staining with toluidine blue (Graziadei
and Monti-Graziadei, 1979). GBC do not express
cytokeratins, but they express NCAM, like the OSN.
They lie above the HBC and are the major proliferat-
ing population in the olfactory epithelium (Caggiano
et al., 1994). This population of cells contains the pro-
genitors that divide and give rise to the OSN. The
progenitors sequentially express the proneuronal genes
Mash1 (these are denominated GBC progenitor cells
or GBCprog) and Ngn1 (denominated GBC immedi-
ate neuronal precurssor cells or GBCinp) (Fig. 3.1b)
(Calof et al., 2002; Cau et al., 2002).
In addition, Bowmans glands are present in the
OE. They consist of acini and ducts that extend from
the lamina propria through the epithelium to discharge
mucus secretions at the apical surface (Getchell and
Getchell, 1992). The mucus layer covering the olfac-
tory epithelium, which is produced by the Bowmans
glands and by the supporting cells, is believed to cre-
ate an optimal environment for OSN function (Getchell
38
et al., 1984). These secretions are lipid rich and also
contain odorant binding proteins, which are small
proteins that belong to a large family of ligand carrier
proteins, the lipocalins (Bignetti et al., 1985; Pevsner
and Snyder, 1990). These proteins may enhance odor-
ant detection by assisting in the solubilization and
transport of the odorants in the mucus.
3.3 Neurogenesis in the Olfactory
Epithelium
The OE provides one of the few examples of contin-
uous neurogenesis in the adult nervous system. The
OSN are directly exposed to pathogens and toxic sub-
stances present in the environment and need to be fre-
quently replaced. These neurons generally live for only
about 3060 days and are continuously replaced from
stem cells localized in the basal region of the epithe-
lium (Graziadei and Monti-Graziadei, 1979; Calof and
Chikaraishi, 1989; Caggiano et al., 1994).
OE neurogenesis can be subdivided in two phases:
the primary and the established neurogenesis (Beites
et al., 2005). During the primary neurogenesis the
olfactory placode, a multipotent group of cells that will
give rise to all cell types in the OE, is determined,
and the establishment of the OE neuronal lineage is
initiated. The interaction between the olfactory pla-
codal epithelium and its associated mesenchyme is
essential for the generation of cellular and molecular
diversity, as well as for the development of appropriate
axon trajectories in the peripheral portion of the olfac-
tory pathway (Balmer and Lamantia, 2005). Signaling
molecules such as retinoic acid (RA), fibroblast growth
factor 8 (FGF8), sonic hedgehog (Shh) and bone mor-
phogenetic protein 4 (BMP4) are involved in this initial
events (Balmer and Lamantia, 2005).
Established neurogenesis is initiated in mice around
E13.5E14.5, when the OE becomes organized into
its mature pattern. The nuclei of the supporting cells
become organized in a single apical layer and there
is a decrease in the overall number of mitotic nuclei,
which are localized primarily to the basal compartment
of the OE. Signaling molecules such as BMP2, 4 and
7; and growth and differentiation factor 11 (GDF11)
exert important roles in this stage (Shou et al., 2000;
Wu et al., 2003). Factors that predominate during the
primary olfactory neurogenesis are likely to determine
B. Malnic and L. Armelin-Correa
that the right amount of cells will populate the OE stem
cell niche, while in the established tissue these factors
play a role in maintaining a tight regulation of the
total number of OE cells (Beites et al., 2005). Indeed,
after olfactory bulbectomy, which induces OSN apop-
tosis, increased neuronal progenitor cell mitoses are
observed (Leung et al., 2007), supporting the idea that
OSN death removes an inhibitory feedback signal that
normally acts to block progenitor cell proliferation.
Commitment and differentiation of the cells that
originate the OSN is controlled by a series of genes
that encode transcription factors of the basic helix-
loop-helix (bHLH) class which are homologues of the
Drosophila proneural genes of the achaete-scute (asc)
and atonal (ato) complex, such as Mash1, neurogenin1
(Ngn1) and NeuroD (Cau et al., 1997, 2002). The
commitment of basal cells to the OSN lineage is ini-
tiated with the expression of Mash1. Accordingly, in
Mash1/ embryos neuronal differentiation in the OE
is blocked. There is increased cell death and cells do
not express Ngn1 and NeuroD (Guillemot et al., 1993;
Cau et al., 1997). The Ngn1 mutant phenotype has
revealed that this transcription factor is required for
NeuroD expression, which in turn promotes neuronal
differentiation of OSN (Cau et al., 2002).
A combination of single-cell RT-PCR and microar-
ray was used to identify transcription factors that are
downstream of Mash1 and are involved in the genera-
tion of further cellular diversity of the OSN (Tietjen
et al., 2003). In these experiments, cDNA libraries
were prepared from isolated single cell progenitors
(expressing Mash1 and the cell division markers cdc2
and Ki67) or mature OSN (expressing OMP and odor-
ant receptor). Probes prepared from these single-cell
libraries were hybridized to an Affymetrix microar-
ray containing a large number of mouse probe sets.
Transcripts enriched in the progenitor cells versus
mature OSN were identified. Some of these genes
(such as Notch1, Lhx2, Hes6) are involved in cell
proliferation and were shown to be dependent on
Mash1 expression, since their expression is abolished
in Mash1/ mice.
There are several transcription factors expressed in
the OE that are not involved in OSN differentiation,
but are required for OSN maturation. Mature OSN
express one single type of odorant receptor (Chess
et al., 1994; Malnic et al., 1999), and olfactory sig-
nal transduction proteins that are required for proper
neuronal function. The expression of odorant receptor
3 Neurogenesis in the Olfactory Epithelium 39

genes is segregated in the dorso-ventral axis of the OE,

so that OSN expressing one given odorant receptor are
contained within one of four distinct zones of the OE
(Ressler et al., 1993; Vassar et al., 1993). Interestingly,
some transcripts enriched in the progenitor cells, such
as Pax6, Sox11 and Eya2, were found to be expressed
in specific zones in E15 OE, and this pattern of expres-
sion was maintained in the Mash1/ mice (Tietjen
et al., 2003). These transcripts may be involved in the
zonal organization of odorant receptor expression in
the OE.
The O/E like proteins are transcription factors that
bind to promoter regions of several genes expressed
in mature OSN, such as OMP, ACIII, Golf and odor-
ant receptors (Kudrycki et al., 1993; Wang and Reed,
1993; Michaloski et al., 2006), and therefore must be
involved in the regulation of the expression of these
genes. Another characterized gene that is expressed
downstream of Mash1 is Zfp423/OAZ (Cheng and
Reed, 2007). This transcription factor is transiently
expressed in newly differentiating neurons and is
required for maturation of the OSN. Zfp423/OAZ binds
to the O/E like proteins and inhibits their binding to
the promoters of the olfactory specific genes (Tsai
and Reed, 1997), thereby preventing the premature
differentiation of the immature OSN.
The newly-generated neurons are result of mitoses
that occur near the basal lamina of the OE, and the pro-
gression of neuronal maturation occurs from the basal
to the apical portion of the tissue (Fig. 3.2). The basal
region of the epithelium contains cells with distinct
proliferating rates: stem cells thought to undergo slow,
asymmetric cell divisions (the HBC), and a popula-
Fig. 3.2 Neuronal regeneration in the olfactory epithelium.
tion of rapidly-dividing transit amplifying progenitors (a) In physiological OSN turnover or under mild injury condi-
which are committed to a neuronal cell fate (the GBC). tions, where only the OSN are ablated, GBC have the capacity
It has been difficult to define exactly which one of to regenerate the missing neurons. (b) Under severe olfactory
epithelium lesions, that ablate the OSN, supporting cells and the
these two distinct cell types represents the stem cell
GBC, HBC are able to regenerate GBC, OSN, Bowmans gland
population of the olfactory epithelium (Calof et al., and supporting cells. Model based on Beites et al. (2005) and
1998). HBC, which undergo slow cell division, a hall- Leung et al. (2007)
mark of other well-characterized adult tissue stem
cell (Weissman, 2000), express cytokeratins (Holbrook progenitor cells which sequentially express the proneu-
et al., 1995), integrins (Carter et al., 2004) and also ral genes Mash1 and Ngn1 (Cau et al., 1997). Studies
respond to epidermal growth factor (EGF), a factor of both developmental and regeneration models sug-
shown to be mitogenic for stem cells in the CNS. gest that the Mash1 expressing cells are daughters of
Besides, when in culture, HBC have demonstrated the the OE stem cell (Gordon et al., 1995). Some retroviral
capacity to generate neurons as well as non-neuronal lineage analyses, including models of injury induced
cells (Sicard et al., 1998; Carter et al., 2004). GBC, OE regeneration as well as cell transplantation experi-
on the other hand, even though cytokeratin-negative, ments, supported the idea that a subpopulation of GBC
contain the two populations of transit amplifying was a multipotent progenitor giving rise not only to
40
OSN, but also to supporting cells (Caggiano et al.,
1994; Huard et al., 1998; Chen et al., 2004).
Recently, the technology of homologous recombi-
nation was used to clarify the controversy that exists
concerning the identity of the OE stem cells. Leung
and cols (2007) performed an in vivo fate mapping
of HBC exploiting the restricted expression of cytok-
eratin K5 gene in post natal HBC. A transgenic
mouse expressing a tamoxifen-inducible Cre recombi-
nase under the control of the cytokeratin K5 promoter
(K5-CreER T2 ) was crossed to a strain containing a
lox-STOP-lox LacZ reporter gene at the Rosa26 locus
(R26R), a locus from which genes can be ubiqui-
tously expressed. Injection of tamoxifen into double-
heterozygous K5-CreERT2 /R26R animals led to the
induction of Cre recombinase in HBC, and resulted
in CreER mediated excision of the lox-flanked STOP
sequence at the LacZ reporter locus. This permanent
activation of the LacZ reporter gene in HBC and all of
their descendents, allows for a HBC-specific lineage
tracing strategy.
Analysis of HBC contribution to the natural OSN
turnover in these double-heterozygous animals indi-
cated that these cells, under normal conditions, remain
quiescent in the OE. Since specific lesions in the OE
lead to different dynamics of cell proliferation and
differentiation (Murray and Calof, 1999), three dif-
ferent OE regeneration models were tested in these
transgenic animals: bulbectomy, exposure to methyl
bromide and injection of methimazole. Animals which
suffered bulbectomy did not show a different HBC
dynamic from that of control animals, suggesting that
HBC remain quiescent under specific ablation of OSN
in OE. In this condition, only mature OSN are lost, and
new neurons are probably regenerated from another
pool of progenitor cells, presumably the GBC (Gordon
et al., 1995) (Fig. 3.2a). But under extensive OE
lesions, as those caused by exposure to methyl bro-
mide or methimazole, which damage all cell types
in the OE including the neuronal progenitors GBC,
transgenic animals showed clusters of marked cells
throughout the OE including GBC, OSN, Bowmans
glands and supporting cells (Fig. 3.2b). OSN and
supporting cells were respectively identified by co-
immunostaining with antibodies anti-OMP and anti-
olfactory sulfotransferase (OST). Bowmans gland
cells were identified based on their peculiar localiza-
tion regarding the neuroepithelium basal lamina. To
determine whether GBC lie in the HBC-initiated path-
way of differentiation into mature neurons, knock-in
B. Malnic and L. Armelin-Correa
mice, bearing the GFP coding sequence replacing
either Mash1 or Ngn1 genes, were crossed with K5-
CreERT2 /R26R animals. These triple heterozygous
animals, once subjected to severe OE lesions, showed
GBC co-expressing GFP and LacZ. These findings are
consistent with in vitro studies (Carter et al., 2004),
and provide in vivo evidence that HBC can give rise to
both neuronal and non-neuronal cell types in the OE.
HBC undergo a regulated transient proliferative burst
to regenerate OE when an extensive cellular depletion
occurs, while under bulbectomy no change is detected
in their proliferation rate (Leung et al., 2007).
The study of OE regeneration in K5-CreERT2 /R26R
animals provides a new model for neural stem cell
dynamics in which distinct cell populations mediate
normal neuronal turnover and neuronal replacement
in traumatic injuries. It also opens the possibility to
perform other cell specific lineage tracing in the OE.
Lineage tracing of GBC at specific commitment points
would help to elucidate if these progenitor cells also
originate supporting cells and Bowmans glands, and
at what point they do so, or if HBC have another cell
intermediate to originate these non-neuronal cell types.
Also it is still a mystery which cells are the true pro-
genitors of OEC. There are studies which suggest that
OEC may originate from stem cells in the OE (Beites
et al., 2005), and it remains to be determined whether
HBC are these progenitors. The complete understand-
ing of the differentiation pathway of neuronal and
non-neuronal cells in the OE should therefore aid in the
potential application of OE stem and progenitor cells
in the treatment of CNS lesions and neurodegenerative
diseases.
The mechanisms of OE regeneration in humans are
however not as well understood as in rodents. Although
the human OE is similar to the rodent OE regard-
ing its cellular and molecular composition, there are
a few differences, which suggest that neurogenesis
of OSN in humans may differ from that in rodents.
First, the molecular and morphological differences that
exist between GBC and HBC in rodents are not found
in humans (Hahn et al., 2005). In humans, the cells
adjacent to the basal lamina show no morphological
differences from the cells located above them. All cells
in the basal layer are round and resemble the rodent
GBC, with no cells showing the morphological char-
acteristics of the rodent HBC. Also, differently from
what is observed in rodents, all of these GBC-like basal
cells express cytokeratin-5. Second, in humans, the
laminar organization of immature and mature neurons
3 Neurogenesis in the Olfactory Epithelium
observed in rodents, does not exist. In rodents, imma-
ture and mature OSN exist in two layers, with the
immature OSN being localized closer to the basal lam-
ina. In humans, the immature and mature OSN were
found to be dispersed throughout the OE (Hahn et al.,
2005).
The fact that human OE biopsies can be obtained
from living human subjects should contribute to the
study of human neuronal differentiation. Because of
its regenerative capacity and the fact that it is read-
ily accessible from its location in the nasal cavity, the
human OE represents a source of mitotically active
neuronal progenitors, which once differentiated into
neurons, could be used in potential therapies of autol-
ogous transplantation for the treatment of neurodegen-
erative diseases (Marshall et al., 2006). Neurospheres
obtained from cultured human OE have already been
shown to differentiate into mature neurons, emphasiz-
ing the potential use of adult human OE as a source of
progenitors for future therapies (Othman et al., 2005;
Zhang et al., 2006).
3.4 The Olfactory Ensheating Cells
Continuous neurogenesis in the OE requires that not
only OSN are replaced, but also that the axons of the
newly formed OSN target the correct glomerulus in
the olfactory bulb. The ability of OSN to regenerate
their axonal connections with the mitral cells in the
bulb throughout life is believed to be in part because
the olfactory axons are ensheated by specialized glial
cells, the olfactory ensheating cells (OEC).
The axons of OSN form loose bundles at the OE
basal lamina. Once these axons have entered the lam-
ina propria they are enwrapped by OEC, which guide
them through the cribriform plate to the olfactory bulb
(Boyd et al., 2003; Franssen et al., 2007). OEC consist
of a thin cytoplasmatic sheet that encloses a tunnel-
like space through which hundreds of unmyelinated
olfactory axons run (Field et al., 2003). Therefore,
the olfactory nerves are constituted by olfactory axons
enclosed by a series of end-to-end OEC and overlaid
by the basal lamina and by olfactory nerve fibrob-
lasts (ONF) (Li et al., 2005). While in the peripheral
nerves Schwann cells enwrap around 20 axons that are
separated one from another by folds of Schwann cells
cytoplasm, in the olfactory nerve, the OEC are not so
41
closely associated with the axons of the OSN. In this
case, hundreds of axons are enclosed by only a thin
OEC cytoplasmatic process (Field et al., 2003). When
the olfactory nerve reaches the olfactory bulb, the basal
lamina and the ONF are reflected off and the OEC
processes open up to interdigitate with the astrocytic
processes which cover the brain, enabling the olfac-
tory axons to enter the olfactory bulbs (Raisman, 1985;
Valverde et al., 1992). Microscopic investigations of
the tubular arrangement of the OEC/ONF during OSN
degeneration and regeneration revealed that it remains
stable, with no indications of cell death, division or
migration, differing completely from Schwann cells
in pheripheral nerve lesions (Williams et al., 2004; Li
et al., 2005). The persistence of the OEC tunnels con-
tributes to the effectiveness of the regeneration of the
olfactory axons.
During development the OEC, like the other cells in
the OE, originate from the olfactory placode (Farbman
and Squinto, 1985). Even though the OEC morpho-
logically resemble Schwann cells, they physiologically
do not produce myelin and they demonstrate a highly
plastic and heterogeneous morphology, being capa-
ble of acquiring in vitro, an astrocyte-like morphology
(Richter and Roskams, 2008). Microarray analysis of
the transcriptional profiling of OEC, Schwann cells
and astrocytes revealed that these cells have overlap-
ping but distinct gene expression profiles, and that
OEC are transcriptionally closer to Schwann cells than
to astrocytes (Vincent et al., 2005). However, OEC
have a transcriptome more similar to astrocytes than
Schwann cells do (Vincent et al., 2005), and while
the co-culture of randomly dispersed Schwann cells
and astrocytes shows cell type-specific territories, co-
cultured OEC and astrocytes intermingle, indicating
that OEC integrate more effectively than Schwann
cells into astrocytic environments (Lakatos et al.,
2000).
Nerve fibers usually require an aligned glial path-
way to establish connections between neurons. During
a spinal cord or dorsal root lesion this pathway is dis-
rupted, and astrocytes immediately seal off the breach,
to recover the external wall of the nervous system,
which avoids invasion of non-neural cells and other
damaging materials (Raisman and Li, 2007). However,
the formation of a fibrous scar or cavity at the site of
injury abrogates the repair of the nerve connections,
because the glial pathway required for the growth of
the nerve fibers are disrupted.
42
It has been shown that axons are capable of sprout-
ing after spinal cord injury, and that transplantation
of Schwann cells amplifies this response, but in these
experiments the axons show minimal capacity to reen-
ter the host CNS (Li and Raisman, 1994; Keyvan-
Fouladi et al., 2005). The fact that there is a continuous
neurogenesis in the olfactory system and that OEC
play a crucial role in this process, maintaining the
olfactory nerve structure and interacting with astro-
cytes in the olfactory bulb, prompted researchers to
study their value as theraupeutic agents for treatment
of CNS injuries (reviewed in Franssen et al., 2007;
Raisman and Li, 2007; Richter and Roskams, 2008).
It was shown that OEC are capable of producing a
series of factors which support axonal growth, such as
nerve growth factor (NGF), brain derived neurotrophic
factor (BDNF), neurotrophin 4/5 (NT-4/5) and also
other growth factors, such as fibroblast growth factor 2
(FGF-2) (reviewed in Richter and Roskams, 2008). A
different number of in vitro assays have shown that co-
culture of not only OSN, but also CNS neurons, with
OEC have led to increased neurite growth (reviewed in
Richter and Roskams, 2008). Besides, OEC produce
several components of the olfactory system extracellu-
lar matrix which may also contribute to neuronal axon
growth.
OEC transplantation consistently induces a more
permissive scar boundary for the extension of growing
axons than that induced by Schwann cell transplan-
tation (Raisman and Li, 2007; Richter and Roskams,
2008). When a lesion occurs, astrocytes and their pro-
cesses form a dense mass around it, and transplantation
of OEC leads to reorganization of the astrocytic scar to
form a newly aligned pathway (Raisman and Li, 2007;
Richter and Roskams, 2008). OEC are also capable
of producing myelin in vitro and in vivo (reviewed in
Richter and Roskams, 2008).
It has been shown that transplanted OEC can inter-
act with host astrocytes to produce a bridge through
the dorsal root entry zone (DREZ) along which axons
are able to regenerate into the dorsal columns (Li
et al., 2004). But these results have been challenged by
studies using labeled OEC and more complex axonal
tracing techniques which suggested that apparent long-
distance afferent in-growth could be the result of
spared fibers, leaking label, or growth fibers only along
physically disrupted injection tracts (Gomez et al.,
2003; Ramer et al., 2004b; Riddell et al., 2004).
B. Malnic and L. Armelin-Correa
OEC transplantation in animals that suffered spinal
cord injuries demonstrated that OEC minimize cavity
and scar formation, induce local angiogenesis and dif-
ferential axon sprouting and regeneration (Ramer et al.,
2004), and improved animals behavioral, breathing
and climbing capacities (Keyvan-Fouladi et al., 2003;
Li et al., 2003). These findings suggest that OEC are
either capable of promoting sprouting or facilitating
plasticity and neuroprotection in injured spinal cord,
which may lead to improved behavioral outcomes, but
the capacity of OEC to promote spinal cord axonal
regeneration is still questioned (Richter and Roskams,
2008).
Several important points must be taken into con-
sideration when trying to understand the potential
effect of OEC in promoting axonal regeneration: the
size and type of lesion inflicted to the nervous tissue,
the type of nervous tract involved and collateral axon
branching and differences in the preparations of OEC
that will be transplanted. There are at least two sources
of OEC for transplantation: the lamina propria of the
OE and the olfactory bulb. OEC may be transplanted
purified or associated with the basal lamina and ONF
which physiologically enclose the olfactory nerve.
These cells maybe cultured or directly transplanted,
and the delay between injury and cell application
also impacts the OEC functions and their ability to
promote regeneration.
Despite some conflicting results the current experi-
mental observations raise hope that transplantation of
OEC will contribute to the treatment of spinal cord and
dorsal root injuries. With these perspectives in mind
some neurosurgical teams have established clinical tri-
als involving autologous transplantation of olfactory
mucosa or OEC in patient with spinal cord injuries
(Lima et al., 2006; Mackay-Sim et al., 2008). Future
investigations should reveal the potential of using OEC
for therapeutic applications in axonal repair.
References
Balmer C, Lamantia A-S (2005) Noses and neurons: induction,
morphogenesis, and neuronal differentiation in the peripheral
olfactory pathway. Dev Dyn 234:464481.
Beites C, Kawauchi S, Crocker C, Calof A (2005) Identification
and molecular regulation of neural stem cells in the olfactory
epithellium. Exp Cell Res 306:309316.
3 Neurogenesis in the Olfactory Epithelium
Berghard A, Buck LB, Liman ER (1996) Evidence for distinct
signaling mechanisms in two mammalian olfactory sense
organs. Proc Natl Acad Sci USA 93:23652369.
Bignetti E, Cavaggioni A, Pelosi P, Persaud K, Sorbi R,
Tirindelli R (1985) Purification and characterisation of
an odorant-binding protein from cow nasal tissue. Eur J
Biochem 149:227231.
Boehm U, Zou Z, Buck L (2005) Feedback loops link odor
and pheromone signaling with reproduction. Cell 123:
683695.
Boyd J, Skihar V, Kawaja M, Doucette R (2003) Olfactory
ensheating cells: historical perspective and therapeutic
potential. Anat Rec B New Anat 271:4960.
Buck LB (2000) The molecular architecture of odor and
pheromone sensing in mammals [comment]. Cell 100:
611618.
Buck LB (2004) Olfactory receptors and odor coding in mam-
mals. Nutr Rev 62:S184S188.
Buck L, Axel R (1991) A novel multigene family may encode
odorant receptors: a molecular basis for odor recognition.
Cell 65:175187.
Caggiano M, Kauer J, Hunter D (1994) Globose basal cells are
neuronal progenitors in the olfactory epithelium: a lineage
analysis using a replication-incompetent retrovirus. Neuron
13:339352.
Calof A, Bonnin A, Crocker C, Kawauchi S, Murray R, Shou
J, Wu H-H (2002) Progenitor cells of the olfactory receptor
neuron lineage. Microsc Res Tech 58:176188.
Calof AL, Chikaraishi DM (1989) Analysis of neurogenesis
in a mammalian neuroepithelium: proliferation and differ-
entiation of an olfactory neuron precursor in vitro. Neuron
3:115127.
Calof A, Mumm J, Rim P, Shou J (1998) The neuronal stem cell
of the olfactory epithelium. J Neurobiol 36:190205.
Carr V, Farbman A, Colletti L, Morgan J (1991) Identification
of a new non-neuronal cell type in rat olfactory epithelium.
Neuroscience 45:433449.
Carter L, MacDonald J, Roskams A (2004) Olfactory horizontal
basal cells demonstrate a conserved multipotent progenitor
phenotype. J Neurosci 24:56705683.
Cau E, Casarosa S, Guillemot F (2002) Mash1 and Ngn1 con-
trol distinct steps of determination and differentiation in
the olfactory sensory neuron lineage. Development 129:
18711880.
Cau E, Gradwohl G, Fode C, Guillemot F (1997) Mash1 acti-
vates a cascade of bHLH regulators in olfactory neuron
progenitors. Development 124:16111621.
Chen X, Fang H, Schwob J (2004) Multipotency of purified,
transplanted globose basal cells in olfactory epithelium.
J Comp Neurol 469:457474.
Cheng L, Reed R (2007) Zfp423/OAZ participates in a devel-
opmental switch during olfactory neurogenesis. Neuron
54:547557.
Chess A, Simon I, Cedar H, Axel R (1994) Allelic inactiva-
tion regulates olfactory receptor gene expression. Cell 78:
823834.
Farbman A, Squinto L (1985) Early development of olfactory
receptor cell axons. Brain Res 351:205213.
Field P, Li Y, Raisman G (2003) Ensheatment of the olfactory
nerves in the adult rat. J Neurocytol 32:317324.
43
Franssen E, de Bree F, Verhaagen J (2007) Olfactory ensheathing
glia: their contribution to primary olfactory nervous sys-
tem regeneration and their regenerative potential following
transplantation into the injured spinal cord. Brain Res Rev
56:236258.
Getchell M, Getchell T (1992) Fine structural aspects of secre-
tion and extrinsic innervation in the olfactory mucosa.
Microsc Res Tech 23:111127.
Getchell T, Margolis F, Getchell M (1984) Perireceptor and
receptor events in vertebrage olfaction. Prog Neurobiol
23:317345.
Glusman G, Yanai I, Rubin I, Lancet D (2001) The complete
human olfactory subgenome. Genome Res 11:685702.
Godfrey PA, Malnic B, Buck LB (2004) The mouse olfac-
tory receptor gene family. Proc Natl Acad Sci USA 101:
21562161.
Gomez V, Averill S, King V, Yang Q, Doncel Perez E, Chacon
S, Ward R, Nieto-Sampedro M, Priestley J, Taylor J (2003)
Transplantation of olfactory ensheathing cells fails to pro-
mote significant axonal regeneration from dorsal roots into
the rat cervical cord. J Neurocytol 32:5370.
Gordon M, Mumm J, Davis R, Holcomb J, Calof A (1995)
Dynamics of MASH1 expression in vitro and in vivo sug-
gest a non-stem cell site of MASH1 action in the olfactory
receptor neuron lineage. Mol Cell Neurosci 6:363379.
Graziadei PPC, Monti-Graziadei GA (1979) Neurogenesis and
neuron regeneration in the olfactory system of mammals.
I. Morphological aspects of differentiation and structural
organization of the olfactory sensory neurons. J Neurocytol
8:118.
Guillemot F, Lo L, Johnson J, Auerbach A, Anderson D, Joyner
A (1993) Mammalian achaete-scute homolog 1 is required
for the early development of olfactory and autonomic neu-
rons. Cell 124:463476.
Hahn C-G, Han L-Y, Rawson N, Mirza N, Borgmann-Winter
K, Lenox R, Arnold S (2005) In vivo and in vitro neuroge-
nesis in human olfactory epithelium. J Comp Neurol 483:
154163.
Holbrook E, Szumowski K, Schwob J (1995) An immunochem-
ical, ultrastructural, and developmental characterization of
the horizontal basal cells of rat olfactory epithelium. J Comp
Neurol 363:129146.
Horowitz LF, Montmayeur J-P, Echelard Y, Buck LB (1999) A
genetic approach to trace neural circuits. Proc Natl Acad Sci
USA 96:31943199.
Huard J, Youngentob S, Goldstein B, Luskin M, Schwob J
(1998) Adult olfactory epithelium contains multipotent pro-
genitors that give rise to neurons and non-neural cells. J
Comp Neurol 400:469486.
Key B, Akeson R (1990) Olfactory neurons express a unique
glycosylated form of the neural cell adhesion molecule
(N-CAM). J Cell Biol 110:17291743.
Keyvan-Fouladi N, Raisman G, Li Y (2003) Functional repair of
the corticospinal tract by delayed transplantation of olfactory
ensheathing cells in adult rats. J Neurosci 23:94289434.
Keyvan-Fouladi N, Raisman G, Li Y (2005) Delayed repair of
corticospinal tract lesions as an assay for the effectiveness of
transplantation of Schwann cells. Glia 51:306311.
Kudrycki K, Stein-Izsak C, Behn C, Grillo M, Akeson R,
Margolis F (1993) Olf-1 binding site: characterization of
44
an olfactory neuron-specific promoter motif. Mol Cell Biol
13:30023014.
Lakatos A, Franklin R, Barnett S (2000) Olfactory ensheathing
cells and Schwann cells differ in their in vitro interactions
with astrocytes. Glia 32:214225.
Leung C, Coulombe P, Reed R (2007) Contribution of olfactory
neural stem cells to tissue maintenance and regeneration. Nat
Neurosci 6:720726.
Li Y, Carlstedt T, Berthold CH, Raisman G (2004) Interaction of
transplanted olfactory-ensheathing cells and host astrocytic
processes provides a bridge for axons to regenerate across
the dorsal root entry zone. Exp Neurol 188:300308.
Li Y, Field P, Raisman G (2005) Olfactory ensheathing cells
and olfactory nerve fibroblasts maintain continuous open
channels for regrowth of olfactory nerve fibres. Glia 52:
245251.
Li Y, Raisman G (1994) Schwann cells induce sprouting in
motor and sensory axons in the adult rat spinal cord. J
Neurosci 14:40504063.
Li Y, Sauve Y, Li D, Lund R, Raisman G (2003) Transplanted
olfactory ensheathing cells promote regeneration of cut adult
rat optic nerve axons. J Neurosci 23:77837788.
Lima C, Pratas-Vital J, Escada P, Hasse-Ferreira A, Capucho
C, Peduzzi J (2006) Olfactory mucosa autografts in human
spinal cord injury: a pilot clinical study. J Spinal Cord Med
29:191203.
Mackay-Sim A, Feron F, Cochrane J, Bassingthwaighte L,
Bayliss C, Davies W, Fronek P, Gray C, Kerr G, Licina P,
Nowitzke A, Perry C, Silburn P, Urquhart S, Geraghty T
(2008) Autologous olfactory ensheathing cell transplanta-
tion in human paraplegia: a 3-year clinical trial. Brain 131:
23762386.
Malnic B, Godfrey PA, Buck LB (2004) The human olfac-
tory receptor gene family. Proc Natl Acad Sci USA 101:
25842589.
Malnic B, Hirono J, Sato T, Buck LB (1999) Combinatorial
receptor codes for odors. Cell 96:713723.
Margolis F (1972) A brain protein unique to the olfactory bulb.
Proc Natl Acad Sci USA 69:12211224.
Marshall C, Lu C, Winstead W, Zhang X, Xiao M, Harding
G, Klueber K, Roisen F (2006) The therapeutic potential
of human olfactory-derived stem cells. Histol Histopatol
21:633643.
Menco BPM, Bruch RC, Dau B, Danho W (1992) Ultrastructural
localization of olfactory transduction components: the G
protein subunit Golf and type III adenylyl cyclase. Neuron
8:441453.
Michaloski J, Galante P, Malnic B (2006) Identification of poten-
tial regulatory motifs in odorant receptor genes by analysis of
promoter sequences. Genome Res 16:10911098.
Mombaerts P, Wang F, Dulac C, Chao S, Nemes A, Mendelsohn
M, Edmondson J, Axel R (1996) Visualizing an olfactory
sensory map. Cell 87:675686.
Murray R, Calof A (1999) Neuronal regeneration: lessons from
the olfactory system. Cell Dev Biol 10:421431.
Niimura Y, Nei M (2003) Evolution of olfactory receptor genes
in the human genome. Proc Natl Acad Sci USA 100:
1223512240.
Niimura Y, Nei M (2005) Comparative evolutionary analysis of
olfactory receptor genes clusters between humans and mice.
Gene 346:1321.
B. Malnic and L. Armelin-Correa
Othman M, Lu C, Klueber K, Winstead W, Roisen F (2005)
Clonal analysis of adult human olfactory neurosphere form-
ing cells. Biotech Histochem 80:189200.
Pevsner J, Snyder S (1990) Odorant binding protein: odorant
transport function in the nasal epithelium. Chemical Senses
15:217222.
Raisman G (1985) Specializedneuroglial arrangement may
explain the capacity of vomeronasal axons to reinnervate
central neurons. Neuroscience 14:237254.
Raisman G, Li Y (2007) Repair of neural pathways by olfactory
ensheating cells. Nat Rev Neurosci 8:312319.
Ramer L, Au E, Richter M, Liu J, Tetzlaff W, Roskams A
(2004a) Peripheral olfactory ensheathing cells reduce scar
and cavity formation and promote regeneration after spinal
cord injury. J Comp Neurol 473:115.
Ramer L, Richter M, Roskams A, Tetzlaff W, Ramer M (2004b)
Peripherally-derived olfactory ensheathing cells do not pro-
mote primary afferent regeneration following dorsal root
injury. Glia 47:189206.
Ressler KJ, Sullivan SL, Buck LB (1993) A zonal organiza-
tion of odorant receptor gene expression in the olfactory
epithelium. Cell 73:597609.
Ressler KJ, Sullivan SL, Buck LB (1994) Information cod-
ing in the olfactory system: evidence for a stereotyped and
highly organized epitope map in the olfactory bulb. Cell 79:
12451255.
Richter M, Roskams A (2008) Olfactory ensheathing cell trans-
plantation following spinal cord injury: hype or hope? Exp
Neurol 209:353367.
Riddell J, Enriquez-Denton M, Toft A, Fairless R, Barnett S
(2004) Olfactory ensheathing cell grafts have minimal influ-
ence on regeneration at the dorsal root entry zone following
rhizotomy. Glia 47:150167.
Shou J, Murray R, Rim P, Calof A (2000) Opposing effects of
bone morphogenetic proteins on neuron production and sur-
vival in the olfactory receptor neuron lineage. Development
127:54035413.
Sicard G, Feron F, Andrieu J, Holley A, Mackay-Sim A (1998)
Generation of neurons from a nonneuronal precursor in
adult olfactory epithelium in vitro. Ann N Y Acad Sci 855:
223225.
Tietjen I, Rihel J, Cao Y, Koentges G, Zakhary L, Dulac C (2003)
Single-cell transcriptional analysis of neuronal progenitors.
Neuron 38:161175.
Tsai R, Reed R (1997) Cloning and functional characterization
of Roaz, a zinc finger protein that interacts with O/E-1 to
regulate gene expression: implications for olfactory neuronal
development. J Neurosci 17:41594169.
Valverde F, Santacana M, Heredia M (1992) Formation
of an Olfactory Glomerulus: Morphological Aspects of
Development and Organization. Neuroscience 49:255275.
Vassar R, Chao S, Sitcheran R, Nunez J, Vosshall L, Axel R
(1994) Topographic organization of sensory projections to
the olfactory bulb. Cell 79:981991.
Vassar R, Ngai J, Axel R (1993) Spatial segregation of odorant
receptor expression in the mammalian olfactory epithelium.
Cell 74:309318.
Verhaagen J, Oestreicher A, Gispen W, Margolis FL (1989) The
expression of the growth associated protein B50/GAP43 in
the olfactory system of neonatal and adult rats. J Neurosci
9:683691.
3 Neurogenesis in the Olfactory Epithelium
Vincent A, Taylor J, Choi-Lundberg D, West A, Chuah M (2005)
Genetic expression profile of olfactory ensheathing cells is
distinct from that of Schwann cells and astrocytes. Glia
51:132147.
Wang M, Reed R (1993) Molecular cloning of the olfactory neu-
ronal transcription factor Olf-1 by genetic selection in yeast.
Nature 364:121126.
Weissman I (2000) Stem cells: units of development, units of
regeneration, and units in evolution. Cell 100:157168.
Williams S, Franklin R, Barnett S (2004) Response of olfac-
tory ensheathing cells to the degeneration and regeneration
of the peripheral olfactory system and the involvement of the
neuregulins. J Comp Neurol 470:5062.
Wu H-H, Ivkovic S, Murray R, Jaramillo S, Lyons K, Johnson J,
Calof A (2003) Autoregulation of neurogenesis by GDF11.
Neuron 37:197207.
45
Young JM, Friedman C, Williams EM, Ross JA, Tonnes-Priddy
L, Trask BJ (2002) Different evolutionary processes shaped
the mouse and human olfactory receptor gene families. Hum
Mol Genet 11:535546.
Yu T, McIntyre J, Bose S, Hardin D, Owen M, McClintock T
(2005) Differentially expressed transcripts from phenotypi-
cally identified olfactory sensory neurons. J Comp Neurol
483:251261.
Zhang X, Klueber K, Guo Z, Cai J, Lu C, Winstead W, Qiu
M, Roisen F (2006) Induction of neuronal differentiation of
adult human olfactory neuroepithelial-derived progenitors.
Brain Res 10731074:109119.
Zhang X, Rodriguez I, Mombaerts P, Firestein S (2004) Odorant
and vomeronasal receptor genes in two mouse genome
assemblies. Genomics 83:802811.
Chapter 4
Cell Diversification During Neural Crest Ontogeny:
The Neural Crest Stem Cells
Elisabeth Dupin, Giordano W. Calloni, and Nicole M. Le Douarin
Contents
4.1 Introduction . . . . . . . . . . . . . . . . . 47
4.2 Formation of the Neural Crest, a Structure
Between CNS and Epidermis in Vertebrate
Embryos . . . . . . . . . . . . . . . . . . 49
4.3 Identification of Neural Crest Progenitors and
Stem Cells by In Vitro Single Cell Cultures . . 49
4.4 Pluripotent Neural Crest Stem Cells in
Tissues and Organs; Developmental Remnant
and Potential Source of Stem Cells for
Regenerative Medicine . . . . . . . . . . . . 51
4.5 In Vivo and In Vitro Demonstration of the
Influence of Environmental Cues on the
Differentiation of Neural Crest Derivatives . . 53
4.5.1 In Vivo Studies . . . . . . . . . . . . 53
4.5.2 In Vitro Studies . . . . . . . . . . . . 53
4.6 Plasticity and Dedifferentiation Ability of Neural
Crest-Derived Differentiated Cells . . . . . . 54
4.7 Concluding Remarks . . . . . . . . . . . . 55
References . . . . . . . . . . . . . . . . . . . . 55
Abstract The neural crest (NC) is a transitory ecto-
dermal structure that forms in the vertebrate embryo
at the junction between the presumptive CNS and epi-
dermis. As it is at the origin of very diverse (neural
and non-neural) cell types in adult tissues, the NC has
attracted for long the interest of developmental biolo-
gists and is a valuable model to investigate stem cell
biology. Here we review a number of data mainly
provided by in vitro single cell culture experiments,
which led to characterizing multipotent stem cells
E. Dupin ()
CNRS UPR2197 Laboratoire Dveloppement, Evolution et
Plasticit du Systme Nerveux, Institut de Neurobiologie Alfred
Fessard, 91198 Gif-sur-Yvette, France
e-mail: dupin@inaf.cnrs-gif.fr
H. Ulrich (ed.), Perspectives of Stem Cells,
DOI 10.1007/978-90-481-3375-8_4, Springer Science+Business Media B.V. 2010
and progenitors in the NC cells (NCC) that undergo
migration during embryogenesis. We focus on one
striking property of the cephalic NCC, i.e., the capac-
ity to yield chondrocytes and bone-forming cells in
addition to skin melanocytes and nerve cells of the
peripheral nervous system (PNS). We also emphasize
the role of environmental cues in ensuring the survival
and directing the differentiation of these progenitors in
their various sites of homing. Finally we also include
recent advances that uncover stem cell properties of
NC-derived cells in the adult body. On one hand,
differentiated cell types of NC origin are prone to ded-
ifferentiate, as shown by in vitro experiments; on the
other hand, undifferentiated multipotent NCC persist
in many tissues and organs. These findings suggest that
a diversity of NC-derived cells could be mobilized to
function as stem cells in adult tissue repair.
Keywords Chondrocyte Clonal culture
Multipotentiality Mesenchymal Neural
Osteoblast Quail embryo
Abbreviations
ET3 endothelin-3
NC neural crest
NCC neural crest cell(s)
NF neural fold
PNS peripheral nervous system
Shh Sonic Hedgehog
4.1 Introduction
The neural crest (NC) is a transitory structure of
the vertebrate embryo. The cells that enter in the
constitution of the crest are pluripotent and endowed
47
48 E. Dupin et al.

with migratory properties. After the NC is formed at

the border of the neural plate, forming an ectoder-
mal area intermediate between the presumptive neural
epithelium and the epidermis (and designated as the
neural fold NF), the NC cells (NCC) loose their
epithelial arrangement and colonize the embryonic tis-
sues to finally settle in elected points in the embryo
where they differentiate in a variety of cell types (see
Le Douarin, 1982; Le Douarin and Kalcheim, 1999 for
reviews).
The invasive properties of the NCC are impres-
sive since there is virtually no tissue in the body
devoid of cells derived from the NC. The NC is
at the origin of the peripheral nervous system (PNS
i.e., neurones of the sympathetic, parasympathetic and
enteric ganglia and plexuses, ganglionic glial cells
and Schwann cells lining peripheral nerves) and glan-
dular cells (carotid-body cells, calcitonin-producing
cells of the ultimobranchial bodies in birds and of the
thyroid gland in mammals, adrenal medullary cells).
The NC is also the source of mesenchymal cells
(designated as mesectoderm or ectomesenchyme).
In lower vertebrates, the mesectoderm arises from
the whole length of the neural axis. In amniotes,
this capacity is restricted to the cephalic neural
tube.
The mesenchymal derivatives of the NC have been
discovered, in 1893, in living embryo of Necturus by
Julia Platt who noticed that cells migrating from the
dorsal aspect of the neural tube contributed to the
skeleton of the mandible (Platt, 1893). In our labo-
ratory, the migration of NCC was followed in inter-
specific chimeras constructed between two species of
birds, the chick (Gallus gallus) and the Japanese quail
(Coturnix coturnix japonica) (Couly et al., 1993; Le Fig. 4.1 Cephalic NCC migration and contribution to the
Livre, 1978). It turned out that the entire facial skele- head skeleton. (a) Migration streams of cephalic NCC to the
naso-frontal bud (NFB) and branchial arches (BA), according to
ton and part of the cranial vault are of NC origin their level of origin (R, rhombomere). (b) Composite origin of
(Fig. 4.1). Moreover, the dermis, connective and fat the avian craniofacial skeleton. The contribution of the cephalic
tissues of the vertebrate head are also NC-derived NC (red), cephalic paraxial mesoderm (blue), and somitic meso-
(Billon et al., 2007; Le Douarin, 1982; Le Douarin and derm (green) is indicated on lateral view of the skull at E14
(upper panel) and on ventral view of E10 chondrocranium
Kalcheim, 1999). (lower panel)
Thus, the NC is a highly pluripotent structure
which, in this respect, can be compared to the
hematopoetic system. The problem was thus raised cells, chondrocytes and osteocytes on the other, could
as to whether all the NC derivatives can arise from have a common precursor in the NC.
one single type of pluripotent stem cells. It was par- In this review, we will envision first the genetic
ticularly interesting to ask whether the neural and pathways, which are responsible for the early speci-
melanocytic derivatives on the one hand, and the fication of the NC itself within the dorsal embryonic
mesectodermal cell types including connective tissue ectoderm. Then we will report the exploration of the
4 Cell Diversification During Neural Crest Ontogeny
differentiative capabilities of single NCC by in vitro
clonal cultures, which revealed the existence, among
cells derived from the NC, of intermediate precursors
able to self-renew.
The persistence in adulthood of such stem-like cells
has been demonstrated in several systems in birds and
mammals and represents a potential source of stem
cells for regenerative medicine.
A fourth part will deal with a notion demonstrated in
vivo and in vitro, that the differentiation of NC deriva-
tives strongly depends upon environmental cues. The
nature of these cues is being investigated in our and
other laboratories and signalling molecules are being
identified, which play a key role in the differentiation
of NC derivatives.
Finally we will discuss the problem of the
reversibility of the differentiated state. This question
was documented, in our laboratory, for melanocytes
and glial cells.
4.2 Formation of the Neural Crest,
a Structure Between CNS
and Epidermis in Vertebrate
Embryos
At the early gastrula stage of vertebrate development,
the ectoderm can be subdivided in three different
regions: the neural plate, its lateral borders (or NF)
and the presumptive epidermis. The neural plate
border cells (which develop at the junction between
the neural and epidermal ectoderm) give rise to NC
and placodal cells (Le Douarin, 1982; Le Douarin and
Kalcheim, 1999; Sauka-Spengler and Bronner-Fraser,
2008). Fate map studies have shown that the NC
originates from the neural plate border, except from
the most anterior NF, which generates olfactory and
lens placodes (Couly and Le Douarin, 1985) and a
variety of epithelial and glandular structures (Couly
et al., 1993). The absence of NCC from this specific
region was recently shown to be due to an inhibitory
signal emanating from the prechordal mesoderm
and mediated by the signalling molecule Dickkopf
(Carmona-Fontaine et al., 2007).
The establishment in the dorsal aspect of the embryo
of definite ectodermal regions including the prospec-
tive NC, occurs already at the late gastrula stage
(Basch et al., 2006) and depends largely upon a
set of signalling molecules and transcription factors
49
whose actions are strikingly coordinated in time and
space. The interplay between these factors is relatively
conserved between different vertebrate species, and
has been especially demonstrated in Xenopus laevis
model. The neural plate border is specified by BMP
and Wnt signals originating in the non-neural ecto-
derm and by FGF signals and BMP antagonists (such
as chordin, noggin and follistatin) which are derived
from the paraxial mesoderm (Garcia-Castro et al.,
2002; LaBonne and Bronner-Fraser, 1998; Marchant
et al., 1998; Mayor et al., 1995, 1997; Monsoro-Burq
et al., 2003). These signalling molecules act in concert
to determine the identity of neural plate border cells
characterized by the expression of Msx1, Msx2, Pax3,
Pax7 and Zic1 transcription factors (for a review, see
Sauka-Spengler et al., 2007). In turn Pax3 and Zic1
act synergistically in a Wnt-dependent manner, up-
regulating some NC-specific marker genes (e.g., Snail2
and FoxD3) in the NF (Hong and Saint-Jeannet, 2007;
Sato et al., 2005). These NC-specific genes (and oth-
ers like c-Myc, Id, AP-2, Twist and Sox9) control later
cellular events like cell proliferation, delamination and
epithelial-mesenchymal transition. This results in the
segregation of the NC from both the future epidermal
ectoderm and the dorsal neural epithelium (for reviews,
see Kuriyama and Mayor, 2008; Sauka-Spengler and
Bronner-Fraser, 2008).
4.3 Identification of Neural Crest
Progenitors and Stem Cells
by In Vitro Single Cell Cultures
The wide diversity of NC-derived cell types raises the
issue of how cell diversification is produced during NC
ontogeny. Are NCC multipotent stem cells or do the
various phenotypes arise from precursors already com-
mitted in the NF? These issues have been for long a
subject of interest for our laboratory and have required
to developing methods in order to investigate the dif-
ferentiation potentials of single NCC. By analogy to
the in vitro assay for colony-forming units, which was
devised in the 1950s onward to decipher the origin of
hemopoetic cell diversity, we have designed an in vitro
clonal analysis of NCC isolated from the quail embryo.
According to the method pioneered by Cohen and
Konisgberg (1975), the NCC can be obtained as adher-
ent cells when they have migrated out from explanted
neural primordium onto culture substrate. Following
50
trypsin treatment, a pure population of NCC can thus
be isolated for subculture experiments.
Clonogenic assays of avian NCC directly isolated
from the embryo as they migrate (for the mesen-
cephalic NC) or after they have spread from neural
tube explants, have provided important information
concerning their developmental potential. The migra-
tory NCC constitute a heterogeneous population with
respect to their proliferation and differentiation poten-
tials. A variety of progenitors can be evidenced in the
avian trunk NCC (Cohen and Konigsberg, 1975; Dupin
and Le Douarin, 1995; Lahav et al., 1998; Sieber-Blum
and Cohen, 1980; Sieber-Blum, 1989, 1991; Trentin
et al., 2004). They are either restricted to the pigment
cell fate, or bipotent, i.e., able to give rise either to both
pigment and glial cells, to glial cells and neurones or
to glial cells and myofibroblasts. Others yield three and
more phenotypes, thus recapitulating the repertoire of
trunk NC derivatives (Trentin et al., 2004).
The in vitro culture method of avian NCC was later
adapted to mammalian cells and similarly led to iden-
tify pluripotent progenitors in mouse and rat trunk
NC (Ito and Sieber-Blum, 1991; Paratore et al., 2001;
Rao and Anderson, 1997; Shah et al., 1994, 1996;
Stemple and Anderson, 1992). Precursors exiting from
the rodent trunk NC are able to yield autonomic neu-
rones, glial cells and myofibroblasts. They were shown
to self-renew in vitro, thus deserving to be considered
as NC stem cells (Stemple and Anderson, 1992).
In vitro clonal assays have also shown the pres-
ence of common progenitors for neurones, pigment
cells as well as fibroblasts and chondrocytes, in the
cardiac NC of the quail (Ito and Sieber-blum, 1991)
and mouse (Youn et al., 2003). This cardiac subset of
the NCC is formed at the post-otic level of the neural
axis and yield migratory cells that colonize the cardiac
outflow tract and have an important role in forma-
tion of heart septation. Even after they have colonized
the posterior branchial arches, post migratory cardiac
NC-derived cells retain common progenitors for neu-
rones and chondrocytes and/or myofibroblasts (Ito and
Sieber-Blum, 1993).
The even greater diversity of cell types originating
from the cephalic as compared to the trunk NC led us to
explore whether the cell types belonging to mesenchy-
mal lineages (e.g., smooth muscle cells, chondrocytes
and osteocytes) were derived from multipotent progen-
itors in the cephalic NC. In clonal cultures carried out
with quail mesencephalic-rhombencephalic NCC, we
E. Dupin et al.
have shown that subsets of clonogenic cells generated
myofibroblasts or chondrocytes together with neu-
ronal, glial and melanocytic cells (Baroffio et al., 1988,
1991; Dupin et al., 1990; Dupin and Le Douarin,
1995; Trentin et al., 2004). These findings provided
first evidence that segregation of neural-melanocytic
and mesenchymal cell lineages is not completed at the
time NCC start to migrate, and indeed, few clono-
genic cells were found to be already committed to
the chondrocytic phenotype. The majority of the pro-
genitors recorded showed diverse combinations of two
and three phenotypes in their progeny. Among these
intermediate progenitors, glial-melanocytic (GM) and
glial-myofibroblastic (GF) bipotent progenitors exhib-
ited stem cell properties as they could self-renew along
successive rounds of in vitro subcloning (Trentin et al.,
2004). Another striking result was that all the inter-
mediate (including bipotent) precursors were able to
yield glial cells, suggesting that the gliogenic differen-
tiation potential could constitute a general marker of
uncommitted NCC.
The existence of a clonogenic cell able to yield
all the phenotypes normally derived from the NC
in vivo, i.e., a cell yielding glia (G), neurones (N),
melanocytes (M), myofibroblasts (F), chondrocytes
(C) and osteoblasts (O): GNMFCO, could not be
proven until recently. We showed first that chondro-
genic cells differentiated from cultured cephalic NCC
if the cells had been harvested after they had migrated
out of the neural tube for only 15 h (Calloni et al.,
2007). Using these conditions (see Fig. 4.2), bona
fide chondrocytes (expressing Sox9 transcription fac-
tor, collagen-2a and chondroitin sulfate markers) dif-
ferentiated and formed aggregates in about 15% of
single NCC cultures. Then the analysis of clonal cul-
tures showed that nearly all chondrogenic progenitors
were multipotent cells that, in addition to chondro-
cytes, gave rise to glia, melanocytes, myofibroblasts
and neurones (Calloni et al., 2007) (Fig. 4.3).
The expression of both neural-melanocytic and
mesenchymal differentiation potencies by a large sub-
set of early cephalic NCC has been further documented
recently by investigating the osteogenic properties of
cultured NCC (Calloni et al., 2009). The NCC iso-
lated after 15 h of migration from explanted mes-
encephalon yielded two types of osteoblasts in day
10-cultures: one, endochondral-like type differenti-
ated in the perichondrium of already formed cartilage
nodules, whereas the other developed independently
4 Cell Diversification During Neural Crest Ontogeny
Fig. 4.2 Method for isolation and culture of quail NCC. The
neural primordium (including premigratory NC) is isolated at
the mesencephalic level in 68 somite-stage (a) or at the trunk
level in 2025 somite-stage (b) quail embryos following enzy-
matic treatment to remove surrounding tissues. The neural tube
(NT) is cultured for 1524 h; during this period NCC migrate
of the presence of chondrocytes and hence can be
assigned to a membranous-type of bone-forming cells.
The presence of osteoblasts, as identified by expres-
sion of the Runx2 gene, was investigated in colonies
derived from cephalic NCC (Fig. 4.3). It turned out that
more than 90% of the clones contained osteoblasts,
together with one or several other NC-derived differen-
tiated cell types. As shown in Fig. 4.4, the clonogenic
NCC endowed with the capacity to yield osteocytes
belong to diverse types of bi- and multipotent pro-
genitors. Upstream of these progenitors, we identi-
fied a highly multipotent NCC that has the ability to
generate all the recorded phenotypes, i.e., glia, neu-
rones, melanocytes, myofibroblasts, chondrocytes and
osteocytes (GNMFCO; see Fig. 4.4) (Calloni et al.,
2009).
These highly multipotent GNMFCO progenitors
present at an early migratory stage in the cephalic
NC are presumably at the origin of all the more
restricted NC precursor cells. Although they have not
been shown so far to self-renew, the GNMFCO pro-
genitors could thus be designated as the NC stem cells
able to generate all types (both neural and mesenchy-
mal/skeletogenic) of NC derivatives in the developing
head.
51
out from the explant (c) and are then harvested to be replated
in secondary cultures on previously established feeder-layers
of growth-arrested 3T3 fibroblasts (d). After 610 days, mass
cultures and colonies derived from single NCC are analysed
by immunocytochemistry and in situ hybridization to identify
differentiated cell types (e)
4.4 Pluripotent Neural Crest Stem Cells
in Tissues and Organs;
Developmental Remnant
and Potential Source of Stem Cells
for Regenerative Medicine
In addition to the differentiated cells derived from the
NC that are present in various organs and tissues, sev-
eral types of NC stem cells and progenitors have been
shown to persist in the adult body. The best function-
ally characterized is a melanocyte stem cell identified
in the permanent lower portion of the mammalian
hair follicle designated as the bulge, which replen-
ishes melanocytes during the hair cycle (Nishimura
et al., 2002). Other NC-derived undifferentiated cells
are present at postnatal stages in birds and rodents
in several NC derivatives, like the PNS ganglia and
nerves (Le Livre and Le Douarin, 1982; Duff et al.,
1991; Dupin, 1984; Hagedorn et al., 1999; Joseph
et al., 2004; Li et al., 2007; Morrison et al., 1999;
Nagoshi et al., 2008; Schweizer et al., 1983; Sextier-
Sainte-claire Deville et al., 1992) and the enteric ner-
vous system (Kruger et al., 2002; Sextier-Sainte-claire
Deville et al., 1994).
52 E. Dupin et al.

Fig. 4.3 NC-derived

phenotypes recorded in the
progeny of quail cephalic
NCC. Colonies derived from
single cephalic NCC are
analysed after 10 days for the
presence of six different cell
types. Immunocytochemistry
to HNK1 (a), neurofilament
proteins and III-tubulin (b),
and smooth muscle actin (d)
is used to identify glial cells,
neurones and
myofibroblasts/smooth
muscle cells, respectively.
Pigmented melanocytes (c)
and chondrocyte aggregates
(e) can be detected in
bright-field and phase-contrast
microscopy. Osteoblasts are
identified by expression of
Runx2 transcription factor (f)
(in situ hybridization)

The persistence of NC stem cells in several other
tissues of adult mammals, such as the skin, carotid
body, bone marrow, heart and cornea, has been demon-
strated in transgenic mice using NC-specific promoter-
Cre and floxed reporter systems, in which NC-derived
cells are permanently labelled (Fernandes et al., 2004;
Nagoshi et al., 2008; Pardal et al., 2007; Sieber-Blum
and Grim, 2004; Sieber-Blum et al., 2004; Tomita
et al., 2005; Wong et al., 2006; Yoshida et al., 2006).
When isolated and cultured in vitro, these NC-derived
progenitors can generate various NC derivatives like
neurones, glial cells and myofibroblasts. Their function
in vivo however remains to be established.
In vivo neurogenesis by rodent NC stem cells in
the carotid body offers a unique example demonstrat-
ing physiological function of adult NC progenitors
in vivo (Pardal et al., 2007). During adaptation to
hypoxia, these cells of the carotid body undergo pro-
liferation, convert into intermediate progenitors and
eventually lead to de novo differentiation of dopamin-
ergic chemosensory neurones that will activate the
brainstem respiratory centre to increase ventilation.
The carotid body is therefore a niche for adult neu-
rogenic stem cells in the PNS (Pardal et al., 2007).
However, unlike the neural stem cells of the subven-
tricular zone in the CNS, NC progenitors in the carotid
body are not permanently active but are turned on dur-
ing adaptive response to changes in the oxygen level of
arterial blood.
The dental pulp also contains NC-derived precur-
sors that differentiate into odontoblasts and are respon-
sible for dentinal repair in adults. Highly proliferative
clonogenic dental pulp stem cells have been isolated
in humans from adult molar and exfoliated deciduous
teeth (Gronthos et al., 2000, 2002; Miura et al., 2003).
These cells can be expanded ex vivo and reconstitute
dentin in transplantation models, thus offering promise
for dental tissue engineering and tooth regeneration in
the next future.
The adult skin is another source where NC-derived
stem cells can be isolated with minimally invasive pro-
cedures. The multipotent NCC populations recently
identified in the murine skin are located in several epi-
dermal and dermal structures of the whisker follicle,
4 Cell Diversification During Neural Crest Ontogeny
Fig. 4.4 Schematic representation of the various progeni-
tors identified by in vitro clonal analysis of quail cephalic
NCC. The progenitors are classified according to the number
of cell phenotypes recorded in the clones following analysis
of the presence of glial cells (G), neurones (N), melanocytes
(M), myofibroblasts (F), chondrocytes (C) and osteoblasts (O)
(see also Fig. 4.3). Subsets of precursors that yield neural and
melanocytic cells only (G, N and/or M) (in yellow) or mesenchy-
mal cell types (F, C and/or O) only (in blue) are indicated. The
great majority of clonogenic NCC generated both neural and
mesenchymal cells (progenitors in grey), including a variety of
osteogenic NCC (progenitors surrounded in light brown). These
experiments led to identify a highly multipotent cell (GNMFCO
progenitor), which is upstream in the hierarchy of all NC-derived
progenitors known so far
which are derived from the cephalic NC. In the back
skin, trunk NC-derived multipotent cells are present
in the hair bulge and share early markers of glial and
melanocytic cell lineages (Wong et al., 2006). These
NC stem cells isolated in rodent and human skin dis-
play neural and mesenchymal (including skeletogenic)
differentiation properties and can be expanded in vitro
(Fernandes et al., 2004; Nagoshi et al., 2008; Sieber-
Blum and Grim, 2004; Sieber-Blum et al., 2004;
Toma et al., 2001, 2005; Wong et al., 2006). The
expanded skin-derived NC progenitors were efficient
to repair nerve tissue when tested in murine models of
spinal cord and nerve injury (Biernaskie et al., 2007;
McKenzie et al., 2006; Sieber-Blum et al., 2006). The
skeletogenic potential of human skin-derived NC pro-
genitors has also been exploited recently to repair bone
fracture after transplantation in immuno-compromised
mice (Lavoie et al., 2008).
The NC-derived stem cells of the adult skin are
easily accessible; they share some characteristics with
pluripotent stem cells without being tumorigenic as are
embryonic stem cells when transplanted into the adult.
53
They therefore represent a potentially attractive source
of adult somatic stem cells for autologous transplanta-
tion and future cell replacement therapy in a variety of
human tissues.
4.5 In Vivo and In Vitro Demonstration
of the Influence of Environmental
Cues on the Differentiation of Neural
Crest Derivatives
4.5.1 In Vivo Studies
Transplantation experiments performed in the 1970s
and 1980s in the avian embryo have established a fate
map of the various NC derivatives along the neural
axis and revealed that the environment into which NCC
home at the term of their migration strongly influ-
ences their fate (Le Douarin, 1982; Le Douarin and
Kalcheim, 1999).
One striking example is the ability of the NC to
give rise either to enteric cholinergic neurones or to
catecholaminergic cells of the adrenal medullary gland
according to the migratory pathway taken by the cells
when they leave the neural tube. Although regionalized
along the anterior-posterior axis, the NCC fates are not
specified before they start migrating. The differentia-
tion choice of NCC depends on the level of the trunk
from which they migrate. This level dictates the migra-
tion pathway and the tissue of the embryo to which
NCC home.
The influence of environmental cues in neurotrans-
mitter choice by sympathetic neurones was also clearly
documented in newborn rat sympathetic neurones,
which can switch their neurotransmitter to acetyl-
choline if they are co-cultured with glial cells or
cardiac muscle (Furshpan et al., 1976; Patterson and
Chun, 1977).
4.5.2 In Vitro Studies
The paramount influence of external factors on NCC
differentiation and their action on particular types of
NC progenitors was underlined by a number of in vitro
culture studies and thanks to the analysis of single
NCC cultures performed in avian and mammalian
species (Le Douarin and Dupin, 2003).
54
In the rat model, Anderson and collaborators have
identified NC stem cells yielding glia, autonomic neu-
rones and myofibroblasts (Stemple and Anderson,
1992) and they have deciphered the molecular path-
ways involved in controlling their fate. BMP2 sig-
nals promote neuronal autonomic development at the
expense of glial cell fate (Shah et al., 1994). The
acquisition of a glial phenotype is controlled by
Neuregulin-1 (Shah et al., 1996) and Notch activation
(Morrison et al., 2000) whereas transforming growth
factor (TGF)- has been suggested to instruct mul-
tipotent NCC to adopt a myofibroblast fate. Signals
that drive multipotent NCC to form sensory neurones
involve activation of Wnt/-catenin signalling, which
is not only required but also sufficient to trigger sen-
sory neurogenesis. Together with BMP, Wnt factors
play an important role in the maintenance of multipo-
tent NC stem cells, as shown recently in the mouse
(Kleber et al., 2005; Lee et al., 2004). These find-
ings have been discussed in detail in a recent review
(Sommer, 2006).
In the avian model, we could demonstrate the strong
implication of the vasoactive peptide endothelin-3
(ET3) in the development of the melanocytic and glial
lineages both in culture and in vivo (Lahav et al., 1996,
1998; Lecoin et al., 1998; Nataf et al., 1996, 1998).
The ET3 peptide is produced by the skin and the gut
wall during the migration of NCC, which express the
receptors of ET3 (ETR), and ET3/ETR signalling path-
way is required cell-autonomously in NCC for proper
development of pigmentation and enteric nerve cells
(Gershon, 1999; McCallion and Chakravarti, 2001). At
early migration stages, ET3 has a strong mitogenic
effect on avian NCC (Lahav et al., 1996). However
it does not act similarly on all types of NC progen-
itors; the particular target of ET3 turned out to be a
bipotent glial-melanocytic stem cell, whose survival,
proliferation and self-renewal were upregulated when
individual NCC were treated in vitro with ET3 (Lahav
et al., 1998). Other types of progenitors, such as a glial-
myofibroblast precursor, were not responsive to this
factor.
In a recent work we sought for the putative fac-
tors involved in growth and differentiation of the
mesenchymal precursors of the cephalic NC, which
are responsible for the development of the facial
skeleton (Calloni et al., 2007). We investigated the
possible role of Sonic hedgehog (Shh) on the differ-
entiation of quail cephalic NCC in vitro. The require-
ment of this morphogen for early NCC survival and
E. Dupin et al.
facial morphogenesis has been previously recognized
(Ahlgren and Bronner-Fraser, 1999; Chiang et al.,
1996). A series of in vitro culture experiments led
to the demonstration that Shh has several effects on
mesencephalic NCC. First, the exposure of these cells
to Shh during the first two days of culture strongly
promoted the chondrocytic cell fate, as assessed by
increased number of Sox9-expressing chondrocyte pre-
cursors and later, of differentiated cartilage cells that
formed numerous aggregates in the NCC cultures
(Calloni et al., 2007). This effect on the differenti-
ation of chondrocytes was further supported by the
results of clonal NCC cultures, showing that Shh
highly enhances the total number of the chondrocyte-
containing colonies.
Second, these experiments also provided evidence
that, among clonogenic cephalic NCC, Shh specifi-
cally increased the number of multipotent progenitors
that are endowed with both neural and mesenchy-
mal (including chondrocytic) differentiation potentials
(Calloni et al., 2007). Notably, this increase of the
proportion of mesenchymal-neural progenitors coin-
cided with a decrease in the rate of the only-neural
ones while the total number of clones remained similar
in Shh-treated and untreated cultures, arguing that Shh
enhances NCC multipotentiality. Recently we found
that Shh also acts at a later stage to enhance the devel-
opment of cultured cephalic NCC into endochondral-
like osteoblasts (Calloni et al., 2009). When NCC are
exposed to Shh between 7 and 10 days of culture,
the differentiation of osteoblasts in the perichondrium
of cartilage nodules is highly promoted whereas Shh
does not act on the dermal-like type of osteoblasts,
which form independently of chondrocytes. By study-
ing the effect of Shh treatment on osteogenic progen-
itors in NCC clonal cultures, we found that it raised
significantly the frequency of the highly multipotent
GNMFCO progenitors (Calloni et al., 2009), thus pro-
viding further evidence for a crucial role of Shh on
multipotent osteo-chondrogenic NCC.
4.6 Plasticity and Dedifferentiation
Ability of Neural Crest-Derived
Differentiated Cells
The plasticity of NCC fate shown in vivo and in
vitro raises the issue as to whether the differentia-
tion state of NCC is stable during development and
4 Cell Diversification During Neural Crest Ontogeny
how differentiated cell phenotypes are maintained
in NC derivatives. The traditional view that cell
differentiation is unidirectional and irreversible has
been challenged by several lines of evidence such
as the transdifferentiation and dedifferentiation pro-
cesses observed in ocular tissues in vivo and in
vitro (reviewed by Blau et al., 2001; Egushi and
Kodama, 1993). Another striking example is the
recently demonstrated reprogramming of adult fibrob-
lasts into pluripotent ES-like cells following forced
expression of only four transcription factors (Okita
et al., 2007; Takahashi and Yamanaka, 2006; Takahashi
et al., 2007).
In the NC lineages, we have shown that differen-
tiated glial and melanocytic cells exhibit phenotype
plasticity in vitro. Schwann cells of the sciatic nerve
and epidermal pigment cells isolated from the quail
until late embryonic stages are able to convert into
each other when treated in culture with ET3, which
displays mitogenic activity on NCC (Lahav et al.,
1996, 1998). Schwann cells in clonal cultures yielded
a multi-phenotypic progeny of melanocytes and myofi-
broblasts in addition to parental-like glial cells (Dupin
et al., 2003; Real et al., 2005). Moreover, when grafted
into the first branchial arch of chick host embryos,
Schwann cell progeny contributed to the smooth mus-
cle wall of cranial blood vessels (Real et al., 2005).
In vitro experiments aimed at challenging the sta-
bility of the pigment cell phenotype have revealed
that, while proliferating, most of isolated pigment cells
can dedifferentiate and reacquire an immature NC-like
state (Dupin et al., 2000; Real et al., 2006). In clonal
cultures, dedifferentiated melanocytes recapitulate the
expression of early NC marker genes and revert to mul-
tipotent NC-like progenitors able to differentiate into
melanocytes, glial cells and myofibroblasts, and which
self-renew along successive subcloning (Real et al.,
2006).
These findings show that the differentiated state
of NC-derived cells is not fixed. If submitted to
new environmental influences or when displaced out-
side their niche, differentiated glial and pigment cells
undergo reversion of their differentiation programme
and recover stem cell properties similar to their mul-
tipotent NC ancestors. This phenotypic plasticity also
suggests that, in several adult tissues and organs, ded-
ifferentiated NC-derived cells as well as persistent
undifferentiated NC stem cells, could be of poten-
tial efficiency in cell replacement after injury or in
diseases.
55
4.7 Concluding Remarks
Although debate still exists as to whether most indi-
vidual NC cells or only a small subset of them are
multipotent after emigrating from the dorsal neural
primordium, several lines of evidence have convinc-
ingly demonstrated the multipotency and self-renewal
capacity of various subsets of NCC in avian and
mammalian species. In the trunk of vertebrates, glial
cells and neurones of the PNS as well as pigment
cells originate from multipotent NC stem cells, which
then undergo fate restrictions to give rise to appro-
priate committed precursors in their various sites of
differentiation. Although the NC and the CNS are
both of ectodermal origin during embryogenesis, the
NCC exhibit a wider range of derivatives than the
neural stem cells of the brain since they not only
form the PNS component cells but also contribute
mesenchymal and skeletogenic cells to the vertebrate
head. One of the significant advances towards deci-
phering the mechanisms of lineage diversification in
the NC has been recently to show that these different
lineages, the neural and mesenchymal ones, actually
are derived from common and multipotent progeni-
tors. This unique characteristic of the cephalic NCC
in higher vertebrates should make this cell popula-
tion of potential interest for investigations in both the
neural stem cell and mesenchymal stem cell fields.
Much remains however to be known with respect to
the molecular regulation of stem cell properties and
differentiation of the various progenitors present in
the target tissues of post-migratory NCC. These issues
are of clinical importance to understand the alterations
in NCC number and function that occur in human
neurocristopathies. The recent discovery of resident
NCC with stem cell properties in adult tissues such
as the skin may offer a source for isolating autolo-
gous NC-derived stem cells with great promise for cell
replacement therapy.
Acknowledgements The authors acknowledge the support of
the Centre National de la Recherche Scientifique, Foundation
Bettencourt Schueller and Association pour la Recherche con-
tre le Cancer. G.W.C. was recipient of a post-doctoral fellowship
from ARC.
References
Ahlgren SC, Bronner-Fraser M (1999) Inhibition of sonic hedge-
hog signaling in vivo results in craniofacial neural crest cell
death. Curr Biol 9:13041314.
56
Baroffio A, Dupin E, Le Douarin NM (1988) Clone-forming
ability and differentiation potential of migratory neural crest
cells. Proc Natl Acad Sci USA 85:53255329.
Baroffio A, Dupin E, Le Douarin NM (1991) Common precur-
sors for neural and mesectodermal derivatives in the cephalic
neural crest. Development 112:301305.
Basch ML, Bronner-Fraser M, Garcia-Castro MI (2006)
Specification of the neural crest occurs during gastrulation
and requires Pax7. Nature 441:218222.
Biernaskie J, Sparling JS, Liu J, Shannon CP, Plemel JR, Xie Y,
Miller FD, Tetzlaff W (2007) Skin-derived precursors gen-
erate myelinating Schwann cells that promote remyelination
and functional recovery after contusion spinal cord injury. J
Neurosci 27:95459559.
Billon N, Iannarelli P, Monteiro MC, Glavieux-Pardanaud C,
Richardson WD, Kessaris N, Dani C, Dupin E (2007) The
generation of adipocytes by the neural crest. Development
134:22832292.
Blau HM, Brazelton TR, Weimann JM (2001) The evolv-
ing concept of a stem cell: entity or function?. Cell 105:
829841.
Calloni GW, Glavieux-Pardanaud C, Le Douarin NM, Dupin
E (2007) Sonic Hedgehog promotes the development of
multipotent neural crest progenitors endowed with both mes-
enchymal and neural potentials. Proc Natl Acad Sci USA
104:1987919884.
Calloni GW, Le Douarin NM, Dupin E (2009) High frequency of
cephalic neural crest cells shows coexistence of neurogenic,
melanogenic and osteogenic differentiation capacities. Proc
Natl Acad Sci USA 106:89478952.
Carmona-Fontaine C, Acuna G, Ellwanger K, Niehrs C, Mayor
R (2007) Neural crests are actively precluded from the ante-
rior neural fold by a novel inhibitory mechanism dependent
on Dickkopf1 secreted by the prechordal mesoderm. Dev
Biol 309:208221.
Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal
H, Beachy PA (1996) Cyclopia and defective axial pattern-
ing in mice lacking Sonic hedgehog gene function. Nature
383:407413.
Cohen AM, Konigsberg IR (1975) A clonal approach to the
problem of neural crest determination. Dev Biol 46:262280.
Couly GF, Coltey PM, Le Douarin NM (1993) The triple ori-
gin of skull in higher vertebrates: a study in quail-chick
chimeras. Development 117:409429.
Couly GF, Le Douarin NM (1985) Mapping of the early neu-
ral primordium in quail-chick chimeras. I. Developmental
relationships between placodes, facial ectoderm, and pros-
encephalon. Dev Biol 110:422439.
Duff RS, Langtimm CJ, Richardson MK, Sieber-Blum M (1991)
In vitro clonal analysis of progenitor cell patterns in dorsal
root and sympathetic ganglia of the quail embryo. Dev Biol
147:451459.
Dupin E (1984) Cell division in the ciliary ganglion of quail
embryos in situ and after back-transplantation into the neu-
ral crest migration pathways of chick embryos. Dev Biol
105:288299.
Dupin E, Baroffio A, Dulac C, Cameron-Curry P, Le Douarin
NM (1990) Schwann-cell differentiation in clonal cultures
of the neural crest, as evidenced by the anti-Schwann cell
myelin protein monoclonal antibody. Proc Natl Acad Sci
USA 87:11191123.
E. Dupin et al.
Dupin E, Glavieux C, Vaigot P, Le Douarin NM (2000)
Endothelin 3 induces the reversion of melanocytes to glia
through a neural crest-derived glial-melanocytic progenitor.
Proc Natl Acad Sci USA 97:78827887.
Dupin E, Le Douarin NM (1995) Retinoic acid promotes the
differentiation of adrenergic cells and melanocytes in quail
neural crest cultures. Dev Biol 168:529548.
Dupin E, Real C, Glavieux-Pardanaud C, Vaigot P, Le Douarin
NM (2003) Reversal of developmental restrictions in neu-
ral crest lineages: transition from Schwann cells to glial-
melanocytic precursors in vitro. Proc Natl Acad Sci USA
100:52295233.
Eguchi G, Kodama R (1993) Transdifferentiation. Curr Opin
Cell Biol 5:10231028.
Fernandes KJ, McKenzie IA, Mill P, Smith KM, Akhavan M,
Barnabe-Heider F, Biernaskie J, Junek A, Kobayashi NR,
Toma JG, Kaplan DR, Labosky PA, Rafuse V, Hui CC, Miller
FD (2004) A dermal niche for multipotent adult skin-derived
precursor cells. Nat Cell Biol 6:10821093.
Furshpan EJ, MacLeish PR, OLague PH, Potter DD (1976)
Chemical transmission between rat sympathetic neurons and
cardiac myocytes developing in microcultures: evidence for
cholinergic, adrenergic, and dual-function neurons. Proc Natl
Acad Sci USA 73:42254229.
Garcia-Castro MI, Marcelle C, Bronner-Fraser M (2002)
Ectodermal Wnt function as a neural crest inducer. Science
297:848851.
Gershon MD (1999) Endothelin and the development of the
enteric nervous system. Clin Exp Pharmacol Physiol 26:
985988.
Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde
A, DenBesten P, Robey PG, Shi S (2002) Stem cell prop-
erties of human dental pulp stem cells. J Dent Res 81:
531535.
Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000)
Postnatal human dental pulp stem cells (DPSCs) in vitro and
in vivo. Proc Natl Acad Sci USA 97:1362513630.
Hagedorn L, Suter U, Sommer L (1999) P0 and PMP22 mark
a multipotent neural crest-derived cell type that displays
community effects in response to TGF-beta family factors.
Development 126:37813794.
Hong CS, Saint-Jeannet JP (2007) The activity of Pax3 and Zic1
regulates three distinct cell fates at the neural plate border.
Mol Biol Cell 18:21922202.
Ito K, Sieber-Blum M (1991) In vitro clonal analysis of quail
cardiac neural crest development. Dev Biol 148:95106.
Ito K, Sieber-Blum M (1993) Pluripotent and developmen-
tally restricted neural-crest-derived cells in posterior visceral
arches. Dev Biol 156:191200.
Joseph NM, Mukouyama YS, Mosher JT, Jaegle M, Crone SA,
Dormand EL, Lee KF, Meijer D, Anderson DJ, Morrison
SJ (2004) Neural crest stem cells undergo multilineage
differentiation in developing peripheral nerves to gener-
ate endoneurial fibroblasts in addition to Schwann cells.
Development 131:55995612.
Kleber M, Lee HY, Wurdak H, Buchstaller J, Riccomagno MM,
Ittner LM, Suter U, Epstein DJ, Sommer L (2005) Neural
crest stem cell maintenance by combinatorial Wnt and BMP
signaling. J Cell Biol 169:309320.
Kruger GM, Mosher JT, Bixby S, Joseph N, Iwashita T,
Morrison SJ (2002) Neural crest stem cells persist in the adult
4 Cell Diversification During Neural Crest Ontogeny
gut but undergo changes in self-renewal, neuronal subtype
potential, and factor responsiveness. Neuron 35:657669.
Kuriyama S, Mayor R (2008) Molecular analysis of neural
crest migration. Philos Trans R Soc Lond B Biol Sci 363:
13491362.
LaBonne C, Bronner-Fraser M (1998) Neural crest induction
in Xenopus: evidence for a two-signal model. Development
125:24032414.
Lahav R, Dupin E, Lecoin L, Glavieux C, Champeval D, Ziller
C, Le Douarin NM (1998) Endothelin 3 selectively promotes
survival and proliferation of neural crest-derived glial and
melanocytic precursors in vitro. Proc Natl Acad Sci USA
95:1421414219.
Lahav R, Ziller C, Dupin E, Le Douarin NM (1996) Endothelin
3 promotes neural crest cell proliferation and mediates a vast
increase in melanocyte number in culture. Proc Natl Acad
Sci USA 93:38923897.
Lavoie JF, Biernaskie JA, Chen Y, Bagli D, Alman B, Kaplan
DR, Miller FD (2009) Skin-derived precursors differentiate
into skeletogenic cell types and contribute to bone repair.
Stem Cells Dev 18:893906.
Le Douarin N (1982) The Neural Crest. Cambridge University
Press, Cambridge.
Le Douarin NM, Dupin E (2003) Multipotentiality of the neural
crest. Curr Opin Genet Dev 13:529536.
Le Douarin N, Kalcheim C (1999) The Neural Crest, 2nd edn.
Cambridge University Press, Cambridge; New York.
Le Lievre CS, Le Douarin NM (1982) The early development
of cranial sensory ganglia and the potentialities of their
component cells studied in quail-chick chimeras. Dev Biol
94:291310.
Le Livre CS (1978) Participation of neural crest-derived cells
in the genesis of the skull in birds. J Embryol Exp Morphol
47:1737.
Lecoin L, Sakurai T, Ngo MT, Abe Y, Yanagisawa M, Le
Douarin NM (1998) Cloning and characterization of a novel
endothelin receptor subtype in the avian class. Proc Natl
Acad Sci USA 95:30243029.
Lee HY, Kleber M, Hari L, Brault V, Suter U, Taketo MM,
Kemler R, Sommer L (2004) Instructive role of Wnt/beta-
catenin in sensory fate specification in neural crest stem cells.
Science 303:10201023.
Li HY, Say EH, Zhou XF (2007) Isolation and characterization
of neural crest progenitors from adult dorsal root ganglia.
Stem Cells 25:20532065.
Marchant L, Linker C, Ruiz P, Guerrero N, Mayor R (1998)
The inductive properties of mesoderm suggest that the neu-
ral crest cells are specified by a BMP gradient. Dev Biol
198:319329.
Mayor R, Guerrero N, Martinez C (1997) Role of FGF
and noggin in neural crest induction. Dev Biol 189:
112.
Mayor R, Morgan R, Sargent MG (1995) Induction of the
prospective neural crest of Xenopus. Development 121:
767777.
McCallion AS, Chakravarti A (2001) EDNRB/EDN3 and
Hirschsprung disease type II. Pigment Cell Res 14:161169.
McKenzie IA, Biernaskie J, Toma JG, Midha R, Miller
FD (2006) Skin-derived precursors generate myelinating
Schwann cells for the injured and dysmyelinated nervous
system. J Neurosci 26:66516660.
57
Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi
S (2003) SHED: stem cells from human exfoliated deciduous
teeth. Proc Natl Acad Sci USA 100:58075812.
Monsoro-Burq AH, Fletcher RB, Harland RM (2003) Neural
crest induction by paraxial mesoderm in Xenopus embryos
requires FGF signals. Development 130:31113124.
Morrison SJ, Perez SE, Qiao Z, Verdi JM, Hicks C, Weinmaster
G, Anderson DJ (2000) Transient Notch activation initiates
an irreversible switch from neurogenesis to gliogenesis by
neural crest stem cells. Cell 101:499510.
Morrison SJ, White PM, Zock C, Anderson DJ (1999)
Prospective identification, isolation by flow cytometry, and
in vivo self-renewal of multipotent mammalian neural crest
stem cells. Cell 96:737749.
Nagoshi N, Shibata S, Kubota Y, Nakamura M, Nagai Y, Satoh
E, Morikawa S, Okada Y, Mabuchi Y, Katoh H, Okada S,
Fukuda K, Suda T, Matsuzaki Y, Toyama Y, Okano H (2008)
Ontogeny and multipotency of neural crest-derived stem cells
in mouse bone marrow, dorsal root ganglia, and whisker pad.
Cell Stem Cell 2:392403.
Nataf V, Grapin-Botton A, Champeval D, Amemiya A,
Yanagisawa M, Le Douarin NM (1998) The expression pat-
terns of endothelin-A receptor and endothelin 1 in the avian
embryo. Mech Dev 75:145149.
Nataf V, Lecoin L, Eichmann A, Le Douarin NM (1996)
Endothelin-B receptor is expressed by neural crest cells
in the avian embryo. Proc Natl Acad Sci USA 93:
96459650.
Nishimura EK, Jordan SA, Oshima H, Yoshida H, Osawa
M, Moriyama M, Jackson IJ, Barrandon Y, Miyachi
Y, Nishikawa S (2002) Dominant role of the niche
in melanocyte stem-cell fate determination. Nature 416:
854860.
Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-
competent induced pluripotent stem cells. Nature 448:313
317.
Paratore C, Goerich DE, Suter U, Wegner M, Sommer L (2001)
Survival and glial fate acquisition of neural crest cells are
regulated by an interplay between the transcription factor
Sox10 and extrinsic combinatorial signaling. Development
128:39493961.
Pardal R, Ortega-Saenz P, Duran R, Lopez-Barneo J (2007) Glia-
like stem cells sustain physiologic neurogenesis in the adult
mammalian carotid body. Cell 131:364377.
Patterson PH, Chun LL (1977) The induction of acetylcholine
synthesis in primary cultures of dissociated rat sympa-
thetic neurons. II. Developmental aspects. Dev Biol 60:
473481.
Platt JB (1893) Ectodermic origin of the cartilage of the head.
Anat Anz 8:506509.
Rao MS, Anderson DJ (1997) Immortalization and controlled in
vitro differentiation of murine multipotent neural crest stem
cells. J Neurobiol 32:722746.
Real C, Glavieux-Pardanaud C, Le Douarin NM, Dupin E (2006)
Clonally cultured differentiated pigment cells can dedifferen-
tiate and generate multipotent progenitors with self-renewing
potential. Dev Biol 300:656669.
Real C, Glavieux-Pardanaud C, Vaigot P, Le-Douarin N, Dupin
E (2005) The instability of the neural crest phenotypes:
Schwann cells can differentiate into myofibroblasts. Int J Dev
Biol 49:151159.
58
Sato T, Sasai N, Sasai Y (2005) Neural crest determination by
co-activation of Pax3 and Zic1 genes in Xenopus ectoderm.
Development 132:23552363.
Sauka-Spengler T, Bronner-Fraser M (2008) A gene regulatory
network orchestrates neural crest formation. Nat Rev Mol
Cell Biol 9:557568.
Sauka-Spengler T, Meulemans D, Jones M, Bronner-Fraser M
(2007) Ancient evolutionary origin of the neural crest gene
regulatory network. Dev Cell 13:405420.
Schweizer G, Ayer-Le Lievre C, Le Douarin NM (1983)
Restrictions of developmental capacities in the dorsal root
ganglia during the course of development. Cell Differ
13:191200.
Sextier-Sainte-Claire Deville F, Ziller C, Le Douarin N (1992)
Developmental potentialities of cells derived from the trun-
cal neural crest in clonal cultures. Brain Res Dev Brain Res
66:110.
Sextier-Sainte-Claire Deville F, Ziller C, Le Douarin NM (1994)
Developmental potentials of enteric neural crest-derived cells
in clonal and mass cultures. Dev Biol 163:141151.
Shah NM, Groves AK, Anderson DJ (1996) Alternative neu-
ral crest cell fates are instructively promoted by TGFbeta
superfamily members. Cell 85:331343.
Shah NM, Marchionni MA, Isaacs I, Stroobant P, Anderson DJ
(1994) Glial growth factor restricts mammalian neural crest
stem cells to a glial fate. Cell 77:349360.
Sieber-Blum M (1989) Commitment of neural crest cells to the
sensory neuron lineage. Science 243:16081611.
Sieber-Blum M (1991) Role of the neurotrophic factors BDNF
and NGF in the commitment of pluripotent neural crest cells.
Neuron 6:949955.
Sieber-Blum M, Cohen AM (1980) Clonal analysis of quail
neural crest cells: they are pluripotent and differentiate
in vitro in the absence of noncrest cells. Dev Biol 80:
96106.
Sieber-Blum M, Grim M (2004) The adult hair follicle: cradle
for pluripotent neural crest stem cells. Birth Defects Res Part
C Embryo Today 72:162172.
Sieber-Blum M, Grim M, Hu YF, Szeder V (2004) Pluripotent
neural crest stem cells in the adult hair follicle. Dev Dyn
231:258269.
Sieber-Blum M, Schnell L, Grim M, Hu YF, Schneider R,
Schwab ME (2006) Characterization of epidermal neural
E. Dupin et al.
crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.
Mol Cell Neurosci 32:6781.
Sommer L (2006) Growth factors regulating neural crest cell fate
decisions. Adv Exp Med Biol 589:197205.
Stemple DL, Anderson DJ (1992) Isolation of a stem cell for
neurons and glia from the mammalian neural crest. Cell
71:973985.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T,
Tomoda K, Yamanaka S (2007) Induction of pluripotent stem
cells from adult human fibroblasts by defined factors. Cell
131:861872.
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by
defined factors. Cell 126:663676.
Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot
A, Kaplan DR, Miller FD (2001) Isolation of multipotent
adult stem cells from the dermis of mammalian skin. Nat Cell
Biol 3:778784.
Toma JG, McKenzie IA, Bagli D, Miller FD (2005) Isolation and
characterization of multipotent skin-derived precursors from
human skin. Stem Cells 23:727737.
Tomita Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I,
Kawaguchi H, Ieda M, Kanakubo S, Shimazaki T, Ogawa S,
Osumi N, Okano H, Fukuda K (2005) Cardiac neural crest
cells contribute to the dormant multipotent stem cell in the
mammalian heart. J Cell Biol 170:11351146.
Trentin A, Glavieux-Pardanaud C, Le Douarin NM, Dupin E
(2004) Self-renewal capacity is a widespread property of var-
ious types of neural crest precursor cells. Proc Natl Acad Sci
USA 101:44954500.
Wong CE, Paratore C, Dours-Zimmermann MT, Rochat A, Pietri
T, Suter U, Zimmermann DR, Dufour S, Thiery JP, Meijer
D, Beermann F, Barrandon Y, Sommer L (2006) Neural
crest-derived cells with stem cell features can be traced
back to multiple lineages in the adult skin. J Cell Biol
175:10051015.
Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y,
Okano H, Tsubota K (2006) Isolation of multipotent neural
crest-derived stem cells from the adult mouse cornea. Stem
Cells 24:27142722.
Youn YH, Feng J, Tessarollo L, Ito K, Sieber-Blum M (2003)
Neural crest stem cell and cardiac endothelium defects in the
TrkC null mouse. Mol Cell Neurosci 24:160170.
Chapter 5

Intermediate Filament Expression in Mouse Embryonic

Stem Cells and Early Embryos

Zhigang Xue, Vivaldo Moura-Neto, Araksya Izmiryan, Sheila Cristina de Souza

Martins, Jean Christophe Larcher, Denise Paulin, and Zhenlin Li

Contents early embryogenesis and differentiation. Finally, we

point out some questions to glial tumors and cytoskele-
5.1 Intermediate Filaments . . . . . . . . . . . 59 tal markers of cells potentially pluripotent present in
5.2 Intermediate Filament Protein Synthesis in these tumors, as nestin and synemin proteins.
Mouse Oocytes and Preimplantation Murine
Embryos . . . . . . . . . . . . . . . . . . 61 Keywords Intermediate filaments Nestin Synemin
5.3 Epithelial Differentiation and Intermediate-Sized
Filaments in Early Postimplantation Embryos . 61 Stem cells Embryonic development Glial tumors
5.4 Intermediate Filaments in Primary Mesenchymal
Cells in Mouse Embryo . . . . . . . . . . . 62 Abbreviations
5.5 Expression of Nestin and Synemin During Early
Embryogenesis and Differentiation . . . . . . 62
5.5.1 Nestin and Synemin Genes . . . . . . . 62 ES cells embryonic stem cells
5.5.2 Nestin Expression . . . . . . . . . . . 63
5.5.3 Synemin Expression . . . . . . . . . . 64 EB embryonic bodies
5.6 Expression of Nestin and Synemin in Tumoral GFAP glial fibrillary acidic protein
Cells of the CNS . . . . . . . . . . . . . . . 67 ICM inner cell mass
5.6.1 Glial Tumors . . . . . . . . . . . . . 67 IF intermediate filament
5.6.2 Nestin in Glioma . . . . . . . . . . . 68 KO knock-out
5.6.3 Synemin Expression in Glioma . . . . . 68
5.6.4 And Now . . . . . . . . . . . . . . . 68 NF neurofilament
References . . . . . . . . . . . . . . . . . . . . 69 PE parietal endoderm
PNS peripheral nervous system
VE visceral endoderm
Abstract In this work we discuss the expression of
intermediate filament protein and synthesis in mouse
oocytes and preimplantation murine embryos. Also
we touch to epithelial differentiation and intermediate- 5.1 Intermediate Filaments
sized filaments in early postimplantation embryos.
After we analyse the intermediate filaments expression
Cellular differentiation in vitro and in vivo is closely
in primary mesenchymal cells in mouse embryo, we
connected with morphological changes based on inter-
look to the expression of nestin and synemin during
mediate filament (IF) protein remodeling. Intermediate
filaments are expressed in cell-type-specific patterns
following and demarcating pathways of embryonic
development and cell differentiation (Herrmann and
Aebi, 2000).
V. Moura-Neto ()
In recent years, intermediate filaments (IFs) have
Instituto de Cincias Biomdicas-Universidade Federal de Rio
de Janeiro, Rio de Janeiro, Brazil attracted much interest, largely because their consti-
e-mail: vivaldo@anato.ufrj.br tutive polypeptide units are specifically expressed in

H. Ulrich (ed.), Perspectives of Stem Cells, 59

DOI 10.1007/978-90-481-3375-8_5, Springer Science+Business Media B.V. 2010
60 Z. Xue et al.

various cell types and thus represent excellent differ-
entiation markers (Zehner, 1991; Oshima, 2007). Data
obtained through biochemical studies and molecular
cloning have allowed the classification of IFs into six
types according to their primary sequence and pro-
tein structure. The expression of most IF types is
characteristic of a given cell type: cytokeratins (IF
types I and II) are produced in epithelia; neurofil-
aments, -internexin (type IV) and peripherin (type
III) in neurons; synemin, nestin (type VI) and syn-
coilin (type IV) in neuroblast and myoblast. On the
other hand, the type III IFs are highly related proteins
which are expressed in different cell types (Peripherin
in neurons, glial fibrillary acidic protein [GFAP] in
glia, desmin in muscle, vimentin with a widespread
distribution).
Tissues without IFs fall apart; they are mechanically
unstable, unable to resist physical stress, and this leads
to cell degeneration. By maintaining the shape and
plasticity of the cell, the intermediate filament network
acts as an integrator within the cell space. The state
of mechanical force imposed on a tissue or a cell can
alter the shape of certain elements of the cytoskeleton
and thus participate to the control of cell functions.
Specific markers of neuronal and glial cell types are
powerful tools for studying the mechanisms generat-
ing cellular diversification in the nervous system. So
far, different types of IFs have been identified in the
nervous system (Table 5.1).
The cloning and sequencing of IF mRNAs start-
ing in 1981 (Lazarides, 1981) revealed the conserved
central coiled-coil 1A, 1B and 2A, 2B rod domains.
Subsequent cDNA sequences of other IFs and gene
structure facilitated evolutionary studies. In 1986,
McKeon discovered that the nuclear lamins were
members of the IF superfamily (type V) but con-
tained extended helical segments not found in other
IF (McKeon et al., 1986). The extended helical seg-
ments of the nuclear lamins were found in IFs for
invertebrates suggesting an evolutionary path from
nuclear lamins to cytoplasmic IFs. By examining the
existence and structure of IF in a variety of differ-
ent animals (Fuchs and Weber, 1994), identification
of 11 IF genes, 5 of which are uniquely essential, in
Caenorhabditis elegans and 65 coding genes in the
human genome contrasts with the absence of cytoplas-
mic IF in Drosophila (Goldman, 2001).

Table 5.1 Intermediate filament expression in mammal embryo nervous system

Earliest stage
IF Cell types detection References IF assembly MW (kDa)
NF-L Neurons E9 Cochard and Paulin (1984) and Homopolymer 70
Hoffman and Lasek (1975)
NF-M Neurons E9.5 Cochard and Paulin (1984) and Heteropolymer 140
Hoffman and Lasek (1975)
NF-H Neurons E10.5 Cochard and Paulin (1984) and Heteropolymer 210
Hoffman and Lasek (1975)
Peripherin Neurons E12 Escurat et al. (1990, 1988) and Homopolymer 61, 58, 55
Portier et al. (1983)
-Internexin Neurons E12 Kaplan et al. (1990) and Pachter Homopolymer 66
and Liem (1985)
Vimentin Glia cells, astrocytes E5 Izmiryan et al. (2006, 2009) Homopolymer 54
Nestin Glia cells, astrocytes, E5 Dahlstrand et al. (1995), Heteropolymer 240
neuroepithelial cells Frederiksen and McKay
(1988), Izmiryan et al. (2009)
and Tohyama et al. (1992)
H synemin Glia cells, astrocytes, E9 Hirako et al. (2003) and Heteropolymer 180
neurons Izmiryan et al., (2006, 2009)
M synemin Glia cells, astrocytes, E5 Hirako et al. (2003) and Heteropolymer 150
neurons Izmiryan et al., (2006, 2009)
L synemin Neurons E11 Izmiryan et al. (2006, 2009) Heteropolymer 41
GFAP Glia cells, astrocytes E10 Galou et al. (1994) Homopolymer 50
5 IF Expression in Mouse Embryonic Stem Cells and Early Embryos
5.2 Intermediate Filament Protein
Synthesis in Mouse Oocytes
and Preimplantation Murine
Embryos
The presence and distribution of IF proteins in mouse
oocytes and preimplantation embryos were studied by
several groups. First, to the setting up of asymmetries
within cells, a process called polarization, which first
takes place during the 8-cell stage at compaction.
Cytoskeletal elements and organelles, such as clathrin
vesicles and endosomes, accumulate first in an apical
focus, while the cell nucleus tends to migrate basally
and gap junctions form. Secondly, to the existence of
asymmetrical (or differentiative) cell divisions giving
rise to the two cell types present in the 16-cell stage
embryo, polarized outer cells and non-polarized inner
cells. This is due to the fact that some elements of
polarity are maintained throughout division and that
the cleavage plane may be roughly orthogonal to the
axis of polarity (Maro et al., 1990).
Among the three types of cytoskeletal elements,
two are clearly present in mouse oocytes: microtubules
and microfilaments, the presence of IFs being more
controversial. In both cases, their organization differs
from that observed in somatic cells. The organiza-
tion and role of the cytoskeletal networks (mainly
microtubules and microfilaments) during oogenesis,
fertilization and preimplantation development of the
mouse are described given the importance of cell
cell interactions and of the subcellular organization in
events leading to the formation of the first two lineages
of the mouse embryo.
In immunoblotting analysis of electrophoretically
separated polypeptides, a distinct doublet of polypep-
tides with Mr of 54K and 57K, reactive with cytok-
eratin antibodies, was detected in oocytes and in
cleavage-stage embryos (Lehtonen et al., 1983b). A
similar doublet of polypeptides, reactive with cytok-
eratin antibodies, was also detected in late morula-
and blastocyst-stage embryos. Immunoblotting with
vimentin antibodies gave negative results in both
cleavage-stage and blastocyst-stage embryos. The
electron microscopy observations show that these early
stages also contain detergent-resistant 10- to 11-nm
filaments. The relative scarcity of these filaments, as
compared to the high intensity in the immunoblotting
and immunofluorescence stainings, speaks in favor of
61
a nonfilamentous pool of cytokeratin in oocytes and
cleavage-stage embryos.
The synthesis of two extra-embryonic endodermal
cytoskeletal proteins (Endo A, Mr = 55,000; Endo B,
Mr = 50,000) was detected by immunoprecipitation at
the 4- to 8-cell stage of preimplantation mouse devel-
opment (Oshima et al., 1983). The first detectable syn-
thesis of both proteins occurs at about the same time as
the earliest allocation of cells to the trophectodermal
lineage (Brulet et al., 1980). Indirect immunofluores-
cence microscopy has been used to detect cytoskeletal
proteins, which allow a distinction between the two-
cell types present in mouse blastocyst: i.e. the cells of
the inner cell mass (ICM) and the outer trophoblastic
cells. Antibodies against prekeratin stain the outer tro-
phoblastic cells but not the ICM in agreement with the
findings on adult tissues that cytokeratins are a marker
for various epithelial cells. Interestingly, vimentin fil-
aments typical of mesenchymal cells as well as of
cells growing in culture seem to be absent in both cell
types of the blastocyst. Thus, cytokeratins of the tro-
phoblastic cells seem to be the first intermediate-sized
filaments expressed in embryogenesis. Antibodies to
tubulin and actin show that microtubules and micro-
filaments are ubiquitous structures, although microfil-
aments have a noticeably different organization in the
two cell types (Paulin et al., 1980).
5.3 Epithelial Differentiation
and Intermediate-Sized Filaments
in Early Postimplantation Embryos
It is concluded that early postimplantation embryonic
development, up to mesoderm formation, is character-
ized by the exclusive presence, in both embryonic ecto-
derm and proximal endoderm, of differentiated epithe-
lial cells containing desmosome-cytokeratin filament
complexes and that other types of intermediate-sized
filaments are not yet expressed.
Two layers of extra-embryonic endoderm, the pari-
etal endoderm (PE) and the visceral endoderm (VE),
arise in mouse embryo shortly after implantation. Both
cell populations apparently originate from the primi-
tive endoderm of the blastocyst. The PE and VE of
mouse conceptuses differ in their expression of IFs:
while both cell types contain cytokeratin, expression
62
of vimentin was only revealed in the cells of the PE
(Lehtonen et al., 1983a). This population of individual,
motile cells seems to be derived from a conventional
epithelium by migration and differentiation. All results
support the idea that vimentin expression is specifically
related to reduced cell-to-cell contact, and to the inde-
pendent existence of a cell following detachment from
an epithelial sheet. The co-expression of cytokeratins
and vimentin was found in the parietal endoderm of the
mouse embryo E8.5-E13.5 (Lane et al., 1983). Among
the six classes of IFs found in vertebrate tissues, the
cytokeratins are considered unique to epithelial tis-
sues, while vimentin is expressed by endothelial and
mesenchymal cells.
The characteristic epithelia formed by the embry-
onic ectoderm and proximal (visceral) endoderm
present well-developed junction complexes and vari-
ous differentiated membrane structures. Several api-
cal differentiations of the proximal endodermal cells,
such as brush border-like microvilli, the endocytotic
labyrinthum, and the supranuclear vacuoles resemble
the organization of epithelial cells of the ileum of
neonatal mammals (Jackson et al., 1981). Both embry-
onic epithelia show typical desmosomes and attached
intermediate-sized filaments of the cytokeratin type
(Franke et al., 1982b). Other types of intermediate-
sized filaments, such as synemin, nestin and vimentin
mRNA, have been detected in E5 embryos by RT-PCR,
and in the embryonic ectoderm and mesoderm at E7
using in situ hybridization (Kawaguchi et al., 2001;
Izmiryan et al., 2009).
5.4 Intermediate Filaments in Primary
Mesenchymal Cells in Mouse Embryo
The cytoskeletal composition of the primitive streak
stage of mouse embryos, i.e. at late day 8 (day 8.5)
of gestation corresponds to the onset of vimentin. At
this stage primary mesenchymal cells are observed
in the posterior part of the embryo, which seem to
migrate toward the anterior region. For most of the
embryo, these mesenchymal cells are separated from
the embryonic ectoderm by a continuous basal lam-
ina. Mesenchymal cells also can form junctions of
the fascia adhaerens-type but appear to be devoid of
desmosomes. Antibodies to cytokeratins reveal strong
Z. Xue et al.
fibrillar fluorescence in cells of the proximal endo-
derm and weak, predominantly subapical staining in
embryonic ectoderm. Correspondingly, antibodies to
desmoplakins, the major proteins of the desmosomal
plaque, show punctate fluorescence in both embryonic
epithelia. These epithelial cells are not significantly
stained with antibodies to other IF proteins such as
vimentin and desmin. However, antibodies to vimentin
show positive fluorescence, often in fibrillar tangles, in
primary mesenchymal cells which in turn are negative
with cytokeratin and desmin antibodies. The obser-
vations indicate that during embryogenesis synthesis
of vimentin occurs, for the first time, in the primitive
streak stage and is restricted to the primary mesenchy-
mal cells. Concomitantly, these cells cease to produce
cytokeratins and desmoplakin (Franke et al., 1982a).
5.5 Expression of Nestin and Synemin
During Early Embryogenesis
and Differentiation
5.5.1 Nestin and Synemin Genes
Upon its identification, nestin was designated as the
prototype of a new IF protein group (type VI) because
it did not fall clearly into any of the previously
described types (Lendahl et al., 1990). Some debate
arose on this classification since nestin gene structure
is closely related to the neurofilament (NF) branch
in having two of its three intron positions in com-
mon with NF genes (Dahlstrand et al., 1992b). The
nestin and synemin genes show structural similarities
to type III (e.g. vimentin, desmin) and especially type
IV (e.g. neurofilaments, alpha-internexin) IF proteins
(Herrmann and Aebi, 2000), which led to its classifi-
cation as a type IV IF protein. Due to a low degree of
protein sequence homology of the rod domain in com-
parison to the five IF protein classes, accordingly, it
had been proposed to re-classify nestin as a type VI
IF protein (Herrmann and Aebi, 2000). Shortly after
nestin was sequenced, the gene structure of synemin
(Bellin et al., 1999; Titeux et al., 2001; Xue et al.,
2004) was also described. According to their sequence
similarities, nestin and synemin were then grouped
with transitin and tanabin, as type VI IF proteins
(Guerette et al., 2007).
5 IF Expression in Mouse Embryonic Stem Cells and Early Embryos
Nestin was first identified with a monoclonal anti-
body (Hockfield and McKay, 1985), and then identified
in neuroepithelial stem cells of rat (Lendahl et al.,
1990). The rat (chromosome 2q34) and human (chro-
mosome 1q23.1) nestin genes contain five exons span-
ning four introns (Dahlstrand et al., 1992b; Lendahl
et al., 1990), whereas the mouse nestin gene (chro-
mosome 3F1) contains eight exons. Transcriptional
regulation of the nestin gene is unique. The 240-
kDa protein nestin contains a short N-terminus and an
unusually long C-terminus. Analyses of the rat nestin
promoter in transgenic mice indicate that the region
upstream of the first exon does not contain any iden-
tifiable regulatory elements (Zimmerman et al., 1994).
In fact, nestin expression in muscle precursor and
neuroepithelial stem cells of the central nervous sys-
tem (CNS) is independently regulated by temporally
and spatially restricted enhancer elements in the first
and second introns, respectively (Zimmerman et al.,
1994). The second intron contains two highly con-
served elements, a mid-brain and a CNS enhancer
element that are activated and independently regulated
in rat and human (Lothian et al., 1999; Yaworsky and
Kappen, 1999) and which are not strong enhancers
in the peripheral nervous system (Zimmerman et al.,
1994). The CNS enhancer is active in neural stem cells
of the developing CNS (Lothian and Lendahl, 1997).
Only limited data are available on the transcriptional
regulation of nestin in other tissues or in vitro.
Synemin is another type VI IF protein. Synemin
was originally identified in avian muscle, along with
desmin and vimentin (Bellin et al., 1999; Granger and
Lazarides, 1980). It has also been found in avian and
cane toad erythrocytes (Centonze et al., 1986; Granger
and Lazarides, 1982), chicken and human lens and the
retina cells (Granger and Lazarides, 1984; Tawk et al.,
2003), in a subpopulation of rodent astrocytes that con-
tains GFAP, vimentin and nestin (Izmiryan et al., 2006;
Sultana et al., 2000), and in neurons (Izmiryan et al.,
2006).
Three synemin isoforms have characterized in
the human (GenBank accession numbers: AJ310521,
AJ310522, and AJ697971) (Titeux et al., 2001)
and mouse (GenBank accession numbers: AJ579700,
AJ579701 and AJ579702) (Xue et al., 2004). This gene
is localized in human chromosome 15 (Chr.15q26.3)
and mouse chromosome 7 (Chr.7B5) (Titeux et al.,
2001; Xue et al., 2004). The synemin gene is one par-
ticular IF gene which produces 3 major mRNAs by
63
alternative splicing, resulting in 3 synemin isoforms of
180 (H or alpha), 150 (M or beta) and 41 (L) kDa. The
structure of mammal synemin gene consists of four
exons and three introns. The isoform M has 5 exons
and 4 introns, because the exon 4b becomes the inton
4 by splicing. In the isoform L, the exon 4 and 4b are
spliced and resulting a reading frame change in its C-
terminal (Xue et al., 2004). All three isoforms have the
same head and rod domains and different tail domain
sequences (Titeux et al., 2001; Xue et al., 2004), but
each is expressed in a developmentally specific man-
ner (Xue et al., 2004). The avian synemin has only one
protein (230 kDa), equivalent to mammal H synemin
isoform (Bellin et al., 1999). As the nestin, the synemin
proteins belong to type VI of IF family.
As is typical for IF proteins, nestin and synemin
are characterized by a central alpha-helical region of
about 310 amino acids flanked by non-a-helical head
and tail domains (Dahlstrand et al., 1992b; Herrmann
and Aebi, 2004; Strelkov et al., 2003). The nestin
and synemin proteins contain a short N-terminus (8
11 amino acids) and an unusually long C-terminus
(Dahlstrand et al., 1992b; Titeux et al., 2001; Xue et al.,
2004), which interacts with other cytoskeleton compo-
nents, such as microfilaments, microtubules and cell
adhesion molecules (Bellin et al., 2001, 1999; Bhosle
et al., 2006; Lendahl et al., 1990; Sun et al., 2008a, b).
Nestin and synemin are unable to self-assemble (Bellin
et al., 1999; Herrmann and Aebi, 2000; Khanamiryan
et al., 2008; Titeux et al., 2001), most likely because
of its very short N-terminus (a domain necessary
for IF assembly) and the property of their 2A and
2B regions of the rod domain (Khanamiryan et al.,
2008); therefore, nestin and synemin require an appro-
priate copolymerization partner, such as vimentin or
desmin (Bellin et al., 1999; Guerette et al., 2007;
Khanamiryan et al., 2008; Marvin et al., 1998; Steinert
et al., 1999; Titeux et al., 2001), to assemble into
heteropolymers.
5.5.2 Nestin Expression
The IF protein nestin has been detected in a multitude
of cellular phenotypes in embryonic and adult tissues
in vivo and in vitro. Cells expressing nestin show char-
acteristic features of progenitor cells, such as multi-
potency, high proliferation, limited self-renewal and
regeneration capacity. After differentiation induction
64
of embryonic stem (ES) cells by tissue-specific growth
and extracellular matrix factors, nestin expression is
significantly upregulated. In a transient ES cell differ-
entiation stage, nestin expression is found in different
subpopulations of potent progenitor cells differentiat-
ing into mesenchymal/mesodermal, neural, pancreatic
endocrine and hepatic cell lineages.
Nestin was abundant in embryonic bodies (EB) and
early EB outgrowths. A significant number of nestin-
positive cells was detected in day 2 and day 6 EB with a
significant concentration of nestin-positive cells at the
ectodermal rim of EB (Wiese et al., 2004).
After spontaneous differentiation or specific differ-
entiation induction into neural, pancreatic and hepatic
cell lineages, a partial and transient co-expression of
nestin with lineage-specific marker proteins, including
glial fibrillary acidic protein (GFAP, neuroglial cells),
C-peptide (pancreatic cells), albumin (hepatic cells)
and desmin (mesenchymal/mesodermal cells). The co-
expression of nestin with lineage-specific marker pro-
teins was only found at time points that define the onset
of cell lineage specification before terminal differenti-
ation. With continued differentiation into specialized
cell types, nestin expression was downregulated in all
differentiated cells.
Nestin-positive neuroglial progenitor cells were
found as early as 3 days after the onset of differen-
tiation of ES cells, indicating that ES cells at first
differentiate into nestin-expressing cells and later into
neuronal and glial cells (Strubing et al., 1995). The
differentiation of ES cells into functional insulin-
producing pancreatic cells occurs also via a transient
stage of progenitor cells expressing C-peptide and
nestin (Wiese et al., 2004). Also, spontaneous dif-
ferentiation of ES cells without selection for nestin-
expressing cells results in hepatic cells that develop
from nestin-positive progenitor cells co-expressing
albumin (Wiese et al., 2004). Altogether, the data from
ES cell in vitro differentiation models suggest that at
least neural, pancreatic and hepatic cells are all derived
via nestin-positive intermediates.
During mammalian embryogenesis, nestin is widely
expressed in a variety of embryonic and fetal tis-
sues. It is first detected in E5 by RT-PCR (Izmiryan
et al., 2009), in the one-cell layer of neural ecto-
derm at day E7 (Kawaguchi et al., 2001; Izmiryan et
al., 2009), in neuroepithelial cells of E7.75 embryos
(Dahlstrand et al., 1995), in presomitic mesoderm and
in the myotome layer of the somites in mice (Sejersen
Z. Xue et al.
and Lendahl, 1993). Importantly, nestin is abundant
during early neurogenesis (Frederiksen and McKay,
1988) and in the peripheral and central nervous sys-
tems, suggesting that nestin-positive cells are critical
for embryonic neural development (Table 5.2).
In adult organisms, nestin-expressing cells are
restricted to defined locations, where they may func-
tion as a (quiescent) cellular reserve capable of
proliferation, differentiation and migration after re-
activation during tissue regeneration.
In addition to nestin expression during embryogen-
esis and in adult tissues, numerous cell types express
nestin after in vitro cultivation. This could suggest that
nestin expression may be a consequence of in vitro
cultivation conditions that are not necessarily represen-
tative of in vivo expression dynamics. Nestin expres-
sion is therefore widespread both in vivo and after in
vitro cultivation of multiple cell lineages. Although,
many questions concerning the functional role and
transcriptional regulation of nestin expression in var-
ious cells and tissues remain open, the data obtained
until today can lead us to propose nestin as a marker of
multi-lineage progenitor cells.
5.5.3 Synemin Expression
The synemins are expressed principally in muscle
and nervous system. The expression of synemins are
dynamic and varied depend the stages of development.
By means of RT-PCR, the genes encoding M syne-
min isoform were already active at E5 when the mouse
embryo has been just implanted (Izmiryan et al., 2009).
H synemin mRNA was found later, in E9 embryos, at
a time when vasculogenesis, somitogenesis, the migra-
tion of neural crest cells and other major activities
are under way in the embryo. L synemin was absent
until E11 (Izmiryan et al., 2009). In toto hybridiza-
tion experiment showed that M synemin mRNA was
principally accumulated within the embryonic ecto-
derm of E7 embryos (Izmiryan et al., 2009). Strong
labelling was found in the cardiac zone of E8 embryos,
while E8.5 embryos showed synemin reactivity in the
cephalic region, the prosencephalon, mesencephalon
and rhombencephalon. M synemin mRNA synthesis
was also detected at E8.5 in the presomitic mesoderm
though not in the segmented somites. At E9.5, M syne-
min mRNA could be found in the cephalic region of
embryos, although it was mostly concentrated in the
5 IF Expression in Mouse Embryonic Stem Cells and Early Embryos 65

Table 5.2 Nestin and synemin expression in mammal embryonic and fetal tissues
Cell types Nestin H synemin M synemin L synemin
Radial glia cells + + +
(Frederiksen and (Hirako et al., 2003; (Hirako et al., 2003; (Izmiryan et al.,
McKay, 1988; Izmiryan et al., Izmiryan et al., 2006)
Tohyama et al., 2006) 2006)
1992)
Astrocytes + + +
(Izmiryan et al., (Hirako et al., 2003; (Hirako et al., 2003; (Izmiryan et al.,
2006) Izmiryan et al., Izmiryan et al., 2006)
2006) 2006)
Schwann cells + + +
(Hockfield and (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
McKay, 1985) 2006) 2006) 2006)
Neuron (CNS) +
(Izmiryan et al., (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
2006) 2006) 2006) 2006)
Neuron (PNS) + + +
(Izmiryan et al., (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
2006) 2006) 2006) 2006)
Neuroepithelial cells + + +
(Dahlstrand et al., (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
1995) 2009) 2009) 2009)
Neural crest cells + + +
(Dahlstrand et al., (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
1995; Hockfield 2009) 2009) 2009)
and McKay,
1985)
Oligodendrocyte + ni ni ni
precursors (Gallo and
Armstrong, 1995)
Sensorial neurons + + +
(Izmiryan et al., (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
2006) 2006) 2006) 2006)
Sympathetic neurons + +
(Izmiryan et al., (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
2006) 2006) 2006) 2006)
Retina + + +
(Walcott and Provis, (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
2003) 2006; Tawk et al., 2006; Tawk et al., 2006)
2003) 2003)
Lens + + +
(Mokry and (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
Nemecek, 1998b) 2006; Tawk et al., 2006; Tawk et al., 2006)
2003) 2003)
Motoneurons ni
(Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
2006) 2006) 2006)
Presomitic mesoderm + +
(Hockfield and (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
McKay, 1985; 2009) 2009) 2009)
Zimmerman
et al., 1994)
Myotome + + +
(Hockfield and (Izmiryan et al., (Izmiryan et al., (Xue et al., 2004)
McKay, 1985; 2009) 2009)
Zimmerman
et al., 1994)
66 Z. Xue et al.

Table 5.2 (continued)

Cell types Nestin H synemin M synemin L synemin
Dermatome + + + ni
(Kachinsky et al., (Izmiryan et al., (Izmiryan et al.,
1994) 2009) 2009)
Striated muscle ni + +
(Xue et al., 2004) (Xue et al., 2004) (Xue et al., 2004)
Smooth muscle ni + +
(Xue et al., 2004) (Xue et al., 2004) (Xue et al., 2004)
Endothelial cells of + + +
developing blood (Mokry and (Izmiryan et al., (Izmiryan et al., (Izmiryan et al.,
vessels Nemecek, 1998a) 2009) 2009) 2009)
Hepatic oval cells + ni ni ni
(Sun and An, 2004)
Hepatic stellate cells ni + + ni
(Schmitt-Graeff (Schmitt-Graeff
et al., 2006; et al., 2006;
Uyama et al., Uyama et al.,
2006) 2006)
Pancreatic epithelial + ni ni ni
progenitor cells (Delacour et al.,
2004; Esni et al.,
2004)
The references are shown in parentheses. ni: no information available.

prosencephalon. At the same stage, the labelling of
H and M synemin extended to the first two branchial
arches and the ventricular zone of the heart. In E11.5
embryos, the limb buds and dorsal root ganglia were
heavily stained in addition to the heart and cephalic
region. At the 25-somite stage, synemins were concen-
trated in the dermomyotomes (Izmiryan et al., 2009).
However, in contrast to the nestin gene, first expressed
in neuroepithelial cells and later activated in somites
originating from the mesoderm at E9 (Lendahl,
1997), the M synemin isoform is present from
both the neuroectoderm and the mesoderm at E7.5
(Table 5.2).
In the nervous system, the synthesis of the three
synemin isoforms was probably selected by cell types,
and their temporal and spatial distributions. The
expression of synemins are dynamic and varied depend
the stages of development. H/M synemins occurred
together with nestin and vimentin in glial progeni-
tors during the early differentiation of the developing
mouse CNS. They are later found in GFAP-labeled
cells. In contrast, the L isoform appeared only in
neurons, together with neurofilaments and beta III-
tubulin in the brain after birth. H/M isoforms were
detected in the medulla oblongata, glia limitans, the
retina and lens from E13, in ependymal cells from
E17 and in adults, where GFAP was also found. The L
synemin was distributed in different neurons in adults,
including the mouse cortex neurons, hippocampus and
the granular neurons of the cerebellum. In contrast,
Purkinje cells were immunostained for the H/M iso-
forms. The L synemin protein was also detected in
the neurons of the motor column of the spinal cord
from E17, newborns, and adult mice. In the periph-
eral nervous system (PNS), L synemin appeared from
E13 where it was confined to the neurons of spinal
ganglia. In the meantime, the H/M synemin isoforms
were found in both the neurons and Schwann cells
of the sensorial ganglia from E11 (Izmiryan et al.,
2006). This complex dynamic pattern of synemin
isoform synthesis may reflect the reorganization of
the cytoskeleton during nervous system development
(Table 5.2).
The complex distribution patterns of the syne-
min isoforms in neural cells raises several questions
about the regulation of the synemin gene during the
determination of glial and neuronal cell lineages in
the CNS and PNS. First, an unexpected finding is the
selective synthesis of the two high molecular weight
(H/M) synemin isoforms in CNS astrocytes, while the
smallest synemin isoform (L) is present only in neu-
rons. This selectivity suggests that the commitment of
5 IF Expression in Mouse Embryonic Stem Cells and Early Embryos
CNS precursor cells to form glia or neuron involves
the direct regulation of the single synemin gene. As
the H/M synemins are present mainly in glial cells and
the L synemin isoform in neurons, it may be partic-
ularly important to identify the mechanisms whereby
the synemin gene is regulated as part of general inves-
tigation of how stem cells differentiate. Second, the
PNS sensory neurons can contain all three isoforms
(H, M, and L), indicating that the production of the
H/M isoforms by glial cells and the L isoform by neu-
rons is not strictly linked to the glia or neuron fates of
precursor cells. Instead, it indicates a flexible program
that can be adjusted to each type of nervous system
and may result from the differentiation of radial glial
cells, which can give rise to both astrocytes and neu-
rons (Bibel et al., 2004). This modulation of synemin
gene regulation within neuron subtypes is also illus-
trated by the presence of the H/M synemin isoforms
in Purkinje cells that lack the L isoform, although it
is present in neighboring granular neurons. We there-
fore believe that the production of synemin proteins is
closely associated with the early specialization of the
cytoplasm of cells in the nervous system.
Out of the nervous system, synemin isoforms are
also expressed in striated muscle (Table 5.2). In muscle
tissues, the synemins are specifically associated with
the desmin and vimentin (Xue et al., 2004). Normally,
the synemin is associated with desmin in the line Z of
the sacromere of skeletal muscle and in the interca-
late disc of cardiac muscle. In the desmin myopathy
patient, the synemin is colocalized with desmin in
the desmin aggregates. Ours studies with desmin and
vimentin knock-out (KO) mouse have demonstrated
that the synemin is specifically associated with the
desmin in the skeletal and cardiac muscle. When the
desmin is absent, the synemin is also absent in the
skeletal and cardiac muscle. In the smooth muscle, the
synemin is associated with both desmin and vimentin.
In desmin KO mouse, the synemin is detected in the
smooth muscle that is associated with the vimentin.
In vimentin KO mouse, the expression of synemin is
decreased in the smooth muscle, but still detected. This
is the synemin associated with the desmin. When both
the desmin and the vimentin are absent, the synemin
is absent in all the muscle tissues (Xue et al., 2004).
The synemin was also detected in human liver hep-
atic stellate cells, where it was co-localized with focal
adhesion proteins in long slender processes (Schmitt-
Graeff et al., 2006; Uyama et al., 2006), and mouse
67
endothelial cells where it was associated with vimentin
(Izmiryan et al., 2009).
However, the transcriptional regulation of synemin
in vivo or in vitro is remained to determine.
5.6 Expression of Nestin and Synemin
in Tumoral Cells of the CNS
5.6.1 Glial Tumors
The theory that tumors derive from stem cells is
now well established. Stem cells occur in the sub-
granular layer of the hippocampus and subependymal
zone, from where they could migrate and colonize
other brain regions. Established neuroectodermal brain
tumors could originate from these migrating stem cells,
and the tumor mass could be continuously replenished
with these cells from those clinically silent regions
(Berger et al., 2004). Nestin and synemin are present
in the cells of these silent regions.
We have immunostained synemin in the ependymal
cells from embryonic and adult brain, and a differ-
ent variant, L synemin, is also present in the mouse
adult neurons of the hippocampus (Izmiryam et al.,
2006). This may suggest that this marker exists in the
ependymal zone earlier during development, present
in cells that could populate the brain regions, and co-
exists in the glial and neuronal cells during the course
of life. Presence of self-renewing neural stem cells in
the CNS of both children and adults suggests that they
play some role in tumorigenesis (Lewis, 1968) and in
resistance to current therapeutic strategies (Pilkington,
2005).
Glial tumors or gliomas originating from astrocytes
are recognized as astrocytomas. Gliomas are the most
frequent primary tumors of the CNS and are an impor-
tant cause of mental impairment and death. They are
among the most difficult neoplasms to treat effectively;
being resistant to different therapeutic procedures. It
is due the combination of their rate of proliferation,
cellular heterogeneity, and capacity to invade dif-
fusely contiguous nervous tissues (Schiffer, 1997).
The classification of glioma subfamilies is based on
specific histopathological characteristics (cellularity,
nuclear atypia, mitotic activity, microvascular prolif-
eration, and necrosis) in agreement with the World
Health Organization (WHO); see Kleihues et al. (2002)
68
and Kleihues et al. (1995). However, the characteris-
tics used to classify these tumors generate complex
interpretations and differences in diagnoses (Daumas-
Duport et al., 2000).
One of the challenges for cancer control is its
early detection. Molecular markers, which could be
signposts to detect tumor cells and indicate their ori-
gin, phenotype, and function, based for instance on
recently developed technologies such as cDNA and
oligonucleotide microarrays on chips, serial analysis
of gene expression (SAGE), and proteomic methods
to detect cellular proteins implicated in a pathologi-
cal transition, are currently in progress (Snirivas et al.,
2001).
5.6.2 Nestin in Glioma
Structural proteins such as cytoskeletal components
have been used for many years to characterize the phe-
notype and tissue origin of the tumor glial cells. GFAP
and neurofilament proteins are useful to identify the
glial and neuronal origin of the cells present in brain
tumors (Schiffer, 1997; Zehner, 1991). Recently, intro-
duction of the nestin protein (Strubing et al., 1995;
Wiese et al., 2004) to decorate tumor cells indicated the
possibility that primitive cells are present in the tumor.
This nestin expression, in isolation or co-existing with
GFAP and vimentin, in glial tumors opens this question
(Bao et al., 2006; Faria et al., 2006; Singh et al., 2004,
2003). In fact, nestin has been detected in all the differ-
ent grades of gliomas. Interestingly, the highest level of
nestin-positive cells (mean 25.3%) is found in glioblas-
toma, the most malignant glial tumor (Ma et al., 2005;
Strojnik et al., 2007). On the other hand, in neuroblas-
toma, another CNS tumor entity, it is very difficult to
confirm a correlation between nestin expression and its
malignancy (Korja et al., 2005; Thomas et al., 2004).
Co-expression of nestin and vimentin in different
astrocytoma cell lines has been related to a migratory
cell phenotype with increased motility and invasive-
ness, which revels a metastatic potential of different
astrocytoma cell lines (Rutka et al., 1999). Moreover,
the high level of nestin in tumor cells indicates a
prognosis for significantly shorter survival of glioma
patients. An intense immunostaining of nestin in tumor
cells may be used to predict the risk of death in patients
Z. Xue et al.
with malignant primary tumors of the CNS (Strojnik
et al., 2007).
Nestin, as indicated above, has been detected in pri-
mary CNS tumors, but not in carcinoma metastases
(Dahlstrand et al., 1992a; Ikota et al., 2006; Tohyama
et al., 1992). More recently, synemin was also detected
in gliomas.
5.6.3 Synemin Expression in Glioma
As shown earlier, H/M synemin expression is related
to glial cell fate during mouse CNS development
(Izmiryan et al., 2006). However, H synemin is not
found in the astrocytes of the adult normal human
brain, H synemin is de novo expressed in response to
pathological situations, such as brain injury (reactive
astrocytes) and gliomas (Jing et al., 2005, 2007). In
glioma cells, synemin interacts with -actinin at the
leading edge (Jing et al., 2005). It can decorate glial
tumor cells in vitro, and in the tumor in vivo.
Recently, it was shown that the presence of synemin
at the leading edge also correlated with a high migra-
tory potential of the cells (Pan et al., 2008). In fact, a
role for synemin in glioma migration was suggested
by its down-regulation, which sharply decreased the
migration of gliomas. Therefore, H/M synemin seems
to be an important glioma marker for its invasive-
ness capacity, but this hypothesis needs experimental
demonstration.
5.6.4 And Now
These are golden days for stem cells. Their contri-
butions to the development and construction of the
human body, to regeneration, and the design of new
therapies have attracted much attention to these cells.
New markers are welcome to identify and follow these
cells guiding the construction of tissues and organs. A
well-exploited example is the neural crest cells, multi-
potent cells that migrate during early development to
construct different tissues and organs (Le Douarin and
Kalcheim, 1999; Trentin et al., 2004).
The cytoskeletal proteins constitute a useful arse-
nal to recognize cells from different tissues, in order to
5 IF Expression in Mouse Embryonic Stem Cells and Early Embryos
analyze cell migration, cell morphology, and morpho-
logical changes (Zehner, 1991).
These proteins are expressed from different genes
and different splices, generating isovariants with dif-
ferent isoelectric points or molecular weights. These
variations can be specifically expressed in the cells.
This is the case for a variant of Tubulin, beta III
tubulin, which is expressed in the neuronal precursor
cells (Menezes and Luskin, 1994). Another example
is GFAP, which is expressed by five different gene
transcripts, generated by alternate transcriptional start
sites or by different splicing, alpha (the predominant
isoform of GFAP), beta, gamma, delta, and kappa
(Blechingberg et al., 2007; Condorelli et al., 1999;
Feinstein et al., 1992; Galea et al., 1995; Zelenika
et al., 1995). One of these, GFAP gamma, seems to be
a potential marker for a restricted astrocytic population
located specifically in the adult human subventricular,
subgranular, and subpial zones (Roelofs et al., 2005).
Furthermore, the antibodies against GFAP delta can
immunostain subpial gliosis (Andreiuolo et al., 2009).
Synemin isotypes, H/M and L, have also differ-
ent cellular distribution in the CNS. This distribution
seems correlated with development, with a precocious
expression in embryonic life. We cannot eliminate
the possibility that synemin could with nestin be a
precocious marker of multipotent cells.
Now, these isoforms of cytoskeletal proteins could
be a point of investigation toward a possible use of
these markers in different periods of development or
at the moment of commitment of the cells.
References
Andreiuolo F, Junier M-P, Hol EM, Miquet C, Chimelli
L, Leonard N, Chneiweiss H, Daumas-Duport C, Varlet
P (2009) GFAP immunostaining improves visualiza-
tion of normal and pathological astrocytic heterogeneity.
Neuropathology 29:3139.
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB,
Dewhirst MW, Bigner DD, Rich JN (2006) Glioma stem
cells promote radioresistance by preferential activation of the
DNA damage response. Nature 444:756760.
Bellin RM, Huiatt TW, Critchley DR, Robson RM (2001)
Synemin may function to directly link muscle cell interme-
diate filaments to both myofibrillar Z-lines and costameres.
J Biol Chem 276:3233032337.
Bellin RM, Sernett SW, Becker B, Ip W, Huiatt TW, Robson
RM (1999) Molecular characteristics and interactions of
the intermediate filament protein synemin. Interactions with
alpha-actinin may anchor synemin-containing heterofila-
ments. J Biol Chem 274:2949329499.
69
Berger F, Gay E, Pelletier L, Tropel P, Wion D (2004)
Development of gliomas: potential role of asymmetrical cell
division of neural stem cells. Lancet Oncol 5:511514.
Bhosle RC, Michele DE, Campbell KP, Li Z, Robson RM (2006)
Interactions of intermediate filament protein synemin with
dystrophin and utrophin. Biochem Biophys Res Commun
346:768777.
Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M,
Goetz M, Barde YA (2004) Differentiation of mouse embry-
onic stem cells into a defined neuronal lineage. Nat Neurosci
7:10031009.
Blechingberg J, Holm IE, Nielsen KB, Jensen TH, Jrgensen
AL, Nielsen AL (2007) Identification and characterization of
GFAPkappa, a novel glial fibrillary acidic protein isoform.
Glia 55:497507.
Brulet P, Babinet C, Kemler R, Jacob F (1980) Monoclonal
antibodies against trophectoderm-specific markers during
mouse blastocyst formation. Proc Natl Acad Sci USA 77:
41134117.
Centonze VE, Ruben GC, Sloboda RD (1986) Structure and
composition of the cytoskeleton of nucleated erythrocytes:
III. Organization of the cytoskeleton of Bufo marinus
erythrocytes as revealed by freeze-dried platinum-carbon
replicas and immunofluorescence microscopy. Cell Motil
Cytoskeleton 6:376388.
Cochard P, Paulin D (1984) Initial expression of neurofilaments
and vimentin in the central and peripheral nervous system of
the mouse embryo in vivo. J Neurosci 4:20802094.
Condorelli DF, Nicoletti VG, Barresi V, Conticello SG, Caruso
A, Tendi EA, Giuffrida Stella AM (1999) Structural features
of the rat GFAP gene and identification of a novel alternative
transcript. J Neurosci Res 56:219228.
Dahlstrand J, Collins VP, Lendahl U (1992a) Expression of
the class VI intermediate filament nestin in human central
nervous system tumors. Cancer Res 52:53345341.
Dahlstrand J, Lardelli M, Lendahl U (1995) Nestin mRNA
expression correlates with the central nervous system pro-
genitor cell state in many, but not all, regions of develop-
ing central nervous system. Brain Res Dev Brain Res 84:
109129.
Dahlstrand J, Zimmerman LB, McKay RD, Lendahl U (1992b)
Characterization of the human nestin gene reveals a close
evolutionary relationship to neurofilaments. J Cell Sci
103:589597.
Daumas-Duport C, Beuvon F, Varlet P, Fallet-Bianco C (2000)
Gliomas: WHO and Sainte-Anne Hospital classifications [in
French]. Ann Pathol 20:413428.
Delacour A, Nepote V, Trumpp A, Herrera PL (2004) Nestin
expression in pancreatic exocrine cell lineages. Mech Dev
121:314.
Le Douarin NM, Kalcheim C (1999) The Neural Crest.
Cambridge University Press, Cambridge, Second Edition.
Escurat M, Djabali K, Gumpel M, Gros F, Portier MM
(1990) Differential expression of two neuronal intermediate-
filament proteins, peripherin and the low-molecular-mass
neurofilament protein (NF-L), during the development of the
rat. J Neurosci 10:764784.
Escurat M, Gumpel M, Lachapelle F, Gros F, Portier MM
(1988) Comparative expression of 2 intermediate filament
proteins, peripherin and the 68 kDa neurofilament protein,
during embryonal development of the rat. C R Acad Sci III
306:447456.
70
Esni F, Stoffers DA, Takeuchi T, Leach SD (2004) Origin of
exocrine pancreatic cells from nestin-positive precursors in
developing mouse pancreas. Mech Dev 121:1525.
Faria J, Romo L, Martins S, Alves T, Mendes FA, de Faria
GP, Hollanda R, Takiya C, Chimelli L, Morandi V, de Souza
JM, Abreu JG, Moura Neto V (2006) Interactive properties
of human glioblastoma cells with brain neurons in cul-
ture and neuronal modulation of glial laminin organization.
Differentiation 74:562572.
Feinstein DL, Weinmaster GA, Milner RJ (1992) Isolation of
cDNA clones encoding rat glial fibrillary acidic protein:
expression in astrocytes and in Schwann cells. J Neurosci Res
32:114.
Franke WW, Grund C, Kuhn C, Jackson BW, Illmensee K
(1982a) Formation of cytoskeletal elements during mouse
embryogenesis. III. Primary mesenchymal cells and the first
appearance of vimentin filaments. Differentiation 23:4359.
Franke WW, Schmid E, Schiller DL, Winter S, Jarasch ED,
Moll R, Denk H, Jackson BW, Illmensee K (1982b)
Differentiation-related patterns of expression of proteins of
intermediate-size filaments in tissues and cultured cells. Cold
Spring Harb Symp Quant Biol 46:431453.
Frederiksen K, McKay RD (1988) Proliferation and differentia-
tion of rat neuroepithelial precursor cells in vivo. J Neurosci
8:11441151.
Fuchs E, Weber K (1994) Intermediate filaments: struc-
ture, dynamics, function, and disease. Annu Rev Biochem
63:345382.
Galea E, Dupouey P, Feinstein DL (1995) Glial fibrillary acidic
protein mRNA isotypes: expression in vitro and in vivo. J
Neurosci Res 41:452461.
Gallo V, Armstrong RC (1995) Developmental and growth
factor-induced regulation of nestin in oligodendrocyte lin-
eage cells. J Neurosci 15:394406.
Galou M, Pournin S, Ensergueix D, Ridet JL, Tchelingerian JL,
Lossouarn L, Privat A, Babinet C, Dupouey P (1994) Normal
and pathological expression of GFAP promoter elements in
transgenic mice. Glia 12:281293.
Goldman RD (2001) Worms reveal essential functions for inter-
mediate filaments. Proc Natl Acad Sci USA 98:76597661.
Granger BL, Lazarides E (1980) Synemin: a new high molec-
ular weight protein associated with desmin and vimentin
filaments in muscle. Cell 22:727738.
Granger BL, Lazarides E (1982) Structural associations of syne-
min and vimentin filaments in avian erythrocytes revealed by
immunoelectron microscopy. Cell 30:263275.
Granger BL, Lazarides E (1984) Expression of the intermediate-
filament-associated protein synemin in chicken lens cells.
Mol Cell Biol 4:19431950.
Guerette D, Khan PA, Savard PE, Vincent M (2007) Molecular
evolution of type VI intermediate filament proteins. BMC
Evol Biol 7:164164.
Herrmann H, Aebi U (2000) Intermediate filaments and their
associates: multi-talented structural elements specifying
cytoarchitecture and cytodynamics. Curr Opin Cell Biol
12:7990.
Herrmann H, Aebi U (2004) Intermediate filaments: molecular
structure, assembly mechanism, and integration into func-
tionally distinct intracellular Scaffolds. Annu Rev Biochem
73:749789.
Hirako Y, Yamakawa H, Tsujimura Y, Nishizawa Y, Okumura M,
Usukura J, Matsumoto H, Jackson KW, Owaribe K, Ohara O
Z. Xue et al.
(2003) Characterization of mammalian synemin, an interme-
diate filament protein present in all four classes of muscle
cells and some neuroglial cells: co-localization and interac-
tion with type III intermediate filament proteins and keratins.
Cell Tissue Res 313:195207.
Hockfield S, McKay RD (1985) Identification of major cell
classes in the developing mammalian nervous system. J
Neurosci 5:33103328.
Hoffman PN, Lasek RJ (1975) The slow component of axonal
transport. Identification of major structural polypeptides of
the axon and their generality among mammalian neurons. J
Cell Biol 66:351366.
Ikota H, Kinjo S, Yokoo H, Nakazato Y (2006) Systematic
immunohistochemical profiling of 378 brain tumors with
37 antibodies using tissue microarray technology. Acta
Neuropathol 111:475482.
Izmiryan A, Cheraud Y, Khanamiryan L, Leterrier JF, Federici
T, Peltekian E, Moura-Neto V, Paulin D, Li Z, Xue ZG
(2006) Different expression of synemin isoforms in glia
and neurons during nervous system development. Glia 54:
204213.
Izmiryan A, Franco CA, Paulin D, Li Z, Xue Z (2009) Synemin
isoforms during mouse development: multiplicity of partners
in vascular and neuronal systems. Exp Cell Res 315:769
783.
Jackson BW, Grund C, Winter S, Franke WW, Illmensee
K (1981) Formation of cytoskeletal elements during
mouse embryogenesis. II. Epithelial differentiation and
intermediate-sized filaments in early postimplantation
embryos. Differentiation 20:203216.
Jing R, Pizzolato G, Robson RM, Gabbiani G, Skalli O (2005)
Intermediate filament protein synemin is present in human
reactive and malignant astrocytes and associates with ruffled
membranes in astrocytoma cells. Glia 50:107120.
Jing R, Wilhelmsson U, Goodwill W, Li L, Pan Y, Pekny M,
Skalli O (2007) Synemin is expressed in reactive astrocytes
in neurotrauma and interacts differentially with vimentin
and GFAP intermediate filament networks. J Cell Sci 120:
12671277.
Kachinsky AM, Dominov JA, Miller JB (1994) Myogenesis and
the intermediate filament protein, nestin. Dev Biol 165:216
228.
Kaplan MP, Chin SS, Fliegner KH, Liem RK (1990) Alpha-
internexin, a novel neuronal intermediate filament pro-
tein, precedes the low molecular weight neurofilament pro-
tein (NF-L) in the developing rat brain. J Neurosci 10:
27352748.
Kawaguchi A, Miyata T, Sawamoto K, Takashita N, Murayama
A, Akamatsu W, Ogawa M, Okabe M, Tano Y, Goldman SA,
Okano H (2001) Nestin-EGFP transgenic mice: visualization
of the self-renewal and multipotency of CNS stem cells. Mol
Cell Neurosci 17:259273.
Khanamiryan L, Li Z, Paulin D, Xue Z (2008) Self-assembly
incompetence of synemin is related to the property of its head
and rod domains. Biochemistry 47:95319539.
Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger
G, Burger PC, Cavenee WK (2002) The WHO classification
of tumors of the nervous system. J Neuropathol Exp Neurol
61:215225.
Kleihues P, Soylemezoglu F, Schauble B, Scheithauer BW,
Burger PC (1995) Histopathology, classification, and grading
of gliomas. Glia 15:211221.
5 IF Expression in Mouse Embryonic Stem Cells and Early Embryos
Korja M, Finne J, Salmi TT, Kalimo H, Karikoski R, Tanner
M, Isola J, Haapasalo H (2005) Chromogenic in situ
hybridization-detected hotspot MYCN amplification asso-
ciates with Ki-67 expression and inversely with nestin
expression in neuroblastomas. Mod Pathol 18:15991605.
Lane EB, Hogan BL, Kurkinen M, Garrels JI (1983) Co-
expression of vimentin and cytokeratins in parietal endoderm
cells of early mouse embryo. Nature 303:701704.
Lazarides E (1981) Intermediate filaments chemical hetero-
geneity in differentiation. Cell 23:649650.
Lehtonen E, Lehto VP, Paasivuo R, Virtanen I (1983a) Parietal
and visceral endoderm differ in their expression of interme-
diate filaments. EMBO J 2:10231028.
Lehtonen E, Lehto VP, Vartio T, Badley RA, Virtanen I (1983b)
Expression of cytokeratin polypeptides in mouse oocytes and
preimplantation embryos. Dev Biol 100:158165.
Lendahl U (1997) Transgenic analysis of central nervous sys-
tem development and regeneration. Acta Anaesthesiol Scand
110:116118.
Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells
express a new class of intermediate filament protein. Cell
60:585595.
Lewis PD (1968) Mitotic activity in the primate subependymal
layer and the genesis of gliomas. Nature 217:974975.
Lothian C, Lendahl U (1997) An evolutionarily conserved region
in the second intron of the human nestin gene directs gene
expression to CNS progenitor cells and to early neural crest
cells. Eur J Neurosci 9:452462.
Lothian C, Prakash N, Lendahl U, Wahlstrom GM (1999)
Identification of both general and region-specific embryonic
CNS enhancer elements in the nestin promoter. Exp Cell Res
248:509519.
Ma DK, Ming GL, Song H (2005) Glial influences on neural
stem cell development: cellular niches for adult neurogene-
sis. Curr Opin Neurobiol 15:514520.
Maro B, Kubiak J, Gueth C, De Pennart H, Houliston E,
Weber M, Antony C, Aghion J (1990) Cytoskeleton orga-
nization during oogenesis, fertilization and preimplantation
development of the mouse. Int J Dev Biol 34:127137.
Marvin MJ, Dahlstrand J, Lendahl U, McKay RD (1998) A
rod end deletion in the intermediate filament protein nestin
alters its subcellular localization in neuroepithelial cells of
transgenic mice. J Cell Sci 111:19511961.
McKeon FD, Kirschner MW, Caput D (1986) Homologies in
both primary and secondary structure between nuclear enve-
lope and intermediate filament proteins. Nature 319:463
468.
Menezes JR, Luskin MB (1994) Expression of neuron-specific
tubulin defines a novel population in the proliferative layers
of the developing telencephalon. J Neurosci 14:53995416.
Mokry J, Nemecek S (1998a) Angiogenesis of extra- and
intraembryonic blood vessels is associated with expression
of nestin in endothelial cells. Folia Biol (Praha) 44:155161.
Mokry J, Nemecek S (1998b) Immunohistochemical detec-
tion of intermediate filament nestin. Acta Medica (Hradec
Kralove) 41:7380.
Oshima RG (2007) Intermediate filaments: a historical perspec-
tive. Exp Cell Res 313:19811994.
Oshima RG, Howe WE, Klier FG, Adamson ED, Shevinsky LH
(1983) Intermediate filament protein synthesis in preimplan-
tation murine embryos. Dev Biol 99:447455.
71
Pachter JS, Liem RK (1985) Alpha-internexin, a 66-kD inter-
mediate filament-binding protein from mammalian central
nervous tissues. J Cell Biol 101:13161322.
Pan Y, Jing R, Pitre A, Williams BJ, Skalli O (2008) Intermediate
filament protein synemin contributes to the migratory prop-
erties of astrocytoma cells by influencing the dynamics of the
actin cytoskeleton. FASEB J 22:31963206.
Paulin D, Babinet C, Weber K, Osborn M (1980) Antibodies as
probes of cellular differentiation and cytoskeletal organiza-
tion in the mouse blastocyst. Exp Cell Res 130:297304.
Pilkington GJ (2005) Cancer stem cells in the mammalian
central nervous system. Cell Prolif 38:423433.
Portier MM, de Nechaud B, Gros F (1983) Peripherin, a new
member of the intermediate filament protein family. Dev
Neurosci 6:335344.
Roelofs RF, Fischer DF, Houtman SH, Sluijs JA, Van Haren
W, Van Leeuwen FW, Hol EM (2005) Adult human sub-
ventricular, subgranular, and subpial zones contain astrocytes
with a specialized intermediate filament cystoskeleton. Glia
52:289300.
Rutka JT, Ivanchuk S, Mondal S, Taylor M, Sakai K, Dirks P, Jun
P, Jung S, Becker LE, Ackerley C (1999) Co-expression of
nestin and vimentin intermediate filaments in invasive human
astrocytoma cells. Int J Dev Neurosci 17:503515.
Schiffer D (1997) Brain Tumors, Biology, Pathology, and
Clinical References. Springer Verlag, Berlin, Second Revised
Edition.
Schmitt-Graeff A, Jing R, Nitschke R, Desmouliere A, Skalli O
(2006) Synemin expression is widespread in liver fibrosis and
is induced in proliferating and malignant biliary epithelial
cells. Hum Pathol 37:12001210.
Sejersen T, Lendahl U (1993) Transient expression of the inter-
mediate filament nestin during skeletal muscle development.
J Cell Sci 106:12911300.
Singh SK, Clarke ID, Hide T, Dirks PB (2004) Cancer stem cells
in nervous system tumors. Oncogene 23:72677273.
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire
J, Dirks PB (2003) Identification of a cancer stem cell in
human brain tumors. Cancer Res 63:58215828.
Srinivas PR, Kramer BS, Srivastava S (2001) Trends in
biomarker research for cancer detection. Lancet Oncol
2:698704.
Steinert PM, Chou YH, Prahlad V, Parry DA, Marekov LN,
Wu KC, Jang SI, Goldman RD (1999) A high molecular
weight intermediate filament-associated protein in BHK-
21 cells is nestin, a type VI intermediate filament protein.
Limited co-assembly in vitro to form heteropolymers with
type III vimentin and type IV alpha-internexin. J Biol Chem
274:98819890.
Strelkov SV, Herrmann H, Aebi U (2003) Molecular architecture
of intermediate filaments. Bioessays 25:243251.
Strojnik T, Rosland GV, Sakariassen PO, Kavalar R, Lah
T (2007) Neural stem cell markers, nestin and musashi
proteins, in the progression of human glioma: correlation
of nestin with prognosis of patient survival. Surg Neurol
68:133143.
Strubing C, Ahnert-Hilger G, Shan J, Wiedenmann B,
Hescheler J, Wobus AM (1995) Differentiation of pluripotent
embryonic stem cells into the neuronal lineage in vitro gives
rise to mature inhibitory and excitatory neurons. Mech Dev
53:275287.
72
Sultana S, Sernett SW, Bellin RM, Robson RM, Skalli O
(2000) Intermediate filament protein synemin is transiently
expressed in a subset of astrocytes during development. Glia
30:143153.
Sun XY, An J (2004) Expression of nestin, an intermediate fila-
ment protein, in human fetal hepatic stem cells. Di Yi Jun Yi
Da Xue Xue Bao 24:207209.
Sun N, Critchley DR, Paulin D, Li Z, Robson RM (2008a)
Human alpha-synemin interacts directly with vinculin and
metavinculin. Biochem J 409:657667.
Sun N, Critchley DR, Paulin D, Li Z, Robson RM (2008b)
Identification of a repeated domain within mammalian alpha-
synemin that interacts directly with talin. Exp Cell Res
314:18391849.
Tawk M, Titeux M, Fallet C, Li Z, Daumas-Duport C,
Cavalcante LA, Paulin D, Moura-Neto V (2003) Synemin
expression in developing normal and pathological human
retina and lens. Exp Neurol 183:499507.
Thomas SK, Messam CA, Spengler BA, Biedler JL, Ross RA
(2004) Nestin is a potential mediator of malignancy in human
neuroblastoma cells. J Biol Chem 279:2799427999.
Titeux M, Brocheriou V, Xue Z, Gao J, Pellissier JF, Guicheney
P, Paulin D, Li Z (2001) Human synemin gene generates
splice variants encoding two distinct intermediate filament
proteins. Eur J Biochem 268:64356449.
Tohyama T, Lee VM, Rorke LB, Marvin M, McKay RD,
Trojanowski JQ (1992) Nestin expression in embryonic
human neuroepithelium and in human neuroepithelial tumor
cells. Lab Invest 66:303313.
Trentin A, Glavieux-Pardanaud C, Le Douarin NM, Dupin E
(2004) Self-renewal capacity is a widespread property of var-
ious types of neural crest precursor cells. Proc Natl Acad Sci
USA 101:44954500.
Z. Xue et al.
Uyama N, Zhao L, Van Rossen E, Hirako Y, Reynaert H,
Adams DH, Xue Z, Li Z, Robson R, Pekny M, Geerts
A (2006) Hepatic stellate cells express synemin, a pro-
tein bridging intermediate filaments to focal adhesions. Gut
55:12761289.
Walcott JC, Provis JM (2003) Muller cells express the neuronal
progenitor cell marker nestin in both differentiated and undif-
ferentiated human foetal retina. Clin Experiment Ophthalmol
31:246249.
Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov
KV, Tarasova Y, Wersto RP, Boheler KR, Wobus
AM (2004) Nestin expression a property of
multi-lineage progenitor cells? Cell Mol Life Sci
61:25102522.
Xue ZG, Cheraud Y, Brocheriou V, Izmiryan A, Titeux M,
Paulin D, Li Z (2004) The mouse synemin gene encodes
three intermediate filament proteins generated by alternative
exon usage and different open reading frames. Exp Cell Res
298:431444.
Yaworsky PJ, Kappen C (1999) Heterogeneity of neural progen-
itor cells revealed by enhancers in the nestin gene. Dev Biol
205:309321.
Zehner ZE (1991) Regulation of intermediate filament gene
expression. Curr Opin Cell Biol 3:6774.
Zelenika D, Grima B, Brenner M, Pessac B (1995) A novel glial
fibrillary acidic protein mRNA lacking exon 1. Brain Res
Mol Brain Res 30:251258.
Zimmerman L, Parr B, Lendahl U, Cunningham M, McKay
R, Gavin B, Mann J, Vassileva G, McMahon A (1994)
Independent regulatory elements in the nestin gene direct
transgene expression to neural stem cells or muscle precur-
sors. Neuron 12:1124.
Chapter 6
Aneuploidy in Embryonic Stem Cells
Rafaela C. Sartore, Priscila B. Campos, Michael J. McConnell, and Stevens K. Rehen
Contents
6.1 Introduction . . . . . . . . . . . . . . . . 74
6.2 A Brief History of Aneuploidy . . . . . . . . 74
6.3 Cell Cycle Checkpoints Maintain Genome
Integrity . . . . . . . . . . . . . . . . . . 74
6.4 Increased Levels of Aneuploidy Indicates
Reduced Checkpoint Fidelity in
Stem/Progenitor Cells . . . . . . . . . . . . 76
6.5 DNA Damage Signaling and Aneuploidy . . . 77
6.6 Does Aneuploidy in Stem and/or Progenitor
Cells Have Consequences for Development and
Disease? . . . . . . . . . . . . . . . . . . 79
6.7 Aneuploidy and Cancer Stem Cells . . . . . 80
6.8 Telomeres and Telomerase Under Genomic
Stability Control . . . . . . . . . . . . . . 80
6.9 Aneuploidy and Cell-Based Therapy . . . . . 81
6.9.1 Mechanical Versus Enzymatic Methods 81
6.9.2 Risks and Benefits of Aneuploidy to
Cell-Based Therapies . . . . . . . . 82
References . . . . . . . . . . . . . . . . . . . . 83
Abstract Aneuploidy is defined as the loss and/or
gain of chromosomes to produce a numerical deviation
from multiples of the haploid chromosomal comple-
ment. This phenomenon can be classified as a net
increase in chromosome number, referred to as hyper-
ploidy, or a net decrease, referred to as hypoploidy.
Biologically, chromosome number can range from
0 (red blood cells) to polyploidy (hepatocytes, neo-
plasms). Human embryonic stem cells (hES) have
S.K. Rehen ()
Instituto de Cincias Biomdicas, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, Brazil
e-mail: srehen@anato.ufrj.br
H. Ulrich (ed.), Perspectives of Stem Cells,
DOI 10.1007/978-90-481-3375-8_6, Springer Science+Business Media B.V. 2010
great potential for use in both basic science and thera-
peutic strategies, including transplantation for regen-
erative medicine. A challenge for cell therapy using
hES is the maintenance of stable cell lines, particularly
following extended passaging. Chromosomal instabil-
ity has recently been reported in hES, resulting from
clonal expansion of aneuploid cells. It includes hyper-
ploidies, particularly trisomies of chromosomes 12, 17
or 20 and, to a lesser extent, chromosome loss. The
generality of this phenomenon is uncertain and it is
currently unclear whether aneuploidy in stem cell lines
is in fact deleterious. What is the physiological signif-
icance or therapeutic risk associated with hES aneu-
ploidy? Here, features of cell cycle and the relevance
of aneuploidy are discussed on regard of its implica-
tions for the physiology and therapeutic purposes of
embryonic stem cells.
Keywords Aneuploidy Cancer stem cells Cell cycle
checkpoint Embryonic stem cells Genome integrity
Abbreviations
AD Alzheimers disease
APC/C anaphase promoting complex-cyclosome
ATM ataxia telangiectasia-mutated protein
kinase
Bub Budding Uninhibited by Benzimidazole
CSC cancer stem cell(s)
Cdc Cell division control protein
Chk Checkpoint kinase
CDK Cyclin-dependent kinase activity
CKI Cyclin Kinase Inhibitors
dNTP deoxyribonucleotide triphosphate(s)
EB Embryoid Bodies
EC cell(s) Embryonal Carcinoma cell(s)
ES cell(s) Embryonic Stem cell(s)
73
74
FISH Fluorescence In Situ Hybridization
G phasesGap phases
pRb Retinoblastoma protein
IVF In Vitro Fertilization
mEF mouse embryonic fibroblast(s)
CENP-E Microtubule-dependent motor
centromere-associated protein E
Mps1 Mitogen-activated protein kinase 1
M phase Mitosis phase
Mad Mitotic arrest deficient
MVA Mosaic Variegated Aneuploidy
NPC neural progenitor cell(s)
OCT-4 Octamer-binding transcription factor 4
p19/ARF p19 alternative reading frame protein
SAC Spindle Assembly Checkpoint
SSEA-1 Stage-Specific Embryonic Antigen 1
S phase Synthesis phase
p53 (53 kilodalton) tumor suppressor protein
6.1 Introduction
The organization of mammalian genomes is such that
an individual cell has two copies of each chromo-
some. At fertilization, each copy comes from a parental
gamete. When this event occurs, typically the result-
ing blastomere is euploid and its genome is diploid
(2n): 2n represents 40 chromosomes in mice and 46
chromosomes in human.
Although euploidy predominates, certain deviations
can be observed. When an entire chromosome comple-
ment is over duplicated, these cells become polyploid
(i.e. 3n is triploid, 4n is tetraploid). However, occur-
ring more frequently during mammalian development
are single chromosome losses and gains, referred to as
aneuploidies, found in embryonic stem (ES) cell and
neural progenitor cell (NPC) populations. Herein, we
review the mechanisms by which altered cell cycle
controls can give rise to aneuploidy, discuss certain
consequences of aneuploidy in human development
and disease, and introduce some of the challenges that
aneuploidy brings to cell-based therapies.
6.2 A Brief History of Aneuploidy
Aneuploidy was first observed during early embryoge-
nesis in sea urchins. As reported by Theodor Boveri
in 1902, the fertilization of one egg by two sperm cells
R.C. Sartore et al.
leads to four, rather than two, blastomeres after the first
mitosis. Given that this unexpected fertilization pro-
cess was initiated with two centrosomes (two sperm
cells) and three chromosomal complements, Boveri
deduced that a quadripolar mitotic spindle might
have been generated which in turn segregated the 3n
genome into four unequal, or at least non-euploid,
fractions. Boveri suggested that aneuploid blastomeres
might have limited developmental potential relative to
euploid blastomeres.
By testing this hypothesis, he found that blas-
tomeres of dispermic eggs differed from each other
in their development. Dispermic-derived blastomeres
arrested during development at the blastula stage,
whereas those derived from normal eggs completed
development. Boveri based the explanation of this
phenomenon on the distribution of the chromosomes.
Furthermore, he suggested that the function of bipolar
mitotic figure is to multiply successively the nucleus in
its totality and transfer of the qualities present in one
nucleus into many nuclei (Boveri, 1902). From this,
Boveri postulated that not only the number but also
the quality of the chromosomes was crucial for nor-
mal development, calling attention to the importance of
maintaining genomic integrity during cell proliferation
(Manchester, 1995; Satzinger, 2008).
Soon after, Boveri identified a correlation between
aneuploidy and tumor formation (Nyberg et al., 2002).
Chromosomal aberrations, including aneuploidy, are
hallmark of many types of cancer. However, it is still
debated whether aneuploidy could be a determinant in
cellular transformation (Rasnick and Duesberg, 1999;
Duesberg et al., 2005) or whether it could even con-
tribute to tumor avoidance (Weaver et al., 2007; Torres
et al., 2008). Indeed, aneuploidy on primary mouse
cells impairs proliferation and altered metabolic prop-
erties. Immortalization, the acquisition of the ability
to proliferate indefinitely, was also abrogated by the
presence of an additional copy of certain chromosomes
(Williams et al., 2008).
6.3 Cell Cycle Checkpoints Maintain
Genome Integrity
In order to generate two daughter cells with identi-
cal genomes and equal size relative to the original
mother cell, a proliferating cell must coordinate
growth with genome duplication and genome
6 Aneuploidy in Embryonic Stem Cells
segregation. Over the course of a cell cycle, genome
duplication is performed during the S (synthesis) phase
and genome segregation occurs during the M (mitosis)
phase. These two phases are separated by two gaps of
variable lengths of time, named G1, located between
the M and S phases, and G2, located between the S
and M phases. These gap phases allow cell growth and
are monitored by surveillance mechanisms known as
checkpoints.
Checkpoints control major transitions in the cell
cycle, verifying that all processes related to each
phase have been appropriately completed before the
next phase is initiated. Furthermore, they function
to ensure that genome integrity is maintained, as
well as to delay crucial events like DNA synthe-
sis or chromosomal segregation into daughter cells
in response to abnormal conditions. For example,
upon detection of DNA damage, cells arrest cell
cycle progression in G1 or G2 phases (Lew and
Burke, 2003; Nyberg et al., 2002). In order to pre-
vent aneuploidy, the spindle assembly checkpoint
(SAC) delays anaphase until all sister chromatids
have been attached to mitotic spindle microtubules,
thus providing time for repairing errors and avoid-
ing chromosomal aberrations (Lew and Burke, 2003).
In the presence of unattached chromosomes to the
mitotic spindle, anaphase is inhibited by kinetochore-
associated mitotic checkpoint machinery including
Bub1, Bub3, BubR1, Mad1, Mad2, Mad3, Mps1 and
CENP-E. More specifically, this accumulation inhibits
the anaphase promoting complex-cyclosome (APC/C),
whose activity is required for securin degradation
and anaphase progression (Musacchio and Salmon,
2007).
Fig. 6.1 Simple
representation of a typical
cell cycle and checkpoints
75
Cell cycle transitions are driven by cyclin-
dependent kinase activity (CDK); checkpoints inhibit
Cdk activity to prevent cell cycle progression. Cdk
complexes are comprised of an enzymatic subunit, the
Cdk core, and an activating subunit called cyclin. The
molecular machinery that promotes and inhibits cellu-
lar proliferation includes fluctuations between cyclin
synthesis and degradation, deactivating phosphoryla-
tion and activating dephosphorylation of cyclin-Cdk
complexes and the presence of cyclin-Cdk inhibitory
proteins, called cyclin kinase inhibitors (CKI), such
as p16, p18, p21 and p27 (van den Heuvel, 2005)
(Fig. 6.1).
Different cyclin-Cdk complexes are formed and
activated in each phase of the cell cycle. In response
to mitogenic and growth factors in early G1 phase,
cyclin D is expressed and its gene product forms a
complex with Cdk4 and Cdk6 to promote G1 phase
progression. The G1/S transition is regulated by the
retinoblastoma tumor suppressor protein (pRb). E2F-
DP-dependent transcription promotes entry into S
phase, and hypophosphorylated Rb represses the E2F-
DP family of transcription factors. Active cyclin D-
Cdk4/6 phosphorylates pRb to allow the transcription
of cyclin E. Cyclin E-Cdk2, in turn, hyperphosphory-
lates pRb, leading to its complete dissociation from
E2F-DP and the derepression of S phase promoting
genes, notably cyclin A. The cyclin A-Cdk2 complex
sustains S phase progression and cyclin A-Cdk1 will
be required later on during the G2 phase. In order to
promote mitosis, cyclin B-Cdk1 is activated and trig-
gers a variety of processes that peak with cytokinesis
(Schwartz and Shah, 2005; Rane and Reddy, 2000)
(Fig. 6.1).
76
In mammalian somatic cells, the fidelity of chro-
mosome transmission is secured by checkpoints, and
when there is an ablation in checkpoint functions, ane-
uploidy can arise (Weaver et al., 2007).Although it
is assumed that all normal mammalian cells maintain
genomic integrity through these control mechanisms,
compelling evidence shows that this is not a common
feature in embryonic stem (ES) cells (Neganova and
Lako, 2008).
6.4 Increased Levels of Aneuploidy
Indicates Reduced Checkpoint
Fidelity in Stem/Progenitor Cells
Despite robust checkpoint control described in sev-
eral cell types, aneuploidy is still observed in certain
stem/progenitor cell populations. Specifically, ~35%
of mouse neural progenitor cells (NPC) are aneuploid
(Rehen et al., 2001). These aneuploid NPC give rise to
aneuploid neurons in mouse and in human (Kingsbury
et al., 2005; Rehen et al., 2005) (Fig. 6.2). Aneuploidy,
Fig. 6.2 Aneuploid neurons are present in the brains of
adult mice. Cells from the cerebral cortex of a male mouse (box
in bottom image) can be hybridized with chromosome paints
(top image) to show variations in the chromosome number (red,
X chromosome; green, Y chromosome; blue, DAPI stain). Note
that the cell on the left has one Y chromosome but two X
chromosomes while the other two cells have a single X and Y
chromosome. Provided courtesy of Marcy Kingsbury
R.C. Sartore et al.
mostly hypoploidy, in normal mouse NPC results from
chromosome missegregation and multipolar spindles
are readily observed in these cells (Rehen et al., 2001;
Yang et al., 2003). Although the exact mechanisms by
which NPC cell cycles permit aneuploidy is not yet
clear, it is likely related to the suspension of the decate-
nation checkpoint as observed in these cells and also
ES cells (Damelin et al., 2005).
In early development, the strict requirements for cell
growth are suspended to allow rapid cell proliferation.
Rapid cell division leads to an unusual cell cycle dis-
tribution: more than 50% of the ES cell populations
observed are found in S phase, whereas smaller frac-
tions (~25%) are found in G1 or in G2/M phases (Stead
et al., 2002; Fluckiger et al., 2006). It is important to
note that early NPC cell cycles are also very rapid
(~8 h), followed by a progressive lengthening of G1
phase to ~24 h by the end of cortical neurogenesis
(Miyama, Cerebral Cortex, 1997).
Rapid ES cell cycle is achieved through the irregu-
lar use of cell cycle control molecules (e.g. cyclins and
CDK) (Fluckiger et al., 2006). ES cells derived from
mice and primates exhibit elevated levels of cyclin E
and cyclin A throughout the whole cell cycle, whereas
the functions of these cyclins are typically restricted
to G1 or S phases (Sancar et al., 2004). Mouse ES
cells also show high Cdk2 activity in a cell cycle-
independent manner, which can be explained in part
by the elevated expressions of cyclin E and cyclin A.
Likewise, the Cdk inhibitors p21 and p27 are almost
undetectable, hence favoring the absence of Cdk2 reg-
ulation throughout the cell cycle (Linke et al., 1996;
Stead et al., 2002) (Fig. 6.3).
Constitutive expression of cyclin E results in kary-
otypic instability in mammalian cells (Spruck et al.,
1999). Similarly, high levels of cyclin E are correlated
to breast, endometrial and skin cancers and also with
aneuploidization (Dutta et al., 1995; Keyomarsi et al.,
1995; Hubalek et al., 2004; Bito et al., 1997).
In addition to cyclins, the Rb protein, whose phos-
phorylation leads to S phase progression, is mainly
found hyperphosphorylated in undifferentiated primate
ES cells (Fluckiger et al., 2006). Moreover, down-
stream E2F-DP target genes, such as B-myb, RRMP2
and cdc2, are continuously transcribed throughout the
ES cell cycle (Stead et al., 2002). Together, consti-
tutive cyclin A/E-Cdk2 activity and a silenced pRb
control provide rapid ES cell division (Stead et al.,
2002; Fluckiger et al., 2006) (Fig. 6.3).
6 Aneuploidy in Embryonic Stem Cells
Fig. 6.3 Embryonic cell
cycle representation
composed basically by M
and S phases. Note
constitutive activation of
cyclin-Cdk complexes typical
of G1/S and S phases and the
predominance of the
hyperphosphorylated form of
pRb. ES cells are refractory to
CKI inhibition (p16, p21,
p27)
The Rb pathway is often inactivated in cancer.
Interestingly, Rb disruption generates chromosomal
instability by destabilizing Mad2 expression, a com-
ponent of SAC. Ablation of Rb function produces
deregulated E2F-DP activity and consequently the
overexpression of Mad2, an E2F-DP target gene, thus
leading to uncontrolled mitosis and finally resulting in
aneuploidy (Hernando et al., 2004).
It was initially thought that cyclin D-associated
activities were absent in ES cells (Savatier et al.,
1996). However, subsequent studies have shown that
cyclin D1 and cyclin D3 are expressed, but at low
levels in murine ES cells. On the other hand, high
activity of cyclin D3-Cdk6 is present, but this com-
plex is not regulated by p16 (Faast et al., 2004)
(Fig. 6.3).
When ES cells are induced to differentiate into
embryoid bodies (EB), G1 phase is lengthened con-
comitantly with the loss of pluripotency markers and
increased levels of differentiation markers (White
et al., 2005). At the same time, the high activity of
Cdk2, cyclin A and cyclin E, whose activities are
cell cycle-independent in ES cells, are reduced and
become cell cycle-regulated upon differentiation. It
is underpinned by elevated levels of p21 and p27
CKIs and the establishment of cell cycle regulated
pRb-E2F activity (White et al., 2005). ES cells also
become sensitive to p16 activity (Savatier et al.,
1996).
Pluripotent cells may shorten their G1 phase and
reject their delaying components to preserve their
pluripotency properties. In line with this idea, it has
been observed that P19 embryonal carcinoma cells are
77
particularly prone to differentiate between the M and S
phases, that is, during G1 phase, and become less sus-
ceptible to differentiate upon S phase (Mummery et al.,
1987a).
Taken together, the emerging data point out that
ES cells guarantee rapid cell division by bypassing
certain mechanisms of cell cycle control and they
are primed to comprise of a lengthened S phase and
shortened gap phases. In other words, they are inces-
santly ready to replicate. Like ES cells, other pluripo-
tent cell types also exhibit an extensive proportion
of cells in S phase. This characteristic is common
to some embryonic carcinoma (EC) cells (Mummery
et al., 1987b; Kranenburg et al., 1995), embryonic
germ cells (Resnick et al., 1992) and embryonic epi-
blast cells (Mac Auley et al., 1993), and is postu-
lated to be a property of stemness (Fluckiger et al.,
2006).
6.5 DNA Damage Signaling and
Aneuploidy
A striking difference has been demonstrated between
somatic and embryonic stem cells: Checkpoints con-
trolling the different phases transitions are neglected
in ES cells.
Cells have a normal load of DNA damage that is
managed by cell cycle checkpoints. In the presence
of DNA damage, entry into S phase is prevented by
the G1/S checkpoint that arrests cell cycle progression
78
at the G1 phase. The checkpoint response is initiated
when the DNA-damage response kinase ATM senses
DNA damage and phosphorylates target molecules
including Chk1/2 and p53. Activated Chk1/2 phospho-
rylates Cdc25A phosphatase, targeting it for ubiquitin-
dependent degradation. In turn, the phosphorylated
form of Cdk2 (inactive) accumulates, caused by the
lack of Cdc25A activity and thus, is unable to trig-
ger DNA replication. The phosphorylation of p53
obstructs its nuclear export and degradation, leading
to its accumulation in the nucleus and the activation of
target genes, notably p21. The Cdk inhibitor, p21, ulti-
mately blocks cell proliferation by inhibiting the cyclin
E-Cdk2 complex, maintaining G1 arrest (Sancar et al.,
2004).
Another signal that promotes G1 arrest and is inde-
pendent of DNA damage occurs by the depletion of
cellular deoxyribonucleotide triphosphates (dNTP). In
this condition, somatic cells inhibit DNA synthesis via
p53 and p21 activities and prevent defects during cell
division (Linke et al., 1996).
In this scenario, the shortage of nucleotides does
not elicited cell cycle arrest in ES cells and they
continue to enter into S phase, even in such a subop-
timal growth condition (Hong and Stambrook, 2004;
Aladjem et al., 1998). Also, when submitted to DNA
lesions, ES cells enter into S phase in a propor-
tion similar to cells that have not been exposed to
any DNA challenges and no accumulation of cells
in G1 or G2 phases is observed (Aladjem et al.,
1998).
The inability of ES cells to undergo cell cycle
arrest stimulated by either dNTP depletion or DNA
damage infers that ES cells do not present a robust p53-
mediated DNA-damage-response pathway (Aladjem et
al., 1998). ES cells do express the p53 protein however,
because it is inefficiently translocated into the nucleus
after DNA damage, it remains predominantly cytoplas-
mic. This is consistent with the undetectable levels of
p21 in these situations. The occurrence of apoptosis
through other p53 independent pathways, confirm that
ES cells have an inefficient p53 response (Aladjem
et al., 1998).
A parallel signaling pathway involving ATM, Chk2,
Cdc25A and Cdk2 is also shown to be corrupted in
ES cells. This is due to the restricted localization
of Chk2 to the centrosomes, rendering it incapable
of phosphorylating its substrates, primarily Cdc25A,
R.C. Sartore et al.
which is responsible for inducing G1 arrest (Hong and
Stambrook, 2004).
Another event that indicates the uncoupling of
checkpoint-apoptosis in ES cells can be observed
by testing the SAC and the checkpoint induced by
DNA double-strand breaks. The treatment of mouse
and human ES cells with drugs that promote micro-
tubule disruption and SAC activation do not hinder
them from achieving mitosis. While prolonged expo-
sure of normal somatic cells to microtubule disrupting
agents induces apoptosis or senescence, embryonic
stem cells bypass mitotic delay and complete cytokine-
sis, resulting in polyploidy/aneuploidy cells which do
not elicit apoptosis. Similarly, mouse ES cells exposed
to double-strands breaks become both aneuploid and
resistant to apoptosis (Mantel et al., 2007). Therefore,
ES cells are allowed to enter into an aneuploid cell
cycle.
In 2005, Damelin suggested that deficiency in
checkpoints could be a feature of progenitor cells. In
his work, the decatenation checkpoint efficiency was
tested by inhibiting topoisomerase II on mouse ES
cells, mouse NPC and human haematopoietic progeni-
tor cells. The decatenation checkpoint delays the entry
into mitosis during the G2 phase if any chromosome
has not yet been appropriately disentangled by topoi-
somerase II (Damelin and Bestor, 2007). Stem and
progenitor cells are observed to enter mitosis even
in the presence of entangled chromosomes, reflecting
a silenced decatenation checkpoint control, possibly
leading to aneuploid daughter cells (Damelin et al.,
2005).
Hence, ES cells have inefficient checkpoints that
allow them to progress through cell cycle even under
abnormal conditions and enable them to escape apop-
tosis. However, as they differentiate into EB, the
checkpoints are restored and aneuploid cells initiate
robust apoptosis, consistent with other somatic cells.
SAC and DNA-damage checkpoint activation then
become coupled to apoptosis (Mantel et al., 2007)
and differentiation of ES cells is accompanied by an
increased efficiency of the decatenation checkpoint
(Damelin et al., 2005). Thus, emerging data suggests
that the switch from aneuploidy tolerance/survival to
aneuploidy intolerance/elimination is discerned upon
differentiation (Mantel et al., 2007) and that check-
point inefficiency could be a feature of the undiffer-
entiated state.
6 Aneuploidy in Embryonic Stem Cells
6.6 Does Aneuploidy in Stem and/or
Progenitor Cells Have Consequences
for Development and Disease?
Aneuploidy in ES cells and NPC might reflect the
necessity of obtaining high quantities of cells dur-
ing early stages of development; another non-mutually
exclusive possibility is that aneuploidy plays an impor-
tant role in normal development. Towards the latter, it
has been proposed that genetically dissimilar cells are
essential during normal development to influence mor-
phogen diffusion and gradient formation (Mantel et al.,
2007; Gurdon and Bourillot, 2001). Only after the
acquisition of a sufficient number of cells the embryo
can eliminate aberrant cells without interfering with
the overall patterning (Mantel et al., 2007).
This idea is supported by studies carried out in
developing embryos generated by in vitro fertilization
(IVF) and assessed by fluorescence in situ hybridiza-
tion (FISH) technique. It revealed a high frequency
of chromosomal mosaicism in normal human preim-
plantation embryos. However, when comparing early
developmental stages with embryos in the blastocyst
stage, it was found that embryos undergo a natural
selection of euploid cells, resulting in a decreased num-
ber of aneuploid cells throughout the blastocyst stage
(Frumkin et al., 2008; Gonzalez-Merino et al., 2003).
Cell biologic mechanisms managed to rescue ane-
uploidy at conception have been observed (Kalousek
and Vekemans, 1996). Trisomic zygotic rescue can
occur when chromosome missegregation generates
aneuploid ES cells. Here, an euploid cell can arise from
a trisomic cell through the loss of the additional chro-
mosome (i.e. trisomic rescue). However, depending
on the lost chromosome, the embryo can remain with
chromosomes of both parental origin (i.e. biparental
disomy) or of only one parental origin (i.e. uniparental
disomy). In these instances, aneuploid ES cells are
confined, in an unspecified way, to the placenta while
the euploid cell line gives rise to the embryo proper
(Kalousek and Dill, 1983; Kalousek, 2000). Thus,
early development has certain mechanisms for distin-
guishing between aneuploid and euploid cells.
Chromosomal abnormalities affecting either one
or a few blastomeres during cleavage manifest as a
mosaic pattern in the developing embryo, in which a
fraction of cells is euploid and the remaining ones are
79
aneuploid (Ambartsumyan and Clark, 2008). Mosaic
Variegated Aneuploidy (MVA) is a detrimental exam-
ple of mosaicism whereby chromosome missegrega-
tion occurs throughout development. MVA (Kajii et al.,
2001; Jacquemont et al., 2002) and several cases of
related syndromes (Hunter, 2003) have been reported
in at least 15 individuals and are recently linked to a
genetic deficit in the SAC protein BubR1 (Kajii et al.,
2001; Ikeuchi et al., 2004).
Similar phenotypes have also been observed for
hereditary deficits that compromise DNA damage sig-
naling (Rolig and McKinnon, 2000; Baker et al., 2007)
and may point out to a common cellular mechanism
that controls the generation and survival of aneuploid
cells during both tumorigenesis and neurogenesis.
Although aneuploidy at fertilization can account for
~35% of spontaneous abortions, this is not always nec-
essarily lethal (Hassold et al., 1980; Griffin, 1996).
Human development is attainable with the gain or loss
of certain chromosomes (e.g. Chromosomes 21, X, Y).
Given that there is a certain level of aneuploidy in the
healthy human brain, it is worth noting that individuals
with trisomy 21 (Downs syndrome) and sex chromo-
some aneuploidies (Klinefelters Syndrome, Turners
Syndrome) frequently present neurological symptoms.
In addition to mental retardation, older Downs syn-
drome patients invariably develop Alzheimers disease
(AD) (Potter, 1991). The amyloid precursor protein
gene, linked to the early onset of AD, is located on
chromosome 21. Thus, an increased gene dosage due
to trisomy 21 might predispose Downs syndrome
patients to AD (Li et al., 1997; Geller and Potter,
1999).
When chromosome missegregation occurs later in
development, both cell types can contribute to the
embryo and mosaic individuals are born. In Downs
syndrome individuals, when development proceeds
from an aneuploid zygote, a beneficial consequence
of mosaicism might be an increased prevalence of the
euploid cell line, which could lessen the severity of the
disease.
In the case of NPCs, loss of heterozygosity due
to aneuploidy can alter gene expression differently
in individual neurons (Kaushal et al., 2003). Such
mosaicism could modulate the structure and/or func-
tion of neural networks.
Despite its characteristic failure in checkpoint
functions, a p53-dependent suppression of nanog
80
expression has been suggested as a mechanism to
avoid genomic instability in ES cells (Lin et al., 2005).
Nanog is a protein exclusively expressed in ES cells
and is essential to maintain self-renewal and pluripo-
tency (Mitsui et al., 2003). It has been demonstrated
that after DNA damage, such as DNA double-strands
break, p53 binds the nanog promoter and represses its
expression to induce differentiation (Lin et al., 2005).
Consequently, efficient p53-dependent cell cycle arrest
and apoptosis can be restored, given that this is a
common phenomenon reached upon differentiation
(Mantel et al., 2007; Damelin et al., 2005). Thus, the
commitment of ES cells to differentiation can be a use-
ful strategy adopted by ES cells to circumvent inherited
genomic errors by subsequent generations.
Another possible strategy adopted by ES cells to
escape genotoxic stress is presented in both mouse
and human ES cells which, in contrast to differenti-
ated cells, are comprised of a higher repair capacity
(Saretzki et al., 2004; Maynard et al., 2008). When
compared to differentiated cells, enzymes that correct
DNA damage are found at elevated levels after damage
has been induced in ES cells, thus demonstrating that
these cells respond more dramatically to DNA stress
(Maynard et al., 2008). These data exemplify another
attempt of ES cells to attenuate genomic alterations
resulting from an inefficient checkpoint surveillance
system.
6.7 Aneuploidy and Cancer Stem Cells
As described in the beginning of this chapter, aneu-
ploidy is usually associated with tumorigenesis. The
idea that cancer arises from a small population of cells
that possess stem cell properties was first proposed 150
years ago, but it is only recently that this hypothesis is
able to be tested. In general, there are three possible
events that can lead to cancer: (1) Cancer arises from
the proliferation of normal cells that have acquired
DNA damage; (2) Cancer originates from mutant tis-
sue stem cells otherwise known as cancer stem cells
(CSC) that have the same properties of stem cells and
can escape from the aging process (Rajaraman et al.,
2006); and (3) through asymmetric division, can gen-
erate one CSC and one differentiated tissue-specific
cancer cell (Wicha et al., 2006).
R.C. Sartore et al.
In most of cases, cancer develops from CSC and
contains alterations such as chromosome transloca-
tions and aneuploidy. Boveris observation of an unde-
fined cell mass at the center of aneuploid blasto-
cysts, known today as a tumor, provided the foun-
dation, which has allowed researchers to later form
a correlation between aneuploidy and cancer. He
also noted that these aneuploid cells had a growth
advantage comparable to wild type cells, and that
this advantage could contribute to tumor progres-
sion (Boveri, 1902). However, one question remains
unclear: are tumor formations always accompanied by
aneuploidy?
In a recent study, Miuras group showed that the
continuous passage in vitro of bone marrow mes-
enchymal stem cells spontaneously transformed them
into malignant cells. This transformation was usually
accompanied by chromosomal abnormalities, includ-
ing chromosome gains (Miura et al., 2006).
On the other hand, many other groups provide evi-
dence that aneuploidy suppresses the formation of spe-
cific kinds of tumors. Individuals with trisomy 21 have
lower risks of developing solid tumors, but are more
prone to developing leukemias (Hasle et al., 2000;
Satge et al., 2003). Weaver also showed that a reduc-
tion in CENP-E leads to spontaneous tumorigenesis
during aging and enhances aneuploidy and transfor-
mation in culture. Contrary to this, aneuploidization
caused by the reduction of CENP-E in cells depleted
of the tumor suppressing protein p19/ARF had dimin-
ished tumor formation. Hence aneuploidy has been
shown to both promote and/or to block tumor forma-
tion (Weaver et al., 2007).
6.8 Telomeres and Telomerase Under
Genomic Stability Control
Telomeres are specialized structures at the end of
eukaryotic chromosomes and protect the ends against
chromosomal fusion, recombination and DNA degra-
dation. In most vertebrates, they are composed of
non-coding TTAGGG repetitive sequences and shorten
during each cell division due to the absence of telom-
erase and the end replication problem. Telomerase is
the enzyme responsible for the maintenance of telom-
eres, but are not present in most somatic cells. This fact
6 Aneuploidy in Embryonic Stem Cells
suggests that its activity is related to undifferentiated
and high proliferating cells such as embryonic stem
cells and cancer cells (Aubert and Lansdorp, 2008).
The concept that cancer cells are immortal comes
from the primary observations made by Gey`s group.
By culturing normal epithelial cells and cells taken
from an invasive carcinoma, they observed that cancer
cells could be cultured with continuous dividing poten-
tial, and then established the oldest carcinoma cell line,
the HeLa cells (Gey et al., 1952). About ten years later,
with improved media for culture and enhanced sterile
conditions, Hayflick published his study on embry-
onic lung fibroblasts, concluding that somatic cells
had a limited number of divisions later known as the
Hayflick limit (Hayflick and Moorhead, 1961). Human
somatic cells usually stop dividing before they reach
this limit, entering into an irreversible growth arrest.
Upon reaching this limit, telomeres may initiate a DNA
damage response through the activation of the p53- and
Rb-dependent checkpoints, leading to senescence and
the inhibition of tumorigenesis. However, when the
p53 and Rb checkpoint arrest pathways are corrupted,
the cells continue to proliferate, resulting in genetic
instability and the survival of cells with chromosomal
rearrangements and/or aneuploidy. This stage, termed
crisis, and its associated genomic instability, leads to
genetic chaos and cell death. Telomere stabilization
from exogenous telomerase or spontaneous expression
could rescue certain cells from crisis by bypassing
senescence, resulting in their immortalization (Akimov
et al., 2005; Bodnar et al., 1998).
Is there a link between telomeres and aneuploidy?
In fact, for many years, researchers have been focusing
on the way in which centromeres have been responsi-
ble for the correct running of cell division, and how one
simple failure could result in abnormal chromosome
segregation, resulting in aneuploidy. On the other hand,
other groups described the importance of telomeres for
the generation of aneuploid cells. The answer lies sim-
ply on the fact that telomeres maintain the integrity of
chromosome ends.
Plentz showed, in a recent work, a link between
telomere shortening and aneuploidy (Plentz et al.,
2005). While in the process of studying mice with
deficient telomerase production, Blasco confirmed that
telomeres were shorter and shorter in each consec-
utive generation. Although these telomerase-deficient
mice were viable for about six generations, many alter-
ations such as chromosomal abnormalities, end-to-end
81
fusions, complete absence of telomeric repeats, as well
as aneuploidy could be observed (Blasco et al., 1997).
Telomeres are involved in almost all types of chro-
mosomal alterations. It is most likely for this rea-
son that Pathak asked the question Centromere or
telomere: who is the boss?, and many groups now
are trying to answer it. For many years, centromeres
have been considered as essential actors of the cell-
division machinery. Today, most groups are consider-
ing the hypothesis that telomere disruption followed
by endomitosis and centrosome amplification, is in
fact the first event in aneuploidization (Pathak, 1995;
Pathak et al., 2002).
6.9 Aneuploidy and Cell-Based Therapy
As recombinant DNA revolutionized drug develop-
ment in the 1990s, cell-based therapeutic technologies
are now poised to drive biotechnology through the next
decade. Perhaps the biggest challenge in advancing
this field would involve the ability to culture embry-
onic stem cells without changing its basic characteris-
tics. Despite the fact that human and mouse ES cells
have been shown to be diploid with normal karyotype
(Thomson et al., 1998; Evans and Kaufman, 1981),
many studies are now reporting that when human
and/or mouse ES cells are cultured in vitro for pro-
longed periods of time, they become susceptible to
genomic alterations such as aneuploidy (Maitra et al.,
2005; Baker et al., 2007).
6.9.1 Mechanical Versus Enzymatic
Methods
There is an interest to optimize ES cell culturing pro-
cedures because of their limitless therapeutic potential
in degenerative diseases. The use of an enzyme was
the first method described for expanding cells in vitro
(Grover, 1961). This technique results in single-cell
dissociation, but this fact may contribute to the gen-
eration of chromosomal alterations. Trypsin is one of
the most common enzyme used to pass cells in cul-
ture (Fig. 6.4). Even though trypsin is used extensively,
many groups are now finding traces suggesting that this
82 R.C. Sartore et al.

Fig. 6.4 Schematic model

of embryonic stem cell
culture. (a) Embryonic stem
cells plated on inactivated
murine embryonic fibroblasts
(mEF) and dissociated by
enzymatic method resulting in
chromosome alterations
during culture. (b)
Maintenance of pluripotency
and chromosome integrity by
mechanical dissociation

passage practice should be monitored. Chans group
showed that when human ES cells are cultured for long
periods of time with the use of trypsin, the cells begin
to acquire chromosomal alterations, most of which
result in trisomy 12/17 (Chan et al., 2008; Gertow
et al., 2007). Although, Thomsons group showed that
human ES cells can be cultured using trypsin for
long periods of time without affecting their karyotype
(Thomson et al., 2008).
In his recent work, Rebuzzinis group monitored a
mouse ES cell line, called UPV04, during 40 passages
while using trypsin. They observed that at passage 6,
100% of cells in metaphase had the correct number
of chromosomes (2n=40) and the correct chromosome
complement. At passage 34, 65% of the cells had the
correct chromosome number but only 50% of them had
the correct chromosome complement. Even with such
chromosomal alterations, the cells presented markers
indicative of an undifferentiated state such as OCT-
4, SSEA-1 and FOM-1 antigens as well as alkaline
phosphatase activity (Rebuzzini et al., 2008).
Sugawaras group analyzed the karyotype of mouse
ES cells lines obtained in Japan, and showed that
abnormal karyotype occurred in 35 out of the 88 mouse
ES cells lines analyzed. They also observed that tri-
somy 11 was related to the acceleration of growth, and
hence cell growth advantage (Sugawara et al., 2006).
This fact correlates to a previous study that relates
higher growth rates with trissomy 8, and in relation to
the differentiation potential of these abnormal cells, no
alteration was observed (Liu et al., 1997; Park et al.,
1998).
Catalinas group showed in a recent study that both
manual and enzymatic methods (Fig. 6.4) for dissociat-
ing human ES maintained a stable karyotype for up to
180 passages in feeders. Surprisingly, when these same
cells were submitted to a non-feeder culture condition
and cultured for up to 30 passages, only one out of
the three lineages studied remained unaltered (Catalina
et al., 2008).
Altogether, these data suggest a straightforward
technique such as cell passage, needs to be highly mon-
itored to ensure chromosomal stability and to assure
the future application of ES cells in therapy and drug
screening.
6.9.2 Risks and Benefits of Aneuploidy to
Cell-Based Therapies
As aneuploidy has been shown to be a common event
in preimplantation embryos, as well as an ordinary
condition in ES cell culture, yet at the same time, it
has been negatively linked to cancer. Thus, a lingering
question remains to be clarified: could aneuploid cells
be compatible for cell-based therapy?
6 Aneuploidy in Embryonic Stem Cells
Monitoring ES cell karyotypes has been deemed
essential in making their application feasible for ther-
apy. Cells with an abnormal number of chromosomes,
usually gain, could present a threat by causing uncon-
trolled proliferation and tumor formation (Zuber et al.,
2002; Liu et al., 1997).
However, contrary to the presumed concept that
aneuploidy is a sign of cellular transformation, high
levels of aneuploidy have been suggested to increase
cell death and suppress tumor development (Weaver
et al., 2003, 2008). Moreover, defined aneuploidy may
be used as the determinant in driving the differentia-
tion process, e.g. trisomy 12 prompts human ES cells
towards the renal phenotype (Gertow et al., 2007). In
this context, the persistence of aneuploid cells within
the neural circuitry of normal brains suggests that some
types of aneuploid cells might further be harmless.
Further research is necessary to determine when and
how aneuploidy can be considered a real barrier to
ES cell-based therapies and whether particular cases of
defined aneuploidies could even be used as a strategy
for obtaining a cell type of interest.
Acknowledgements We thank Stacie Ngo Abdalla for
manuscript editing. This work was supported by grants from
Faperj Fundao Carlos Chagas Filho de Amparo Pesquisa
do Estado do Rio de Janeiro (S.R.), CNPq Conselho Nacional
para o Desenvolvimento Cientfico e Tecnolgico (R.S., P.B.,
S.R.), Pew Latin American Program in Biomedical Sciences
(S.R.) and Brazilian Ministry of Health/DECIT (S.R.).
References
Akimov SS, Ramezani A, Hawley TS, Hawley RG (2005)
Bypass of senescence, immortalization, and transforma-
tion of human hematopoietic progenitor cells. Stem Cells
23:14231433.
Aladjem MI, Spike BT, Rodewald LW, Hope TJ, Klemm M,
Jaenisch R, Wahl GM (1998) ES cells do not activate p53-
dependent stress responses and undergo p53-independent
apoptosis in response to DNA damage. Curr Biol 8:145155.
Ambartsumyan G, Clark AT (2008) Aneuploidy and early
human embryo development. Hum Mol Genet 17:R10R15.
Aubert G, Lansdorp PM (2008) Telomeres and aging. Physiol
Rev 88:557579.
Baker DE, Harrison NJ, Maltby E, Smith K, Moore HD, Shaw
PJ, Heath PR, Holden H, Andrews PW (2007) Adaptation to
culture of human embryonic stem cells and oncogenesis in
vivo. Nat Biotechnol 25:207215.
Bito T, Ueda M, Ito A, Ichihashi M (1997) Less expression
of cyclin E in cutaneous squamous cell carcinomas than
83
in benign and premalignant keratinocytic lesions. J Cutan
Pathol 24:305308.
Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM,
DePinho RA, Greider CW (1997) Telomere shortening and
tumor formation by mouse cells lacking telomerase RNA.
Cell 91:2534.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin
GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998)
Extension of life-span by introduction of telomerase into
normal human cells. Science 279:349352.
Boveri T (1902) ber mehrpolige Mitosen als Mittel zur
Analyse des Zellkerns. Verhandlungen der physikalisch-
medizinischen Gesellschaft zu Wrzburg 35:3790.
Catalina P, Montes R, Ligero G, Sanchez L, Cueva TD, Bueno C,
Leone PE, Menendez P (2008) Human ESCs predisposition
to karyotypic instability: Is a matter of cell culture adapta-
tion or differential vulnerability among hESC lines due to
inherent properties? Mol Cancer 7:7676.
Chan EM, Yates F, Boyer LF, Schlaeger TM, Daley GQ (2008)
Enhanced plating efficiency of trypsin-adapted human
embryonic stem cells is reversible and independent of tri-
somy 12/17. Cloning Stem Cells 10:107118.
Damelin M, Bestor TH (2007) The decatenation checkpoint. Br
J Cancer 96:201205.
Damelin M, Sun YE, Sodja VB, Bestor TH (2005) Decatenation
checkpoint deficiency in stem and progenitor cells. Cancer
Cell 8:479484.
Duesberg P, Li R, Fabarius A, Hehlmann R (2005) The chromo-
somal basis of cancer. Cell Oncol 27:293318.
Dutta A, Chandra R, Leiter LM, Lester S (1995) Cyclins
as markers of tumor proliferation: immunocytochemical
studies in breast cancer. Proc Natl Acad Sci USA 92:
53865390.
Evans MJ, Kaufman MH (1981) Establishment in culture
of pluripotential cells from mouse embryos. Nature 292:
154156.
Faast R, White J, Cartwright P, Crocker L, Sarcevic B, Dalton S
(2004) Cdk6-cyclin D3 activity in murine ES cells is resistant
to inhibition by p16(INK4a). Oncogene 23:491502.
Fluckiger AC, Marcy G, Marchand M, Negre D, Cosset FL,
Mitalipov S, Wolf D, Savatier P, Dehay C (2006) Cell
cycle features of primate embryonic stem cells. Stem Cells
24:547556.
Frumkin T, Malcov M, Yaron Y, Ben-Yosef D (2008) Elucidating
the origin of chromosomal aberrations in IVF embryos
by preimplantation genetic analysis. Mol Cell Endocrinol
282:112119.
Geller LN, Potter H (1999) Chromosome missegregation and tri-
somy 21 mosaicism in Alzheimers disease. Neurobiol Dis
6:167179.
Gertow K, Cedervall J, Unger C, Szoke K, Blennow E, Imreh
MP, Ahrlund-Richter L (2007) Trisomy 12 in HESC leads
to no selective in vivo growth advantage in teratomas, but
induces an increased abundance of renal development. J Cell
Biochem 100:15181525.
Gey GO, Coffman W, Kubicek MT (1952) Tissue culture studies
of the growth properties of cervical carcinoma and normal
epithelium. Cancer Res 12:264265.
Gonzalez-Merino E, Emiliani S, Vassart G, Van den Bergh M,
Vannin AS, Abramowicz M, Delneste D, Englert Y (2003)
Incidence of chromosomal mosaicism in human embryos at
84
different developmental stages analyzed by fluorescence in
situ hybridization. Genet Test 7:8595.
Griffin DK (1996) The incidence, origin, and etiology of aneu-
ploidy. Int Rev Cytol 167:263296.
Grover JW (1961) The enzymatic dissociation and reproducible
reaggregation in vitro of 11-day embryonic chick lung. Dev
Biol 3:555568.
Gurdon JB, Bourillot PY (2001) Morphogen gradient interpreta-
tion. Nature 413:797803.
Hasle H, Clemmensen IH, Mikkelsen M (2000) Risks of
leukaemia and solid tumours in individuals with Downs
syndrome. Lancet 355:165169.
Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura
J, Matsuyama A, Wilson C, Yamane JA, Jacobs PA (1980) A
cytogenetic study of 1000 spontaneous abortions. Ann Hum
Genet 44:151178.
Hayflick L, Moorhead PS (1961) The serial cultivation of human
diploid cell strains. Exp Cell Res 25:585621.
Hernando E, Nahl Z, Juan G, Diaz-Rodriguez E, Alaminos
M, Hemann M, Michel L, Mittal V, Gerald W, Benezra R,
Lowe SW, Cordon-Cardo C (2004) Rb inactivation promotes
genomic instability by uncoupling cell cycle progression
from mitotic control. Nature 430:797802.
Hong Y, Stambrook PJ (2004) Restoration of an absent G1
arrest and protection from apoptosis in embryonic stem
cells after ionizing radiation. Proc Natl Acad Sci USA 101:
1444314448.
Hubalek MM, Widschwendter A, Erdel M, Gschwendtner A,
Fiegl HM, Mller HM, Goebel G, Mueller-Holzner E, Marth
C, Spruck CH, Reed SI, Widschwendter M (2004) Cyclin
E dysregulation and chromosomal instability in endometrial
cancer. Oncogene 23:41874192.
Hunter A (2003) High risk of malignancy in mosaic variegated
aneuploidy syndrome. Am J Med Genet 117A:199199.
Ikeuchi T, Yang ZQ, Wakamatsu K, Kajii T (2004) Induction
of premature chromatid separation (PCS) in individuals
with PCS trait and in normal controls. Am J Med Genet
127A:128132.
Jacquemont S, Boceno M, Rival JM, Mechinaud F, David A
(2002) High risk of malignancy in mosaic variegated ane-
uploidy syndrome. Am J Med Genet 109:1721.
Kajii T, Ikeuchi T, Yang ZQ, Nakamura Y, Tsuji Y, Yokomori
K, Kawamura M, Fukuda S, Horita S, Asamoto A (2001)
Cancer-prone syndrome of mosaic variegated aneuploidy and
total premature chromatid separation: report of five infants.
Am J Med Genet 104:5764.
Kalousek DK (2000) Pathogenesis of chromosomal mosaicism
and its effect on early human development. Am J Med Genet
91:3945.
Kalousek DK, Dill FJ (1983) Chromosomal mosaicism con-
fined to the placenta in human conceptions. Science 221:
665667.
Kalousek DK, Vekemans M (1996) Confined placental
mosaicism. J Med Genet 33:529533.
Kaushal D, Contos JJ, Treuner K, Yang AH, Kingsbury MA,
Rehen SK, McConnell MJ, Okabe M, Barlow C, Chun J
(2003) Alteration of gene expression by chromosome loss in
the postnatal mouse brain. J Neurosci 23:55995606.
Keyomarsi K, Conte D Jr, Toyofuku W, Fox MP (1995)
Deregulation of cyclin E in breast cancer. Oncogene 11:
941950.
R.C. Sartore et al.
Kingsbury MA, Friedman B, McConnell MJ, Rehen SK, Yang
AH, Kaushal D, Chun J (2005) Aneuploid neurons are func-
tionally active and integrated into brain circuitry. Proc Natl
Acad Sci USA 102:61436147.
Kranenburg O, de Groot RP, Van der Eb AJ, Zantema A (1995)
Differentiation of P19 EC cells leads to differential modula-
tion of cyclin-dependent kinase activities and to changes in
the cell cycle profile. Oncogene 10:8795.
Lew DJ, Burke DJ (2003) The spindle assembly and spindle
position checkpoints. Annu Rev Genet 37:251282.
Li J, Xu M, Zhou H, Ma J, Potter H (1997) Alzheimer prese-
nilins in the nuclear membrane, interphase kinetochores, and
centrosomes suggest a role in chromosome segregation. Cell
90:917927.
Lin T, Chao C, Saito S, Mazur SJ, Murphy ME, Appella E, Xu
Y (2005) p53 induces differentiation of mouse embryonic
stem cells by suppressing Nanog expression. Nat Cell Biol 7:
165171.
Linke SP, Clarkin KC, Di Leonardo A, Tsou A, Wahl GM (1996)
A reversible, p53-dependent G0/G1 cell cycle arrest induced
by ribonucleotide depletion in the absence of detectable
DNA damage. Genes Dev 10:934947.
Liu X, Wu H, Loring J, Hormuzdi S, Disteche CM, Bornstein
P, Jaenisch R (1997) Trisomy eight in ES cells is a common
potential problem in gene targeting and interferes with germ
line transmission. Dev Dyn 209:8591.
Mac Auley A, Werb Z, Mirkes PE (1993) Characterization
of the unusually rapid cell cycles during rat gastrulation.
Development 117:873883.
Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V,
Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN, Noaksson
K, Hyllner J, Schulz TC, Zeng X, Freed WJ, Crook J,
Abraham S, Colman A, Sartipy P, Matsui S, Carpenter M,
Gazdar AF, Rao M, Chakravarti A (2005) Genomic alter-
ations in cultured human embryonic stem cells. Nat Genet
37:10991103.
Manchester KL (1995) Theodor Boveri and the origin of malig-
nant tumours. Trends Cell Biol 5:384387.
Mantel C, Guo Y, Lee MR, Kim MK, Han MK, Shibayama
H, Fukuda S, Yoder MC, Pelus LM, Kim KS, Broxmeyer
HE (2007) Checkpoint-apoptosis uncoupling in human and
mouse embryonic stem cells: a source of karyotpic instabil-
ity. Blood 109:45184527.
Maynard S, Swistowska AM, Lee JW, Liu Y, Liu ST, Da
Cruz AB, Rao M, de Souza-Pinto NC, Zeng X, Bohr
VA (2008) Human embryonic stem cells have enhanced
repair of multiple forms of DNA damage. Stem Cells 26:
22662274.
Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M,
Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003)
The homeoprotein Nanog is required for maintenance of
pluripotency in mouse epiblast and ES cells. Cell 113:
631642.
Miura M, Miura Y, Padilla-Nash HM, Molinolo AA, Fu B,
Patel V, Seo BM, Sonoyama W, Zheng JJ, Baker CC,
Chen W, Ried T, Shi S (2006) Accumulated chromoso-
mal instability in murine bone marrow mesenchymal stem
cells leads to malignant transformation. Stem Cells 24:
10951103.
Mummery CL, van den Brink CE, de Laat SW (1987a)
Commitment to differentiation induced by retinoic acid in
6 Aneuploidy in Embryonic Stem Cells
P19 embryonal carcinoma cells is cell cycle dependent. Dev
Biol 121:1019.
Mummery CL, van Rooijen MA, van den Brink SE, de Laat SW
(1987b) Cell cycle analysis during retinoic acid induced dif-
ferentiation of a human embryonal carcinoma-derived cell
line. Cell Differ 20:153160.
Musacchio A, Salmon ED (2007) The spindle-assembly check-
point in space and time. Nat Rev Mol Cell Biol 8:
379393.
Neganova I, Lako M (2008) G1 to S phase cell cycle tran-
sition in somatic and embryonic stem cells. J Anat 213:
3044.
Nyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002)
Toward maintaining the genome: DNA damage and replica-
tion checkpoints. Annu Rev Genet 36:617656.
Park JI, Yoshida I, Tada T, Takagi N, Takahashi Y, Kanagawa H
(1998) Trisomy 8 does not affect differentiative potential in
a murine parthenogenetic embryonic stem cell line. Jpn J Vet
Res 46:2935.
Pathak S (1995) Centromere or telomere: who is the boss?
Anticancer Res 15:25492550.
Pathak S, Multani AS, Furlong CL, Sohn SH (2002) Telomere
dynamics, aneuploidy, stem cells, and cancer (review). Int J
Oncol 20:637641.
Plentz RR, Schlegelberger B, Flemming P, Gebel M, Kreipe
H, Manns MP, Rudolph KL, Wilkens L (2005) Telomere
shortening correlates with increasing aneuploidy of chro-
mosome 8 in human hepatocellular carcinoma. Hepatology
42:522526.
Potter H (1991) Review and hypothesis: Alzheimer dis-
ease and Down syndromechromosome 21 nondisjunc-
tion may underlie both disorders. Am J Hum Genet 48:
11921200.
Rajaraman R, Guernsey DL, Rajaraman MM, Rajaraman SR
(2006) Stem cells, senescence, neosis and self-renewal in
cancer. Cancer Cell Int 6:2525.
Rane SG, Reddy EP (2000) Cell cycle control of pancreatic beta
cell proliferation. Front Biosci 5:D1D19.
Rasnick D, Duesberg PH (1999) How aneuploidy affects
metabolic control and causes cancer. Biochem J 340:
621630.
Rebuzzini P, Neri T, Mazzini G, Zuccotti M, Redi CA,
Garagna S (2008) Karyotype analysis of the euploid
cell population of a mouse embryonic stem cell line
revealed a high incidence of chromosome abnormalities
that varied during culture. Cytogenet Genome Res 121:
1824.
Rehen SK, McConnell MJ, Kaushal D, Kingsbury MA, Yang
AH, Chun J (2001) Chromosomal variation in neurons of the
developing and adult mammalian nervous system. Proc Natl
Acad Sci USA 98:1336113366.
Rehen SK, Yung YC, McCreight MP, Kaushal D, Yang AH,
Almeida BS, Kingsbury MA, Cabral KM, McConnell MJ,
Anliker B, Fontanoz M, Chun J (2005) Constitutional
aneuploidy in the normal human brain. J Neurosci 25:
21762180.
Resnick JL, Bixler LS, Cheng L, Donovan PJ (1992) Long-
term proliferation of mouse primordial germ cells in culture.
Nature 359:550551.
Rolig RL, McKinnon PJ (2000) Linking DNA damage and
neurodegeneration. Trends Neurosci 23:417424.
85
Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004)
Molecular mechanisms of mammalian DNA repair and
the DNA damage checkpoints. Annu Rev Biochem 73:
3985.
Saretzki G, Armstrong L, Leake A, Lako M, von Zglinicki T
(2004) Stress defense in murine embryonic stem cells is
superior to that of various differentiated murine cells. Stem
Cells 22:962971.
Satge D, Sasco AJ, Lacour B (2003) Are solid tumours differ-
ent in children with Downs syndrome? Int J Cancer 106:
297298.
Satzinger H (2008) Theodor and Marcella Boveri: chromosomes
and cytoplasm in heredity and development. Nat Rev Genet
9:231238.
Savatier P, Lapillonne H, van Grunsven LA, Rudkin BB,
Samarut J (1996) Withdrawal of differentiation inhibitory
activity/leukemia inhibitory factor up-regulates D-type
cyclins and cyclin-dependent kinase inhibitors in mouse
embryonic stem cells. Oncogene 12:309322.
Schwartz GK, Shah MA (2005) Targeting the cell cycle: a new
approach to cancer therapy. J Clin Oncol 23:94089421.
Spruck CH, Won KA, Reed SI (1999) Deregulated
cyclin E induces chromosome instability. Nature 401:
297300.
Stead E, White J, Faast R, Conn S, Goldstone S, Rathjen J,
Dhingra U, Rathjen P, Walker D, Dalton S (2002) Pluripotent
cell division cycles are driven by ectopic Cdk2, cyclin A/E
and E2F activities. Oncogene 21:83208333.
Sugawara A, Goto K, Sotomaru Y, Sofuni T, Ito T (2006)
Current status of chromosomal abnormalities in mouse
embryonic stem cell lines used in Japan. Comp Med 56:
3134.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA,
Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic
stem cell lines derived from human blastocysts. Science
282:11451147.
Thomson A, Wojtacha D, Hewitt Z, Priddle H, Sottile V,
Di Domenico A, Fletcher J, Waterfall M, Corrales NL,
Ansell R, McWhir J (2008) Human embryonic stem
cells passaged using enzymatic methods retain a normal
karyotype and express CD30. Cloning Stem Cells 10:
89106.
Torres EM, Williams BR, Amon A (2008) Aneuploidy: cells
losing their balance. Genetics 179:737746.
Van den Heuvel S (2005) Cell-cycle regulation. Worm Book Sep
21:116.
Weaver BA, Bonday ZQ, Putkey FR, Kops GJ, Silk AD,
Cleveland DW (2003) Centromere-associated protein-E is
essential for the mammalian mitotic checkpoint to prevent
aneuploidy due to single chromosome loss. J Cell Biol
162:551563.
Weaver BA, Silk AD, Cleveland DW (2008) Low rates of
aneuploidy promote tumorigenesis while high rates of ane-
uploidy cause cell death and tumor suppression. Cell Oncol
30:453.
Weaver BA, Silk AD, Montagna C, Verdier-Pinard P, Cleveland
DW (2007) Aneuploidy acts both oncogenically and as a
tumor suppressor. Cancer Cell 11:2536.
White J, Stead E, Faast R, Conn S, Cartwright P, Dalton S
(2005) Developmental activation of the Rb-E2F pathway
and establishment of cell cycle-regulated cyclin-dependent
86
kinase activity during embryonic stem cell differentiation.
Mol Biol Cell 16:20182027.
Wicha MS, Liu S, Dontu G (2006) Cancer stem cells: an old idea
a paradigm shift. Cancer Res 66:18831890; discussion
18951886.
Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker
CA, Housman DE, Amon A (2008) Aneuploidy affects pro-
liferation and spontaneous immortalization in mammalian
cells. Science 322:703709.
R.C. Sartore et al.
Yang AH, Kaushal D, Rehen SK, Kriedt K, Kingsbury MA,
McConnell MJ, Chun J (2003) Chromosome segregation
defects contribute to aneuploidy in normal neural progenitor
cells. J Neurosci 23:1045410462.
Zuber MA, Koschny R, Koschny T, Froster UG (2002)
Gain of chromosome 7 detected by comparative genomic
hybridization accumulates with age in patients with
glioblastoma multiforme. Cancer Genet Cytogenet 136:
9294.
Chapter 7
Retrotransposition and Neuronal Diversity
Maria C. N. Marchetto, Fred H. Gage, and Alysson R. Muotri
Contents
7.1 Introduction . . . . . . . . . . . . . . . . . 87
7.2 Silencing and Activation of L1 Retrotransposons 89
7.3 L1 Targets in Neuronal
Progenitor Cells . . . . . . . . . . . . . . . 91
7.4 Environmental Regulation of L1 Activity
in the Brain . . . . . . . . . . . . . . . . . 93
7.5 L1 Activity and Disease . . . . . . . . . . . 93
7.6 Evolutionary Consequences of L1 Impact
in Neuronal Genomes . . . . . . . . . . . . 94
References . . . . . . . . . . . . . . . . . . . . 95
Abstract The generation of specialized cell types,
such as neurons, derived from stem cells has been
proposed as a groundbreaking technology for regener-
ative medicine. Unfortunately, subtype-specific differ-
entiation of functional neurons is extremely difficult.
Understanding the mechanisms of neuronal diversi-
fication is not only relevant for the proper differ-
entiation of complex neuronal types but might also
shed light on normal brain development and cognitive
diversification. The recent finding that LINE-1 (Long
Interspersed Nucleotide Elements-1, or L1) retroele-
ments are active in somatic neuronal progenitor cells
has provided a potential additional mechanism for gen-
erating neuronal diversification. L1 retrotransposition
in the nervous system challenges the idea of static neu-
ronal genomes, adding a new element for neuronal
A.R. Muotri ()
University of California at San Diego, School of Medicine,
Department of Pediatrics/Rady Childrens Hospital San Diego
and Department of Cellular and Molecular Medicine, La Jolla,
CA 92093, MC 0695, USA
e-mail: muotri@ucsd.edu
H. Ulrich (ed.), Perspectives of Stem Cells,
DOI 10.1007/978-90-481-3375-8_7, Springer Science+Business Media B.V. 2010
plasticity. However, the extent of the impact of L1 on
the neuronal genome is unknown. In this chapter we
will discuss the potential influence of L1 retrotrans-
position during brain development and the evolution-
ary pressures that may have selected this unexpected
machinery of diversity in neuronal precursor cells.
Keywords Brain evolution Genetic mosaicism L1
retrotransposon Neural stem cells Selfish gene
Abbreviations
LINE-1 or L1 Long Interspersed Nucleotide
Elements-1
NSC neural stem cell(s)
EGFP enhanced green fluorescent protein
EN endonuclease
NRSE neuron-restrictive silencer element
RT reverse transcriptase
TPRT Target Primed Reverse Transcription
UTR untranslated terminal repeats
7.1 Introduction
It is known for more than a century, through the work
of Camilo Golgi and Santiago Ramon y Cajal, that
neurons are specialized cells with a huge diversity of
shapes and connections. It is estimated that the human
brain contains more than 10,000 different morpho-
logical types of neurons. However, neuronal diversity
cannot be defined only by morphology or anatomic
position. Similar cells, located at the same brain region,
may have distinct electrophysiological properties and
unique connection within other neurons. Moreover,
87
88 M.C.N. Marchetto et al.

neurons are extremely plastic cells, allowing extraor-

dinary response upon micro and macro environmental
stimulation. Any attempt to understand how the brain
works must take into account neuronal diversity. Such
diversity is likely the reason why each one of us is
unique; even genetically identical twins have different
preferences or opinions. But the fundamental mech-
anisms by which neural stem cells produce such a
variety of neuronal types are slowly being revealed.
In contrast to the single mechanism for the pro-
duction of antibodies (VDJ recombination) in the
immune system, several molecular mechanisms con-
tribute to the generation of neuronal diversity (Muotri
and Gage, 2006). Those mechanisms not only act
on the DNA, but also act on the RNA and pro-
tein level, allowing epigenetic modifications to take
place. Among these mechanisms, alternative splic-
ing, promoter usage, alternate polyadenylation, RNA
editing and post-translation modifications are all part
of the genetic tool box present in neuronal precur-
sor cells. However, even with such repertoire, this
is still not enough to justify the observed constella-
tion of neuronal types. New mechanisms are likely
to be uncovered. We anticipate that novel strategies
for the neuronal diversity contribution are hidden in
non-coding regions of the genome (Cao et al., 2006;
Muotri and Gage, 2006). We have recently shown that
an engineered human L1 element could retrotranspose
in neuronal precursor cells, changing neuronal-related
gene expression, which, in turn, can influence neu-
ronal cell fate in vitro (Muotri et al., 2005). Long
dismissed as selfish or junk DNA, retroelements
are thought to be intracellular parasites from our dis- Fig. 7.1 A model for generation of neuronal diversity by
L1 retrotransposition. In neural stem cells, Sox2 expression
tant evolutionary past. Together with their mutated is correlated with a repression of L1 retrotransposons and
relatives, retroelement sequences constitute 45% of neuronal genes. During early phases of neuronal differentiation,
the mammalian genome, with L1 alone representing there is a reduction in the expression of Sox2 and other neuronal
20%. Full-length L1s are ~6 Kb long, and encode two stem cell genes. As a result, L1 transcription can be activated,
allowing subsequent retrotransposition into neuronal genes such
ORFs (ORF1 and ORF2), which code for an RNA- as for the Psd-93 gene (Muotri et al., 2005). The resulting retro-
binding protein with nucleic acid chaperone activity transposition events can alter gene expression, which, in turn,
and a 150-kDa protein with both endonuclease (EN) can influence the phenotype of the resulting cell. The functional
and reverse transcriptase (RT) activities (Fig. 7.1). L1s variability in gene expression induced by L1 retrotransposition
could also contribute, in principle, to the high cell death rate
mobilize via an RNA intermediate to integrate them- observed in adult neurogenesis, where only a few newly born
selves into genomic DNA at the target site (Fig. 7.1). neurons successfully integrate into the pre-existing neuronal
Most of the L1s in the mammalian genome are unable network
to retrotransposed due to mutations. Consequently,
only 80150 retrotransposition competent L1s capa-
ble of autonomous retrotransposition are located in the The fact that L1 can retrotranspose in a defined
human genome (Brouha et al., 2003; Sassaman et al., window of neuronal differentiation, changing the
1997). genetic information in single neurons in an arbitrary
7 Retrotransposition and Neuronal Diversity
fashion, could allow the brain to develop in distinctly
different ways. These characteristics of variety and
flexibility may contribute to the uniqueness of an indi-
vidual brain. L1 retrotransposition causes a neuronal
genetic mosaicism, i.e., the presence of more than
one genetically distinct neuronal type. Such mosaicism
might be undetectable unless closely inspected. In
fact, genetic mosaicism is frequently overlooked or
interpreted as normal variation caused by stochas-
tic developmental factors or unequal influence of
the environment. However, depending on the mosaic
nature, frequency, environmental cues, and tissues
of origin, even subtle alterations in gene expression
can contribute to detectable phenotypic alterations in
the organism. Normal processes, such as aging, the
generation of immune diversity, and the pheno-
typic variability between monozygotic twins (such
as schizophrenia) can be due to somatic genetic
mosaicism (Dipple and McCabe, 2000; Machin, 1996;
Vijg, 2000). The stochastic nature of retrotransposon
activity, and the large number of genes that this process
may affect, could produce an ample spectrum of neu-
ronal diversity, which may affect behavior, cognition
and disease risk.
7.2 Silencing and Activation of L1
Retrotransposons
L1 retrotransposons can threaten the structure and reg-
ulate the expression of the genome in different ways,
such as creating new splicing forms, promoter activa-
tion, skipping exons or gene inactivation among others
(Gilbert et al., 2005; Kazazian, 2004). Such a variety of
strategies make L1 retrotransposons the most creative
force shaping the genomes during evolution.
Deleterious retrotransposition events in the germ
line or in early development have resulted in a variety
of genetic disorders, and a somatic L1 retrotransposi-
tion in man has resulted in a sporadic case of colon
cancer (Kazazian, 1998; Miki et al., 1992; Ostertag
and Kazazian, 2001). In plants and other organisms in
which transposition is not restricted to the germ line,
somatic activity of transposable elements provides the
opportunity for a phenotypic variability that can some-
times be stunning with regard to individual genome
flexibility (Lisch, 2002). In contrast, retrotransposons
are frequently assumed to be silenced in mammalian
89
somatic tissues. This assumption is based on sev-
eral arguments. First, there is no detectable or low
level of retrotransposon expression in most somatic tis-
sues. However, only a few tissues have been subjected
to meticulous analysis, including subtype cell differ-
ences. Second, the somatic silencing of L1s fits well in
the selfish DNA hypothesis, where the mobile ele-
ments exist merely to propagate themselves, so there is
no reason to transpose in somatic cells. Finally, there
is a clear detection bias of somatic retrotransposition
since only visible mutants, usually leading to human
diseases, such as cancer, are detectable (Kazazian,
2001; Kazazian et al., 1988). The lack of experimen-
tal data and the paucity of natural evidence for somatic
L1 retrotransposition have led to the view that L1
activity is restricted to early embryonic and germ line
cells, suggesting that intrinsic factors may be present
or absent in certain cell types responsible for trans-
position (Mathias and Scott, 1993; Prak et al., 2003).
Nonetheless, retrotransposon silencing could be phys-
iologically attenuated. DNA methylation is likely the
most effective and global strategy against retrotrans-
poson mobility (Yoder et al., 1997). Accordingly, DNA
methyltransferase-1 (Dmnt1)-deficient mouse embryos
have much higher levels of IAP (Intracisternal A-
particle) retrotransposon transcripts than their wild-
type littermates (Walsh et al., 1998). Repression of
retrotransposition is removed under definite conditions
during a specific developmental window. One example
is the specific induction of IAP elements in the stem
cells of the male germ line at undifferentiated stages
when they are de-methylated. This leads to the hypoth-
esis that, a similar mechanism may be found in somatic
tissues.
One useful approach to track somatic retrotrans-
position is the analysis of the L1-enhanced green
fluorescent protein (EGFP) transgenic animal (Muotri
et al., 2005; Prak et al., 2003). These mice were
engineered to carry an active L1 retrotransposon
with an EGFP indicator cassette that only expresses
EGFP after retrotransposition and de novo insertions
(Fig. 7.2). Because the assay uses the strong and ubiq-
uitous CMV promoter, it is expected to express EGFP
in a large spectrum of somatic cells, if retrotranspo-
sition indeed occurs. Obviously, the system will not
detect truncated or silenced insertions of the reporter
cassette. Of the several somatic tissues analyzed by
immunohistochemistry, brain tissue was the only tis-
sue where EGFP expression was detected, specifically
90 M.C.N. Marchetto et al.

in neurons (Muotri et al., 2005). The in vitro cellu-
lar assay indicates that L1 retrotransposition actually
happened in precursor cells rather than postmitotic
neurons. Therefore, neuronal precursor cells may have
a greater frequency of L1 retrotransposition than other
cell types and/or this finding may be due to the long
life of neurons, in contrast to the continuous renewal
of other cell types. Either way, the presence of EGFP-
positive cells indicates that somatic L1 silencing is
incomplete in the brain. This observation suggests that
L1 retrotransposons might be activated in neuronal
precursor cells and the resultant retrotransposition
events could alter the expression of neuronal genes.
Such mechanism could, presumably, generate a large
spectrum of genetically distinct neurons, adding to
the great neuronal variation that is currently observed
in the adult CNS. L1 activation is likely regulated
by host factors in equilibrium: too much L1 retro-
transposition can cause cell damage and induce the
cells to die (Haoudi et al., 2004); too little can limit
neuronal diversity. The identification of neuronal host
factors responsible for L1 repression and/or activa-
tion will be extremely important to understand how
retrotransposition is regulated.

Fig. 7.2 Detection of L1

retrotransposition in the
brains of transgenic mice.
The structure of the
L1RP -EGFP transgene is
indicated at the top of the
figure. The
retrotransposition-competent
human L1 (L1RP ) contains a
5 untranslated region (UTR)
that harbors an internal
promoter, two open reading
frames (ORF1 and ORF2; not
drawn to scale), and a 3 UTR
that ends in a poly (A) tail.
The EGFP retrotransposition
indicator cassette consists of a
backward copy of the EGFP
gene whose expression is
controlled by the human
cytomegalovirus major
immediate early promoter
(pCMV) and the herpes
simplex virus thymidine
kinase polyadenylation
sequence (pA). This
arrangement ensures that
EGFP expression will only
become activated upon L1
retrotransposition. The black
arrows indicate PCR primers
flanking the intron present in
the EGFP gene
7 Retrotransposition and Neuronal Diversity
Fig. 7.3 A model for L1 retrotransposition. After transcrip-
tion, the L1 RNA forms a complex with ORF1 and ORF2 in
the cytoplasm (RNP complex). The complex travels back to the
nucleus where the endonuclease domain of ORF2 (EN) nicks
the target site on the genomic DNA. The de novo insertion prob-
ably occurs by Target Primed Reverse Transcription (TPRT) and
L1 expression is dependent on the activation of its
own 5 untranslated terminal repeat (UTR) sequence,
which acts as a promoter. The human L1 5 UTR is 1 kb
long, harboring one YY1-binding site that is required
for proper transcriptional initiation (Athanikar et al.,
2004; Swergold, 1990) two Sox (sex determining
region of Y-chromosome, SRY, related HMG-box)
binding sites (Tchenio et al., 2000) and a runt-domain
transcription factor 3 (RUNX3) binding site (Yang
et al., 2003). Interestingly, none of these factors is
germ-cell specific, suggesting the presence of other,
unknown factors. Sox proteins are expressed in a
variety of tissues, including NSC and testis (Wegner,
1999). The lack of Sox2 allowed activation of neu-
ronal genes and differentiation, suggests that Sox2
may function as a repressor of differentiation in neu-
ral stem cells (Graham et al., 2003). We demonstrated
that a decrease in Sox2 expression during the early
stages of neuronal differentiation is correlated with an
increase in both L1 transcriptional activity and retro-
transposition (Muotri et al., 2005). We propose that
L1 retrotransposons are silenced in NSC due to Sox2-
mediated transcriptional repression. Down-regulation
of Sox2 accompanies chromatin modifications, such as
DNA de-methylation and histone acetylation, which
91
the new insertion is frequently mutated on its 5 UTR. Other
retroelements, such as Alus, can hijack the L1 machinery and
retrotranspose into the genome. Once inserted, the new insertion
can impact the genome in different ways, such as by the creation
of new insertions and deletions
may trigger neuronal differentiation (Fig. 7.3). Such
a mechanism preserves genetic stability in NSC but
allow instability to happen in neuronal committed
cells.
7.3 L1 Targets in Neuronal
Progenitor Cells
To cause a significant impact on neuronal genomes,
new L1 insertions must target important regulatory
regions or genes that are being expressed at the
moment of neuroblast differentiation. Likely, only the
combination of multiple L1 events, and not an even-
tual catastrophic insertion in single neurons will be
ultimately responsible for any change in the neu-
ronal network. But L1 retrotransposition is a dan-
gerous situation for the cell, since L1 insertions
can hit essential genes that may induce cell death
or even target oncogenes, leading to a neoplastic
transformation.
Despite the low number of examples, the sequence
data from target insertional sites in rat neuroblasts
92
were often close to or inside neuron-associated genes
(Muotri et al., 2005). Even with a small sample,
two L1 insertions were located in the same gene,
indicating that the integration process might not
be completely random. Some of these target genes
included an olfactory receptor, ion channel-associated
genes and a cadherin receptor (Muotri et al., 2005).
An L1 insertion in the promoter region of the Psd-
93 gene, encoding a post-synaptic density protein
involved in different aspect of synapse formation,
significantly increased gene expression level and, con-
sequently, accelerated neuronal maturation in culture.
Despite the fact that randomness seems to be the
best way for L1 to survive during evolution when
they are active in germ cells, somatic insertions might
be controlled by local microvariations in DNA chro-
matin structure that depend on different host factors
in specific subsets of cell types. Thus, we propose
that L1 insertions in the nervous system are some-
how guided to specific gene targets. In a similar way,
the yeast Ty1 transposon is highly nonrandom in
vivo, being preferentially inserted upstream of tRNA
genes (Bachman et al., 2004; Devine and Boeke,
1996).
In the L1-EGFP transgenic mice, we followed
the retrotransposition of a single human L1 element
and retrotransposition was detected by EGFP expres-
sion. However, the indicator cassette did not reflect
a direct measurement of the 3,000 estimated endoge-
nous active L1 retrotransposons (DeBerardinis et al.,
1998; Goodier et al., 2001). Moreover, as pointed out
before, the L1 retrotransposition assay did not report
EGFP-truncated or silenced insertions. Additionally,
it certainly did not account for the indirect, in trans,
L1-mediated insertions of Alus, retrotransposition-
defective L1s, and other non-autonomous RNAs.
Virtually any RNA molecule can be subject to retro-
transposition if hijacked by L1 machinery. In this
regard, every single developing neuron can potentially
carry L1-mediated events, and if part of the resultant
insertions occurs in genes expressed during neuronal
development, altering gene expression, then it is pos-
sible that brain development could be significantly
affected by L1 retrotransposition. It has been proposed
that stochastic gene expression might be a fundamental
part of development and differentiation and, where it is
advantageous, these stochastic patterns are retained in
the adult organism (Fiering et al., 2000). We speculate
that these new L1 retrotransposition events are stably
M.C.N. Marchetto et al.
integrated into the genomes of individual neurons dur-
ing the entire life of the organism. These insertions
then act in a stochastic fashion, working as control-
ling elements, fine-tuning to increase the probability
that genes will be differentially transcribed. The model
is consistent with neuroblast differentiation, in which
similar cells are subjected to the same environmental
stimuli but do not respond uniformly. Thus, new inser-
tions in neurons represent genomic scars that may
have the potential to influence the fate of the resultant
cells and, consequently, the function of the neuronal
network.
The study of the human L1 5 UTR promoter dur-
ing neuronal differentiation revealed that L1 activation
occurs in the initial stages of cell differentiation. That
is exactly the same time that several neuronal genes,
such as NeuroD1, are upregulated and several cell
cycle genes are downregulated (Hsieh et al., 2004a;
Zhao and Gage, 2002). Additionally, the strong anti-
mitotic small modulatory neuron-restrictive silencer
element (NRSE) dsRNA, responsible for the neuronal
fate of NSC, is expressed in initial steps of differ-
entiation, activating several NRSE-containing neuron-
specific genes and stopping the cell cycle (Kuwabara
et al., 2004). These data suggest that there is an
orchestrated regulation during neuronal differentia-
tion, avoiding an eventual cell transformation. Such
an idea conforms with the low incidence of neu-
roblastomas (Zhu and Parada, 2002) but does not
exclude the possibility that an abnormal L1 retrotrans-
position leads to a neoplastic transformation in CNS
cells.
Taken together, a specific regulation of L1 retro-
transposon activity that takes into account its non-
random neuronal insertion and a specific window of
time during cell differentiation may turn a potentially
harmful phenomenon into a useful one. The problem
now, as with most novel scientific debates, is one of
quantification and significance. Future technologies for
single-cell endogenous L1 activity assays will bring
new insights into the problem. Moreover, the genera-
tion of three-dimensional brain mapping depicting the
occurrence of L1 retrotransposition will allow the visu-
alization of preferential target neuronal subtypes. The
comparison of normal brains with brains where L1
activity is mis-regulated, will provide the structural
organization for the design of algorithms that predict
eventual retrotransposition-affected neuronal networks
or systems.
7 Retrotransposition and Neuronal Diversity
7.4 Environmental Regulation of L1
Activity in the Brain
L1 somatic retrotransposition may be regulated by
environmental signals. Because adult neurogenesis can
be influenced by a wide variety of environmental and
behavioral cues, and since adult neuroblasts can sup-
port L1 transposition, it is possible that L1 retrotrans-
position is also affected during neurogenesis. Recently,
we described the effects of voluntary running (a potent
neurogenic activity) in the L1-EGFP transgenic ani-
mals. Surprisingly, the number of EGFP-positive cells
increased about twofold in the runners brains (Muotri
et al., 2009). It is still premature to speculate how run-
ning can induce L1 retrotransposition or the survival of
L1-inserted cells. It could be an indirect observation,
since running increases the number of cell divisions
in neurogenic areas, thus increasing the chances of L1
insertions in newborn cells or that another factor, such
as hormones, could be acting to stimulate L1 transcrip-
tion, as previously suggested (Morales et al., 2002;
Trelogan and Martin, 1995).
Interestingly, EGFP-positive cells were increased
not only in the adult neurogenic areas but also in
non-neurogenic areas, such as the cortex or amyg-
dala. Because preliminary data suggest that L1 retro-
transposition does not occur in postmitotic neurons
(Muotri et al., 2005), this finding indicates that run-
ning not only increased the number of new inser-
tions in the brain but also activated EGFP expression
in mature neurons from silenced L1 insertions. The
mechanism for this activation is unknown; however,
the L1-EGFP insertion is likely working as a real
time gene-trap system, indicating that several regions
of the neuronal genome can be re-expressed dur-
ing exercise. Such re-expression is probably taking
place by chromatin remodeling factors that expose
the silenced DNA region to transcription factors. A
similar phenomenon was observed in neural clones
that harbor L1-EGFP insertions (Muotri et al., 2005).
After single-cell cloning in the presence of FGF-2,
the EGFP expression are silenced, generating EGFP-
variegated clones in which EGFP expression will be
completely attenuated after a prolonged period in cul-
ture. Curiously, EGFP expression could be restored
only during neuronal differentiation, suggesting that
the L1-inserted loci are activated in the neuronal but
not in the glial lineage. Immunostaining revealed that
93
most of the EGFP-positive cells co-localized with neu-
ronal markers, but not with glial markers, in both
treatments, consistent with recent reports demonstrat-
ing that epigenetic modifications accompany neuronal
differentiation of NSC (Hsieh and Gage, 2004; Hsieh
et al., 2004b).
Barbara McClintock first proposed the genomic
shocks hypothesis, saying that environmental factors
could induce transposition of controlling elements
(McClintock, 1984). In fact, a recent finding demon-
strated that the copy number of the plant BARE-1
retrotransposon correlates with a sharp microclimate
divergence, suggesting stress-induced mobilization of
retroelements in response to environmental stimuli
(Kalendar et al., 2000). The experiment with the
L1-EGFP animal illustrates how neuronal genomes
might modify themselves when confronted with a new
or unfamiliar environment. Some of these changes
are irreversible (L1 insertions) but others may be
reversible (EGFP re-expression), resetting specific
sequences of the genome to its early state. The fact that
genetic modifications caused by L1 elements specif-
ically happen in neurons is certainly associated with
the enormous somatic plasticity observed in the ner-
vous system. The implications of these observations
are potentially important for the definition of indi-
vidual organisms as well as the meaning of neural
plasticity. Such plasticity predicts that the brain and
the neuronal network will never be the same after a
new experience, even in genetically identical twins.
Moreover, individuals lacking factors involved with
chromatin remodeling might have abnormal genome
reprogramming that can give rise to a wide range of
altered phenotypes.
7.5 L1 Activity and Disease
The idea that retroviral elements are relevant for cer-
tain brain disorders has its origins in the search for
a viral pathogen for schizophrenia in the early 1970s
(Torrey and Peterson, 1976). The research brought
evidences that endogenous retrovirus transcripts were
expressed in the brains and cerebrospinal fluids of
affected individuals (Frank et al., 2005; Karlsson
et al., 2001; Kim et al., 1999; Yee and Yolken,
1997). Interestingly, RNA expression in libraries gen-
erated from the frontal cortex regions of individuals
94
with schizophrenia revealed an increased level of L1
sequences, in particular from ORF2 sequences (Yolken
et al., 2000). Furthermore, retrotransposition activity,
measured by the presence of processed pseudogenes
without introns, is higher in genomic DNA from
individuals with schizophrenia. Accordingly, individ-
uals with schizophrenia and other psychiatric disor-
ders have increased levels of RT activity as com-
pared to unaffected controls (Yolken et al., 2000).
Taken together, this evidence identifies retroelements
as important somatic components of neuropsychiatric
diseases that are consistent with genetic, environmen-
tal, and neurodevelopmental aspects of the disease
process observed in schizophrenia, autism and oth-
ers syndromes where brain waves may fall out of
synchrony during the process of perception.
Successful identification of susceptible target genes
seems unlikely to lead to gene therapy for psychi-
atric illnesses. However, it may be important to deter-
mine the potential biochemical pathways involved in
producing particular symptoms, which could be down-
stream of a group of genes with individually weak
effects. The characterization of the role of mobile ele-
ments in the etiopathogenesis of psychotic diseases
might lead to new methods for diagnosis, treatment,
and prevention.
7.6 Evolutionary Consequences of L1
Impact in Neuronal Genomes
One of the most remarkable findings from the sequenc-
ing of the human genome is that retrotransposable ele-
ments make up a significant portion of the human DNA
(Deininger et al., 2003). Based on reverse transcriptase
(RT) phylogeny, L1 elements are most closely related
to the group II introns of mitochondria and eubacte-
ria (Cavalier-Smith, 1991; Xiong and Eickbush, 1990).
These studies revealed that the RT enzyme is extremely
old and that retroelements can be viewed as relics or
molecular fossils of the first primitive replication sys-
tems in the progenote. The origin of retroelements
possibly traces back to the conversion of RNA-based
systems, the RNA World (Orgel, 2004), to modern
DNA-based systems. Current models suggest that
these mobile introns of eubacteria were transmitted to
eukaryotes during the initial fusion of the eubacterial
and archaebacterial genomes or during the symbiosis
M.C.N. Marchetto et al.
that gave rise to the mitochondria, generating the
modern-day spliceosomal introns (Zimmerly et al.,
1995). Further acquisition of an endonuclease enzyme
and a promoter sequence certainly represented impor-
tant steps in the evolution of L1 retrotransposons, pro-
viding autonomy for L1s to insert into many locations
throughout the genome.
The apparent lack of obvious function of retroele-
ments in the genome suggests that transposable ele-
ments are selfish DNA, acting as parasites in the
genome in order to propagate themselves. This idea
has long puzzled scientists and inspired the con-
cept of junk DNA to illustrate the idea that such
sequences were mere evolutionary remnants (Doolittle
and Sapienza, 1980; Orgel and Crick, 1980). However,
the recognition that retrotransposons can actively
reshape the genome is slowly challenging this termi-
nology. Moreover, the mammalian genome has suf-
fered waves of transposon bombardment, but the con-
stant, single lineage of L1 history reveals that active
L1 were never absent from mammals genomes dur-
ing evolution, suggesting an inextricable link between
L1 and their host (Furano et al., 2004). The relation-
ship between transposons and their hosts is probably
not entirely antagonistic, as several host genes have a
high degree of homology to one or more transposable
elements. Evidence in the literature points to a somatic
function for L1 transcripts, involving cell prolifera-
tion (Kuo et al., 1998), differentiation (Mangiacasale
et al., 2003) and early embryo development (Pittoggi
et al., 2003). Moreover, it is difficult to reconcile why
the genome would need so many copies of retrotrans-
posons and whether this expansion has any correlation
with retrotransposition itself. The restricted activity
of retrotransposons in germ or early embryonic cells
apparently fits well with the selfish DNA concept,
since new insertions will be passed to the next gen-
erations, but somatic insertions pose a conundrum.
According to the symbiotic theory, it is advantageous
to any transposable element to promote host mating,
securing the propagation of the master elements to
the next generations. From this perspective, it is not
surprising that advantageous insertional events in the
brain, resulting in a better (cultural and social) fitness
of the individual organism, also can contribute to the
host mating.
The evolution of the CNS provided a notable selec-
tive advantage, as information about the environment
could be processed rapidly and would allow organisms
7 Retrotransposition and Neuronal Diversity
to more readily meet the challenges of ever-changing
environmental conditions. Moreover, epigenetic mod-
ification allowed the non-genetic transfer of informa-
tion or transmission of culture at an unprecedented
magnitude. Such specialization is highly dependent
on the cognitive levels acquired by the species that
are directly linked to the complexity of the neuronal
network. Therefore, the advantages gained by retain-
ing the mechanisms for somatic retrotransposition may
outweigh the cost of a less plastic nervous system. In
fact, such strategy expands the number of function-
ally distinct neurons that could be produced from a
given stem cell gene pool (Muotri and Gage, 2006).
This characteristic of variety and flexibility may con-
tribute to the uniqueness of an individual brain, even
between genetically identical twins. Mobile elements
in the brain may be part of the conserved core pro-
cess responsible for evoking facilitated complex non-
random phenotypical variation on which selection may
act. It is remarkable to imagine that the brain is a
consequence of ancient retrotransposition in eukary-
otic cells. Such possibility had not, at any time, been
considered before, but it was suggested to us by the
experimental results.
The identification of L1 elements as potential cre-
ative somatic shapers of transcriptional complexity in
neuronal genomes may be an important phenomenon
for developmental neurosciences. The hypothesis that
L1 activity is responsible for fine-tuning neuronal
wiring waves requires the merger of different fields
and may consequently open new ways of consider-
ing individual differences and the neuronal correlates
of human cognition. Rigorous experimental proof of
this model will require attenuation of retrotransposi-
tion activity from the mammalian genome and com-
paring their behavior to that of wild type animals.
Nonetheless, the experimental approach presents a
major methodological challenge for molecular biolo-
gists, since a canonical single-gene knockout strategy
is no longer suitable. On the other hand, the study
of abnormal activation of L1 retrotransposition in the
brain may elucidate complex neurological syndromes,
permitting an understanding of diseases at a different
level.
Acknowledgments M.C.N.M. and F.H.G. are supported by the
Lookout Fund and the National Institutes of Health: National
Institute on Aging and National Institute of Neurological
Disease and Stroke. The authors would like to thank M.L. Gage
for editorial comments.
95
References
Athanikar JN, Badge RM, Moran JV (2004) A YY1-binding
site is required for accurate human LINE-1 transcription
initiation. Nucleic Acids Res 32:38463855.
Bachman N, Eby Y, Boeke JD (2004) Local definition of Ty1 tar-
get preference by long terminal repeats and clustered tRNA
genes. Genome Res 14:12321247.
Brouha B, Schustak J, Badge RM, Lutz-Prigge S, Farley AH,
Moran JV, Kazazian HH Jr. (2003) Hot L1s account for the
bulk of retrotransposition in the human population. Proc Natl
Acad Sci USA 100:52805285.
Cao X, Yeo G, Muotri AR, Kuwabara T, Gage FH (2006)
Noncoding RNAs in the mammalian central nervous system.
Annu Rev Neurosci 29:77103.
Cavalier-Smith T (1991) Intron phylogeny: a new hypothesis.
Trends Genet 7:145148.
DeBerardinis RJ, Goodier JL, Ostertag EM, Kazazian HH Jr.
(1998) Rapid amplification of a retrotransposon subfamily is
evolving the mouse genome. Nat Genet 20:288290.
Deininger PL, Moran JV, Batzer MA, Kazazian HH Jr. (2003)
Mobile elements and mammalian genome evolution. Curr
Opin Genet Dev 13:651658.
Devine SE, Boeke JD (1996) Integration of the yeast retro-
transposon Ty1 is targeted to regions upstream of genes
transcribed by RNA polymerase III. Genes Dev 10:
620633.
Dipple KM, McCabe ER (2000) Phenotypes of patients with
simple Mendelian disorders are complex traits: thresh-
olds, modifiers, and systems dynamics. Am J Hum Genet
66:17291735.
Doolittle WF, Sapienza C (1980) Selfish genes, the phenotype
paradigm and genome evolution. Nature 284:601603.
Fiering S, Whitelaw E, Martin DI (2000) To be or not to be
active: the stochastic nature of enhancer action. Bioessays
22:381387.
Frank O, Giehl M, Zheng C, Hehlmann R, Leib-Mosch C,
Seifarth W (2005) Human endogenous retrovirus expression
profiles in samples from brains of patients with schizophrenia
and bipolar disorders. J Virol 79:1089010901.
Furano AV, Duvernell DD, Boissinot S (2004) L1 (LINE-1)
retrotransposon diversity differs dramatically between mam-
mals and fish. Trends Genet 20:914.
Gilbert N, Lutz S, Morrish TA, Moran JV (2005) Multiple fates
of L1 retrotransposition intermediates in cultured human
cells. Mol Cell Biol 25:77807795.
Goodier JL, Ostertag EM, Du K, Kazazian HH Jr. (2001) A
novel active L1 retrotransposon subfamily in the mouse.
Genome Res 11:16771685.
Graham V, Khudyakov J, Ellis P, Pevny L (2003) Sox2 func-
tions to maintain neural progenitor identity. Neuron 39:
749765.
Haoudi A, Semmes OJ, Mason JM, Cannon RE (2004)
Retrotransposition-competent human LINE-1 induces apop-
tosis in cancer cells with intact p53. J Biomed Biotechnol
2004:185194.
Hsieh J, Gage FH (2004) Epigenetic control of neural stem cell
fate. Curr Opin Genet Dev 14:461469.
Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH
(2004a) Histone deacetylase inhibition-mediated neuronal
96
differentiation of multipotent adult neural progenitor cells.
Proc Natl Acad Sci USA 101:1665916664.
Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH (2004b)
Histone deacetylase inhibition-mediated neuronal differenti-
ation of multipotent adult neural progenitor cells. Proc Natl
Acad Sci USA 101:1665916664.
Kalendar R, Tanskanen J, Immonen S, Nevo E, Schulman AH
(2000) Genome evolution of wild barley (Hordeum spon-
taneum) by BARE-1 retrotransposon dynamics in response
to sharp microclimatic divergence. Proc Natl Acad Sci USA
97:66036607.
Karlsson H, Bachmann S, Schroder J, McArthur J, Torrey EF,
Yolken RH (2001) Retroviral RNA identified in the cere-
brospinal fluids and brains of individuals with schizophrenia.
Proc Natl Acad Sci USA 98:46344639.
Kazazian HH Jr. (1998) Mobile elements and disease. Curr Opin
Genet Dev 8:343350.
Kazazian HH (2001) Retrotransposon insertions in germ cells
and somatic cells. Dev Biol (Basel) 106:307313.
Kazazian HH Jr. (2004) Mobile elements: drivers of genome
evolution. Science 303:16261632.
Kazazian HH Jr., Wong C, Youssoufian H, Scott AF, Phillips
DG, Antonarakis SE (1988) Haemophilia A resulting from
de novo insertion of L1 sequences represents a novel mecha-
nism for mutation in man. Nature 332:164166.
Kim HS, Takenaka O, Crow TJ (1999) Isolation and phylogeny
of endogenous retrovirus sequences belonging to the HERV-
W family in primates. J Gen Virol 80:26132619.
Kuo KW, Sheu HM, Huang YS, Leung WC (1998) Expression of
transposon LINE-1 is relatively human-specific and function
of the transcripts may be proliferation-essential. Biochem
Biophys Res Commun 253:566570.
Kuwabara T, Hsieh J, Nakashima K, Taira K, Gage FH (2004)
A small modulatory dsRNA specifies the fate of adult neural
stem cells. Cell 116:779793.
Lisch D (2002) Mutator transposons. Trends Plant Sci 7:
498504.
Machin GA (1996) Some causes of genotypic and phenotypic
discordance in monozygotic twin pairs. Am J Med Genet
61:216228.
Mangiacasale R, Pittoggi C, Sciamanna I, Careddu A, Mattei
E, Lorenzini R, Travaglini L, Landriscina M, Barone C,
Nervi C, Lavia P, Spadafora C (2003) Exposure of normal
and transformed cells to nevirapine, a reverse transcriptase
inhibitor, reduces cell growth and promotes differentiation.
Oncogene 22:27502761.
Mathias SL, Scott AF (1993) Promoter binding proteins of
an active human L1 retrotransposon. Biochem Biophys Res
Commun 191:625632.
McClintock B (1984) The significance of responses of the
genome to challenge. Science 226:792801.
Miki Y, Nishisho I, Horii A, Miyoshi Y, Utsunomiya J, Kinzler
KW, Vogelstein B, Nakamura Y (1992) Disruption of the
APC gene by a retrotransposal insertion of L1 sequence in
a colon cancer. Cancer Res 52:643645.
Morales JF, Snow ET, Murnane JP (2002) Environmental fac-
tors affecting transcription of the human L1 retrotrans-
poson. I. Steroid hormone-like agents. Mutagenesis 17:
193200.
Muotri AR, Gage FH (2006) Generation of neuronal variability
and complexity. Nature 441:10871093.
M.C.N. Marchetto et al.
Muotri AR, Zhao C, Marchetto MC, Gage FH (2009)
Environmental influence on L1 retrotransposons in the adult
hippocampus. Hippocampus 19:10021007.
Orgel LE (2004) Prebiotic chemistry and the origin of the RNA
world. Crit Rev Biochem Mol Biol 39:99123.
Orgel LE, Crick FH (1980) Selfish DNA: the ultimate parasite.
Nature 284:604607.
Ostertag EM, Kazazian HH Jr. (2001) Biology of mammalian L1
retrotransposons. Annu Rev Genet 35:501538.
Pittoggi C, Sciamanna I, Mattei E, Beraldi R, Lobascio AM,
Mai A, Quaglia MG, Lorenzini R, Spadafora C (2003) Role
of endogenous reverse transcriptase in murine early embryo
development. Mol Reprod Dev 66:225236.
Prak ET, Dodson AW, Farkash EA, Kazazian HH Jr. (2003)
Tracking an embryonic L1 retrotransposition event. Proc
Natl Acad Sci USA 100:18321837.
Sassaman DM, Dombroski BA, Moran JV, Kimberland ML,
Naas TP, DeBerardinis RJ, Gabriel A, Swergold GD,
Kazazian HH Jr. (1997) Many human L1 elements are
capable of retrotransposition. Nat Genet 16:3743.
Swergold GD (1990) Identification, characterization, and cell
specificity of a human LINE-1 promoter. Mol Cell Biol
10:67186729.
Tchenio T, Casella JF, Heidmann T (2000) Members of the SRY
family regulate the human LINE retrotransposons. Nucleic
Acids Res 28:411415.
Torrey EF, Peterson MR (1976) The viral hypothesis of
schizophrenia. Schizophr Bull 2:136146.
Trelogan SA, Martin SL (1995) Tightly regulated, developmen-
tally specific expression of the first open reading frame from
LINE-1 during mouse embryogenesis. Proc Natl Acad Sci
USA 92:15201524.
Vijg J (2000) Somatic mutations and aging: a re-evaluation.
Mutat Res 447:117135.
Walsh CP, Chaillet JR, Bestor TH (1998) Transcription of IAP
endogenous retroviruses is constrained by cytosine methyla-
tion. Nat Genet 20:116117.
Wegner M (1999) From head to toes: the multiple facets of Sox
proteins. Nucleic Acids Res 27:14091420.
Xiong Y, Eickbush TH (1990) Origin and evolution of retroele-
ments based upon their reverse transcriptase sequences.
Embo J 9:33533362.
Yang N, Zhang L, Zhang Y, Kazazian HH Jr. (2003) An
important role for RUNX3 in human L1 transcription and
retrotransposition. Nucleic Acids Res 31:49294940.
Yee F, Yolken RH (1997) Identification of differentially
expressed RNA transcripts in neuropsychiatric disorders.
Biol Psychiatry 41:759761.
Yoder JA, Walsh CP, Bestor TH (1997) Cytosine methylation
and the ecology of intragenomic parasites. Trends Genet
13:335340.
Yolken RH, Karlsson H, Yee F, Johnston-Wilson NL, Torrey
EF (2000) Endogenous retroviruses and schizophrenia. Brain
Res Brain Res Rev 31:193199.
Zhao X, Gage FH (2002) Expressing the central nervous system.
Neurochem Res 27:953954.
Zhu Y, Parada LF (2002) The molecular and genetic basis of
neurological tumours. Nat Rev Cancer 2:616626.
Zimmerly S, Guo H, Perlman PS, Lambowitz AM (1995) Group
II intron mobility occurs by target DNA-primed reverse
transcription. Cell 82:545554.
Chapter 8

Directing Differentiation of Embryonic Stem Cells

into Distinct Neuronal Subtypes

Noelle Ammon, Nathaniel Hartman, and Laura Grabel

Contents learned about the conditions that promote emergence

of these lineages in the embryo.
8.1 Introduction . . . . . . . . . . . . . . . . . 98
8.2 Identifying the Desired ESC-Derived Cell Type Keywords Embryonic stem cell Neural stem cell
for Transplantation . . . . . . . . . . . . . 98 Neurodegenerative disease Neuron
8.3 Generating Neural Progenitors: Back to the
Embryo . . . . . . . . . . . . . . . . . . . 100 Abbreviations
8.4 Midbrain Dopaminergic Neurons . . . . . . . 102
8.5 GABAergic Interneurons . . . . . . . . . . . 104
8.6 Spinal Cord Motor Neurons . . . . . . . . . 106 5-HT 5-hydroxytryptamine (serotonin)
8.7 Serotonergic Neurons . . . . . . . . . . . . 108
8.8 Basal Forebrain Cholinergic Neurons . . . . . 109
AA ascorbic acid
8.9 Conclusions . . . . . . . . . . . . . . . . . 110 AADC amino acid decarboxylase
References . . . . . . . . . . . . . . . . . . . . 110 ACh acetylcholine
AD Alzheimers disease
ALS Amyotrophic Lateral Sclerosis
Abstract There is great interest in testing the effi- BAC bacterial artificial chromosome
cacy of treating neurodegenerative diseases using BDNF brain derived neurotrophic factor
embryonic stem cell derivatives. A first step towards BMP bone morphogenic protein
this goal is demonstrating that embryonic stem cells cAMP cyclic adenosine monophosphate
can produce, in culture, the specific cell types lost CB calbindin
in the various diseases. We describe here currently CCK cholecystokinin
used approaches for generating neural stem cells, CGE caudal ganglionic eminences
CNS central nervous system
as well as specific neuronal subtypes, from mouse
CR calretinin
and human embryonic stem cells. Based upon their
DA dopaminergic
demonstrated role in neurodegenerative disease and
DV dorsal-ventral
the reports documenting their derivation from embry-
EGF epidermal growth factor
onic stem cells, we focus on dopaminergic neurons, EPL primitive ectoderm-like
GABAergic interneurons, spinal cord motor neurons, ESC embryonic stem cell(s)
serotonergic neurons, and basal forebrain cholinergic FACS fluorescence activated cell sorting
neurons. Protocols are all based upon what has been FGF fibroblast growth factor
GABA -aminobutyric acid
GAD glutamic acid decarboxylase
GDNF glial cell derived neurotrophic factor
Glu glutamate
L. Grabel ()
Hall-Atwater Laboratories, Biology Department, Wesleyan hESC human embryonic stem cell(s)
University, Middletown, CT, USA Hh hedgehog
e-mail: lgrabel@wesleyan.edu iPS induced pluripotent stem cell

H. Ulrich (ed.), Perspectives of Stem Cells, 97

DOI 10.1007/978-90-481-3375-8_8, Springer Science+Business Media B.V. 2010
98 N. Ammon et al.

MAP2 microtubule associated protein 2 also necessary to identify the most appropriate cell
mDA midbrain dopaminergic type for transplant and to be able to generate large
mES cells mouse embryonic stem cells numbers of relatively pure populations of these cells
MGE medial ganglionic eminences readily in vitro. Our goal in this chapter is to summa-
MN motor neuron(s) rize the existing literature documenting the derivation
MPTP 1-methyl-4-phenyl-1,2,3,6- of some neuronal subtypes: midbrain dopaminergic
tetrahycopyridine neurons, forebrain -aminobutyric acid (GABA)ergic
NGF nerve growth factor interneurons, and motor neurons. We also briefly
NPY neuropeptide Y describe the derivation of serotonergic and cholinergic
NSC neural stem cell(s) neurons. These subtypes were chosen because of their
NT-4 neurotrophin 4
potential therapeutic uses; dopaminergic midbrain
PD Parkinsons disease
neurons for PD, inhibitory GABAergic interneurons
PDD Parkinsonian disease dementia
for epilepsy, serotonergic neurons for psychiatric
pMN motor neuron progenitor(s)
disorders, cholinergic neurons for Alzheimers disease
PV parvalbumin
RA retinoic acid (AD), and motor neurons for ALS or spinal cord injury
SCI spinal cord injuries (SCI). We will focus on the derivation of neurons
Shh sonic hedgehog from ESC, though glial derivatives can also be readily
SMA Spinal Muscular Atrophy generated.
SNi substantia nigra This chapter is not meant to provide specific proto-
SSRI serotonin selective reuptake inhibitors cols for the derivation of neurons, but rather to outline
SST somatostatin the conceptual framework behind the approaches used.
TGF- transforming growth factor Citations of the original research literature should pro-
TH tyrosine hydroxylase vide the reader with the necessary references to design
TLE temporal lobe epilepsy a relevant experimental approach for production of the
VIP vasoactive intestinal peptide desired neuronal cell type (see Table 8.1).
VMAT2 vesicular monoamine transporter 2

8.2 Identifying the Desired ESC-Derived

8.1 Introduction Cell Type for Transplantation

With the report of the isolation of the first human
embryonic stem cells (hESC) (Thomson et al., 1998),
now over 10 years ago, their potential use as ther-
apeutic agents became apparent, and an exciting
new era in stem cell biology began. There has been
particular interest in using ESC-derived neural pro-
genitors and neurons for the treatment of a variety
of neurodegenerative diseases and central nervous
system injuries that have proven somewhat intractable
to other treatment approaches. Candidate disorders for
ESC-based cell therapy include Parkinsons Disease
(PD), amyotrophic lateral sclerosis (ALS), and spinal
cord lesions. There are many scientific roadblocks that
must be overcome before we can approach the clinic,
including genetic compatibility of transplant material
with the patient, the tendency of ESC to form tumors
in vivo, and the contamination of many existing hESC
lines with animal products (Gruen and Grabel, 2006).
Work along all of these fronts is proceeding, but it is
Ideally a stem cell-based therapy would replace the
dead or damaged tissue with viable cells that could
integrate and function after transfer to the brain. Given
ESC as the starting material, it is not clear at what
stage in their trajectory from pluripotent stem cells or
neural stem cells (NSC) to differentiated neurons they
should be transplanted. The answer to this question
will depend, to some extent, on the specific disease
under treatment, but some considerations generally
apply. It may be desirable to have a self-renewing
source of the desired cell type, particularly if the disor-
der is characterized by an ongoing neurodegenerative
environment, which is likely to promote the death
of transplanted derivatives as well as the endogenous
neuronal population. Transplantation of undifferenti-
ated ESC, however, frequently results in the formation
of teratocarcinomas, tumors that contain undifferenti-
ated ESC as well as differentiated derivatives of all
three primary germ layers (Brustle et al., 1997; Deacon
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes 99

Table 8.1 Overview of selected protocols for derivation of specific neuronal subtypes from mouse and human embryonic stem
cells
Desired Cell Growth factor and other Molecular markers Molecular References
neuron type source molecular treatments (intermediates) markers (mature)
Dopaminergic mES AA, cAMP, and FGF2 Lmx1a and Msx1, TH Lee et al.
then AA, FGF2, then Nurr1 and (2000)
FGF8, and Shh, then Pitx3
only AA and cAMP

hES Wnt1 (from MS5 Lmx1a and Msx1, TH Perrier et al.

stromal cells) then then Nurr1 and (2004)
AA, BDNF, cAMP, Pitx3
FGF8, GDNF, Shh,
and TGF3 then AA,
BDNF, FGF8, and
Shh then AA, BDNF,
cAMP, GDNF, and
TGF3

Serotonergic mES FGF4 and Shh then GATA2, Pet1, and 5-HT and SERT Barberi et al.
FGF2, FGF4, and Shh Lmx1b (2003)
then only AA and
BDNF

hES BDNF, FGF1, Forskolin, GATA2, Pet1, and TPH and 5-HT Kumar et al.
GDNF, and 5HT Lmx1b (2008)

Cholinergic mES FGF2 and RA then Nkx2.1 and Lhx8 ChAT Okada et al.
BMP4, Shh, or Wnt3a (2008)

GABAergic mES FGF2 then FGF2, FGF8, GAD67 GAD67 and Barberi et al.
(cortical) and Shh then BDNF GABA (2003)
and NT-4

mES FGF2 then only N2B27 GAD67 GAD67, GABA, Okabe et al.
and FCS and calbindin (1996)

GABAergic mES BMP2, RA, Shh, and MAP-2 Lim2, GAD67, Murashov
(spinal) Wnt3a and GABA et al.
(2004)

Motor mES RA then Shh (or Pax6, Nkx6.1, and HB9 Wichterle
synthetic Hh agonist) Olig2 et al.
(2002)

hES RA and Shh then BDNF, Pax6, Sox1, Olig2 HB9 and Islet1/2 Li et al.
GDNF, and IGF-1 then ChAT (2005)

hES AA, BDNF, RA, and Nkx6.1 and Olig2 HB9, ChAT, and Lee et al.
Shh then AA, BDNF Lhx3 (2007)
and GDNF

iPS RA and Shh Pax6 and Olig2 HB9, Islet1/2, Dimos et al.
(human) and ChAT (2008)

et al., 1998; Wernig et al., 2004). This is obviously not transplanted is proliferative, however, it remains capa-
a desired outcome, and most approaches have therefore ble of forming tumors consisting of predominantly
focused on using NSC, neural progenitors, or differ- NSC and progenitors (Carpentino et al., 2008). If ter-
entiated neurons for grafts. As long as the cell type minally differentiated neurons are grafted, tumor risk
100
decreases, but these cells are no longer capable of self-
renewal and may not even survive in certain areas of
the brain. The relative threat of neural tumor forma-
tion by proliferative progenitors would depend upon
the host environment and whether it could support
appropriate terminal differentiation. Here the niche
into which the ESC-derived grafts are transplanted
may be key, and this will vary for different disor-
ders. Evidence suggests that both NSC and specific
progenitors are able to respond to certain host environ-
ments and integrate and function following transplant,
and some examples are discussed in subsequent sec-
tions. If the host environment is unable to support
the differentiation and integration of transplanted NSC
or progenitors, terminally differentiated neurons may
provide the most suitable graft material, although a
limited lifespan in vivo may necessitate multiple grafts
administered periodically. Thus, in selecting the cell
type for transplant, one must weigh relative tumor risk
with the ability of the cells to survive and function in
the host environment. As we identify factors that pro-
mote successful transplant incorporation, a subject of
intense investigation, these may be used to facilitate
functional integration under unfavorable conditions.
8.3 Generating Neural Progenitors: Back
to the Embryo
Directing the differentiation of pluripotential ESC to
specific neuronal subtypes begins with their transition
to multipotential NSC that can differentiate into oligo-
dendrocytes, astrocytes, or neurons (Temple, 2001).
Successful methodologies for producing NSC and neu-
rons from either mouse or human ESC have been
based on a thorough understanding of the conditions
Fig. 8.1 From ESC to
neuron. Production of
neurons or glia from
embryonic stem cells involves
production of NSC
N. Ammon et al.
that promote the emergence of these cell types in
the embryo and during adult neurogenesis (Cai and
Grabel, 2007) (Fig. 8.1). During mammalian embryo-
genesis the epiblast, or primitive ectoderm, is the
source of all three primary germ layers; ectoderm,
mesoderm and endoderm. The signals that direct the
ectoderm to make neurectoderm versus epidermis or
other lineages have been studied extensively in many
species (Stern, 2005). Initial analyses suggested that
the absence of instructions, a so-called default state,
led to specification of neurectoderm. In particular,
organizer regions associated with neural induction con-
tain molecules that inhibit both bone morphogenic
protein (BMP) (Sasai et al., 1995, 1994; Smith and
Harland, 1992) and Wnt signaling (Wilson et al.,
2001). Additional studies have implicated a positive
role for Notch (Ross et al., 2003) as well as fibrob-
last growth factor (FGF) (De Robertis and Kuroda,
2004) and Wnts (Baker et al., 1999) in the initial
phase of neural specification, and Hedgehog(Hh) in
the proliferation and survival of embryonic and adult
neural stem cells. Studies suggest that a similar array
of factors, including Hh and Wnts, supports the sur-
vival and proliferation of NSC in the two regions
of adult neurogenesis, the subventricular zone of the
lateral ventricles and the subgranular zone of the hip-
pocampus dentate gyrus (Ahn and Joyner, 2005; Lie
et al., 2005; Machold et al., 2003; Palma et al., 2005).
Protocols for generating NSC from ESC can be
divided into two classes, those that rely upon an embry-
oid body intermediate, and those that do not (Cai and
Grabel, 2007). When ESC are removed from their
feeder layer, and placed in suspension culture, they
form aggregates, which within a few days consist of
an outer layer of hypoblast-like cells (extraembry-
onic visceral endoderm) surrounding an epiblast-like
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
core (Martin et al., 1977; Maye et al., 2004; Rathjen
et al., 2002). At early stages these embryoid bodies
resemble the anterior pre-streak stage embryo, with
the epiblast-like core able to generate derivatives of all
three primary germ layers (Keller, 2005). The interac-
tion of the distinct lineages within the embryoid body
provides the positive and negative instructions needed
to promote neurectoderm differentiation. This lineage
commitment can be enhanced in vitro by a number of
treatments (Maye et al., 2004; Rathjen et al., 2002).
The signaling molecule Indian hedgehog, secreted by
the visceral endoderm, promotes neurectoderm differ-
entiation in vitro (Maye et al., 2004), as does the addi-
tion of retinoic acid (RA) (Bain et al., 1995; Gottlieb,
2002; Guan et al., 2001). RA addition also increases
the yield of neural derivatives, including motor neu-
rons (Bibel et al., 2004; Li et al., 2005; Wichterle
et al., 2002). Conditioned medium from a hepatocel-
lular carcinoma cell line can promote the homoge-
neous differentiation of primitive ectoderm-like (EPL)
cells from mouse ESC in suspension culture (Rathjen
et al., 2002, 1999). The conditioned medium contains
unidentified signals that induce neurectoderm differen-
tiation at the expense of mesodermal and endodermal
derivatives.
An additional protocol combines use of an embry-
oid body intermediate with a neural lineage-specific
selection step (Guan et al., 2001; Okabe et al., 1996).
The selection step deprives the cells of growth fac-
tors and signaling molecules, mimicking a default state
under which non-neural lineages die and NSC survive.
These conditions remove inhibitory factors for neu-
ral differentiation, such as BMPs, as well as signals
required for the differentiation and survival of other
lineages. Early stage embryoid bodies are plated on
adhesive substrates in a minimal serum-free medium.
During several days of culture, non-neurectoderm
derivatives die, leaving a neurectoderm-derived neural
progenitor population (Okabe et al., 1996). Subsequent
plating on laminin substrates in the presence of FGF2
and epidermal growth factor (EGF) promotes NSC
proliferation, and at this stage cultures may be 8095%
NSC (Okabe et al., 1996).
NSC can be generated directly from ESC without an
embryoid body intermediate by plating the cells at very
low clonal densities (120 cells/well) under feeder-free
conditions in serum-free defined medium (Tropepe et
al., 2001). This approach uses a neurosphere-based
101
suspension assay for production of NSC (Smukler et
al., 2006). Production of NSC, however, is a rare event
under these conditions, with only 0.2% of ESC form-
ing neurospheres. More efficient direct differentiation
of ESC to NSC can also be obtained under serum-free
conditions in monolayer culture at moderate cell den-
sities (Ying et al., 2003). By 5 days, up to 75% of
the cells express NSC markers. The transition to NSC
is accompanied by the death of significant numbers
of cells in the cultures, suggesting that these condi-
tions are effective, at least in part, due to selective
survival of the NSC. Under these conditions, emerging
NSC form rosettes, in which cells elongate and align
radially, in a manner reminiscent of neural tube for-
mation, as well as of neurectoderm differentiation in
ESC embryoid bodies (Fig. 8.1b). These ESC-derived
NSC share morphological and biochemical character-
istics of radial glia, the NSC of the embryonic neural
tube. Co-culture of ESC at very low, clonal densities on
stromal cell lines (Table 8.1) also promotes NSC pro-
duction (Barberi et al., 2003; Kawasaki et al., 2000).
The identity of the factor, or factors, provided by the
stromal cells has not been determined (Kawasaki et al.,
2000).
Given the goal to produce a variety of neuronal
cell types for transplantation therapies, it is impor-
tant to determine whether the ESC-derived NSC have
restricted potential or can generate all desired progen-
itors and differentiated cell types. During embryoge-
nesis, the potential of emerging NSC, as measured
by their ability to generate different classes of neu-
rons both in vitro (Fig. 8.2) and in vivo, is dependent
upon embryonic age as well as their site of ori-
gin along both the rostral/caudal and dorsal/ventral
axes. Identification of neuronal subtype is based upon
the expression profile of region specific transcrip-
tion factors and neurotransmitters. Care must be taken
to not rely on a single marker, including neuro-
transmitters, since, for example, GABA characterizes
interneurons of both the telencephalon and spinal
cord.
What is known about the potential of NSC gen-
erated by the various protocols described? The NSC
produced using the RA protocol may have restricted
pluripotency, in one case to make dorsal, Pax6-positive
progenitors that differentiate in vitro into pyramidal
neurons (Bibel et al., 2004). When these progenitors
were transplanted to the embryonic chick neural tube,
102
Fig. 8.2 Mouse
ESC-derived NSC and
neurons. Nestin+ neural stem
cells derived using the
embryoid body-based defined
medium protocol, (a);
Sox1-GFP+ rosettes of
ESC-derived NSC formed
using the monolayer defined
medium protocol, (b);
III-tubulin+, (c); MAP2+
ESC-derived neurons, (d);
glutamatergic (Glu) neuron,
E; cluster of GABAergic
III-tubulin+ neurons
they showed limited capacity to differentiate, com-
pared to ESC-derived neural progenitors generated in
the absence of RA (Plachta et al., 2004). RA has
also been demonstrated to caudalize the neural tube,
as well as ESC-derived neurons (Okada et al., 2004).
Figure 8.1c, d shows III tubulin-positive and MAP2-
positive neurons as well as a glutamatergic and
GABAergic neurons derived from mouse ESC.
Although not investigated extensively, the other
neurogenesis protocols appear to produce progenitors
with anterior, dorsal properties. Treatment with growth
factors or expression of transcription factors known
to provide regional identity during embryogenesis can
be used to direct differentiation toward specific pro-
genitor and neuron fates. For example, treatment of
ESC-derived neural progenitors with Wnt3A promotes
a dorsal telencephalic fate, whereas sonic hedgehog
(Shh) promotes a ventral fate (Watanabe et al., 2005).
Approaches for the derivation of specific subtypes of
neurons, as well as the role of these cells in neuro-
logical disorders are described below. Progress in stem
cell-based therapeutics to treat these conditions is also
summarized.
8.4 Midbrain Dopaminergic Neurons
Motor control, coordinated by the striatum, is influ-
enced by dopamine release from neurons of the sub-
stantia nigra (SNi). Dopaminergic (DA) neurons of the
SNi are derived from the developing midbrain where
N. Ammon et al.
early post-mitotic neurons express tyrosine hydrox-
ylase (TH), which catalyzes the production of DA
(Marin et al., 2005). Degeneration of SNi DA neurons
has been linked to PD, and defective dopaminergic
transmission in the nucleus accumbens can contribute
to the development of depression, schizophrenia and
drug addiction (Nestler, 2000; Sulzer, 2007). As DA
neurons in the SNi deteriorate, levels of dopamine
release in the striatum plummet. This leads to motor
deficits and cognitive impairments at later stages.
Patients exhibit rigidity, tremor and dyskinesia (Samii
et al., 2004). Current treatment with L-dopa can help
reduce impairments, but eventually the drug loses effi-
cacy and the disease progresses. Understanding the
factors that influence DA neuron differentiation may
lead to better cell therapies for the treatment of PD.
Several growth factors contribute to the produc-
tion of DA neurons. During the development of
the forebrain, Shh and FGF8, which is dependent
upon Shh for its induction (Aoto et al., 2002),
are both expressed at the midbrain-hindbrain bor-
der, specifying mesencephalic progenitors (Abeliovich
and Hammond, 2007). Expression of both FGF8 and
Shh continues into adulthood in the SNi, with a
decrease of FGF8 during the onset of PD (Tanaka
et al., 2001). Also, Transforming Growth Factor-
(TGF-) family members are expressed in the floor
plate during early midbrain development and can pro-
mote the survival of cultured mesencephalic DA neu-
rons. TGF- and Shh appear to act independently of
each other for the induction of midbrain dopaminer-
gic (mDA) neurons (Roussa and Krieglstein, 2004).
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
Taken together, Shh, FGF8 and TGF- temporal and
regional expression in the early midbrain overlap,
and these factors appear to establish a niche envi-
ronment for the differentiation of DA neurons in the
midbrain.
At this point, there is a second wave of expression
of regional markers that influences the differentia-
tion of neurons in the ventral midbrain into mature
DA neurons. Precursor cells are still dividing as the
homeobox transcription factors Lmx1a and Msx1 are
expressed. Lmx1a is a crucial transcription factor for
the emergence of DA neurons in the midbrain. Loss
of function studies for Lmx1using RNAi have shown a
reduction of DA neurons in the midbrain, while gain
of function studies using ESC yield an increase of
DA neurons in vitro (Andersson et al., 2006). As DA
progenitors become post-mitotic another set of tran-
scription factors are expressed including Nurr1 and
Pitx3. Nurr1 is expressed in DA neurons in adulthood
and is responsible for vesicle packaging and axonal
transport of dopamine. When neuronal progenitors are
co-cultured with Nurr1-overexpressing astrocytes, a
fourfold increase in DA markers is observed (Chung
et al., 2002). Pitx3 expression increases following
Nurr1, is highly expressed by DA neurons in the adult
SNi, and contains a high-affinity binding site for the
TH promoter, which is expressed by mature mDA
neurons (Cazorla et al., 2000).
The pathways for in vivo differentiation of mid-
brain DA neurons can be applied in vitro to direct ESC
toward a DA neuron fate. Early methods for producing
neural progenitors from mouse ESC lines required the
addition of FGF2 and EGF, which bias the population
of neural progenitors towards a forebrain fate (Okabe
et al., 1996). Later methods utilized cAMP and ascor-
bic acid (AA) to bias in favor of a ventral phenotype
(Lee et al., 2000). As Shh and FGF8 are expressed by
the midbrain-hindbrain organizer, several studies have
shown that addition of these factors during neural dif-
ferentiation in vitro increases the yield of DA neurons
substantially (Geeta et al., 2008; Sonntag et al., 2007).
However, only 40% of the neurons were TH positive in
these cultures, possibly due to the inclusion of FGF2
in the protocols. Eliminating FGF2 and co-culturing
neural progenitors with Wnt1-overexpressing stromal
cells has been shown to dramatically increase TH-
positive populations (Castelo-Branco et al., 2003).
During early ventral midbrain development, Shh,
FGF8 and the TGF- family members cooperate to
103
establish a niche for the development of DA pre-
cursors. Neural progenitors exposed to all three fac-
tors as well as glial cell derived neurotrophic factor
(GDNF) and brain derived neurotrophic factor (BDNF)
can differentiate into cultures that are 80% DA neu-
rons (Perrier et al., 2004). It is clear from these
studies that ESC-derived neural progenitors rely on
similar environmental cues as neural progenitors in
vivo.
Utilizing transgenic approaches, recent studies have
been able to purify, via fluorescence activated cell
sorting (FACS), populations of neural progenitors that
express Pitx3. Mouse ESC-derived neural progeni-
tors that express eGFP under the Pitx3 promoter were
enriched from 2 to 3% of the total population to
over 90% after FACS (Hedlund et al., 2008). The
resulting population also expressed Nurr1, Engrailed-
1, Lmx1a, TH, l-aromatic amino acid decarboxy-
lase (AADC), and vesicular monoamine transporter
2 (VMAT2), all of which are expressed in mature
midbrain DA neurons. Future strategies for the deriva-
tion of midbrain DA neurons should focus on mark-
ers that are expressed in committed DA progenitors,
such as Limx1a. Once a transgenic line is created
with fluorescent protein expression under the con-
trol of a promoter like Limx1a, cells can be sorted
and expanded in order to produce larger numbers of
pure post-mitotic neurons that express markers for
midbrain DA neurons. The resulting cell population
would provide a highly enriched graft of DA neu-
rons that could potentially restore function and alle-
viate symptoms of PD when transplanted into the
striatum.
Animal models for PD were developed in the 1980s
when it was discovered that the drug 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively
destroys mDA neurons in rodents and primates result-
ing in hypokinesia and rigidity (Doudet et al., 1985).
Initially, cell replacement therapy for PD was tested
on patients who had ingested MPTP and devel-
oped Parkinsonian symptoms. Bilaterally grafting fetal
mesencephalic progenitors to the caudate putamen
enabled the patients to regain motor function and
reduce their L-dopa regimen (Widner et al., 1992).
However, transplantation of the same cell population
in Parkinsonian patients has yielded mixed results due
to poor grafts or subsequent inflammation (Piccini
et al., 2005). Because of the ethical complications
concerning the origin of the graft population and
104
the difficulty of obtaining a substantial amount of
DA neurons from fetal ventral mesencephalic tis-
sue for transplant, deriving a pure population of DA
neurons from ESC offers more promise for a cell
therapy.
8.5 GABAergic Interneurons
-aminobutyric acid (GABA)ergic neurons are the
major inhibitory neurons in the mammalian nervous
system. By activating GABA receptors on target glu-
tamatergic neurons, they provide inhibitory regulation
in neuronal circuits. GABAergic neurons are typi-
cally categorized based on their morphology, location,
synaptic targets (whether they synapse on the soma,
axons, or dendrites of target cells), and the calcium
binding proteins and neuropeptides they express. In
this chapter, we focus on the expression of calcium
binding proteins and neuropeptides as indicators of
interneuronal subtype generated in in vitro culture. For
a more detailed description of categorization based on
morphology and synaptic targets refer to Freund and
Buzsaki (Freund and Buzsaki, 1996).
All GABAergic neurons express the enzyme glu-
tamic acid decarboxylase (GAD), which is necessary
for synthesis of the inhibitory neurotransmitter GABA.
Different subtypes of interneurons express the calcium
binding proteins parvalbumin (PV), calretinin (CR),
or calbindin (CB) (Freund and Buzsaki, 1996). While
PV interneurons tend to be large and have elaborate
axons, CR positive interneurons are smaller, bipolar,
and are usually vertically oriented (Tamamaki
et al., 2003; Wonders and Anderson, 2006).
Interneurons are also classified based upon expression
of the neuropeptides somatostatin (SST), vasoactive
intestinal peptide (VIP), cholecystokinin (CCK) or
neuropeptide Y (NPY) (Markram et al., 2004).
As the main role of GABAergic interneurons is to
regulate neuronal activity by providing inhibition of
circuit firing, damage to or loss of these neurons can
result in uncontrolled activity of excitatory neurons.
This hyperactivity can lead to excitotoxicity and neu-
ronal death, uncontrolled body movement, and other
neurological and psychological disorders.
Many studies indicate that loss of GABAergic
interneurons is central to seizures originating in the
hippocampus in human cases of temporal lobe epilepsy
N. Ammon et al.
(TLE). In the hippocampus, a tri-synaptic circuit exists
in which excitatory signals coming from the entorhi-
nal cortex activate granule cells in the dentate gyrus.
These cells then synapse onto pyramidal cells in the
CA3 region, which in turn activate pyramidal cells in
the CA1 region. Excitatory signals from CA1 are sent
back to the entorhinal cortex and the relay continues
(Danglot et al., 2006). The GABAergic interneurons
present in the hippocampus are damaged as a result of
overactivation in this circuit (Magloczky et al., 2000).
As the loss of interneurons results in lessened inhibi-
tion of other excitatory neurons of the hippocampus,
firing continues and the interneurons are further dam-
aged (Morimoto et al., 2004). The result is seizures,
which can vary in severity and ultimately lead to
hippocampal sclerosis. GABAergic interneurons gen-
erated in vitro could be used to replace the damaged
neurons in the hippocampus, thereby alleviating the
seizures. There is hope that GABAergic interneurons
generated from human ESC could provide the neu-
ronal replacement cells to treat human patients with
TLE.
Changes in the number of specific types of
GABAergic interneurons as well as errors in their
function have also been implicated in psychological
diseases such as schizophrenia and bipolar disorder
(Benes and Berretta, 2001). In cases of schizophre-
nia, analysis of post-mortem human brain tissue
has indicated a decrease in PV- and SST-expressing
interneurons in various layers of the prefrontal cor-
tex (Hashimoto et al., 2003; Morris et al., 2008).
Another study of post-mortem tissue from both
schizophrenic and bipolar disorder patients showed
variations in number of interneurons representative
of all three calcium binding protein subtypes when
compared to control subjects (Sakai et al., 2007).
These data suggest that transplantation of healthy, in
vitro-derived GABAergic interneurons could provide
possible therapy for patients with either psychological
disorder.
Huntingtons disease is a genetic disorder caused by
a mutation in the huntingtin gene which results in an
abnormally high number of CAG repeats in the coding
sequence of the gene and aggregation of the mutant
protein product in affected cells (Walker, 2007). In
Huntingtons patients, the GABAergic medium spiny
projection neurons of the striatum (comprising about
95% of total striatal neurons) are progressively lost.
Many events have been proposed that ultimately cause
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
the death of these neurons, including excitotoxicity
and disruption of cellular metabolism (Gil and Rego,
2008). This disease is manifested by cognitive and
emotional deficits as well as progressive loss of coor-
dination and uncontrolled body movements (chorea),
eventually leading to death (Walker, 2007).
In addition to the documented degeneration of
motor neurons in the neurodegenerative disorders ALS
and spinal muscular atrophy (SMA), there has recently
been interest in the effect of these diseases on interneu-
rons present in the adult spinal cord (Stephens et al.,
2006). The GABAergic neurons of the ventral horn
of the spinal cord appear to be damaged to a similar
extent as the motor neurons in this region, indicating
that in order to provide cell therapy for these diseases,
the inhibitory neurons that regulate motor neuron
function will also need to be generated and introduced
into patients for successful treatment.
During mammalian embryonic development, cor-
tical GABAergic interneurons are born in ventral
regions of the telencephalon called the medial and cau-
dal ganglionic eminences (MGE and CGE) (Sussel
et al., 1999; Wonders and Anderson, 2006). The
interneurons, which make up about 20% of the total
cortical neuron population, then migrate tangentially
towards their final locations in the cortex. Many
steps are required for the specification of cortical
GABAergic interneurons. Activation and maintenance
of the homeodomain-containing transcription factor
Nkx2.1 in interneuron precursors comprising the MGE
is required for their initial specification, as Nkx2.1
mutant mice lack a proper MGE and have a dra-
matic reduction in GABA positive interneurons (Sussel
et al., 1999). Shh, secreted from the prechordal
mesendoderm, is involved in the activation of Nkx2.1
(Ericson et al., 1995) during early patterning and main-
tenance of the subpallium (Gulacsi and Anderson,
2006) during interneuron neurogenesis (E12 to E16 in
mouse). Proper dorsal/ventral patterning of the MGE
also involves strict regulation of gradients of BMPs
and FGF8, as loss of BMP inhibition leads to lack of
Nkx2.1 expression (Anderson et al., 2002). A recent
study on the role of Nkx2.1 in cortical interneuron
specification demonstrates that it directly activates
the LIM-homeodomain transcription factor Lhx6 (Du
et al., 2008), which is expressed in migrating PV and
SST positive interneurons and is believed to be essen-
tial for proper GABAergic interneuron migration from
the MGE and incorporation into the cortex (Liodis
105
et al., 2007). Interestingly, CR-positive interneurons
appear to be specified independent of Nkx2.1 activity
as they are still generated in Nkx2.1 mutant mice
(Nery et al., 2002). Strong evidence exists that PV-
and SST-positive interneurons derive from the MGE,
whereas CR-positive interneurons derive from the
Nkx2.1-negative CGE (Xu et al., 2004). CR-positive
interneurons appear to be more dependent on activity
of the distal-less homeobox transcription factor Dlx1/2
for their specification, as they are nearly eliminated
in Dlx1/2 null mice (Xu et al., 2004) while SST- and
NPY-positive interneurons are only reduced in num-
ber. Dlx1/2 is required in all interneurons arising from
both the MGE and CGE for proper migration into cor-
tical regions (Anderson et al., 1999; Panganiban and
Rubenstein, 2002).
GABAergic interneurons present in the mammalian
spinal cord are specified during neural tube pattern-
ing in response to opposing gradients of BMPs from
the overlying ectoderm and roof plate and Shh from
the notochord (Briscoe and Ericson, 1999; Helms
and Johnson, 2003; Jessell, 2000). The dorsal half of
the spinal cord contains eight populations of neurons
referred to as dI1 through dI6 (from dorsal to ventral)
and dILA and dILB , which are subsequently born in
the region of dI4 through dI6. The ventral spinal cord
is comprised of five domains of neurons; three pools
of ventral interneurons called V0 through V3 and a
pool of motor neurons (MN). Of the thirteen total neu-
ronal populations present in the spinal cord, only the
dI4, dI6, dILA , V0, and V1 neurons are GABAergic
inhibitory neurons (Glasgow et al., 2005; Wenner et al.,
2000). The early transcription factor profile of the pro-
genitors for the six dorsal populations includes Math1
(expressed by dI1 progenitors), Ngn1/2 (expressed by
dI2 and dI6 progenitors), Dbx2 (expressed by dI6
progenitors), and Mash1 and Pax7 (expressed by dI3
through dI5 progenitors) (Helms and Johnson, 2003;
Muller et al., 2002; Wilson and Maden, 2005). The
progenitors of V0 and V1 GABAergic neurons in the
ventral neural tube express Dbx2, Pax6, Irx3, and
Dbx1 (the latter only in V0 progenitors)(Briscoe and
Ericson, 2001). The early and late born GABAergic
neurons themselves go on to express varied transcrip-
tion factors such as Lbx1, Isl1/2, Lim1/2, Lmx1b,
Brn3a, Evx1/2, and En1 (Muller et al., 2002; Wilson
and Maden, 2005). Pax2 and transcription factors,
such as Ptf1, Lhx1, and Lhx5 that regulate its expres-
sion in the spinal cord, are necessary for GABAergic
106
specification of spinal cord progenitors (Cheng et al.,
2004, 2005; Glasgow et al., 2005; Pillai et al., 2007).
Based on knowledge of how GABAergic interneu-
rons develop during embryogenesis, protocols for in
vitro differentiation from pluripotent ESC have been
developed to enrich for these neurons. One such
protocol makes use of the embryoid body interme-
diate method of deriving NSC from mouse ESC
(Okabe et al., 1996). Following expansion of NSC
in FGF2, growth factors were withdrawn and cells
were differentiated in neurobasal medium supple-
mented with B27 and fetal calf serum (Okabe et al.,
1996; Westmoreland et al., 2001). This differentiation
method resulted in cultures containing cells that were
positive for the GAD67 protein, which is necessary
for production and packaging of GABA. Sequential
treatment of ESC-derived NSC with Shh and FGF8 fol-
lowed by withdrawal of these mitogens and treatment
with neurotrophin 4 (NT-4) and BDNF has improved
upon this method, resulting in greater than 60% of
total neurons generated that were positive for GABA
(Barberi et al., 2003).
Efforts have also been made to derive GABAergic
interneurons specific to the dorsal spinal cord
(Murashov et al., 2004). By mimicking the patterning
of spinal interneuron progenitors using BMP signal-
ing and treatment with Wnts, which are believed to act
downstream of BMP in dorsal spinal cord specification
(Muroyama et al., 2002), ESC can be differentiated
into Lim2-positive neurons that also express GAD67
at a rate of about 55% of total neurons produced
(Murashov et al., 2004). This protocol involves sequen-
tial treatment with RA to induce a caudalized fate,
recombinant Shh, BMP2, and Wnt3a. Importantly,
treatment of the NSC with these factors individu-
ally was observed to be inefficient at inducing dorsal
GABAergic interneuron fate. The order and timing of
treatment is critical to reproducing the appropriate in
vivo environment.
Cell transplantation studies for Huntingtons dis-
ease began in the 1980s using fetal striatal cells and
were shown to generate healthy grafts and improve
motor function in both rodent and primate models
of Huntingtons disease and later in human patients
(Freeman et al., 2000, 1995; Hantraye et al., 1992;
Isacson et al., 1986; Kendall et al., 1998). The use of
fetal derived striatal neurons for transplantation faces
many ethical issues and is not practical for widespread
use in the treatment of Huntingtons disease. Human
N. Ammon et al.
and mouse adult NSC have also been used in animal
models of Huntingtons disease and have demonstrated
varying degrees of graft incorporation and differentia-
tion of the cells into more mature neurons (Clelland
et al., 2008). Adult NSC need to be obtained from
human patients by biopsy and are not as plentiful
or malleable in culture as ESC. This puts further
emphasis on our need to better understand the in vivo
development of striatal GABAergic neurons so that
ESC-derived neurons can efficiently be generated for
transplantation approaches.
Transplantation paradigms for treatment of epilepsy
have focused mainly on transplanting multipotent NSC
obtained from embryonic mouse or human brain tis-
sue into the hippocampus or other brain regions to
alleviate seizure activity. Replacement of GABA inhi-
bition in epileptic hippocampi seems to be critical for
preventing seizures by inhibiting the overactivity of
the hippocampal circuitry characteristic of epilepsy.
Previous studies have demonstrated that transplanted
embryonic or adult NSC are able to survive to vari-
ous extents, differentiate into a selection of neuronal
subtypes including astrocytes and some GABAergic
neurons, and provide initial relief of seizure activity
(Chu et al., 2004; Hattiangady et al., 2008; Shetty and
Hattiangady, 2007). As ESC can proliferate in culture
to generate large numbers of cells, and many pro-
tocols have been devised to differentiate these ESC
into defined neuronal lineages, transplantation studies
using ESC-derived NSC from mouse and human have
been increasing. Interestingly, it appears that the spe-
cific location into which NSC are transplanted plays
a role in determining what type of neurons they will
mature into, as the host environment may send sig-
nals to the transplanted cells to instruct their fate
(Carpentino et al., 2008). This indicates that NSC may
need to be patterned into specific progenitors (such as
GABAergic progenitors) or into more mature neurons
in order to generate the cells of interest following trans-
plantation. To date, there are no reports of ESC-derived
mature GABAergic neurons transplanted into epilepsy
animal models.
8.6 Spinal Cord Motor Neurons
Motor neurons are generally cholinergic neurons and
project from the four sections of the spinal cord
(cervical, thoracic, lumbar, and sacral) and innervate
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
musculature of the body needed for locomotion
(somatic motor neurons) or various smooth muscle
organs and glands (visceral motor neurons). These
neurons either project from the brain stem to other
motor neurons of the spinal cord, or directly synapse
onto their target muscles to produce neuromuscu-
lar junctions where neurotransmitters such as acetyl-
choline are released. Loss of muscle innervation
by motor neurons can lead to muscle atrophy, and
improper regulation of motor neuron activity can cause
uncontrolled muscle contraction.
Cell replacement therapy using ESC-derived motor
neurons has potential for treatment of patients suffer-
ing from motor neuron diseases such as ALS and SMA
as well as SCI (Hedlund et al., 2007). ALS, a late-
onset disease typically affecting middle-aged people,
is characterized by loss of motor neurons in the cere-
bral cortex, brain stem, and spinal cord (Boillee et al.,
2006; Mitchelle and Borasio, 2007). In SMA, which
mostly affects infants, the spinal cord motor neurons
selectively degenerate (Lunn and Wang, 2008). As
these neurons degenerate, the muscles that they inner-
vate lose function, resulting in paralysis and muscle
deterioration. While many drug therapies have been
proposed, most offer little or no benefit to ALS and
SMA patients. Cell therapy, however, could combine
transplantation of ESC-derived neurons with growth
factor treatments, potentially holding more promise
(Boillee et al., 2006; Hedlund et al., 2007; Lunn and
Wang, 2008).
SCIs occur mostly in young adults, however they
can affect people of all ages. In SCI, compression
to or lesion of the spinal cord can occur at various
levels of the spinal cord, often resulting in paralysis
below the point of injury. Following initial damage,
inflammation, ischemia, and glial scarring occur, ulti-
mately leading to loss of myelination and neuronal
death (Schwab and Bartholdi, 1996). The ability to
implant healthy neurons near the site of SCI and direct
growth of their axons to restore neurological and motor
activity is the ultimate goal of stem cell therapy for
SCI.
Motor neurons are specified in the ventral half of
the posterior developing neural tube in response to
patterning cues emanating from the floor plate and
the underlying notochord. The main factor involved
in patterning the ventral neural tube is Shh, which is
produced by both the notochord and floor plate and is
distributed in a gradient along the dorsal ventral axis
107
of the neural tube, with the highest concentration in
the most ventral region (Roelink et al., 1995). The
varying concentrations of Shh present along the DV
axis are responsible for activation of specific home-
obox transcription factors in each of the five domains
of the ventral neural tube termed V0, V1, V2, MN,
and V3 (from dorsal to ventral). The combination of
transcription factors indicates the type of neuron that
will develop from progenitors in each region (Briscoe
et al., 2000; Wilson and Maden, 2005). The homeobox
transcription factors Pax6 and Nkx2.2 are expressed
in response to Shh, with Pax6 expressed in a high
to low gradient from dorsal to ventral and Nkx2.2
expressed in the ventral-most region where Pax6 is
absent (Ericson et al., 1997; Price and Briscoe, 2004).
Motor neurons are specified from progenitors (pMN)
in the ventral neural tube that express the transcription
factors Olig2, Nkx6.1, and Pax6 (Briscoe et al., 2000;
Wilson and Maden, 2005). As these motor neurons
mature, they go on to express the molecular markers
MNR2 and HB9 (Wilson and Maden, 2005).
In vitro protocols aimed at directing ESC toward
a motor neuron fate take advantage of the depen-
dence of motor neuron progenitors on distinct con-
centrations of Shh. A protocol established by the
Jessell group (Wichterle et al., 2002) demonstrated
that in order to generate motor neurons from mouse
ESC, three steps were necessary. First, ESC are dif-
ferentiated into neuroectoderm via embryoid body
formation. These neurectodermal cells are then cau-
dalized by RA treatment to give them spinal cord
identity. Lastly, a ventral motor neuron identity is
induced by treatment with either Shh peptide or a
synthetic Hh agonist (Wichterle et al., 2002). Similar
protocols have been developed working with human
ESC (Lee et al., 2007; Li et al., 2005) with some
modifications, including treatment with BDNF and
GDNF (Lee et al., 2007). Recently, motor neurons
have been derived, using the above guidelines, from
induced pluripotent stem (iPS) cells that were gener-
ated from fibroblast cells of an elderly patient with
ALS (Dimos et al., 2008). This work will allow fur-
ther study of the progression of motor neuron diseases
like ALS and improve our ability to produce the proper
cell types and conditions needed for effective cell
therapy.
Several transplantation studies using animal mod-
els of ALS, SMA, and SCI have had some success in
functional incorporation of transplanted ESC-derived
108
motor neurons and other stem cells including mes-
enchymal stem cells and adult NSC (Deshpande
et al., 2006; Harper et al., 2004; Hedlund et al.,
2007; Mazzini et al., 2008; Vercelli et al., 2008).
In a recent study using a rat motor neuron disease
model in which ventral motor neurons are destroyed
by the Neuroadapted Sindbis virus, mouse ESC-
derived motor neurons transplanted into the spinal cord
were able to survive and form functional neuromus-
cular junctions with skeletal muscle (Deshpande et
al., 2006). Some treated rats even exhibited hindlimb
improvement. In another study using human ESC-
derived motor neurons transplanted into rodent spinal
cord, cells were able to form healthy grafts, send
processes out from the graft site, and continued to
mature in vivo (Lee et al., 2007). Further stud-
ies are needed to demonstrate whether these human
ESC-derived neurons can make functional connections
with the musculature and relieve symptoms of motor
neuron diseases.
8.7 Serotonergic Neurons
The dorsal Raphe nuclei are comprised of about 20,000
serotonergic (5-HT) neurons that project to most areas
of the brain. Because of their extensive innervation,
5-HT neurons can influence many modes of behav-
ior and homeostasis. Disruptions of 5-HT signaling
characterize many neuropsychiatric disorders such as
depression, schizophrenia and bipolar disorder. Recent
reports have shown dystrophic 5-HT axons in patients
with PD, frontal lobe dementia and Lewy-body demen-
tia (Azmitia and Nixon, 2008). As conventional med-
ication does little to repair or replace atrophied 5-HT
neurons, cell therapy may become an invaluable tool.
Neural progenitors caudal to the midbrain-hindbrain
border give rise to the pool of 5-HT neurons in the
adult brain. Similar to the induction of DA progenitors,
Shh and FGF8 play roles in 5-HT neuron specification,
as does FGF4 (Ye et al., 1998). Together, these fac-
tors define the initial inductive center of 5-HT neuronal
differentiation. Pet1 is an important transcription fac-
tor that marks early 5-HT progenitors into adulthood.
Mutations of Pet1 in mice lead to a severe reduction of
5-HT neurons (Hendricks et al., 2003). In addition, the
ablation of Lmx1b leads to the absence of 5-HT neu-
rons in the brain (Ding et al., 2003). These two factors
N. Ammon et al.
are dependent upon Gata2, which, in chicks, is suffi-
cient for the induction of both Pet1 and Lmx1b (Craven
et al., 2004). Not all neurons from this developmental
niche differentiate into 5-HT neurons. The repression
of this fate is dependent on the action of the transcrip-
tion factor Phox2b, as loss of function of Phox2b leads
to a vast increase in the number of 5-HT neurons in the
brain (Pattyn et al., 2003).
Thus far, there have been few attempts to dif-
ferentiate 5-HT neurons from ES cells lines. Most
protocols that influence the production of DA neurons
result in the formation of a small number (23%) of
5-HT neurons. This is most likely due to the addi-
tion of Shh and FGF8, crucial in both DA and 5-HT
differentiation. Induction of 5-HT neurons from mono-
layer ESC cell lines occurs in the presence of Shh,
FGF2 and BDNF (Ying et al., 2003). However, these
conditions produce only small numbers of 5-HT neu-
rons. From mouse ESC, optimal enrichment for 5-HT
neurons resulted from co-culture on a stromal cell layer
and treatment with Shh and FGF4 several days before
exposure to FGF8 (Barberi et al., 2003). Recently,
5-HT neurons were derived from human ESC with
a somewhat greater efficiency (Kumar et al., 2008).
Sixty percent of the neurons in these cultures were
5-HT positive.
Clues of how to derive purer populations of 5-HT
neurons from ESC may be gained from observing how
these cells arise during development. DA and 5-HT
neurons share similar initial induction steps. From here
the two populations diverge. DA neuron differentia-
tion relies upon the expression of Pitx3 and Lmx1a,
whereas 5-HT neurons require the expression of Pet1
and Lmx1b, both of which are controlled by Gata2.
Future strategies targeting 5-HT populations could
focus on both driving the expression of 5-HT pro-
genitor transcription factors, through proper timing of
FGF4 and/or other growth factors or by introducing
genetic modifications that increase the expression of
Pet1 and Lmx1b, and inhibiting the production of DA
progenitors.
5-HT neurons have not been widely used in
transplantation studies. It has been shown, however,
that 5-HT increases the proliferation and survival of
transplanted neuroepithelial cells into the primordial
neocortex of rats (Petrova and Otellin, 2007). Thus
transplantation of 5-HT neurons could function to
improve engraftment of other cell populations, such
as DA neurons in patients with PD or GABAergic
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
interneurons in patients with TLE. It is also possible
that 5-HT neurons may be used to treat neuropsychi-
atric disorders without the risk of side effects from
conventional treatments. As increases in synaptic 5-
HT are linked to the function of serotonin selective
reuptake inhibitors (SSRIs), transplanting 5-HT neu-
rons directly into brain regions like the hippocampus
and the anterior cingulated cortex may decrease the
severity of the disorder when drug regimens fail.
8.8 Basal Forebrain Cholinergic Neurons
Cholinergic neurons in the CNS contribute to
the regulation of cortical activation, behavioral
arousal and memory formation. Located in the basal
forebrain, clusters of cholinergic neurons project axons
into the limbic system, including the amygdala and
the hippocampus, and all cortical areas. Cholinergic
neurons play an important role in learning and mem-
ory formation whereby acetylcholine (ACh) release
can contribute to long-term potentiation and affect the
plasticity of synapses of neurons in the hippocam-
pus (Sarter and Bruno, 2000). Activity of cholinergic
neurons is increased during arousal and attention.
Diseases associated with memory deficits and
dementia often involve the degeneration of choliner-
gic neurons. In Parkinsonian Disease Dementia (PDD),
cholinergic deficits appear early in the disease and
are widespread throughout the brain. The loss of
cholinergic innervation in the cortex and hippocam-
pus is often associated with the formation of Lewy
Bodies, aggregates of protein in the cell bodies of
neurons. Plaque formation, a build up of amyloid-
peptide, is minimal in PDD patient brains when
compared to the brains of patients with AD (Farlow
and Cummings, 2008). Plaque formation during the
progression of AD increases in the medial tempo-
ral lobe and cortical structures. In addition, cognitive
deficits in patients with AD appear when cholin-
ergic transduction declines. The neuropathology of
AD shows an increased loss of cholinergic neu-
rons in the basal forebrain and hippocampus lead-
ing to deficits in cognition, memory, language and
behavior.
Most cholinergic neurons in the forebrain, found in
the striatum, can be classified into two distinct groups:
cholinergic interneurons and cholinergic projection
109
neurons. Cholinergic progenitors arise in the MGE, a
region that expresses the transcription factor Nkx2.1
and is exposed to Shh released from the prechordal
plate. In addition to cholinergic progenitors, the MGE
gives rise to the majority of GABAergic interneu-
rons, as previously described. Nkx2.1 represses dor-
sal transcription factors like Pax6 while enhancing
the expression of the LIM-homeodomain transcription
factors Lhx6 and Lhx8 (also known as Lhx7 and L3)
(Sussel et al., 1999). While Lhx6 is critical for the for-
mation of GABAergic interneurons and is expressed
by cholinergic progenitors, only Lhx8 is critical to
the development of forebrain cholinergic neurons.
Lhx8 deficient mice completely lose both the cholin-
ergic interneurons and cholinergic projection neurons
of the striatum (Fragkouli et al., 2005; Zhao et al.,
2003).
To date, protocols for the efficient derivation of
cholinergic neurons from ESC are lacking. It has been
observed that several factors can increase the pop-
ulation of cholinergic neurons from varying starting
cell populations. Fetal basal forebrain tissue from rats
exposed in utero to BNDF followed by later addition of
nerve growth factor (NGF) increased the emergence of
cholinergic neurons (Knusel et al., 1991). Endogenous
adult NSC were induced to a cholinergic neuron fate
with both NGF and RA in rats with a lesioned basal
forebrain (Calza et al., 2003). In mouse ESC, RA
also increases the yield of cholinergic neurons while
expanding the population of GAD67 positive interneu-
rons (Okada et al., 2008). As both cholinergic and
GABAergic neurons develop in the same regional
niche in vitro, factors that encourage the differentia-
tion of one cell type should also bias in favor of the
differentiation of the other in vitro. In order to select
for cholinergic neurons over GABAergic interneurons,
it will be necessary to identify the conditions that
increase the expression of Lhx8. In addition, pure
population of cholinergic neurons could be induced
using a bacterial artifical chromosome (BAC) encod-
ing Lhx8. As cholinergic progenitors are derived, cell
sorting techniques could be employed to enhance the
cholinergic population for transplantation.
ESC-derived cholinergic neurons could provide a
valuable tool for treating patients with AD, as the
loss of cholinergic innervation leads to the most pro-
nounced symptoms of the disease. Using mouse adult
NSC, cortical grafts differentiated into cholinergic
neurons and improved working memory in a mouse
110
model of AD (Wang et al., 2006). Additionally, human
NSC transplantation also leads to a reduction in work-
ing memory error as the transplanted cells migrate
through the cortex and hippocampus (Marutle et al.,
2007). While transplanting NSC improves the cogni-
tive abilities in mouse models, the grafted cells also
differentiate into glia and neurons that are not cholin-
ergic. By focusing on producing pure populations of
cholinergic neurons for engraftment, the beneficial
effects of ESC-derived transplants could be increased.
8.9 Conclusions
The next decade may well see the application of human
ESC-based therapies. It is likely that neurological dis-
orders will be among the first conditions treated, and
with care taken to avoid the potential pitfalls, we will
hopefully enter a new era of regenerative medicine.
References
Abeliovich A, Hammond R (2007) Midbrain dopamine neuron
differentiation: factors and fates. Dev Biol 304: 447454.
Ahn S, Joyner AL (2005) In vivo analysis of quiescent adult
neural stem cells responding to Sonic hedgehog. Nature 437:
894897.
Anderson RM, Lawrence AR, Stottmann RW, Bachiller D,
Klingensmith J (2002) Chordin and noggin promote orga-
nizing centers of forebrain development in the mouse.
Development 129: 49754987.
Anderson S, Mione M, Yun K, Rubenstein JL (1999) Differential
origins of neocortical projection and local circuit neurons:
role of Dlx genes in neocortical interneurogenesis. Cereb
Cortex 9: 646654.
Andersson E, Tryggvason U, Deng Q, Friling S, Alekseenko Z,
Robert B, Perlmann T, Ericson J (2006) Identification of
intrinsic determinants of midbrain dopamine neurons. Cell
124: 393405.
Aoto K, Nishimura T, Eto K, Motoyama J (2002) Mouse GLI3
regulates Fgf8 expression and apoptosis in the developing
neural tube, face, and limb bud. Dev Biol 251: 320332.
Azmitia EC, Nixon R (2008) Dystrophic serotonergic axons in
neurodegenerative diseases. Brain Res 1217: 185194.
Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI (1995)
Embryonic stem cells express neuronal properties in vitro.
Dev Biol 168: 342357.
Baker JC, Beddington RS, Harland RM (1999) Wnt signaling
in Xenopus embryos inhibits bmp4 expression and activates
neural development. Genes Dev 13: 31493159.
Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H,
Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N, Tabar V,
Harrison NL, Beal MF, Moore MAS, Studer L (2003) Neural
N. Ammon et al.
subtype specification of fertilization and nuclear transfer
embryonic stem cells and application in parkinsonian mice.
Nat Biotechnol 21: 12001206.
Benes FM, Berretta S (2001) GABAergic interneurons: implica-
tions for understanding schizophrenia and bipolar disorder.
Neuropsychopharmacology 25: 127.
Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M,
Goetz M, Barde YA (2004) Differentiation of mouse embry-
onic stem cells into a defined neuronal lineage. Nat Neurosci
7: 10031009.
Boillee S, Velde CV, Cleveland DW (2006) ALS: a disease of
motor neurons and ther nonneuronal neighbors. Neuron 52:
3959.
Briscoe J, Ericson J (1999) The specification of neuronal identity
by graded sonic hedgehog signalling. Semin Cell Dev Biol
10: 353362.
Briscoe J, Ericson J (2001) Specification of neuronal fates in the
ventral neural tube. Curr Opin Neurobiol 11: 4349.
Briscoe J, Pierani A, Jessell TM, Ericson J (2000) A home-
odomain protein code specifies progenitor cell identity and
neuronal fate in the ventral neural tube. Cell 101: 435445.
Brustle O, Spiro AC, Karram K, Choudhary K, Okabe S, McKay
RD (1997) In vitro-generated neural precursors participate in
mammalian brain development. Proc Natl Acad Sci U S A
94: 1480914814.
Cai C, Grabel L (2007) Directing the differentiation of embry-
onic stem cells to neural stem cells. Dev Dyn 236:
32553266.
Calza L, Giuliani A, Fernandez M, Pirondi S, DIntino G,
Aloe L, Giardino L (2003) Neural stem cells and choliner-
gic neurons: regulation by immunolesion and treatment with
mitogens, retinoic acid, and nerve growth factor. Proc Natl
Acad Sci U S A 100: 73257330.
Carpentino JE, Hartman NW, Grabel LB, Naegele JR (2008)
Region-specific differentiation of embryonic stem cell-
derived neural progenitor transplants into the adult mouse
hippocampus following seizures. J Neurosci Res 86:
512524.
Castelo-Branco G, Wagner J, Rodriguez FJ, Kele J, Sousa K,
Rawal N, Pasolli HA, Fuchs E, Kitajewski J, Arenas E
(2003) Differential regulation of midbrain dopaminergic neu-
ron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc Natl
Acad Sci U S A 100: 1274712752.
Cazorla P, Smidt MP, OMalley KL, Burbach JP (2000) A
response element for the homeodomain transcription fac-
tor Ptx3 in the tyrosine hydroxylase gene promoter. J
Neurochem 74: 18291837.
Cheng L, Arata A, Mizuguchi R, Qian Y, Karunaratne A, Gray
PA, Arata S, Shirasawa S, Bouchard M, Luo P, et al. (2004)
Tlx3 and Tlx1 are post-mitotic selector genes determining
glutamatergic over GABAergic cell fates. Nat Neurosci 7:
510517.
Cheng L, Samad OA, Xu Y, Mizuguchi R, Luo P, Shirasawa S,
Goulding M, Ma Q (2005) Lbx1 and Tlx3 are opposing
switches in determining GABAergic versus glutamatergic
transmitter phenotypes. Nat Neurosci 8: 15101515.
Chu K, Kim M, Jung KH, Jeon D, Lee ST, Kim J, Jeong SW,
Kim SU, Lee SK, Shin HS et al. (2004) Human neural stem
cell transplantation reduces spontaneous recurrent seizures
following pilocarpine-induced status epilepticus in adult rats.
Brain Res 1023: 213221.
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
Chung S, Sonntag KC, Andersson T, Bjorklund LM, Park JJ,
Kim DW, Kang UJ, Isacson O, Kim KS (2002) Genetic engi-
neering of mouse embryonic stem cells by Nurr1 enhances
differentiation and maturation into dopaminergic neurons.
Eur J Neurosci 16: 18291838.
Clelland CD, Barker RA, Watts C (2008) Cell therapy in
Huntington disease. Neurosurg Focus 24: E9.
Craven SE, Lim KC, Ye W, Engel JD, de Sauvage F, Rosenthal A
(2004) Gata2 specifies serotonergic neurons downstream of
sonic hedgehog. Development 131: 11651173.
Danglot L, Triller A, Marty S (2006) The development of
hippocampal interneurons in rodents. Hippocampus 16:
10321060.
De Robertis EM, Kuroda H (2004) Dorsal-ventral patterning and
neural induction in Xenopus embryos. Annu Rev Cell Dev
Biol 20: 285308.
Deacon T, Dinsmore J, Costantini LC, Ratliff J, Isacson O (1998)
Blastula-stage stem cells can differentiate into dopaminergic
and serotonergic neurons after transplantation. Exp Neurol
149: 2841.
Deshpande DM, Kim Y-S, Martinez T, Carmen J, Dike S,
Shats I, Rubin LL, Drummond J, Krishnan C, Hoke A, et al.
(2006) Recovery from paralysis in adult rats using embryonic
stem cells. Ann Neurol 60: 3244.
Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto
H, Chung W, Croft GF, Saphier G, Leibel R, Goland R,
et al. (2008) Induced pluripotent stem cells generated from
patients with ALS can be differentiated into motor neurons.
Science 321: 12181221.
Ding YQ, Marklund U, Yuan W, Yin J, Wegman L, Ericson J,
Deneris E, Johnson RL, Chen ZF (2003) Lmx1b is essential
for the development of serotonergic neurons. Nat Neurosci 6:
933938.
Doudet D, Gross C, Lebrun-Grandie P, Bioulac B (1985) MPTP
primate model of Parkinsons disease: a mechanographic and
electromyographic study. Brain Res 335: 194199.
Du T, Xu Q, Ocbina PJ, Anderson SA (2008) Nkx2.1 specifies
cortical interneuron fate by activating Lhx6. Development
135: 15591567.
Ericson J, Muhr J, Placzek M, Lints T, Jessell T, Edlund T (1995)
Sonic hedgehog induces the differentiation of ventral fore-
brain neurons: a common signal for ventral patterning within
the neural tube. Cell 81: 747756.
Ericson J, Rashbass P, Schedl A, Brenner-Morton S,
Kawakami A, Heyningen Vv, Jessell T, Briscoe J (1997)
Pax6 controls progenitor cell identity and neuronal fate in
response to graded Shh signaling. Cell 90: 169180.
Farlow MR, Cummings J (2008) A modern hypothesis: the dis-
tinct pathologies of dementia associated with Parkinsons
disease versus Alzheimers disease. Dement Geriatr Cogn
Disord 25: 301308.
Fragkouli A, Hearn C, Errington M, Cooke S, Grigoriou M,
Bliss T, Stylianopoulou F, Pachnis V (2005) Loss of fore-
brain cholinergic neurons and impairment in spatial learning
and memory in LHX7-deficient mice. Eur J Neurosci 21:
29232938.
Freeman TB, Cicchetti F, Hauser RA, Deacon TW, Li XJ, Hersch
SM, Nauert GM, Sanberg PR, Kordower JH, Saporta S, et al.
(2000) Transplanted fetal striatum in Huntingtons disease:
phenotypic development and lack of pathology. Proc Natl
Acad Sci U S A 97: 1387713882.
111
Freeman TB, Sanberg PR, Isacson O (1995) Development of the
human striatum: implications for fetal striatal transplantation
in the treatment of Hungtingtons disease. Cell Transplant 4:
539545.
Freund T, Buzsaki G (1996) Interneurons of the Hippocampus.
Hippocampus 6: 347470.
Geeta R, Ramnath RL, Rao HS, Chandra V (2008) One year
survival and significant reversal of motor deficits in parkin-
sonian rats transplanted with hESC derived dopaminergic
neurons. Biochem Biophys Res Commun 373: 258264.
Gil JM, Rego AC (2008) Mechanisms of neurodegeneration in
Huntingtons disease. Eur J Neurosci 27: 28032820.
Glasgow SM, Henke RM, Macdonald RJ, Wright CV, Johnson
JE (2005) Ptf1a determines GABAergic over glutamatergic
neuronal cell fate in the spinal cord dorsal horn. Development
132: 54615469.
Gottlieb DI (2002) Large-scale sources of neural stem cells.
Annu Rev Neurosci 25: 381407.
Gruen L, Grabel L (2006) Concise review: scientific and ethi-
cal roadblocks to human embryonic stem cell therapy. Stem
Cells 24: 21622169.
Guan K, Chang H, Rolletschek A, Wobus AM (2001) Embryonic
stem cell-derived neurogenesis. Retinoic acid induction and
lineage selection of neuronal cells. Cell Tissue Res 305:
171176.
Gulacsi A, Anderson SA (2006) Shh maintains Nkx2.1 in the
MGE by a Gli3-independent mechanism. Cereb Cortex 16:
i89i95.
Hantraye P, Riche D, Maziere M, Isacson O (1992) Intrastriatal
transplantation of cross-species fetal striatal cells reduces
abnormal movements in a primate model of Huntington
disease. Proc Natl Acad Sci U S A 89: 41874191.
Harper JM, Krishnan C, Darman JS, Deshpande DM, Peck S,
Shats I, Backovic S, Rothstein JD, Kerr DA (2004) Axonal
growth of embryonic stem cell-derived motoneurons in vitro
and in motoneuron-injured adult rats. Proc Natl Acad Sci
U S A 101: 71237128.
Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z,
Sampson AR, Lewis DA (2003) Gene expression deficits
in a subclass of GABA neurons in the prefrontal cortex of
subjects with schizophrenia. J Neurosci 23: 63156326.
Hattiangady B, Rao MS, Shetty AK (2008) Grafting of striatal
precursor cells into hippocampus shortly after status epilep-
ticus restrains chronic temporal lobe epilepsy. Exp Neurol
212: 468481.
Hedlund E, Hefferan MP, Marsala M, Isacson O (2007) Cell
therapy and stem cells in animal models of motor neuron
disorders. Eur J Neurosci 26: 17211737.
Hedlund E, Pruszak J, Lardaro T, Ludwig W, Vinuela A,
Kim KS, Isacson O (2008) Embryonic stem cell-derived
Pitx3-enhanced green fluorescent protein midbrain dopamine
neurons survive enrichment by fluorescence-activated cell
sorting and function in an animal model of Parkinsons
disease. Stem Cells 26: 15261536.
Helms AW, Johnson JE (2003) Specification of dorsal spinal
cord interneurons. Curr Opin Neurobiol 13: 4249.
Hendricks TJ, Fyodorov DV, Wegman LJ, Lelutiu NB, Pehek
EA, Yamamoto B, Silver J, Weeber EJ, Sweatt JD, Deneris
ES (2003) Pet-1 ETS gene plays a critical role in 5-HT neu-
ron development and is required for normal anxiety-like and
aggressive behavior. Neuron 37: 233247.
112
Isacson O, Dunnett SB, Bjorklund A (1986) Graft-induced
behavioral recovery in an animal model of Huntington dis-
ease. Proc Natl Acad Sci U S A 83: 27282732.
Jessell TM (2000) Neuronal specification in the spinal cord:
inductive signals and transcriptional codes. Nat Rev Genet
1: 2029.
Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y,
Nakanishi S, Nishikawa SI, Sasai Y (2000) Induction of
midbrain dopaminergic neurons from ES cells by stromal
cell-derived inducing activity. Neuron 28: 3140.
Keller G (2005) Embryonic stem cell differentiation: emer-
gence of a new era in biology and medicine. Genes Dev 19:
11291155.
Kendall AL, Rayment FD, Torres EM, Baker HF, Ridley RM,
Dunnett SB (1998) Functional integration of striatal allo-
grafts in a primate model of Huntingtons disease. Nat Med
4: 727729.
Knusel B, Winslow JW, Rosenthal A, Burton LE, Seid DP,
Nikolics K, Hefti F (1991) Promotion of central choliner-
gic and dopaminergic neuron differentiation by brain-derived
neurotrophic factor but not neurotrophin 3. Proc Natl Acad
Sci U S A 88: 961965.
Kumar M, Kaushalya SK, Gressens P, Maiti S, Mani S (2008)
Optimized derivation and functional characterization of 5-
HT neurons from human embryonic stem cells. Stem Cells
Dev.
Lee H, Shamy GA, Elkabetz Y, Schofield CM, Harrison
NL, Panagiotakos G, Socci ND, Tabar V, Studer L
(2007) Directed differentiation and transplantation of human
embryonic stem cell-derived motoneurons. Stem Cells 25:
19311939.
Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD
(2000) Efficient generation of midbrain and hindbrain neu-
rons from mouse embryonic stem cells. Nat Biotechnol 18:
675679.
Li XJ, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, Pearce
RA, Zhang SC (2005) Specification of motoneurons from
human embryonic stem cells. Nat Biotechnol 23: 215221.
Lie DC, Colamarino SA, Song HJ, Desire L, Mira H,
Consiglio A, Lein ES, Jessberger S, Lansford H, Dearie
AR, et al. (2005) Wnt signalling regulates adult hippocampal
neurogenesis. Nature 437: 13701375.
Liodis P, Denaxa M, Grigoriou M, Akufo-Addo C, Yanagawa Y,
Pachnis V (2007) Lhx6 activity is required for the neuronal
migration and specification of cortical interneuron subtypes.
J Neurosci 27: 30783089.
Lunn MR, Wang CH (2008) Spinal muscular atrophy. Lancet
371: 21202133.
Machold R, Hayashi S, Rutlin M, Muzumdar MD, Nery S,
Corbin JG, Gritli-Linde A, Dellovade T, Porter JA, Rubin
LL, et al. (2003) Sonic hedgehog is required for progenitor
cell maintenance in telencephalic stem cell niches. Neuron
39: 937950.
Magloczky Z, Wittner L, Borhegyi Z, Halasz P, Vajda J,
Czirjak S, Freund T (2000) Changes in the distribution and
connectivity of interneurons in the epileptic human dentate
gyrus. Neuroscience 96: 725.
Marin F, Herrero MT, Vyas S, Puelles L (2005) Ontogeny of
tyrosine hydroxylase mRNA expression in mid- and fore-
brain: neuromeric pattern and novel positive regions. Dev
Dyn 234: 709717.
N. Ammon et al.
Markram H, Toledo-Rodriguez M, Wang Y, Gupta A,
Silberberg G, Wu C (2004) Interneurons of the neocortical
inhibitory system. Nat Rev Neurosci 5: 793807.
Martin GR, Wiley LM, Damjanov I (1977) The development of
cystic embryoid bodies in vitro from clonal teratocarcinoma
stem cells. Dev Biol 61: 230244.
Marutle A, Ohmitsu M, Nilbratt M, Greig NH, Nordberg A,
Sugaya K (2007) Modulation of human neural stem cell
differentiation in Alzheimer (APP23) transgenic mice by
phenserine. Proc Natl Acad Sci U S A 104: 1250612511.
Maye P, Becker S, Siemen H, Thorne J, Byrd N, Carpentino J,
Grabel L (2004) Hedgehog signaling is required for the dif-
ferentiation of ES cells into neurectoderm. Dev Biol 265:
276290.
Mazzini L, Mareschi K, Ferrero I, Vassallo E, Oliveri G,
Nasuelli N, Oggioni GD, Testa L, Fagioli F (2008) Stem
cell treatment in Amyotrophic Lateral Sclerosis. J Neurol Sci
265: 7883.
Mitchelle J, Borasio G (2007) Amyotrophic lateral sclerosis.
Lancet 369: 20312041.
Morimoto K, Fahnestock M, Racine RJ (2004) Kindling and sta-
tus epilepticus models of epilepsy: rewiring the brain. Prog
Neurobiol 73: 160.
Morris HM, Hashimoto T, Lewis DA (2008) Alterations in
somatostatin mRNA expression in the dorsolateral prefrontal
cortex of subjects with schizophrenia or schizoaffective dis-
order. Cereb Cortex 18: 15751587.
Muller T, Brohmann H, Pierani A, Heppenstall PA, Lewin
GR, Jessell TM, Birchmeier C (2002) The homeodomain
factor lbx1 distinguishes two major programs of neu-
ronal differentiation in the dorsal spinal cord. Neuron 34:
551562.
Murashov AK, Pak ES, Hendricks WA, Owensby JP, Sierpinski
PL, Tatko LM, Fletcher PL (2004) Directed differentiation
of embyronic stem cells into dorsal interneurons. FASEB J
2: 252254.
Muroyama Y, Fujihara M, Ikeya M, Kondoh H, Takada S (2002)
Wnt signaling plays an essential role in neuronal specifica-
tion of the dorsal spinal cord. Genes Dev 16: 548553.
Nery S, Fishell G, Corbin JG (2002) The caudal ganglionic emi-
nence is a source of distinct cortical and subcortical cell
populations. Nat Neurosci 5: 12791287.
Nestler EJ (2000) Genes and addiction. Nat Genet 26: 277281.
Okabe S, Forsberg-Nilsson K, Spiro AC, Segal M, McKay RD
(1996) Development of neuronal precursor cells and func-
tional postmitotic neurons from embryonic stem cells in
vitro. Mech Dev 59: 89102.
Okada Y, Matsumoto A, Shimazaki T, Enoki R, Koizumi A,
Ishii S, Itoyama Y, Sobue G, Okano H (2008) Spatio-
temporal recapitulation of central nervous system develop-
ment by murine ES cell-derived neural stem/progenitor cells.
Stem Cells 26: 30863098.
Okada Y, Shimazaki T, Sobue G, Okano H (2004) Retinoic-acid-
concentration-dependent acquisition of neural cell identity
during in vitro differentiation of mouse embryonic stem cells.
Dev Biol 275: 124142.
Palma V, Lim DA, Dahmane N, Sanchez P, Brionne TC,
Herzberg CD, Gitton Y, Carleton A, Alvarez-Buylla A, Ruiz
i Altaba A (2005) Sonic hedgehog controls stem cell behav-
ior in the postnatal and adult brain. Development 132:
335344.
8 Directing Differentiation of ESC into Distinct Neuronal Subtypes
Panganiban G, Rubenstein JL (2002) Developmental functions
of the Distal-less/Dlx homeobox genes. Development 129:
43714386.
Pattyn A, Vallstedt A, Dias JM, Samad OA, Krumlauf R, Rijli
FM, Brunet JF, Ericson J (2003) Coordinated temporal and
spatial control of motor neuron and serotonergic neuron gen-
eration from a common pool of CNS progenitors. Genes Dev
17: 729737.
Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N,
Harrison NL, Studer L (2004) Derivation of midbrain
dopamine neurons from human embryonic stem cells. Proc
Natl Acad Sci U S A 101: 1254312548.
Petrova ES, Otellin VA (2007) Serotonin is involved in the reg-
ulation of histogenetic processes in rat embryonic neocortex.
Bull Exp Biol Med 143: 372375.
Piccini P, Pavese N, Hagell P, Reimer J, Bjorklund A, Oertel
WH, Quinn NP, Brooks DJ, Lindvall O (2005) Factors
affecting the clinical outcome after neural transplantation in
Parkinsons disease. Brain 128: 29772986.
Pillai A, Mansouri A, Behringer R, Westphal H, Goulding M
(2007) Lhx1 and Lhx5 maintain the inhibitory-
neurotransmitter status of interneurons in the dorsal
spinal cord. Development 134: 357366.
Plachta N, Bibel M, Tucker KL, Barde YA (2004)
Developmental potential of defined neural progenitors
derived from mouse embryonic stem cells. Development
131: 54495456.
Price SR, Briscoe J (2004) The generation and diversification of
spinal motor neurons: signals and responses. Mech Dev 121:
11031115.
Rathjen J, Haines BP, Hudson KM, Nesci A, Dunn S, Rathjen
PD (2002) Directed differentiation of pluripotent cells to
neural lineages: homogeneous formation and differenti-
ation of a neurectoderm population. Development 129:
26492661.
Rathjen J, Lake JA, Bettess MD, Washington JM, Chapman G,
Rathjen PD (1999) Formation of a primitive ectoderm
like cell population, EPL cells, from ES cells in response
to biologically derived factors. J Cell Sci 112 (Pt 5):
601612.
Roelink H, Porter J, Chiang C, Tanabe Y, Chang D, Beachy P,
Jessell T (1995) Floor plate and motor neuron induction
by different concentrations of the amino-terminal cleav-
age product of sonic hedgehog autoproteolysis. Cell 81:
445455.
Ross SE, Greenberg ME, Stiles CD (2003) Basic helix-loop-
helix factors in cortical development. Neuron 39: 1325.
Roussa E, Krieglstein K (2004) Induction and specification of
midbrain dopaminergic cells: focus on SHH, FGF8, and
TGF-beta. Cell Tissue Res 318: 2333.
Sakai T, Oshima A, Nozaki Y, Ida I, Haga C, Akiyama H,
Nakazato Y, Mikuni M (2007) Changes in density of
calcium-binding-protein-immunoreactive GABAergic neu-
rons in prefrontal cortex in schizophrenia and bipolar disor-
der. Neuropathology 28: 143150.
Samii A, Nutt JG, Ransom BR (2004) Parkinsons disease.
Lancet 363: 17831793.
Sarter M, Bruno JP (2000) Cortical cholinergic inputs mediat-
ing arousal, attentional processing and dreaming: differential
afferent regulation of the basal forebrain by telencephalic and
brainstem afferents. Neuroscience 95: 933952.
113
Sasai Y, Lu B, Steinbeisser H, De Robertis EM (1995)
Regulation of neural induction by the Chd and Bmp-4 antag-
onistic patterning signals in Xenopus. Nature 377: 757.
Sasai Y, Lu B, Steinbeisser H, Geissert D, Gont LK, De Robertis
EM (1994) Xenopus chordin: a novel dorsalizing factor
activated by organizer-specific homeobox genes. Cell 79:
779790.
Schwab ME, Bartholdi D (1996) Degeneration and regenera-
tion of axons in the lesioned spinal cord. Physiol Rev 76:
319370.
Shetty AK, Hattiangady B (2007) Concise review: prospects of
stem cell therapy for temporal lobe epilepsy. Stem Cells 25:
23962407.
Smith WC, Harland RM (1992) Expression cloning of noggin, a
new dorsalizing factor localized to the Spemann organizer in
Xenopus embryos. Cell 70: 829840.
Smukler SR, Runciman SB, Xu S, van der Kooy D (2006)
Embryonic stem cells assume a primitive neural stem cell
fate in the absence of extrinsic influences. J Cell Biol 172:
7990.
Sonntag KC, Pruszak J, Yoshizaki T, van Arensbergen J,
Sanchez-Pernaute R, Isacson O (2007) Enhanced yield of
neuroepithelial precursors and midbrain-like dopaminergic
neurons from human embryonic stem cells using the bone
morphogenic protein antagonist noggin. Stem Cells 25:
411418.
Stephens B, Guiloff RJ, Navarrete R, Newman P, Nikhar N,
Lewis P (2006) Widespread loss of neuronal populations in
the spinal ventral horn in sporadic motor neuron disease. A
morphometric study. J Neurol Sci 244: 4158.
Stern CD (2005) Neural induction: old problem, new findings,
yet more questions. Development 132: 20072021.
Sulzer D (2007) Multiple hit hypotheses for dopamine neuron
loss in Parkinsons disease. Trends Neurosci 30: 244250.
Sussel L, Marin O, Kimura S, Rubenstein JL (1999) Loss of
Nkx2.1 homeobox gene function results in a ventral to dorsal
molecular respecification within the basal telencephalon: evi-
dence for a transformation of the pallidum into the striatum.
Development 126: 33593370.
Tamamaki N, Yanagawa Y, Tomioka R, Miyazaki J-I, Obata K,
Kaneko T (2003) Green fluorescent protein expression and
colocalization with calretinin, parvalbumin, and somatostatin
in the GAD67-GFP knock-on mouse. J Comp Neurol 467:
6079.
Tanaka A, Kamiakito T, Hakamata Y, Fujii A, Kuriki K,
Fukayama M (2001) Extensive neuronal localization and
neurotrophic function of fibroblast growth factor 8 in the
nervous system. Brain Res 912: 105115.
Temple S (2001) The development of neural stem cells. Nature
414: 112117.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA,
Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic
stem cell lines derived from human blastocysts. Science 282:
11451147.
Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der
Kooy D (2001) Direct neural fate specification from embry-
onic stem cells: a primitive mammalian neural stem cell stage
acquired through a default mechanism. Neuron 30: 6578.
Vercelli A, Mereuta OM, Garbossa D, Muraca G, Mareschi K,
Rustichelli D, Ferrero I, Mazzini L, Madon E, Fagioli F
(2008) Human mesenchymal stem cell transplantation
114
extends survival, improves motor performance and decreases
neuroinflammation in mouse model of amyotrophic lateral
sclerosis. Neurobiol Dis 31: 395405.
Walker FO (2007) Huntingtons disease. Lancet 369: 218228.
Wang Q, Matsumoto Y, Shindo T, Miyake K, Shindo A,
Kawanishi M, Kawai N, Tamiya T, Nagao S (2006) Neural
stem cells transplantation in cortex in a mouse model of
Alzheimers disease. J Med Invest 53: 6169.
Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki S,
Kawasaki H, Watanabe Y, Mizuseki K, Sasai Y (2005)
Directed differentiation of telencephalic precursors from
embryonic stem cells. Nat Neurosci 8: 288296.
Wenner P, ODonovan MJ, Matise MP (2000) Topographical
and physiological characterization of interneurons that
express engrailed-1 in the embryonic chick spinal cord. J
Neurophysiol 84: 26512657.
Wernig M, Benninger F, Schmandt T, Rade M, Tucker KL,
Bussow H, Beck H, Brustle O (2004) Functional integration
of embryonic stem cell-derived neurons in vivo. J Neurosci
24: 52585268.
Westmoreland JJ, Hancock CR, Condie BG (2001) Neuronal
development of embryonic stem cells: a model of
GABAergic neuron differentiation. Biochem Biophys Res
Commun 284: 674680.
Wichterle H, Lieberam I, Porter JA, Jessell TM (2002) Directed
differentiation of embryonic stem cells into motor neurons.
Cell 110: 385397.
Widner H, Tetrud J, Rehncrona S, Snow B, Brundin P,
Gustavii B, Bjorklund A, Lindvall O, Langston JW (1992)
N. Ammon et al.
Bilateral fetal mesencephalic grafting in two patients
with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP). N Engl J Med 327: 15561563.
Wilson L, Maden M (2005) The mechanisms of dorsoven-
tral patterning in the vertebrate neural tube. Dev Biol 282:
113.
Wilson SI, Rydstrom A, Trimborn T, Willert K, Nusse R, Jessell
TM, Edlund T (2001) The status of Wnt signalling regulates
neural and epidermal fates in the chick embryo. Nature 411:
325330.
Wonders CP, Anderson SA (2006) The origin and spec-
ification of cortical interneurons. Nat Rev Neurosci 7:
687695.
Xu Q, Cobos I, Cruz EDL, Rubenstein JL, Anderson SA (2004)
Origins of cortical interneuron subtypes. J Neurosci 24:
26122622.
Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A
(1998) FGF and Shh signals control dopaminergic and sero-
tonergic cell fate in the anterior neural plate. Cell 93:
755766.
Ying QL, Stavridis M, Griffiths D, Li M, Smith A (2003)
Conversion of embryonic stem cells into neuroectodermal
precursors in adherent monoculture. Nat Biotechnol 21:
183186.
Zhao Y, Marin O, Hermesz E, Powell A, Flames N, Palkovits M,
Rubenstein JL, Westphal H (2003) The LIM-homeobox gene
Lhx8 is required for the development of many cholinergic
neurons in the mouse forebrain. Proc Natl Acad Sci U S A
100: 90059010.
Chapter 9

Neurotransmitters as Main Players in the Neural

Differentiation and Fate Determination Game

Katia K. Yuahasi, Katia N. Gomes, Marcelo Campos, Arthur A. Nery, Ariane

Nunes-Alves, Cleber A. Trujillo, and Henning Ulrich

Contents (peaks or waves) leads to initiation of a calcium-

dependent gene expression program essential for the
9.1 Introduction . . . . . . . . . . . . . . . . . 116 progress to the next differentiation stage. We dis-
9.2 An Overview of Neurogenesis . . . . . . . . 116 cuss the evidence pointing to participation of GABA,
9.3 Models of Neuronal Differentiation . . . . . . 118 glutamate, cholinergic and purinergic receptors in neu-
9.3.1 Mesenchymal Stem Cells (MSC) . . . . 118
ronal differentiation of various stem and progenitor
9.3.2 Neural Stem Cells (NSC) . . . . . . . . 119
9.3.3 Embryonic Stem (ES) and Embryonal cell models.
Carcinoma (EC) Cells . . . . . . . . . 119
9.4 Participation of Neurotransmitters in Neural Keywords Calcium signalling Neural
Differentiation . . . . . . . . . . . . . . . . 120 differentiation Neurotransmitter Phenotype
9.4.1 -Aminobutyric Acid (GABA) . . . . . 120
fate Stem cells
9.4.2 Acetylcholine . . . . . . . . . . . . . 121
9.4.3 Glutamate . . . . . . . . . . . . . . 122 Abbreviations
9.4.4 Purines . . . . . . . . . . . . . . . . 124
9.5 Calcium Signaling and Neuronal Differentiation 125
9.6 Conclusions . . . . . . . . . . . . . . . . . 128 ADSC adipose-derived stem cells
References . . . . . . . . . . . . . . . . . . . . 128 AMPA -amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid
BMSC bone marrow stromal cells
Abstract The wide phenotypic variety of individ- CICR calcium-induced calcium release
ual cells in the CNS and the enormous complexity of EB embryoid bodies
the network formed between these cells results from [Ca2+ ]i free intracellular calcium concentration
specific development programs which direct differenti- EC cells embryonal carcinoma cells
ation of stem and progenitor cells into numerous types EGF epidermal growth factor
of neurons as well as into astrocytes and oligodendro- ER endoplasmic reticulum
cytes. It is believed that in addition to activation of ES cells embryonic stem cells
FGF fibroblast growth factor
intrinsic genetic programs, extrinsic factors are needed
GABA -aminobutyric acid
for the progress of differentiation and neural pheno-
GluR glutamate receptor(s)
type determination. Recent studies have revealed that
ICM inner cell mass
in addition to classical growth factors, neurotransmit-
IP3 inositol-1,4,5-triphosphate
ters have morphogenic functions. Calcium signaling is MPEP methyl-6-(phenylethynyl)-pyridine
triggered by activation of ion channels or metabotropic mGluR metabotropic glutamate receptor subtype(s)
receptors that codified in a frequency of transients MSC mesenchymal stem cell(s)
nAChR nicotinic acetylcholine receptor(s)
H. Ulrich () NMDA N-methyl-D-aspartate
Departamento de Bioqumica, Instituto de Quimica, NSC Neural stem cells
Universidade de So Paulo, So Paulo, Brazil RA retinoic acid
e-mail: henning@iq.usp.br RyR ryanodine receptor(s)

H. Ulrich (ed.), Perspectives of Stem Cells, 115

DOI 10.1007/978-90-481-3375-8_9, Springer Science+Business Media B.V. 2010
116 K.K. Yuahasi et al.

9.1 Introduction effort has been devoted to characterize intrinsic and

extrinsic factors involved in proliferation and differ-
entiation programs of stem cells and neural progeni-
The development of the central nervous system is
tors. Several soluble and membrane-bound molecules,
one of the most important and complex morpho-
including growth factors, hormones and neurotrans-
genetic events occurring in the embryo. This process
mitters, have been implied in the extrinsic regulation
is the result of the standard events which begins with
of these processes (for a review see Cameron et al.,
proliferation and specification of neural cell identity
1998).
(neuronal or glial), followed by cell migration and mat-
While classic roles of neurotransmitters and their
uration, neurite extension and nerve guidance, synapse
receptors in the adult brain related to synaptic com-
formation, plasticity and network implementation and
munication are well understood, the comprehension
finally concludes with the refinement of synaptic con-
of their functions in the development of the nervous
nections through the elimination of axonal branches
system is only beginning and has been more inten-
or cell death (Frisn et al., 1998). In this way, the
sively investigated during the last years (for a review
mature nervous system arises from few embryonic
see Nguyen et al., 2001). It has become evident that
cells, reaches billions of cells in adult life and forms
in order to achieve its enormous complexity, the devel-
trillions of high precision circuits.
oping neuronal system must rely on cellular interac-
Due to the immense complexity of this system, the
tions and subsequent intracellular signal transduction.
general opinion had been that birth of new neurons or
Neurotransmitters are part of diverse classes of dif-
replacement of lost ones did not exist in the adult cen-
fusible factors capable to activate membrane-bound
tral nervous system, according to the neuronal doctrine
receptors, thereby regulating the progress of neuronal
elaborated by Santiago Ramon y Cajal at the end of
differentiation and determining the final phenotypic
the nineteenth century. For many following years only
fate. In view of that, neurotransmitters function as
a handful of biologists, including Joseph Altman and
morphogens during development.
Michael Kaplan, suggested the existence of adult neu-
In this chapter we will review experimental evi-
rogenesis (Altman and Das, 1965; Kaplan and Hinds,
dence pointing to the participation of neurotransmitter
1977). However, during the last decades, the study
receptors and their agonists in the developing neuronal
of neurogenesis in adult brains of birds (Nottebohm
system as well as in neuronal differentiation of stem
and Goldman, 1983), rodents (Kuhn et al., 1996),
cell models. The selected neurotransmitter receptors
primates (Gould et al., 1999) and finally in humans
whose functions will be discussed are -aminobutyric-
(Eriksson et al., 1998) overturned this ancient dogma
acid (GABA), glutamate, acetylcholine and purinergic
of neuroscience.
receptors. Without any doubts, the selected recep-
The discovery of neurogenesis in some regions of
tors and ligands are components of diverse classes
the adult brain, in the subgranular zone of the dentate
of interconnected extrinsic factors responsible for the
gyrus and the earlier part of the subventricular zone
immense complexity of brain development.
(Ming and Song, 2005), and advances in the study of
embryonic and adult stem cells brought new insights
regarding the use of these cells for the treatment of neu-
rological injuries of traumatic or degenerative nature.
However, although the capacity of stem cells of gen- 9.2 An Overview of Neurogenesis
erating new neurons has raised numerous expectations
in therapeutic intervention, the complex mechanisms The diversity of neural cells in embryonic development
underlying proliferation and differentiation of stem arises from proliferative regions in the neural tube.
cells, such as the elucidation of the factors involved New neurons are continuously added to the neural cir-
in the establishment of the individual neuronal phe- cuits in two restricted areas of the adult mammalian
notypes, need yet to be consolidated. Recent studies brain: the subventricular zone and the hippocampal
indicate that the final neuronal fate can be experi- subgranular zone. Outside of these two regions, prolif-
mentally determined by in vitro manipulation of the erating cells give rise to new glia but not to neurons
environment of stem cell cultures. Therefore, much in the adult CNS. The molecular mechanisms and
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
the cross-talk between multiple signaling pathways,
transcription factors and cellcell interactions control-
ling cell fate have lately attracted much attention.
Many studies revealed interactions among astrocytes,
radial glial cells and neural progenitor cells. These
interactions can promote and control neurogenesis
and gliogenesis of resident precursor cells in their
niches (Doetsch, 2003a). The mechanisms underlying
these remarkable changes in progenitor behavior and
fate during development are not understood, but are
thought to include both changes in the intrinsic prop-
erties of neural progenitors, as well as changes in their
signaling environment (Guillemot, 2007).
The direction of cell fates into neurons or glia is
also related to symmetric and asymmetric cell divi-
sion, characterized by equal and unequal cytoplasmic
distributions to progeny. Symmetric or proliferative
cell division results in a rapid expansion of stem cells
and progenitors. Neurogenesis starts by changing from
symmetric to asymmetric and neurogenic division,
whereby stem cells produce another stem cell and a
neuron or a neural progenitor. Asymmetric division
is important for the generation of cellular diversity in
the mammalian cortex (Fishell and Kriegstein, 2003;
Miyata et al., 2004). The transition to gliogenesis
Fig. 9.1 Novel hypothesis on the lineage of neural stem
cells. In 2001, Alvarez-Buylla and coworkers proposed a uni-
fied hypothesis based on oak tree model of the neural stem cells
lineage, abandoning the classical pine tree model. According
to the pine tree model, neuroepithelial cells in the neural tube
generate two separate groups of committed glial and neuronal
117
involves a return to the symmetric division of progeni-
tors, controlled by intrinsic and extrinsic factors.
Glial cells and in particular radial glia are now con-
sidered as a source of neuronal progenitors (Song et al.,
2002). According to Doetsch (2003b), radial glial cells
are a widespread non-neuronal cell type in the devel-
oping CNS, appearing at the onset of neurogenesis
and giving rise, directly or through progenitor forma-
tion, to most neurons of the cortex. Radial glial cells,
defined by their radial morphology and glial properties,
appear during early brain development where they sup-
port migrating neurons (Levitt and Rakic, 1980). In the
embryonic telencephalon, radial glial cells reach the
inner ventricular zone, extend radial process at the pial
surfaces of the neural tube and divide at the ventricular
surface (Cameron and Rakic, 1991). Gtz and Huttner
(2005) have published a comprehensive review of this
complex mechanism of neurogenesis.
At this point, with these recent findings that glia
can act as neural stem cells (Goldman, 2003), we
are experiencing one of the biggest revolutions in
the field of neural stem cell research. The traditional
opinion was that neuroepithelial cells from the early
neural tube generate two separate groups of glial and
neuronal progenitors. The current accepted view of
progenitors. According to the oak tree model, neuroepithelial
cells originate radial glia which then differentiates into astro-
cytes. Radial glia can divide asymmetrically to produce neurons
and glia and might produce these cells directly or through transit
amplifying cells
118
Fig. 9.2 In vitro models
for the study of
neurogenesis. P19 embryonal
carcinoma, embryonic stem,
neural progenitor and
mesenchymal stem cells have
the capacity to differentiate in
vitro into neural phenotypes.
Neurotransmitter receptors are
functional along the process
of differentiation
neurogenesis proposes that neuroepithelial cells at the
base of the lineage develop into radial glia and then
into astrocytes. Neurons and glia are produced by
asymmetric division of radial glial cells, which occurs
directly or through intermediated precursors. Radial
glial cells disappear after birth in mammals, when
they are thought to transform into astrocytes (Fig. 9.1)
(Alvarez-Buylla et al., 2001).
Even with recent data providing evidence that neu-
ral stem cells have some characteristics of glia, it is
extremely improbable that all glial cells are neural
stem cells. The quest of highly efficient cell-specific
markers and isolation methods together with investiga-
tions into the mechanisms of extrinsic factors, such as
neurotransmitters, growth factors and their receptors,
and induction of intracellular calcium transients con-
tributing to neurogenesis promises to reveal how these
cells decide their fates.
9.3 Models of Neuronal Differentiation
Understanding the mechanism controlling neuronal
differentiation and behavior has raised much interest
over recent years. A large variety of extrinsic and
intrinsic factors are being characterized regarding their
functions during neuronal development. However, due
to the enormous complexity of such studies in vivo,
K.K. Yuahasi et al.
in vitro stem cell models were established to study
the actions of these factors in a simplified environ-
ment. This chapter will focus on mesenchymal stem
cells (MSC), neural stem cells (NSC), embryonic stem
(ES) and embryonal carcinoma (EC) cells as models
for neuronal differentiation (Fig. 9.2).
9.3.1 Mesenchymal Stem Cells (MSC)
MSC, recognized as adherent stromal cells, are derived
from embryonic mesoderm. The existence of stem
cells for non-hematopoietic cells in bone marrow
was proposed over 100 years ago, but the isola-
tion and differentiation of marrow stromal cells into
osteoblasts, chondroblasts, adipocytes and myoblasts
was only recently demonstrated (reviewed by Prockop,
1997). Non-hematopoietic precursors from bone
marrow stroma have been referred to as colony-
forming-unit fibroblasts, MSC or bone marrow stro-
mal cells (BMSC). Although BMSC can naturally be
expected to be a source of surrounding tissue of bone,
cartilage, and fat, several recent reports demonstrate
that these cells, under specific experimental conditions,
can differentiate into muscle, glia and hepatocytes
(Ferrari et al., 1998; Azizi et al., 1998; Peterson et al.,
1999; Sanchez-Ramos et al., 2000).
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
Cultures of mesenchymal stem cells have also been
studied for capacity of differentiation into cell types of
non-mesenchymal origin, including neurons (reviewed
by Jackson et al., 2007). This trans-differentiation
potential has not only been observed for bone-marrow
stem cells but also for adipose-derived stem cells
(ADSC). These cells are characterized by their capa-
bility to differentiate into adipocytes, osteoblasts,
myocytes, chondrocytes, endothelial cells and car-
diomyocytes (Zuk et al., 2002; Gimble et al., 2007).
Cultures of ADSC can also be induced to form neu-
rospheres (Kang et al., 2004; Nagase et al., 2007) and
further differentiate into cells with neuronal morphol-
ogy or even Schwann cells (Safford et al., 2002; Xu
et al., 2008). Intracerebral transplantation of human
ADSC improved neurological deficits after cerebral
ischemia in rats (Kang et al., 2003). In fact, ADSC may
be a better source for cell therapy as bone-marrow stem
cells as they can be easily obtained in large amounts by
liposuction from subcutaneous adipose tissue (Franco
Lambert et al., 2009).
9.3.2 Neural Stem Cells (NSC)
Since they were first described by Reynolds and Weiss
(1992), neural stem and progenitor cells have been the
focus of growing attention due to their potential in the
study of neurogenesis and development of future ther-
apy in neurological disorders. In this study, Reynolds
and Weiss isolated striatum cells from adult mouse
brain and induced them to proliferate as free-floating
spherical expansion (neurospheres). Neurospheres are
clonal expansions of self-renewing neural progenitor
cells that form tridimensional cellular aggregates in
the presence of epidermal growth factor (EGF) and
fibroblast growth factor (FGF)-2 (Tropepe et al., 1999;
Ostenfeld and Svendsen, 2004; Kelly et al., 2005).
In their undifferentiated stage, cells originally express
nestin (stemness marker) and following attachment
to culture dishes, they change their morphology and
antigenic properties to those of neurons and astro-
cytes. Such cells keep their potential differentiation
into neurons, astrocytes and oligodendrocytes both
in vitro and in vivo following transplantation into
developing or adult brain. It is believed that the inter-
action between multiple cell types occurring during
neurosphere differentiation facilitates maturation of
119
neurons. For instance, astrocytes (one of three cell
types obtained during neurosphere differentiation) pro-
mote formation and function of excitatory synapses
(Haber et al., 2006).
9.3.3 Embryonic Stem (ES) and Embryonal
Carcinoma (EC) Cells
Pluripotent embryonic carcinoma (EC) and embry-
onic stem (ES) cells possess characteristics of early
embryonic cells (Kahan and Ephrussi, 1970; Evans and
Kaufman, 1981; Martin, 1981). ES and EC cells are
able to undergo unlimited self-renewal and differenti-
ation into the three primary germ layers (endoderm,
mesoderm, and ectoderm) of the embryo. EC cells
being part of teratocarcinomas (malignant germ cell
tumors) were used for decades as in vitro models for
developmental processes. The in vivo counterpart of
EC cells are ES cells, the inner cell mass (ICM) of the
blastocyst stage. These cells originate all cells of the
adult body. Mouse EC cells are similar to cells of the
ICM (Gachelin et al., 1977; Solter and Knowles, 1978)
and contribute to differentiation into somatic cell types
when injected into the ICM. Thus, EC and ES cells
are a versatile in vitro tool for understanding molecular
and cellular controls in early mammalian neurogenesis
(Tropepe et al., 2001; Wichterle et al., 2002; Mizuseki
et al., 2003; Ying et al., 2003; Watanabe et al., 2005).
A widely used model for in vitro differentiation is
the P19 mouse embryonic carcinoma cell line, which
can be induced to neuroectodermal differentiation by
culturing free floating embryoid bodies (EB) in serum-
free defined medium containing retinoic acid (RA)
(McBurney et al., 1982; Jones-Villeneuve et al., 1982).
The obtained differentiated phenotypes are neurons,
astrocytes, oligodendrocytes and fibroblast-like cells
(Jones-Villeneuve et al., 1982). Like EC cells, ES cells
can be induced to differentiation into neural pheno-
types in the presence of RA and by culturing them
as floating or hanging drop cell suspension (Guan
et al., 2001). Mechanisms of RA-triggered neural dif-
ferentiation of ES and EC cells are largely unknown.
Nevertheless, gene expression of several transcription
factors and signaling molecules such as sonic hedge-
hog, transcription factors Pax6 and Mash1 and the
signaling molecule Wnt-1 was induced in ES cells in
the presence of RA. The transcription factors Neuro D,
Math1 and NSCL2 were expressed in differentiating
120 K.K. Yuahasi et al.

mouse EC cells following RA application (reviewed 9.4.1 -Aminobutyric Acid (GABA)

by Guan et al., 2001).
-aminobutyric-acid (GABA) is the main fast-
acting inhibitory neurotransmitter in the adult cortex
9.4 Participation of Neurotransmitters (Connors et al., 1988; Krnjevic and Schwartz, 1967),
in Neural Differentiation and it is also one of the most abundant neurotrans-
mitters detected in mammalian brain development
(Cicirata et al., 1991; Miranda-Contreras et al., 1998,
Neurotransmitters and their receptors have well char-
1999, 2000; Nguyen et al., 2001). GABA receptors
acterized functions in neuromodulation, but their par-
are divided into ionotropic GABAA and GABAC sub-
ticipation in directing neural differentiation has only
types and metabotropic GABAB receptors (reviewed
recently been studied. Experimental evidence strongly
by Chebib and Johnston, 1999). The abundance of
suggests that neurotransmitter receptors and their ago-
GABA and its receptors in the early developing rodent
nists could behave as growth regulators during neu-
CNS has raised speculations regarding the role of
ronal differentiation and development (Brezun and
this transmitter in immature neural cell proliferation,
Daszuta, 2000; Butler et al., 1999; Cameron et al.,
migration, differentiation and survival. For instance,
1998; Fiszman et al., 1999; Haydar et al., 2000; Lauder
immunoreactivity for GABA together with glutamate
et al., 1998; LoTurco et al., 1995; Ma et al., 1998,
was detected in the developing cortical marginal zone
2000; Wang et al., 1996; Weiss et al., 1998). As
and subplate cells as examples of transient popula-
we shall discuss, neurotransmitters and their recep-
tions in the developing neuronal system (Del Rio
tors are expressed throughout development where they
et al., 1992). Moreover, GABA was present at the
control cellular processes such as metabolism, pro-
beginning of hypothalamus development (Van den Pol,
liferation, cell survival, migration and differentiation.
1997) pointing to functions of this neurotransmitter in
With on-going neuronal differentiation and matura-
neurogenesis of several regions prior to formation of
tion completed by the refinement of synaptic contacts,
synaptic interactions.
neurotransmitters take over new functions as medi-
Nowadays, it is well established that GABA exerts
ators of synaptic communication (Buznikov et al.,
trophic actions during neural development which may
1996). Since many neurotransmitters and hormones
involve stimulation of all three types of GABA recep-
are known to induce signaling cascades in progeni-
tors (reviewed by Barker et al., 1998; Lauder et al.,
tors and stem cells, a major question remains how
1998). These findings have initiated further studies in
these multiple external signals cooperate throughout
order to elucidate GABA release and GABA-receptor
development of the neuronal system into its immense
interactions during neuronal differentiation of embry-
diversity. Nicolas Spitzer and co-workers have shown
onic and adult stem cells into specific neural phe-
ion channel and receptor activation codes for a spe-
notypes. In young neurons and neuronal precursors,
cific follow-up of [Ca2+ ]i transients as a frequency of
GABA is an excitatory neurotransmitter, becoming
calcium waves that trigger the progress of neural dif-
inhibitory only later (Ben-Ari et al., 1989; Owens
ferentiation (reviewed by Spitzer et al., 2004). This
et al., 1996; Rivera et al., 1999). GABAA receptor sub-
phenotypic transition depends on activation of neuron-
unit expression is differentially regulated during brain
specific gene expression programs at defined check
development, with each subunit exhibiting a unique
points along differentiation.
regional and temporal developmental expression pro-
However, most of the available data of neurotrans-
file. GABAA receptor activity also acts facilitating
mitters and their receptors role in neuronal differenti-
ciliary neurotrophic factor-induced astrocyte differen-
ation as morphogens, by paracrine or autocrine mech-
tiation of neural progenitors isolated from fetal rat
anisms, have been obtained using simplified in vitro
brain (Yoneyama et al., 2007). Although the signifi-
systems. This limitation arises from the complexity of
cance of the differential expression of GABAA recep-
in vivo development, and therefore the participation
tor subunits is not completely understood, it seems that
of neurotransmitter receptors during animal develop-
subunit switching in certain brain regions is essential
ment can only be studied in transgenic and knock-out
for normal development (Culiat et al., 1994; Gnther
animals or by using pharmacological approaches.
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
et al., 1995). Furthermore, GABAB receptors have
been recently related to modulation of proliferation
and differentiation of neural progenitor cells isolated
from fetal mouse brain (Fukui et al., 2008).
In the same way, the differentiation of embry-
onic or adult tissue-specific stem cells is regulated by
a microenvironment of interactions between several
neurotransmitters, growth factors and others trophic
factors defining a specific neuronal phenotype. Cells
with neuronal-like morphology obtained by in vitro
differentiation of embryonic or mesenchymal stem
cells express functional GABA receptors. Moreover,
GABA-evoked receptor activity increases during pro-
gressing differentiation of mouse neurospheres, sug-
gesting that functional properties of neurons derived
from fetal mouse neurospheres are compatible with
those of neuronal precursors in vivo (Pagani et al.,
2006).
9.4.2 Acetylcholine
Acetylcholine receptors are classified into the two
classes of ionotropic nicotinic and metabotropic mus-
carinic receptors based on structural and pharmaco-
logical properties. Neuronal nicotinic receptors are
assembled by five homo-or heteromeric receptor sub-
units (2-10 and 2-4), while muscarinic subtypes
are comprised by five metabotropic receptors coupling
to Gq/11 (M1, M3 and M5 receptors) or Gi/o pro-
teins (M2, M4). While M1-M4 subtypes are found
throughout all tissues, M5 types are only expressed in
the neuronal system. Nicotinic acetylcholine receptors
(nAChR) are widely distributed all over the mam-
malian CNS (Quik et al., 2000; Zoli et al., 1995) with
high expression levels in adult and developing human
brain (Court et al., 1995, 1997; Gotti et al., 1997;
Kinney et al., 1993; Paterson and Nordberg, 2000). The
activity of these ionotropic receptors contributes to a
wide range of brain processes from cognitive functions
such as learning, memory formation or reward (Levin,
1992) to cellular events such as neurodegeneration
(Zoli et al., 1999) and neural development (Coronas
et al., 2000; Lauder, 1993).
Nicotinic receptor subunits are among the first
membrane proteins to appear during CNS devel-
opment. Synthesis of acetylcholine initiates dur-
ing motoneuron development upon cessation of cell
121
division (Vaca, 1988). The release of acetylcholine
through growth cones of migrating cholinergic neu-
rons suggests trophic roles for this neurotransmitter in
establishing correct synaptic connections. Consistent
with this, choline acetyltransferase immunoreactivity
was detectable in pre-migratory or early migratory
neurons of rat spinal cord (Phelps et al., 1990). Subunit
expression and functional properties of nicotinic recep-
tors change during neural differentiation and matura-
tion. In this context, Schneider and coworkers (2002)
provided proof for functional nAChR in fetal mouse
cerebral cortex as early as on E10. Transient eleva-
tion of intracellular calcium concentration ([Ca2+ ]i )
in response to application of nicotinic agonists were
markedly prolonged in cells from early embryonic
stages when compared to cells from later stages of
development. The effects of nicotine on developing
neurons have been intensively studied during hip-
pocampal formation which is characterized by high
expression of 7-subtype nAChR (Adams et al., 2002).
Due to the high permeability of this ion channel for
calcium ions, high nicotine concentrations may be
cytotoxic and therefore reduce neurogenesis.
The participation of nicotinic and muscarinic recep-
tors in neurogenesis has also been studied in simplified
systems suggesting that this neurotransmitter can act
as morphogen alone or in combination with other
extrinsic factors. For instance, mouse ES and P19 EC
cells synthesize acetylcholine (Paraoanu et al., 2007;
Parnas and Lilian, 1995) and the expression of the
nicotinic and muscarinic receptor subtypes changes
during RA-induced neuronal differentiation of P19
cells (Resende et al., 2008a, b). Embryonic P19 cells
already expressed various nicotinic receptor subunits
including the 7 type and responded to nicotine appli-
cation with transient elevations of free intracellular
calcium concentration ([Ca2+ ]i ). Nicotinic receptor
subunits were differentially expressed along neuronal
differentiation of P19 EC to neurons and increases
in nicotinic agonist-induced [Ca2+ ]i responses were
noted when cells acquired neuronal phenotypes. This
increased response to nicotine resulted from higher
overall nAChR expression and activation of ryanodine-
sensitive intracellular calcium release and voltage-
gated calcium channels (Resende et al., 2008a).
Nicotinic receptor stimulation inhibited the prolifer-
ation of embryonic P19 EC cells in a dose-dependent
manner. P19 neural progenitor cells following 4 days
of induction to differentiation by RA responded to
122
nicotine application with proliferation stimulation and
augmented differentiation to neurons as judged by
increased expression of high molecular mass neurofil-
ament. Activation of ryanodine-sensitive intracellular
calcium release has been suggested as a possible mech-
anism for nicotine-promoted proliferation stimulation
in neural progenitors. This mechanism, also known
as calcium-induced calcium release (CICR), was not
present in embryonic P19 cells, in agreement with the
inability of nicotine to stimulate proliferation (Resende
et al., 2008b).
Similar mechanisms, which also involve liberation
of intracellular calcium, have been shown for mus-
carinic receptor induced proliferation and differenti-
ation in P19 cells. In contrast to nAChR, muscarinic
receptors were not functional in embryonic P19 cells.
In progenitor cells, muscarine induced proliferation
by activation of Gq/11 -protein coupled M1, M3 and
M5 receptors and intracellular calcium stores whereas
Gi/o -protein coupled M2 receptor activity mediated
neuronal differentiation (Resende et al., 2008b).
Functions of the cholinergic system have also been
revealed in other cell types, including neuroblastoma
cells used as model of differentiation into choliner-
gic phenotypes and sensory neurons which express
components of the cholinergic system during early
development (Biagoni et al., 2000). Neurite outgrowth
induced in neuroblastoma cells by RA or dibutyril-
cAMP depended on the capability of the cells to
synthesize acetylcholine. Besides the impaired acetyl-
choline synthesis of the mouse neuroblastoma clone
N18TG2, this cell line was unable to establish synaptic
contacts. Following hybridization of this neuroblas-
toma cell with a glial component, the generated neu-
roblastoma X glioma hybrid cell line showed a marked
increase in acetylcholine synthesis and was also capa-
ble to proceed with neurite outgrowth (Hamprecht,
1977). As further confirmation for the importance
of acetylcholine production during differentiation of
neuroblastoma cells, N18TG2 cells could be stimu-
lated to fiber outgrowth following transfection with
cDNAs coding for choline acetyltransferase and gene
expression of synapsin I (Bignami et al., 1997). As
a mechanism for differentiation induction, the group
of Augusti-Tocco suggested an autocrine loop by
which muscarinic receptor activity stimulates acetyl-
choline synthesis and neuron-specific gene expres-
sion (Augusto-Tocco et al., 2006). Acetylcholine and
muscarinic receptor activity were attributed to play
K.K. Yuahasi et al.
roles in sensory dorsal root ganglion development.
Coordination of neuronal and glial differentiation,
including Schwann cell formation, is also believed to
involve cholinergic receptor activity. Biagioni and co-
workers discuss in this context that neuron-produced
acetylcholine acts on muscarinic receptors expressed
by Schwann cells, thereby inducing transient changes
in [Ca2+ ]i . Schwann cells participate in regulation of
neuronal differentiation and axon outgrowth (Biagioni
et al., 2000). The example of acetylcholine synthe-
sis in sensory neurons shows that cholinergic receptor
function is important for development of neuronal-glial
networks.
9.4.3 Glutamate
Glutamate is the main mediator of excitatory neu-
rotransmisson in the adult human brain (Gass and
Olive, 2008) and can be found in several areas of the
mouse CNS during neurogenesis (Miranda-Contreras
et al., 1998, 1999, 2000). Glutamatergic receptors are
classified by their structural properties into ionotropic
and metabotropic receptors. There are eight types of
metabotropic receptors: mGluR1-8. Ionotropic recep-
tors are subdivided into three families: -amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors, N-methyl-D-aspartic acid (NMDA) recep-
tors and kainate receptors (Pin and Duvoisin, 1995).
Several studies have already confirmed the role
of glutamatergic receptors in regulating prolifera-
tion during neurogenesis where glutamate may have
proliferation-inducing or inhibiting effects depending
on the developmental status and localization of glu-
tamate action. Application of glutamate or GABA
to organotypic slice cultures stimulated proliferation
in the ventricular zone by shortening the cell cycle,
whereas proliferation in the subventricular zone was
decreased (Haydar et al., 2000). Based on these results,
the authors concluded that relative contributions of the
subventricular zone to neocortical growth may be regu-
lated by differential effects of glutamate and GABA on
proliferation. Proliferation stimulating effects of glu-
tamate in E12-22 rat striatum neuroblasts were inhib-
ited in the presence of N-methyl-D-aspartate (NMDA)
receptor antagonists (Sadikot et al., 1998). Activation
of glutamate receptors in cortical oligodendrocyte
progenitor (O-2A) cells suppressed proliferation and
blocked lineage progression from the O-2A stage to the
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
pro-oligodendroblast stage (Gallo et al., 1996) suggest-
ing that glutamate also participated in the regulation of
glial cell differentiation.
Roles for glutamate receptors during neurogene-
sis include cell survival and migration processes. For
instance, activation of NMDA- or kainate receptors
enhanced survival of immature granule neurons of
P7-P8 rat cerebellum (Balazs et al., 1988, 1990). In
agreement, blockade of NMDA receptors resulted in
increased apoptotic elimination of granule neurons in
P7-P11 rat cerebellum (Monti and Contestabile, 2000),
augmented cell death within the dentate gyrus of
neonatal rats (Gould et al., 1994) and inhibited migra-
tion of O-2A cells in P0 rat neurohypophysial explants
(Wang et al., 1996). Enhancement of NMDA receptor
activity promoted neurite outgrowth of rat cerebellar
granule cells (Pearce et al., 1987) and increased the rate
of migration of granule cells of the developing mouse
cerebellum (Komuro and Rakic, 1993). Furthermore,
increase of endogenous extracellular glutamate con-
centration by inhibition of its uptake also augmented
cell migration rates. These and subsequent studies sug-
gested that glutamate, acting through NMDA receptors
and subsequent calcium signaling, is a chemoattractive
factor for neurons in mouse developing cortex, signal-
ing cells to migrate from the ventricular and subven-
tricular zones into the cortical plate (Behar et al., 1999,
reviewed by Nacher and McEwen, 2006). Glutamate-
induced signaling is essential for dendritic and axonal
growth (Mattsson et al., 1988a, b) as well as for
the refinement and reorganization of synaptic contacts
(Aruffo et al., 1987; Scheetz and Constantine-Paton,
1994).
The effects of glutamatergic signaling on neu-
ral gene expression have been studied in hippocam-
pal development. Glutamate promote neurogenesis
in adult hippocampal precursor cells by augmenting
[Ca2+ ]i levels which was blocked by NMDA recep-
tor antagonists (Deisseroth et al., 2004). In agreement,
neurospheres from adult mice hippocampus express-
ing NR1, NR2A and NR2B subunits of the NMDA
receptor were induced to augmented gene expression
of c-fos and c-jun, and neuronal differentiation by sus-
tained exposure to NMDA (Kitayama et al., 2004).
However, in dentate gyrus of one week old rats inhi-
bition of NMDA receptors led to an increase in neu-
rogenesis (Poulsen et al., 2005). These contradictory
findings of glutamate-induced NMDA receptor activ-
ity on neurogenesis could be in part due to differences
123
in expression levels and responsiveness of such recep-
tors in developing neurons. Finally, non-NMDA-type
ionotropic and metabotropic glutamate receptors may
modulate effects of NMDA receptor activation on
neurogenesis (reviewed by Nacher and McEwen,
2006).
The participation of glutamatergic system has also
been studied during in vitro neuronal differentiation
using P19 EC and ES cells. The concentration of
beta-citryl-L-glutamate, catalyzing the formation of
L-glutamate, increased (Narahara et al., 2000), and
many glutamatergic receptors, such as kainate recep-
tor subunits (GluR5, GluR6 and GluR7) as well as
the AMPA receptor subunits (GluR2 and GluR3), had
their expression changed during the course of differ-
entiation of P19 EC cells into neurons (Minakami
et al., 1995; Heck et al., 1997; Lee et al., 2003).
Gene expression of metabotropic glutamate receptors
(mGluR2, mGluR4 and mGluR8) were detectable in
undifferentiated P19 cells and in all stages of differ-
entiation. However, expression of mGluR3, mGluR7
and mGluR8 became visible during the course of dif-
ferentiation (Heck et al., 1997). Functional NMDA-,
kainate- and AMPA receptors were present follow-
ing three days of differentiation induction by RA.
Glutamate-induced [Ca2+ ]i transients augmented dur-
ing on-going differentiation (Lee et al., 2003; Ulrich
and Majumder, 2006). There is also evidence of gluta-
mates regulatory role during ES cell differentiation.
For instance, gene expression of the NMDA recep-
tor subunits NR1, NR2A and NR2B increased during
neural differentiation of mouse ES cells (Qu et al.,
2003). Gene expression of metabotropic mGluR5 was
the only among metabotropic glutamate receptors
detectable in mouse ES cells. Blockade of this recep-
tor decreased the expression of Oct-4 and Nanog,
two transcription factors involved in maintaining the
undifferentiated state of ES cells, and of alkaline phos-
phatase, a marker of ES cells (Cappuccio et al., 2005).
Mouse ES cells differentiated with serum-free
N2 and B27 medium expressed mGluR5 through-
out the entire differentiation period. Blockade of
the metabotropic glutamatergic subtype mGluR5
by methyl-6-(phenylethynyl)-pyridine (MPEP) dur-
ing differentiation of ES cells into neuron-like cells
resulted in an increase of 3-tubulin-positive cells
with GABAergic phenotype. At the end of differ-
entiation, percentages of cells expressing 3-tubulin
were identical when cells had been differentiated in
124
the presence or absence of MPEP. These data sug-
gest that metabotropic glutamate receptor blockade
accelerated differentiation of ES cells into neuron-like
cells (Sarichelou et al., 2008). ES cells differentiat-
ing into embryoid bodies (EB) progressively stopped
expressing mGluR5 but augmented mGluR4 gene
and protein expression. Activation of mGluR4 dur-
ing neural induction of ES cells with RA or medium
supplemented with insulin, transferrin, selenium and
fibronectin increased the expression of the early neu-
ral markers Dlx-2 and nestin (Cappuccio et al., 2006),
and differentiation into neurons with a GABAergic
phenotype (Sarichelou et al., 2008). This work also
demonstrated that subtype-selective glutamate recep-
tor ligands might be used for in vitro production of
GABAergic neurons from ES cell cultures.
Depending on the activated subtype (mGluR4 or
mGluR5), metabotropic glutamate receptors either
stimulated or inhibited the progress of neuronal differ-
entiation. Activation of mGluR5 favored self-renewal
of ES cells and maintenance of the cells in their undif-
ferentiated state, whereas differentiation of ES cells
into embryoid bodies was associated with mGluR4
activation (Melchiorri et al., 2007). Consequently,
mGluR5 expression with inhibitory action on neuro-
genesis decreased following induction to differentia-
tion, while mGluR4 expression was enhanced during
on-going differentiation.
9.4.4 Purines
In the 1970s, the first direct evidence was presented
for ATP action as a fast transmitter. This suggested the
concept of purinergic nerves and purinergic neuro- mocytoma cells and cultured cortical and hippocampal
transmission (Burnstock, 1972, 1978). Following an
initially slow acceptance, purinergic signaling in neu-
rotransmission and neuronal development is a rapidly
expanding field. However, due to the absence of spe-
cific agonists and antagonists for many purinergic
receptor subtypes, most cellular functions of these
receptors are not yet understood.
Two types of purinoreceptors (P1 and P2) are
distinguished based on their ligand specificity for
adenosine or ATP and ADP, respectively. Four sub-
types of P1 adenosine receptors have been cloned
(A1 and A3 inhibiting and A2a and A2b stimulating
adenylyl cyclase, respectively) (reviewed by Fredholm
et al., 2001). P2 receptors are divided into two
K.K. Yuahasi et al.
families: ionotropic, ligand-gated ion channel recep-
tors assembled by P2X1,2,3,4,5,6,7 subunits and the
metabotropic P2Y family of G protein-coupled recep-
tors consisting of P2Y1,2,4,6,11,12,13,14 subtypes
(reviewed by North, 2002).
ATP is released by a variety of cell types, includ-
ing neurons, glia, endothelium and blood cells.
ATP acts as a primary neurotransmitter or as a
co-transmitter with glutamate, acetylcholine, nora-
drenaline, 5-hydroxytryptamine, dopamine or GABA.
In the synaptic cleft, ATP is enzymatically con-
verted to ADP, AMP and adenosine (Burnstock, 2004).
Adenosine release is observed in neurons, astrocytes
and microglia. It is known to exert neuroprotective
function by protecting against excessive glutamate
and P2X7 receptor mediated calcium fluxes (Schubert
and Kreutzberg, 1993; Dona et al., 2009). Adenosine
reverts deleterious membrane depolarization by acti-
vating Go protein-coupled A1 receptors followed by
augmented K+ conductance and opening of chlo-
ride channels (reviewed by Schubert and Kreutzberg,
1993). However, it seems that adenosine negatively
interferes with the progress of neuronal development.
A1 subtype gene expression and receptor concentra-
tions in the brains of prenatal rodents are consid-
erably less than postnatal levels. In addition, during
neonatal period A1 receptors are expressed on axons
and growth cones and their activation interferes with
axon and white matter formation. Treatment with an
A1 receptor agonist led to axonal loss and reduced
expression of myelin basic protein in treated animals
(Turner et al., 2002). In agreement, A1 receptor activa-
tion also resulted in inhibition of nerve-growth factor
(NGF)-induced neurite outgrowth in PC12 pheochro-
neurons (Thevananther et al., 2001). Early develop-
mental expression and subsequent alterations in the
pattern of A2a adenosine receptors within the rat stria-
tum also suggested functions for this P1 receptor in
differentiation and migration events (Weaver, 1993).
Several studies also indicated functions of puriner-
gic signaling by P2 (P2X and P2Y) receptors during
development. Metabotropic P2Y1, P2Y2, P2Y4 and
P2Y6 subtypes were expressed during rat embryoge-
nesis, with the P2Y4 receptor being the most abundant
one during brain development (Cheung et al., 2003).
The P2Y1 subtype seems to be crucial in early neu-
rogenesis including neurulation and initiation of eye
development of frog embryos (Bogdanov et al., 1997;
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
Mass et al., 2007) and gastrulation of the chicken
embryo (Lasberg, 1990). In rat embryos of age E14-18,
propagation of calcium waves through radial glial cells
in the proliferative cortical ventricular zone was medi-
ated by P2Y1 receptors. Disruption of these calcium
waves decreased proliferation of the ventricular zone,
demonstrating the importance of this receptor during
embryonic neurogenesis (Weissman et al., 2004).
Ionotropic purinergic P2X also exert several func-
tions during brain development. Cheung et al. (2005)
detected P2X3 and P2X2 receptors first in the embry-
onic rat CNS on E11 and E14, respectively. P2X7
receptors were also expressed from E14 onwards, and
the existence of functional P2X7 receptors in the mid-
brain synaptic terminals as well as in synaptosomes
and axodendritic prolongations of cerebellar gran-
ule cells has also been documented (Miras-Portugal
et al., 2003). These receptors mediate apoptotic cell
death by inducing cytotoxicity at high ATP concen-
trations released during inflammation or upon tissue
trauma (Le Feuvre et al., 2002). Since apoptosis is
a common event in developing brain, activation of
P2X7 receptors could participate in cell death during
neurogenesis.
Purinergic receptors were also studied in cell
lines. Using P19 embryonal carcinoma cells as an
in vitro model for early neural development, our
laboratory has shown that P2Y1, P2Y2 and P2X4
subtypes are important in regulating cell prolifera-
tion and differentiation by activating intracellular cal-
cium pathways (Resende et al., 2008c). ATP dose-
dependently stimulated proliferation of P19 embry-
onic and progenitor cells and accelerated differentia-
tion as judged by augmented expression of neuron-
specific proteins. These cellular effects were blocked
when cells were treated with purinergic receptor
antagonists or with inhibitors of intracellular inositol-
1,4,5-triphosphate (IP3 )-mediated calcium mobiliza-
tion. Moreover, purinergic receptor subtype activity
contributed to the acquisition of defined neural phe-
notypes. For instance, inhibition of P2Y1 receptor
activity along differentiation of P19 cells resulted
in loss of NMDA receptor activity. Furthermore,
reduction of cholinergic receptor responses in dif-
ferentiated P19 cells was due to inhibition of
P2Y2 receptor activity during differentiation (Resende
et al., 2007). In agreement with growth stimulation
effects demonstrated in P19 cells (Resende et al.,
2008c), P2Y1 and P2Y2 receptor activation enhanced
125
EGF- and FGF-2-induced neurosphere proliferation
(Mishra et al., 2006).
Extracellular purines stimulated the synthesis and
release of polypeptides with trophic actions (Rathbone
et al., 1999; Neary et al., 2004), and regulated differ-
entiation and neurite outgrowth in various cell lines
(Aono et al., 1990; Gysbers and Rathbone, 1992,
1996). However, elucidation of the exact roles of many
purinergic receptors depends on the development of
subtype agonists and antagonists as well as on down-
regulation of receptor expression by RNA interference
and in vivo studies with knock-out animals.
9.5 Calcium Signaling and Neuronal
Differentiation
Changes in intracellular calcium concentration pro-
vide a signaling mechanism responsible for controlling
numerous cellular events, including metabolism, cell
death, proliferation and differentiation. The ability of a
simple ion such as calcium to acquire crucial functions
in cell biology emerges from the use of an exten-
sive molecular repertoire of signaling components that
can be assembled in combinations to create signals
with widely different spatial and temporal profiles. Its
signaling function is activated when its cytosolic con-
centration [Ca2+ ]i is transiently elevated from 10100
nM to roughly 1000 nM due to spontaneous events or
as result of receptor-activated calcium flux. The coor-
dination of calcium signaling occurs through intrinsic
mechanisms of calcium influx into the cell and lib-
eration or capture of calcium by intracellular stores
(reviewed by Berridge et al., 2000). The main internal
calcium store is the membrane system of the endoplas-
mic reticulum (ER). The ER contains various special-
ized channels for calcium release, of which IP3 and
ryanodine receptor (RyR) channels have been stud-
ied most extensively. The activity of RyR intracellular
calcium channels is furthermore influenced by [Ca2+ ]i
levels, a mechanism known as calcium-induced cal-
cium release (Berridge, 1993; Clapham, 1995). The
entry of external calcium occurs through calcium chan-
nel proteins in the plasma membranes activated by
different mechanisms: (i) voltage-operated channels
activated by membrane depolarization, (ii) receptor-
operated channels opening in response to binding of
an extracellular ligand, (iii) store-operated channels
126
opening in response to depletion of internal calcium
stores, (iv) and mechano-sensitive ion channels
(reviewed by Berridge et al., 2006).
The cellular mechanism employed for removal of an
excess of calcium from the cytoplasm is based on acti-
vation of various pumps and exchangers. The plasma
membrane Ca2+ -ATPase pumps (Pozzan et al., 1994)
as well as Na+ /Ca2+ exchangers remove calcium from
the cell into the extracellular space whereas the sarco-
endoplasmic reticulum ATPase pumps return calcium
back into internal stores (Blaustein and Lederer, 1999).
Mitochondria also play an important function in regu-
lating cytosolic calcium levels. These organelles have
a low affinity, but high-capacity rapid Ca2+ uniporter
that can significantly reduce [Ca2+ ]i levels (Rizzuto
et al., 1993). When the cell returns to the resting state,
a mitochondrial Na+ /Ca2+ exchanger pumps the large
load of calcium back into the cytoplasm, from which
it either returns to the ER or is removed from the cell
(Duchen, 1999; Bernadi, 1999). In response to stimuli,
both entry and release channels mediate calcium fluxes
into the cytoplasm. Since these channels have short
open-times, they only introduce brief pulses of calcium
concentration elevations that then form a small plume
around the mouth of the channel before diffusing into
the cytoplasm. Thus, calcium signals are usually pre-
sented as brief local spikes. In some cases, individual
spikes are sufficient to trigger a cellular response such
as neurotransmitter release.
When longer periods of signaling are necessary,
spikes are repeated to originate waves with differ-
ent frequencies (Berridge et al., 2000; Spitzer et al.,
2000). The cell responds to these stimuli by differential
gene expression programs. Wave frequencies and peak
heights are internally encoded in the localization and
activity states. It is possible that calcium signals gener-
ated at the cell membrane cross the nuclear envelope to
trigger gene expression as shown for hippocampal neu-
rons (Eder and Bading, 2007). However, in other cell
types, i.e. neuroblastoma cells and primary rat sensory
neurons, the nuclear envelope is thought to insulate the
nucleus from larger activity-induced cytoplasmic cal-
cium transients (Al-Mohanna et al., 1994). As a final
result of the translocation of the calcium-induced sig-
naling into the nucleus by incoming calcium transients
or other signaling factors, down-stream genes are acti-
vated which implement cellular responses promoting
differentiation.
K.K. Yuahasi et al.
Functional consequences of changes of cal-
cium spike frequency during development of the
GABAergic phenotype were studied by the group
of Dr. Nicolas Spitzer. Developmental expression
of GABA in Xenopus spinal neurons was calcium-
dependent and involved mRNA transcription (Gu and
Spitzer, 1995; Spitzer et al., 1993). Increased expres-
sion of glutamic acid decarboxylase, responsible for
GABA synthesis, was observed with rising [Ca2+ ]i
levels. Gene expression of glutamic acid decarboxy-
lase was down-regulated when neurons were grown
in a calcium-free medium, but augmented follow-
ing imposing specific frequencies of calcium tran-
sients in cultured neurons (Spitzer et al., 2000). In
agreement with this study, depolarization and sub-
sequent calcium influx through voltage-operated cal-
cium channels also defined neurotransmitter choice
in other systems. Depolarization mediated calcium
influx during early differentiation of cultured sen-
sory neurons led to an increased number of tyrosine
hydroxylase-immunoreactive cells (Brosenitsch et al.,
1998). Accordingly, calcium influx also mediated dif-
ferentiation of chick ciliary ganglion and mouse spinal
neurons (Nishi and Berg, 1981; Ishida and Deguchi,
1983). Thus, spikes and waves of cytosolic calcium
contribute in an important manner to the phenomena
of proliferation and neuronal differentiation (Fig. 9.3).
Ligand-gated ion channels and metabotropic receptors
(glutamate, purinergic, GABA and cholinergic recep-
tors among many others) contribute to the generation
of [Ca2+ ]i transients (waves and spikes) and signal-
ing during development and neuronal fate acquisition.
Essential functions for calcium have been shown for
neuronal motility (Zheng and Poo, 2007), intercellu-
lar communication (Braet et al., 2004), cell fate and
developmental patterning (Whitaker and Smith, 2008;
Leclerc et al., 2006). However, despite their well-
known functions in the mature CNS, the role of these
receptors in the modulation of the calcium waves and
spikes during development, directing differentiation
and phenotype acquisition, has been the subject of
recent studies (Spitzer et al., 1994, 2004; Spitzer and
Borodinsky, 2008).
Receptor-induced calcium waves are not only
important for inducing signal transduction in the stim-
ulated cell. These signals are also essential for com-
munication between neurons and glial cells in the
adult nervous system and the developing brain. Such
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game 127

Fig. 9.3 Direction

of proliferation and neural
differentiation by
neurotransmitter-induced
pattern of intracellular
calcium spikes and waves.
The progress of neuronal
differentiation is directed
through the different types
of intracellular calcium
transients (spikes and waves)
induced at defined time points
(check points) during
development. This calcium
signaling then activates
transcription factors
responsible for expression
of ion channels specific
for the next, advanced
differentiation stage. These
channels generate patterns
of intracellular calcium spikes
and waves, which again
promote differentiation
through activation
of calcium-dependent
transcription factors and final
maturation into neurons
expressing specific
neurotransmitters
and receptors

intercellular calcium signals are transient elevations
in [Ca2+ ]i , which in analogy to action potentials, are
propagated to neighboring cells. The velocity of sig-
nal propagation is in the order of tens of micrometers
per second, i.e. two to three orders of magnitude
slower than action potential propagation (Braet et al.,
2004). The transmission of calcium signals as part of
intercellular communication involves paracrine mech-
anisms by calcium-induced release of neurotransmit-
ters into the extracellular space, which then bind to
receptors on neighboring cells, as well as activation
of downstream calcium signaling or diffusion of the
128
calcium-mobilizing messenger IP3 (Saez et al., 1989)
or calcium itself, through gap junction channels.
The importance of intracellular calcium release in
modulation of proliferation and differentiation has also
been shown in various works. Proliferation and dif-
ferentiation induction of P19 cells by metabotropic
purinergic and muscarinic activation involved libera-
tion of calcium from intracellular pools, since these
effects were abolished when formation of IP3 was
inhibited (Resende et al., 2008b, c). Moreover, cal-
cium influx from the extracellular space into embry-
onic P19 cells led to decreased proliferation activity.
However, in P19 progenitor cells nicotinic recep-
tor activity promoted proliferation and differentiation
since at this differentiation state nicotinic receptor
activation involved activation of intracellular RyR
calcium-channels as calcium-induced calcium release
(CICR). Cooperation of L-type calcium channels with
RyR2 was essential for neuronal differentiation of ES
cells (Yu et al., 2008). Moreover, pituitary adeny-
late cyclase-activating peptide ligand/type 1 receptor-
mediated activation of the phospholipase C-/IP3 -
dependent signaling pathway promoted proliferation
and gliogenesis of mouse cortex neural progenitor cells
(Nishimoto et al., 2007).
Other neurotransmitters are also important for trig-
gering calcium signals during neuronal development.
For example, calcium spikes initiated by activation
of GABA and metabotropic glutamate receptors in
embryonic cells contribute to the determination of the
final phenotype such as specification of neurotransmit-
ter expression (Root et al., 2008). In fact, non-synaptic
(paracrine actions) signaling of GABA presents con-
comitant effects which provides the timing and instruc-
tion for cells to properly differentiate and integrate
into an existing synaptic network (reviewed by Bordey,
2007).
Molecular mechanisms of developmental neural
diseases such as autism are suggested to involve
changes in calcium signaling. Mutations in several
voltage-operated calcium channels and ligand-gated
ion channels (GABA and NMDA receptors) affecting
neuronal excitability, calcium signaling and conse-
quently neurogenesis have been associated with autism
and related diseases (reviewed by Krey and Dolmetsch,
2007). Such defects in developmental signal transmis-
sion could contribute to neuroanatomical alterations
observed in patients with autism spectrum disorder
including augmented cell packing density, decreases in
K.K. Yuahasi et al.
neuron size and functional neural networks (DiCicco-
Bloom et al., 2006).
9.6 Conclusions
A large amount of evidence, mostly derived from
diverse in vitro models for neurogenesis indicate that
neurotransmitters and other molecules act on cal-
cium entry into the cell and on intracellular calcium
fluxes throughout development. The early appearance
of these signaling factors and their wide distribution
throughout development of a variety of animal species
led to suggest that neurotransmitters act as extrinsic
factors that trigger differentiation and define final phe-
notypes of these cells. Although the mechanism of
how these multiple external signals are integrated and
cooperate, is far from being understood, it is clear
that neuronal diversity is codified by this system, and
that any change to the pattern of calcium signaling at
defined time points will interfere with the final out-
come of the differentiation process. Recent studies
of mechanisms of developmental related neurological
diseases have identified mutations in neurotransmitter
receptors and other ion channels. More studies may
elucidate changes in developmental signaling related
to neurological disorder and identify new targets for
therapeutic intervention. Furthermore, the promising
therapeutic applications of embryonic, neural and mes-
enchymal stem cells in regeneration therapy require the
elucidation of the composition of niches favoring cell
survival and differentiation at the localization of cell
transplantation. The understanding of the mechanisms
of how stem cells differentiate into defined species of
neurons will also contribute to cell regeneration ther-
apy of neurodegenerative illnesses such as Parkinsons
disease, for which prior in vitro differentiation to a
homogeneous neural phenotype is crucial.
References
Adams CE, Broide RS, Chen Y, Winzer-Serhan UH, Henderson
TA, Leslie FM, Freedman R (2002) Development of the
alpha7 nicotinic cholinergic receptor in rat hippocampal
formation. Brain Res Dev Brain Res 139:175187.
Al-Mohanna FA, Caddy KW, Bolsover SR (1994) The nucleus
is insulated from large cytosolic calcium ion changes. Nature
367:745750.
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
Altman J, Das GD (1965) Autoradiographic and histological evi-
dence of postnatal hippocampal neurogenesis in rats. J Comp
Neurol 124:319335.
Alvarez-Buylla A, Garcia-Verdugo JM, Tramontin AD (2001)
An unified hypothesis on the lineage of neural stem cells.
Nat Rev Neurosci 2:287293.
Aono K, Nakanishi N, Yamada S (1990) Increase in intracellu-
lar Ca2+ level and modulation of nerve growth factor action
on pheochromocytoma PC12h cells by extracellular ATP.
Meikai Daigaku Shigaku Zasshi 19:221229.
Aruffo C, Ferszt R, Hildebrandt AG, Crvos-Navarro J (1987)
Low doses of L-monosodium glutamate promote neu-
ronal growth and differentiation in vitro. Dev Neurosci 9:
228239.
Augusti-Tocco G, Biagioni S, Tata AM (2006) Acetylcholine
and regulation of gene expression in developing systems.
J Mol Neurosci 30:4548.
Azizi SA, Stokes D, Augelli BJ, DiGirolamo C, Prockop DJ
(1998) Engraftment and migration of human bone marrow
stromal cells implanted in the brains of albino rats similar-
ities to astrocyte grafts. Proc Natl Acad Sci USA 95:3908
3913.
Balazs R, Hack N, Jorgensen OS (1990) Selective stimulation
of excitatory amino acid receptor subtypes and the survival
of cerebellar granule cells in culture: effect of kainic acid.
Neuroscience 37:251258.
Balazs R, Jorgensen OS, Hack N (1988) N-Methyl-D-aspartate
promotes the survival of cerebellar granule cells in culture.
Neuroscience 27:437451.
Barker JL, Behar T, Li YX, Liu QY, Ma W, Maric D, Maric I,
Schaffner AE, Serafini R, Smith SV, Somogyi R, Vautrin JY,
Wen XL, Xian H (1998) GABAergic cells and signals in CNS
development. Perspect Dev Neurobiol 5:305322.
Behar TN, Scott CA, Greene CL, Wen X, Smith SV, Maric
D, Liu QY, Colton CA, Barker JL (1999) Glutamate acting
at NMDA receptors stimulates embryonic cortical neuronal
migration. J Neurosci 19:44494461.
Ben-Ari Y, Cherubini E, Corradetti R, Gaiarsa JL (1989) Giant
synaptic potentials in immature rat CA3 hippocampal neu-
rones. J Physiol 416:303325.
Bernadi P (1999) Mitochondrial transport of cations: chan-
nels, exchangers, and permeability transition. Physiol Rev
79:11271155.
Berridge MJ (1993) Inositol trisphosphate and calcium sig-
nalling. Nature 361:315325.
Berridge MJ (2006) Calcium microdomains: organization and
function. Cell Calcium 40:405412.
Berridge MJ, Lipp P, Bootman MD (2000) The versatility and
universality of calcium signaling. Nat Rev Mol Cell Biol
1:1121.
Biagioni S, Tata AM, De Jaco A, Augusti-Tocco G (2000)
Acetylcholine synthesis and neuron differentiation. Int J Dev
Biol 44:689697.
Bignami F, Bevilacqua P, Biagioni S, De Jaco A, Casamenti F,
Felsani A, Augusti-Tocco G (1997) Cellular acetylcholine
content and neuronal differentiation. J Neurochem 69:
13741381.
Blaustein MP, Lederer WJ (1999) Sodium/calcium exchange: Its
physiological implications. Physiol Rev 79:763854.
Bogdanov YD, Dale L, King BF, Whittock N, Burnstock G
(1997) Early expression of a novel nucleotide receptor in
129
the neural plate of Xenopus embryos. J Biol Chem 272:
1258312590.
Bordey A (2007) Enigmatic GABAergic networks in adult
neurogenic zones. Brain Res Rev 53:124134.
Braet K, Cabooter L, Paemeleire K, Leybaert L (2004) Calcium
signal communication in the central nervous system. Biol
Cell 96:7991.
Brezun JM, Daszuta A (2000) Serotonin may stimulate gran-
ule cell proliferation in the adult hippocampus, as observed
in rats grafted with foetal raphe neurons. Eur J Neurosci
12:391396.
Brosenitsch TA, Salgado-Commissariat D, Kunze DL, Katz DM
(1998) A role for L-type calcium channels in developmen-
tal regulation of transmitter phenotype in primary sensory
neurons. J Neurosci 18:10471055.
Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:
509581.
Burnstock G (1978) A basis for distinguishing two types of
purinergic receptor. In: Cell Membrane Receptors for Drugs
and Hormones: A Multidisciplinary Approach (Straub, RW
and Bolis, L, eds), pp. 107118. Raven Press, New York.
Burnstock G (2004) Cotransmission. Curr Opin Pharmacol
4:4752.
Butler AK, Uryu K, Rougon G, Chesselet MF (1999) N-Methyl-
Daspartate receptor blockade affects polysialylated neural
cell adhesion molecule expression and synaptic density dur-
ing striatal development. Neuroscience 89:11691181.
Buznikov GA, Shmukler YB, Lauder JM (1996) From oocyte
to neuron: do neurotransmitters function in the same
way throughout development? Cell Mol Neurobiol 16:
537559.
Cameron HA, Hazel TG, McKay RD (1998) Regulation
of neurogenesis by growth factors and neurotransmitters.
J Neurobiol 36:287306.
Cameron RS, Rakic P (1991) Glial cell lineage in the cerebral
cortex: a review and synthesis. Glia 4:124137.
Cappuccio I, Spinsanti P, Porcellini A, Desiderati F, De Vita T,
Storto M, Capobianco L, Battaglia G, Nicoletti F, Melchiorri
D (2005) Endogenous activation of mGlu5 metabotropic glu-
tamate receptors supports self-renewal of cultured mouse
embryonic stem cells. Neuropharmacology 49(Suppl 1):
196205.
Cappuccio I, Verani R, Spinsanti P, Niccolini C, Gradini R,
Costantino S, Nicoletti F, Melchiorri D (2006) Context-
dependent regulation of embryonic stem cell differ-
entiation by mGlu4 metabotropic glutamate receptors.
Neuropharmacology 51:606611.
Chebib M, Johnston GA (1999) The  ABC of GABA receptors:
a brief review. Clin Exp Pharmacol Physiol 26:937940.
Cheung KK, Chan WY, Burnstock G (2005) Expression of
P2X purinoceptors during rat brain development and their
inhibitory role on motor axon outgrowth in neural tube
explant cultures. Neuroscience 133:937945.
Cheung KK, Ryten M, Burnstock G (2003) Abundant and
dynamic expression of G protein-coupled P2Y receptors in
mammalian development. Dev Dyn 228:254266.
Cicirata F, Meli C, Castorina C, Serapide MF, Sorrenti V, Di
Giacomo C, Gambera G, Vanella A (1991) Neurotransmitter
amino acid levels in rat thalamus and cerebral cortex after
cerebellectomy. Int J Dev Neurosci 9:365369.
Clapham DE (1995) Calcium signaling. Cell 80:259268.
130
Connors BW, Malenka RC, Silva LR (1988) Two inhibitory
postsynaptic potentials, and GABAA and GABAB receptor-
mediated responses in neocortex of rat and cat. J Physiol
406:443468.
Coronas V, Durand M, Chabot JG, Jourdan F, Quirion R (2000)
Acetylcholine induces neuritic outgrowth in rat primary
olfactory bulb cultures. Neuroscience 98:213219.
Court JA, Lloyd S, Johnson M, Griffiths M, Birdsall NJ, Piggott
MA, Oakley AE, Ince PG, Perry EK, Perry RH (1997)
Nicotinic and muscarinic cholinergic receptor binding in
the human hippocampal formation during development and
aging. Brain Res Dev Brain Res 101:93105.
Court JA, Perry EK, Spurden D, Griffiths M, Kerwin JM, Morris
CM, Johnson M, Oakley AE, Birdsall NJ, Clementi F et al.
(1995) The role of the cholinergic system in the develop-
ment of the human cerebellum. Brain Res Dev Brain Res 90:
159167.
Culiat CT, Stubbs LJ, Montgomery CS, Russell LB, Rinchik EM
(1994) Phenotypic consequences of deletion of the gamma 3,
alpha 5, or beta 3 subunit of the type A gamma-aminobutyric
acid receptor in mice. Proc Natl Acad Sci USA 91:
28152818.
Deisseroth K, Singla S, Toda H, Monje M, Palmer TD, Malenka
RC (2004) Excitation-neurogenesis coupling in adult neural
stem/progenitor cells. Neuron 42:535552.
Del Rio JA, Soriano E, Ferrer I (1992) Development of GABA-
immunoreactivity in the neocortex of the mouse. J Comp
Neurol 326:501526.
DiCicco-Bloom E, Lord C, Zwaigenbaum L, Courchesne E,
Dager SR, Schmitz C, Schultz RT, Crawley J, Young LJ
(2006) The developmental neurobiology of autism spectrum
disorder. J Neurosci 26:68976906.
Doetsch F (2003a) A niche for adult neural stem cells. Curr Opin
Genet Dev 13:543550.
Doetsch F (2003b) The glial identity of neural stem cells. Nat
Neurosci 6:11271134.
Don F, Ulrich H, Persike DS, Conceio IM, Blini JP,
Cavalheiro EA, Fernandes MJ (2009) Alteration of puriner-
gic P2X4 and P2X7 receptor expression in rats with
temporal-lobe epilepsy induced by pilocarpine. Epilepsy Res
83:157167.
Duchen MR (1999) Contributions of mitochondria to animal
physiology: from homeostatic sensor to calcium signalling
and cell death. J Physiol 516:117.
Eder A, Bading H (2007) Calcium signals can freely cross
the nuclear envelope in hippocampal neurons: somatic cal-
cium increases generate nuclear calcium transients. BMC
Neurosci 8:5757.
Eriksson PS, Perfilieva E, Bjrk-Eriksson T, Alborn AM,
Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in
the adult human hippocampus. Nat Med 4:13131317.
Evans MJ, Kaufman MH (1981) Establishment in culture
of pluripotential cells from mouse embryos. Nature 292:
154156.
Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E,
Stornaiuolo A, Cossu G, Mavilio F (1998) Muscle regenera-
tion by bone marrow-derived myogenic precursors. Science
279:15281530.
Fishell G, Kriegstein AR (2003) Neurons from radial glia:
the consequences of asymmetric inheritance. Curr Opin
Neurobiol 13:3441.
K.K. Yuahasi et al.
Fiszman ML, Borodinsky LN, Neale JH (1999) GABA induces
proliferation of immature cerebellar granule cells grown in
vitro. Brain Res Dev Brain Res 115:18.
Franco Lambert AP, Fraga Zandonai A, Bonatto D, Cantarelli
Machado D, Pgas Henriques J (2009) Differentiation of
human adipose-derived adult stem cells into neuronal tissue:
does it work? Differentiation 77:221228.
Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden
J (2001) International Union of Pharmacology. XXV.
Nomenclature and classification of adenosine receptors.
Pharmacol Rev 53:527552.
Frisn J, Johansson CB, Lothian C, Lendahl U (1998) Central
nervous system stem cells in the embryo and adult. Cell Mol
Life Sci 54:935945.
Fukui M, Nakamichi N, Yoneyama M, Ozawa S, Fujimori S,
Takahata Y, Nakamura N, Taniura H, Yoneda Y (2008)
Modulation of cellular proliferation and differentiation
through GABA(B) receptors expressed by undifferentiated
neural progenitor cells isolated from fetal mouse brain. J Cell
Physiol 216:507519.
Gachelin G, Kemler R, Kelly F, Jacob F (1997) PCC4, a new cell
surface antigen common to multipotential embryonal car-
cinoma cells, spermatozoa, and mouse early embryos. Dev
Biol 57:199209.
Gallo V, Zhou JM, McBain CJ, Wright P, Knutson PL,
Armstrong RC (1996) Oligodendrocyte progenitor cell pro-
liferation and lineage progression are regulated by gluta-
mate receptor-mediated K+ channel block. J Neurosci 16:
26592670.
Gass JT, Olive MF (2008) Glutamatergic substrates of
drug addiction and alcoholism. Biochem Pharmacol 75:
218265.
Gimble JM, Katz AJ, Bunnell BA (2007) Adipose-derived stem
cells for regenerative medicine. Circ Res 100:12491260.
Goldman S (2003) Glia as neural progenitor cells. Trends
Neurosci 26(11):590596.
Gotti C, Fornasari D, Clementi F (1997) Human neuronal
nicotinic receptors. Prog Neurobiol 53:199237.
Gould E, Cameron HA, McEwen BS (1994) Blockade of NMDA
receptors increases cell death and birth in the developing rat
dentate gyrus. J Comp Neurol 340:551565.
Gould E, Reeves AJ, Fallah M, Tanapat P, Gross CG, Fuchs
E (1999) Hippocampal neurogenesis in adult Old World
primates. Proc Natl Acad Sci USA 96:52635267.
Gu X, Spitzer NC (1995) Distinct aspects of neuronal differenti-
ation encoded by frequency of spontaneous Ca2+ transients.
Nature 375:784787.
Guan K, Chang H, Rolletschek A, Wobus AM (2001) Embryonic
stem cell-derived neurogenesis. Retinoic acid induction and
lineage selection of neuronal cells. Cell Tissue Res 305:
171176.
Guillemot F (2007) Cell fate specification in the mammalian
telencephalon. Prog Neurobiol 83:3752.
Gysbers JW, Rathbone MP (1992) Guanosine enhances
NGFstimulated neurite outgrowth in PC12 cells.
NeuroReport 3:9971000.
Gysbers JW, Rathbone MP (1996) GTP and guanosine synergis-
tically enhance NGF-induced neurite outgrowth from PC12
cells. Int J Dev Neurosci 14:19 34.
Gtz M, Huttner WB (2005) The cell biology of neurogenesis.
Nat Rev Mol Cell Biol 6:777788.
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
Gnther U, Benson J, Benke D, Fritschy JM, Reyes G, Knoflach
F, Crestani F, Aguzzi A, Arigoni M, Lang Y, Bluethmann
H, Mohler H, Luscher B (1995) Benzodiazepine-insensitive
mice generated by targeted disruption of the gamma 2 sub-
unit gene of gamma-aminobutyric acid type A receptors.
Proc Natl Acad Sci USA 92:77497753.
Haber M, Zhou L, Murai KK (2006) Cooperative astrocyte
and dendritic spine dynamics at hippocampal excitatory
synapses. J Neurosci 26:88818891.
Hamprecht B (1977) Structural, electrophysiological, biochem-
ical, and pharmacological properties of neuroblastoma-
glioma cell hybrids in cell culture. Int Rev Cytol 49:99170.
Haydar TF, Wang F, Schwartz ML, Rakic P (2000) Differential
modulation of proliferation in the neocortical ventricular and
subventricular zones. J Neurosci 20:57645774.
Heck S, Enz R, Richter-Landsberg C, Blohm DH (1997)
Expression of eight metabotropic glutamate receptor sub-
types during neuronal differentiation of P19 embryocarci-
noma cells: a study by RT-PCR and in situ hybridization.
Brain Res Dev Brain Res 101:8591.
Ishida I, Deguchi T (1983) Effect of depolarizing agents on
choline acetyltransferase and acetylcholinesterase activities
in primary cell cultures of spinal cord. J Neurosci 3:
18181823.
Jackson L, Jones DR, Scotting P, Sottile V (2007) Adult mes-
enchymal stem cells: differentiation potential and therapeutic
applications. J Postgrad Med 53:121127.
Jones-Villeneuve EM, McBurney MW, Rogers KA, Kalnins VI
(1982) Retinoic acid induces embryonal carcinoma cells to
differentiate into neurons and glial cells. J Cell Biol 94:
253262.
Kahan BW, Ephrussi B (1970) Developmental potentialities of
clonal in vitro cultures of mouse testicular teratoma. J Natl
Cancer Inst 44:10151036.
Kang SK, Lee DH, Bae YC, Kim HK, Baik SY, Jung JS (2003)
Improvement of neurological deficits by intracerebral trans-
plantation of human adipose tissue-derived stromal cells after
cerebral ischemia in rats. Exp Neurol 183:355366.
Kang SK, Putnam LA, Ylostalo J, Popescu IR, Dufour J,
Belousov A, Bunnell BA (2004) Neurogenesis of Rhesus
adipose stromal cells. J Cell Sci 117:42894299.
Kaplan MS, Hinds JW (1977) Neurogenesis in the adult rat: elec-
tron microscopic analysis of light radioautographs. Science
197:10921094.
Kelly CM, Tyers P, Borg MT, Svendsen CN, Dunnett SB, Rosser
AE (2005) EGF and FGF-2 responsiveness of rat and mouse
neural precursors derived from the embryonic CNS. Brain
Res Bull 68:8394.
Kinney HC, ODonnell TJ, Kriger P, White WF (1993) Early
developmental changes in [3H] nicotine binding in the
human brainstem. Neuroscience 55:11271138.
Kitayama T, Yoneyama M, Tamaki K, Yoneda Y (2004)
Regulation of neuronal differentiation by N-methyl-D-
aspartate receptors expressed in neural progenitor cells
isolated from adult mouse hippocampus. J Neurosci Res
76:599612.
Komuro H, Rakic P (1993) Modulation of neuronal migration by
NMDA receptors. Science 260:9597.
Krey JF, Dolmetsch RE (2007) Molecular mechanisms of
autism: a possible role for Ca2+ signaling. Current Opin
Neurobiol 17:112119.
131
Krnjevic K, Schwartz S (1967) The action of gamma-
aminobutyric acid on cortical neurons. Exp Brain Res 3:
320336.
Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis
in the dentate gyrus of the adult rat: age-related decrease of
neuronal progenitor proliferation. J Neurosci 16:20272033.
Laasberg T (1990) Ca2(+) -mobilizing receptors of gastrulating
chick embryo. Comp Biochem Physiol C 97:912.
Lauder JM (1993) Neurotransmitters as growth regulatory sig-
nals: role of receptors and second messengers. Trends
Neurosci 16:233240.
Lauder JM, Liu J, Devaud L, Morrow AL (1998) GABA as a
trophic factor for developing monoamine neurons. Perspect
Dev Neurobiol 5:247259.
Leclerc C, Nant I, Webb SE, Miller AL, Moreau M (2006)
Calcium transients and calcium signalling during early neu-
rogenesis in the amphibian embryo Xenopus laevis. Biochim
Biophys Acta 1763:11841191.
Le Feuvre RA, Brough D, Iwakura Y, Takeda K, Rothwell
NJ (2002) Priming of macrophages with lipopolysaccha-
ride potentiates P2X7-mediated cell death via a caspase-1-
dependent mechanism, independently of cytokine produc-
tion. J Biol Chem 277:32103218.
Lee YH, Lin CH, Hsu LW, Hu SY, Hsiao WT, Ho YS (2003)
Roles of ionotropic glutamate receptors in early developing
neurons derived from the P19 mouse cell line. J Biomed Sci
10:199207.
Levin ED (1992) Nicotinic systems and cognitive function.
Psychopharmacology (Berl) 108:417431.
Levitt P, Rakic P (1980) Immunoperoxidase localization of glial
fibrillary acidic protein in radial glial cells and astrocytes
of the developing rhesus monkey brain. J Comp Neurol
193:815840.
LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein
AR (1995) GABA and glutamate depolarize cortical pro-
genitor cells and inhibit DNA synthesis. Neuron 15:
12871298.
Ma W, Liu QY, Maric D, Sathanoori R, Chang YH, Barker JL
(1998) Basic FGF-responsive telencephalic precursor cells
express functional GABA(A) receptor/Cl channels in vitro.
J Neurobiol 35:277286.
Ma W, Maric D, Li BS, Hu Q, Andreadis JD, Grant GM, Liu
QY, Shaffer KM, Chang YH, Zhang L, Pancrazio JJ, Pant
HC, Stenger DA, Barker JL (2000) Acetylcholine stimulates
cortical precursor cell proliferation in vitro via muscarinic
receptor activation and MAP kinase phosphorylation. Eur J
Neurosci 12:12271240.
Martin GR (1981) Isolation of a pluripotent cell line from
early mouse embryos cultured in medium conditioned by
teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:
76347638.
Mass K, Bhamra S, Eason R, Dale N, Jones EA (2007)
Purine-mediated signalling triggers eye development. Nature
449:10581062.
Mattson MP, Dou P, Kater SB (1988b) Outgrowth-regulating
actions of glutamate in isolated hippocampal pyramidal neu-
rons. J Neurosci 8:20872100.
Mattson MP, Lee RE, Adams ME, Guthrie PB, Kater SB (1988a)
Interactions between entorhinal axons and target hippocam-
pal neurons: a role for glutamate in the development of
hippocampal circuitry. Neuron 1:865876.
132
McBurney MW, Jones-Villeneuve EM, Edwards MK, Anderson
PJ (1982) Control of muscle and neuronal differentiation
in a cultured embryonal carcinoma cell line. Nature 299:
165167.
Melchiorri D, Cappuccio I, Ciceroni C, Spinsanti P, Mosillo P,
Sarichelou I, Sale P, Nicoletti F (2007) Metabotropic gluta-
mate receptors in stem/progenitor cells. Neuropharmacology
53:473480.
Minakami R, Iida K, Hirakawa N, Sugiyama H (1995) The
expression of two splice variants of metabotropic glutamate
receptor subtype 5 in the rat brain and neuronal cells during
development. J Neurochem 65:15361542.
Ming GL, Song H (2005) Adult neurogenesis in the mam-
malian central nervous system. Annu Rev Neurosci 28:
223250.
Miranda-Contreras L, Bentez-Diaz PR, Mendoza-Briceo RV,
Delgado-Saez MC, Palacios-Pr EL (1999) Levels of amino
acid neurotransmitters during mouse cerebellar neurogene-
sis and in histotypic cerebellar cultures. Dev Neurosci 21:
147158.
Miranda-Contreras L, Mendoza-Briceo RV, Palacios-Pr EL
(1998) Levels of monoamine and amino acid neurotrans-
mitters in the developing male mouse hypothalamus and
in histotypic hypothalamic cultures. Int J Dev Neurosci
16:403412.
Miranda-Contreras L, Ramrez-Martens LM, Bentez-Diaz PR,
Pea-Contreras ZC, Mendoza-Briceo RV, Palacios-Pr E
(2000) Levels of amino acid neurotransmitters during mouse
olfactory bulb neurogenesis and in histotypic olfactory bulb
cultures. Int J Dev Neurosci 18:8391.
Miras-Portugal MT, Daz-Hernndez M, Girldez L, Hervs C,
Gmez-Villafuertes R, Sen RP, Gualix J, Pintor J (2003)
P2X7 receptors in rat brain: presence in synaptic terminals
and granule cells. Neurochem Res 28:15971605.
Mishra SK, Braun N, Shukla V, Fllgrabe M, Schomerus
C, Korf HW, Gachet C, Ikehara Y, Svigny J, Robson
SC, Zimmermann H (2006) Extracellular nucleotide sig-
naling in adult neural stem cells: synergism with growth
factor-mediated cellular proliferation. Development 33:
675684.
Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, Ogawa
M (2004) Asymmetric production of surface-dividing and
non-surface-dividing cortical progenitor cells. Development
131:31333145.
Mizuseki K, Sakamoto T, Watanabe K, Muguruma K, Ikeya
M, Nishiyama A, Arakawa A, Suemori H, Nakatsuji N,
Kawasaki H, Murakami F, Sasai Y (2003) Generation of neu-
ral crest-derived peripheral neurons and floor plate cells from
mouse and primate embryonic stem cells. Proc Natl Acad Sci
USA 100:58285833.
Monti B, Contestabile A (2000) Blockade of the NMDA recep-
tor increases developmental apoptotic elimination of granule
neurons and activates caspases in the rat cerebellum. Eur J
Neurosci 12:31173123.
Nacher J, McEwen BS (2006) The role of N-methyl-D-asparate
receptors in neurogenesis. Hippocampus 16:267270.
Nagase T, Matsumoto D, Nagase M, Yoshimura K, Shigeura T,
Inoue M, Hasegawa M, Yamagishi M, Machida M (2007)
Neurospheres from human adipose tissue transplanted into
cultured mouse embryos can contribute to craniofacial mor-
phogenesis: a preliminary report. J Craniofac Surg 18:4953.
K.K. Yuahasi et al.
Narahara M, Tachibana K, Kurisu N, Kanazawa M, Miyake
M (2000) Immunohistochemical and chemical changes of
beta-citryl-L-glutamate in the differentiation of bovine lens
epithelial cells into lens fiber cells. Biol Pharm Bull 23:
704707.
Neary JT, Kang Y, Shi YF (2004) Signaling from nucleotide
receptors to protein kinase cascades in astrocytes.
Neurochem Res 29:2037 2042.
Nguyen L, Rigo JM, Rocher V, Belachew S, Malgrange B,
Rogister B, Leprince P, Moonen G (2001) Neurotransmitters
as early signals for central nervous system development. Cell
Tissue Res 305:187202.
Nishi R, Berg DK (1981) Effects of high K+ concentrations on
the growth and development of ciliary ganglion neurons in
cell culture. Dev Biol 87:301307.
Nishimoto M, Furuta A, Aoki S, Kudo Y, Miyakawa H, Wada
K (2007) PACAP/PAC1 autocrine system promotes pro-
liferation and astrogenesis in neural progenitor cells. Glia
55:317327.
North RA (2002) Molecular physiology of P2X receptors.
Physiol Rev 82:10131067.
Nottebohm F, Goldman SA (1983) Neuronal production, migra-
tion, and differentiation in a vocal control nucleus of the
adult female canary brain. Proc Natl Acad Sci USA 80:
23902394.
Ostenfeld T, Svendsen CN (2004) Requirement for neuroge-
nesis to proceed through the division of neuronal progen-
itors following differentiation of epidermal growth factor
and fibroblast growth factor-2-responsive human neural stem
cells. Stem Cells 22:798811.
Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996)
Excitatory GABA responses in embryonic and neonatal
cortical slices demonstrated by gramicidin perforated-patch
recordings and calcium imaging. J Neurosci 16:64146423.
Pagani F, Lauro C, Fucile S, Catalano M, Limatola C, Eusebi
F, Grassi F (2006) Functional properties of neurons derived
from fetal mouse neurospheres are compatible with those of
neuronal precursors in vivo. J Neurosci Res 83:14941501.
Paraoanu LE, Steinert G, Koehler A, Wessler I, Layer PG (2007)
Expression and possible functions of the cholinergic system
in a murine embryonic stem cell line. Life Sci 80:23752379.
Parnas D, Linial M (1995) Cholinergic properties of neurons dif-
ferentiated from an embryonal carcinoma cell-line (P19). Int
J Dev Neurosci 13:767781.
Paterson D, Nordberg A (2000) Neuronal nicotinic receptors in
the human brain. Prog Neurobiol 61:75111.
Pearce IA, Cambray-Deakin MA, Burgoyne RD (1987)
Glutamate acting on NMDA receptors stimulates neurite
outgrowth from cerebellar granule cells. FEBS Lett 223:
143147.
Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan
AK, Murase N, Boggs SS, Greenberger JS, Goff JP (1999)
Bone marrow as a source of hepatic oval cells. Science 284:
11681170.
Phelps PE, Barber RP, Brennan LA, Maines VM, Salvaterra PM,
Vaughn JE (1990) Embryonic development of four differ-
ent subsets of cholinergic neurons in rat cervical spinal cord.
J Comp Neurol 291:926.
Pin JP, Duvoisin R (1995) The metabotropic glutamate
receptors: structure and functions. Neuropharmacology 34:
126.
9 Neurotransmitters as Main Players in the Neural Differentiation and Fate Determination Game
Poulsen FR, Blaabjerg M, Montero M, Zimmer J (2005)
Glutamate receptor antagonists and growth factors modu-
late dentate granule cell neurogenesis in organotypic, rat
hippocampal slice cultures. Brain Res 1051:3549.
Pozzan T, Rizzuto R, Volpe P, Meldolesi J (1994) Molecular and
cellular physiology of intracellular calcium stores. Physiol
Rev 74:595636.
Prockop DJ (1997) Marrow stromal cells as stem cells for non-
hematopoietic tissues. Science 276:7174.
Qu Y, Vadivelu S, Choi L, Liu S, Lu A, Lewis B, Girgis
R, Lee CS, Snider BJ, Gottlieb DI, McDonald JW (2003)
Neurons derived from embryonic stem (ES) cells resemble
normal neurons in their vulnerability to excitotoxic death.
Exp Neurol 184:326336.
Quik M, Polonskaya Y, Gillespie A, Jakowec M, Lloyd GK,
Langston JW (2000) Localization of nicotinic receptor sub-
unit mRNAs in monkey brain by in situ hybridization.
J Comp Neurol 425:5869.
Rathbone MP, Middlemiss PJ, Gysbers JW, Andrew C, Herman
MA, Reed JK, Ciccarelli R, Di Iorio P, Caciagli F (1999)
Trophic effects of purines in neurons and glial cells. Prog
Neurobiol 59:663 690.
Resende RR, Alves AS, Britto LR, Ulrich H (2008b) Role
of acetylcholine receptors in proliferation and differentia-
tion of P19 embryonal carcinoma cells. Exp Cell Res 314:
14291443.
Resende RR, Britto LR, Ulrich H (2008c) Pharmacological
properties of purinergic receptors and their effects on pro-
liferation and induction of neuronal differentiation of P19
embryonal carcinoma cells. Int J Dev Neurosci 26:763777.
Resende RR, Gomes KN, Adhikari A, Britto LR, Ulrich
H (2008a) Mechanism of acetylcholine induced cal-
cium signaling during neuronal differentiation of P19
embryonal carcinoma cells in vitro. Cell Calcium 43:
107121.
Resende RR, Majumder P, Gomes KN, Britto LR, Ulrich H
(2007) P19 embryonal carcinoma cells as in vitro model
for studying purinergic receptor expression and modula-
tion of N-methyl-D-aspartate-glutamate and acetylcholine
receptors during neuronal differentiation. Neuroscience 146:
11691181.
Reynolds BA, Weiss S (1992) Generation of neurons and astro-
cytes from isolated cells of the adult mammalian central
nervous system. Science 255:17071710.
Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa
K, Pirvola U, Saarma M, Kaila K (1999) The K+ /Cl co-
transporter KCC2 renders GABA hyperpolarizing during
neuronal maturation. Nature 397:251255.
Rizzuto R, Brini M, Murgia M, Pozzan T (1993) Microdomains
with high Ca2+ close to IP3-sensitive channels that are sensed
by neighboring mitochondria. Science 262:744747.
Root CM, Velzquez-Ulloa NA, Monsalve GC, Minakova E,
Spitzer NC (2008) Embryonically expressed GABA and
glutamate drive electrical activity regulating neurotransmitter
specification. J Neurosci 28:47774784.
Sadikot AF, Burhan AM, Blanger MC, Sasseville R (1998)
NMDA receptor antagonists influence early development of
GABAergic interneurons in the mammalian striatum. Brain
Res Dev Brain Res 105:3542.
Safford KM, Hicok KC, Safford SD, Halvorsen YD, Wilkison
WO, Gimble JM, Rice HE (2002) Neurogenic differentiation
133
of murine and human adipose-derived stromal cells.
Biochem Biophys Res Commun 294:371379.
Sez JC, Connor JA, Spray DC, Bennett MV (1989) Hepatocyte
gap junctions are permeable to the second messenger, inosi-
tol 1,4,5-trisphosphate, and to calcium ions. Proc Natl Acad
Sci USA 86:27082712.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C,
Stedeford T, Willing A, Freeman TB, Saporta S, Janssen W,
Patel N, Cooper DR, Sanberg PR (2000) Adult bone mar-
row stromal cells differentiate into neural cells in vitro. Exp
Neurol 164:247256.
Sarichelou I, Cappuccio I, Ferranti F, Mosillo P, Ciceroni C, Sale
P, Stocchi F, Battaglia G, Nicoletti F, Melchiorri D (2008)
Metabotropic glutamate receptors regulate differentiation of
embryonic stem cells into GABAergic neurons. Cell Death
Differ 15:700707.
Scheetz AJ, Constantine-Paton M (1994) Modulation of NMDA
receptor function: implications for vertebrate neural develop-
ment. FASEB J 8:745752.
Schneider AS, Atluri P, Shen Q, Barnes W, Mah SJ, Stadfelt
D, Goderie SK, Temple S, Fleck MW (2002) Functional
nicotinic acetylcholine receptor expression on stem and pro-
genitor cells of the early embryonic nervous system. Ann N
Y Acad Sci 971:135138.
Schubert P, Kreutzberg GW (1993) Cerebral protection by
adenosine. Acta Neurochir Suppl (Wien) 57:8088.
Solter D, Knowles BB (1978) Monoclonal antibody defining a
stage-specific mouse embryonic antigen (SSEA-1). Proc Natl
Acad Sci USA 75:55655569.
Song H, Stevens CF, Gage FH (2002) Astroglia induce neuroge-
nesis from adult neural stem cells. Nature 417:3944.
Spitzer NC, Borodinsky LN (2008) Implications of activity-
dependent neurotransmitter-receptor matching. Philos Trans
R Soc Lond B Biol Sci 363:13931399.
Spitzer NC, Debaca RC, Allen KA, Holliday J (1993) Calcium
dependence of differentiation of GABA immunoreactivity in
spinal neurons. J Comp Neurol 337:168175.
Spitzer NC, Gu X, Olson E (1994) Action potentials, calcium
transients and the control of differentiation of excitable cells.
Curr Opin Neurobiol 4:7077.
Spitzer NC, Lautermilch NJ, Smith RD, Gomez TM (2000)
Coding of neuronal differentiation by calcium transients.
BioEssays 22:811817.
Spitzer NC, Root CM, Borodinsky LN (2004) Orchestrating
neuronal differentiation: patterns of Ca2+ spikes specify
transmitter choice. Trends Neurosci 27:415421.
Thevananther S, Rivera A, Rivkees SA (2001) A1 adenosine
receptor activation inhibits neurite process formation by Rho
kinase-mediated pathways. NeuroReport 12:30573063.
Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van
der Kooy D (2001) Direct neural fate specification from
embryonic stem cells: A primitive mammalian neural stem
cell stage acquired through a default mechanism. Neuron
30:6578.
Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van
der Kooy D (1999) Distinct neural stem cells proliferate
in response to EGF and FGF in the developing mouse
telencephalon. Dev Biol 208:166188.
Turner CP, Yan H, Schwartz M, Othman T, Rivkees SA (2002)
A1 adenosine receptor activation induces ventriculomegaly
and white matter loss. Neuroreport 13:11991204.
134
Ulrich H, Majumder P (2006) Neurotransmitter receptor
expression and activity during neuronal differentiation of
embryonal carcinoma and stem cells: from basic research
towards clinical applications. Cell Prolif 39:281300.
Vaca K (1988) The development of cholinergic neurons. Brain
Res 472:261286.
van den Pol AN (1997) GABA immunoreactivity in hypothala-
mic neurons and growth cones in early development in vitro
before synapse formation. J Comp Neurol 383:178188.
Wang C, Pralong WF, Schulz MF, Rougon G, Aubry JM,
Pagliusi S, Robert A, Kiss JZ (1996) Functional N-methyl-
D-aspartate receptors in O-2A glial precursor cells: a crit-
ical role in regulating polysialic acid-neural cell adhe-
sion molecule expression and cell migration. J Cell Biol
135:15651581.
Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki
S, Kawasaki H, Watanabe Y, Mizuseki K, Sasai Y (2005)
Directed differentiation of telencephalic precursors from
embryonic stem cells. Nat Neurosci 8:288296.
Weaver DR (1993) A2a adenosine receptor gene expression
in developing rat brain. Brain Res Mol Brain Res 20:
313327.
Weiss ER, Maness P, Lauder JM (1998) Why do neurotrans-
mitters act like growth factors? Perspect Dev Neurobiol
5:323335.
Weissman TA, Riquelme PA, Ivic L, Flint AC, Kriegstein AR
(2004) Calcium waves propagate through radial glial cells
and modulate proliferation in the developing neocortex.
Neuron 43:647661.
Whitaker M, Smith J (2008) Introduction. Calcium signals
and developmental patterning. Phil Trans R Soc B 363:
13071310.
K.K. Yuahasi et al.
Wichterle H, Lieberam I, Porter JA, Jessell TM (2002) Directed
differentiation of embryonic stem cells into motor neurons.
Cell 110:385397.
Xu Y, Liu Z, Liu L, Zhao C, Xiong F, Zhou C, Li Y, Shan
Y, Peng F, Zhang C (2008) Neurospheres from rat adipose-
derived stem cells could be induced into functional Schwann
cell-like cells in vitro. BMC Neurosci 9:2131.
Ying QL, Stavridis M, Griffiths D, Li M, Smith A (2003)
Conversion of embryonic stem cells into neuroectodermal
precursors in adherent monoculture. Nat Biotechnol 21:
183186.
Yoneyama M, Fukui M, Nakamichi N, Kitayama T, Taniura
H, Yoneda Y (2007). Activation of GABA(A) receptors
facilitates astroglial differentiation induced by ciliary neu-
rotrophic factor in neural progenitors isolated from fetal rat
brain. J Neurochem 100:16671679.
Yu H-M, Wen J, Wang R (2008) Critical role of type 2 ryanodine
receptor in mediating activity-dependent neurogenesis from
embryonic stem cells. Cell Calcium 43:417431.
Zheng JQ, Poo M (2007) Calcium signaling and neuronal motil-
ity. Annu Rev Cell Dev Biol 23:375404.
Zoli M, Le Novre N, Hill JA Jr, Changeux JP (1995)
Developmental regulation of nicotinic ACh receptor subunit
mRNAs in the rat central and peripheral nervous systems.
J Neurosci 15:19121939.
Zoli M, Picciotto MR, Ferrari R (1999) Increased neurodegen-
eration during ageing in mice lacking high-affinity nicotine
receptors. EMBO J 18:12351244.
Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno
H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH (2002)
Human adipose tissue is a source of multipotent stem cells.
Mol Biol Cell 13:42794295.
Chapter 10
Rhythmic Expression of Notch Signaling in Neural
Progenitor Cells
Hiromi Shimojo, Toshiyuki Ohtsuka, and Ryoichiro Kageyama
Contents
10.1 Introduction . . . . . . . . . . . . . . . . .
10.2 Activator-Type bHLH Genes . . . . . . . . .
10.3 Repressor-Type bHLH Genes . . . . . . . .
10.4 Notch Signaling . . . . . . . . . . . . . . .
10.5 Dynamic Expression in Neural Progenitor Cells
10.6 Oscillatory Versus Persistent Hes1 Expression .
10.7 Conclusions . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .
Abstract The activator-type basic helix-loop-helix
(bHLH) genes such as Mash1 and Neurogenin2 (Ngn2)
promote neuronal differentiation and induce expres-
sion of Notch ligands such as Delta1, which activate
Notch signaling of neighboring cells. Activation of
Notch signaling induces expression of the repressor-
type bHLH genes such as Hes1 and Hes5, which main-
tain neural progenitor cells by antagonizing activator-
type bHLH genes. Thus, differentiating neurons keep
their neighboring cells as neural progenitor cells via
Notch signaling. How, then, are neural progenitor cells
maintained before formation of such neurons? A recent
study revealed that Hes1 expression occurs rhythmi-
cally in neural progenitor cells, and that Ngn2 and
Delta1 are also expressed in an oscillatory manner
by these cells. Inhibition of Notch signaling, a condi-
tion known to induce neuronal differentiation, leads to
down-regulation of Hes1 and sustained up-regulation
of Ngn2 and Delta1, suggesting that Hes1 oscillation
regulates Ngn2 and Delta1 oscillations. It is likely that
R. Kageyama ()
Institute for Virus Research, Kyoto University, Kyoto, Japan
e-mail: rkageyam@virus.kyoto-u.ac.jp
H. Ulrich (ed.), Perspectives of Stem Cells,
DOI 10.1007/978-90-481-3375-8_10, Springer Science+Business Media B.V. 2010
Delta1 oscillations reciprocally activate Notch signal-
ing between neighboring neural progenitor cells. These
135 results also suggest that oscillatory expression of Ngn2
136 is not sufficient but sustained up-regulation is required
137 for neuronal differentiation and that Ngn2 oscillation
138
is advantageous for activation of Notch signaling by
139
140 inducing Delta1 expression without promoting neu-
141 ronal differentiation.
142
Keywords bHLH gene Hes1 Neural progenitor
cell Notch signaling Oscillatory expression
Abbreviations
bHLH basic helix-loop-helix
GABA -aminobutyric acid
Ngn2 Neurogenin 2
NICD Notch intracellular domain
Stat3-P Phosphorylated Stat3
10.1 Introduction
In the developing mammalian nervous system, neu-
roepithelial cells undergo self-renewal repeatedly by
symmetric division (Fig. 10.1). As the number of neu-
roepithelial cells increases, the wall of the neural tube
grows thicker, and neuroepithelial cells are elongated
to become radial glial cells (Fig. 10.1). Radial glial
cells have a cell body in the inner side (called the
ventricular zone) of the neural tube and a long pro-
cess (called a radial fiber) reaching the outer surface. It
was long thought that radial glial cells are specialized
glial cells that guide neuronal migration. However, it
has been shown that radial glial cells divide and gen-
erate neurons (Alvarez-Buylla et al., 2001; Anthony
135
136
Fig. 10.1 Neural stem/progenitor cells and their differ-
entiation. Initially, neuroepithelial cells are formed. These
cells undergo self-renewal repeatedly by symmetric division.
As development proceeds, neuroepithelial cells are elongated
to become radial glial cells, which have a cell body in the
inner side (called the ventricular zone) of the neural tube
and a long process (called a radial fiber) reaching the outer
et al., 2004; Fujita, 2003; Malatesta et al., 2000;
Noctor et al., 2001). Each radial glial cell gives rise
to two radial glial cells by symmetric division, one
radial glial cell and one neuron/neuronal precursor
by asymmetric division, or two neurons/neuronal pre-
cursors by symmetric neurogenic division. Neuronal
precursors further divide and produce more neurons.
After production of neurons, some radial glial cells
give rise to oligodendrocytes and ependymal cells
(Fig. 10.1). Radial glial cells finally differentiate into
astrocytes (Fig. 10.1). Both neuroepithelial cells and
radial glial cells are considered as embryonic neural
stem/progenitor cells (here, we call neural progeni-
tor cells). Embryonic neural progenitor cells change
their differentiation ability during development: ini-
tially undergoing self-renewal only and then differenti-
ation into neurons and other cell types, and finally into
astrocytes.
It has been shown that neural development is antag-
onistically regulated by activator-type and repressor-
type basic helix-loop-helix (bHLH) genes (Bertrand
et al., 2002; Kageyama et al., 2005; Ross et al., 2003).
The former genes promote neuronal differentiation,
whereas the latter genes maintain neural progeni-
tor cells by antagonizing the former. Recent studies
revealed that both activator-type and repressor-type
bHLH genes are expressed in an oscillatory man-
ner in neural progenitor cells and that this dynamic
expression is important for maintenance of these cells
(Shimojo et al., 2008). In this chapter, we describe how
H. Shimojo et al.
surface. Radial glial cells give rise to neurons/neuronal pre-
cursors. After production of neurons, some radial glial cells
give rise to oligodendrocytes and ependymal cells. Radial glial
cells finally differentiate into astrocytes. Both neuroepithelial
cells and radial glial cells are considered as embryonic neural
stem/progenitor cells
dynamic bHLH gene expression regulates proliferation
and differentiation of neural progenitor cells.
10.2 Activator-Type bHLH Genes
Activator-type bHLH genes include Mash1, Math and
Neurogenin (Ngn), homologs of Drosophila proneu-
ral genes achaete-scute complex and atonal (Bertrand
et al., 2002; Kageyama et al., 2005; Ross et al.,
2003). They are expressed by differentiating neurons
in the developing nervous system. These bHLH factors
form heterodimers through the helix-loop-helix (HLH)
domain with a ubiquitously expressed bHLH factor,
E47, a product of a homolog of Drosophila proneu-
ral gene daughterless, and bind to the E box sequences
(CANNTG) through the basic regions, which are rich
in basic amino acid residues (Johnson et al., 1992).
These heterodimers activate gene expression, and thus
these bHLH factors are categorized as the activator
type (Fig. 10.2a).
Activator-type bHLH genes not only induce the
pan-neuronal gene expression but also promote the
neuronal subtype specification. In Drosophila, the
proneural genes achaete and atonal regulate speci-
fication of different subtypes of the peripheral ner-
vous system: external sensory organs and chordotonal
organs, respectively. It was previously shown that the
basic regions are involved in specification of different
10 Rhythmic Expression of Notch Signaling in Neural Progenitor Cells
Fig. 10.2 Activator-type and repressor-type bHLH factors.
(a) Activator-type bHLH factors form heterodimers and activate
expression of neuronal-specific genes by binding to the E box.
(b) Repressor-type bHLH factors recruit a homolog of Groucho,
neuronal subtypes (Chien et al., 1996). Mammalian
homologs of achaete and atonal also regulate speci-
fication of different neuronal subtypes. For example,
Mash1 regulates specification of GABAergic interneu-
rons in the ventral telencephalon, while Ngn2 regulates
specification of the glutamatergic pyramidal neurons
in the dorsal telencephalon (Fode et al., 2000; Parras
et al., 2002). In the absence of Ngn2, Mash1 is ectopi-
cally expressed and generates ectopic GABAergic
interneurons in the dorsal telencephalon. However,
it has been shown that activator-type bHLH genes
alone are not sufficient to generate diverse subtypes
of neurons. For example, retinal progenitor cells give
rise to six subtypes of neurons, which are aligned in
specific layers, but activator-type bHLH genes alone
cannot regulate specification of these retinal neurons
(Hatakeyama et al., 2001). Although Mash1 is required
for formation of bipolar neurons in the retina, misex-
pression of Mash1 alone does not evoke an increase
in bipolar cell genesis. Similarly, although the home-
odomain gene Chx10 is required for formation of
bipolar neurons in the retina, misexpression of Chx10
alone does not generate bipolar neurons but induces
formation of non-neuronal cells in the inner nuclear
layer, where bipolar neurons normally reside. In con-
trast, misexpression of both Mash1 and Chx10 effi-
ciently induces bipolar cell formation (Hatakeyama
et al., 2001). It is likely that homeodomain genes regu-
late the layer identity while activator-type bHLH genes
determine the neuronal fate specific for each layer.
137
a co-repressor, and repress expression of genes such as activator
bHLH genes (left). Repressor-type bHLH factors also antag-
onize the activity of activator-type bHLH factors by forming
non-functional heterodimers (right)
Activator-type bHLH factors not only stimulate
neuronal-specific gene expression but also repress
glial-specific gene expression. Expression of glial fib-
rillary acidic protein (GFAP), an astrocyte-specific
gene, is up-regulated by Stat1/3 and Smad1, which are
bridged by the co-activator p300, but Ngn1 sequesters
the p300/Smad complex from the glial promoters and
thereby inhibits glial-specific gene expression (Sun
et al., 2001). Thus, it is likely that activator-type
bHLH factors reinforce the neuronal fate specifica-
tion by inhibiting the alternative fate. In agreement
with this notion, in Mash1;Math3 double knock-out
or Mash1;Ngn2 double knock-out mice, the cells that
should normally differentiate into neurons adopted the
glial fate instead, indicating that there is a fate switch
from neurons to glia in the absence of activator-type
bHLH genes (Tomita et al., 2000; Nieto et al., 2001).
Thus, activator-type bHLH genes regulate neuronal
versus glial cell fate determination.
10.3 Repressor-Type bHLH Genes
Repressor-type bHLH genes include Hes genes,
homologs of Drosophila hairy and Enhancer of split
(Sasai et al., 1992; Kageyama et al., 2007). Among
the Hes family members, Hes1, Hes3 and Hes5 are
highly expressed in the developing nervous system.
Neuroepithelial cells initially express Hes1 and Hes3,
138
but then Hes3 expression is down-regulated whereas
Hes5 expression occurs (Hatakeyama et al., 2004).
Radial glial cells express mostly Hes1 and Hes5. Hes
factors form homodimers or heterodimers with Hes-
related factors such as Hey1 through the HLH domain
and bind to the N box (CACNAG) or the class C site
(CACG(C/A)G) with a higher affinity than to the E box
(CANNTG), unlike other bHLH factors (Fig. 10.2b)
(Sasai et al., 1992). Hes factors contain the sequence
Trp-Arg-Pro-Trp (the WRPW domain) at the carboxy-
terminus, which recruits the co-repressor TLE/Grg, a
homolog of Drosophila Groucho (Fig. 10.2b) (Paroush
et al., 1994; Fisher et al., 1996; Grbavec and Stifani,
1996). Thus, Hes factors function as transcriptional
repressors. The target genes for Hes factors include
activator-type bHLH genes such as Mash1. For exam-
ple, Hes1 represses Mash1 expression by directly
binding to the Mash1 promoter (Chen et al., 1997).
Hes factors also form heterodimers through the HLH
domain with activator-type bHLH factors, but these
heterodimers cannot bind to the DNA and therefore
are non-functional (Fig. 10.2b) (Sasai et al., 1992).
Thus, Hes1 antagonizes Mash1 by two different mech-
anisms: repressing the expression at the transcriptional
level and inhibiting the activity by forming a non-
functional heterodimer.
Misexpression of Hes1, Hes3 or Hes5 in the embry-
onic brain inhibits neuronal differentiation and main-
tains radial glial cells, whereas in Hes1;Hes5 dou-
ble knock-out mice, many radial glial cells are not
maintained and prematurely differentiate into neu-
rons (Ishibashi et al., 1994; Ohtsuka et al., 2001;
Hatakeyama et al., 2004). In these mutant embryos,
neuroepithelial cells and some radial glial cells are
still maintained, suggesting that Hes3 may compen-
sate for Hes1 and Hes5 deficiency. In agreement with
this notion, in Hes1;Hes3;Hes5 triple knock-out mice
many neuroepithelial cells prematurely differentiate
into neurons as early as on E8.5 in contrast to wild-type
embryos, where neuroepithelial cells do not differen-
tiate into neurons at this stage (Hatakeyama et al.,
2004). Furthermore, in the triple knock-out mice, vir-
tually all radial glial cells prematurely differentiate
into neurons in the spinal cord and the hindbrain
by E10.0 at the expense of the later-born cell types
such as later born neurons and astrocytes (Hatakeyama
et al., 2004). Thus, Hes1, Hes3 and Hes5 are essen-
tial to generate a sufficient number and a full spectrum
of cells by maintaining neural progenitor cells until
later stages. The premature neuronal differentiation in
H. Shimojo et al.
Hes-mutant mice is associated with up-regulation of
activator-type bHLH genes such as Mash1 and Math3
(Hatakeyama et al., 2004). Thus, it is likely that Hes
genes regulate the normal timing of differentiation by
preventing premature onset of activator-type bHLH
genes.
10.4 Notch Signaling
In differentiating neurons, activator-type bHLH genes
up-regulate expression of Notch ligands such as
Delta1, which then activates Notch, a transmembrane
protein, on neighboring cells (Fig. 10.3) (Artavanis-
Tsakonas et al., 1999; Selkoe and Kopan, 2003). Upon
activation by Notch ligands, the Notch intracellular
domain (NICD) is released from the transmembrane
region and moves into the nucleus, where NICD forms
a complex with the DNA-binding protein RBPj. RBPj
alone is a transcriptional repressor, but the NICD-
RBPj complex is a transcriptional activator and induces
expression of the repressor-type bHLH genes Hes1
and Hes5 (Fig. 10.3) (Jarriault et al., 1995; Ohtsuka
et al., 1999). Hes1 and Hes5 antagonize the expression
and activity of activator-type bHLH genes, thereby
inhibiting neuronal differentiation (Fig. 10.3). Thus,
Fig. 10.3 Notch signaling and lateral inhibition. Activator-
type bHLH factors induce expression of Notch ligands such
as Delta1, which activate Notch signaling in neighboring cells.
Upon activation by Notch ligands, the Notch intracellular
domain (NICD) is released from the transmembrane region and
moves into the nucleus, where NICD forms a complex with
the DNA-binding protein RBPj. RBPj alone is a transcriptional
repressor, but the NICD-RBPj complex is a transcriptional acti-
vator and induces expression of the repressor-type bHLH genes
Hes1 and Hes5. Hes1 and Hes5 antagonize the expression and
activity of activator-type bHLH genes, thereby inhibiting neu-
ronal differentiation and maintaining neural progenitor cells
10 Rhythmic Expression of Notch Signaling in Neural Progenitor Cells
differentiating neurons inhibit neighboring cells from
differentiating into the same cell types by activation of
Notch signaling. This Notch signaling-mediated cell-
cell regulation is called lateral inhibition. This regu-
lation is essential for maintenance of neural progenitor
cells, because in the absence of Notch signaling all
neural progenitor cells prematurely differentiate into
neurons and are depleted without generating later-born
cell types. These results also indicate that activator-
type bHLH genes are important for maintenance of
neural progenitor cells, because they induce expres-
sion of Notch ligands, which activate Notch signaling
in neighboring cells. In agreement with this notion,
in Mash1;Math3 double knock-out mice, expression
of a Notch ligand is lost in the hindbrain, leading to
inactivation of Notch signaling and premature loss of
neural progenitor cells (Ohsawa et al., 2005). Thus,
activator-type bHLH genes not only promote neu-
ronal differentiation cell-autonomously but also main-
tain neural progenitor cells non-cell-autonomously via
Notch signaling.
Before neurons are formed, neural progenitor cells
proliferate extensively, but even at this early stage
Notch signaling seems to be important for maintenance
of neural progenitor cells. Without any neurons, how
is Notch signaling active? It has been recently shown
that neural progenitor cells also express activator-type
bHLH genes and Notch ligands, suggesting that these
cells mutually activate Notch signaling. How does
the Notch signaling operate in neural progenitor cells
before neuronal formation? Why do neural progeni-
tor cells remain undifferentiated although they express
activator-type bHLH genes? We discuss these issues in
the next section.
10.5 Dynamic Expression in Neural
Progenitor Cells
In neural progenitor cells, Hes1 expression is variable:
some cells express Hes1 at high levels while others
express it at lower levels. It was previously shown
that Hes1 expression oscillates with a period of about
2 h in many cell types such as fibroblasts (Fig. 10.4)
(Hirata et al., 2002). Hes1 represses its own expression
by directly binding to the Hes1 promoter (Takebayashi
et al., 1994). This negative feedback leads to dis-
appearance of both hes1 mRNA and Hes1 protein,
because they are extremely unstable, allowing the next
139
round of expression. In this way, Hes1 autonomously
starts oscillatory expression (Fig. 10.4) (Hirata et al.,
2002). Real-time imaging analysis indicated that Hes1
expression also oscillates in each neural progenitor cell
with a period of about 23 h (Fig. 10.5) (Masamizu
et al., 2006; Shimojo et al., 2008). Interestingly, when
the Hes1 expression level is high, the expression lev-
els of the activator-type bHLH factor Ngn2 and the
Notch ligand Delta1 are low, and vice versa in neu-
ral progenitor cells, suggesting that Ngn2 and Delta1
expression also oscillate in these cells but with an
opposite phase to Hes1 oscillation (Shimojo et al.,
2008). Real-time imaging showed that Ngn2 and
Delta1 expression indeed oscillate in neural progen-
itor cells but are persistent in post-mitotic neurons,
which lose Hes1 expression (Fig. 10.5) (Shimojo et al.,
2008). Thus, Hes1 oscillation drives Ngn2 and Delta1
oscillations in neural progenitor cells, whereas Ngn2
and Delta1 expression are persistently up-regulated
when Hes1 expression disappears in post-mitotic neu-
rons. It is likely that Delta1 oscillation periodically
activates Notch signaling between neighboring neural
progenitor cells. This reciprocal regulation is particu-
larly important for maintenance of neural progenitor
cells before formation of neurons, which persistently
express Delta1.
Why do neural progenitor cells that express Ngn2
in an oscillatory manner remain undifferentiated? One
likely answer is that Ngn2 oscillation can induce
expression of only subsets of downstream genes such
as Delta1 while sustained Ngn2 expression is required
for expression of a full spectrum of downstream genes.
For example, Delta1 is expressed in the ventricular
zone, indicating that Delta1 expression occurs rapidly
after Ngn2 expression (Castro et al., 2006). In con-
trast, another downstream gene, Rnd2, is expressed
mainly in the subventricular and intermediate zones,
suggesting that Rnd2 expression occurs rather late after
Ngn2 expression (Heng et al., 2008). Expression of
such late-responding genes may depend on sustained
Ngn2 expression and thus may not occur when Ngn2
expression oscillates.
Hes1 oscillation is also important for efficient cell
proliferation. Sustained Hes1 expression represses cell
cycle regulators such as cyclin D1 and cyclin E2
and inhibits cell cycle progression (Baek et al., 2006;
Strm et al., 2000; Shimojo et al., 2008). However,
Hes1 also promotes G1-S transition by repressing
expression of cyclin-dependent kinase inhibitors (CKI)
(Castella et al., 2000; Kabos et al., 2002; Murata et al.,
140 H. Shimojo et al.

Fig. 10.4 Oscillatory

expression of Hes1. Hes1
expression oscillates with a
period of about 2 h in many
cell types such as fibroblasts.
Hes1 represses its own
expression by directly binding
to the Hes1 promoter. This
negative feedback leads to
disappearance of both hes1
mRNA and Hes1 protein,
because they are extremely
unstable, allowing the next
round of expression. In this
way, Hes1 autonomously
starts oscillatory expression

2005). Thus, Hes1 both inhibits and promotes cell

cycle progression, suggesting that oscillatory expres-
sion is required for efficient cell cycle progression.
In agreement with this notion, cells in the boundaries
such as the isthmus, the roof plate and the floor plate
that express Hes1 persistently proliferate slowly, com-
pared to neural progenitor cells whose Hes1 expression
oscillates (Hirata et al., 2001; Baek et al., 2006). Thus,
different expression mode of Hes1 (oscillatory ver-
sus persistent) leads to different characteristics of cells
(rapidly versus slowly dividing).

Fig. 10.5 Dynamic expression in neural progenitor cells.
Hes1 expression oscillates with a period of about 23 h in
neural progenitor cells. In these cells, Ngn2 and Delta1 expres-
sion also oscillate but with an opposite phase to Hes1 oscil-
lation. However, Ngn2 and Delta1 expression are persistent in
post-mitotic neurons, which lose Hes1 expression. It is likely
that Delta1 oscillation periodically activates Notch signaling
between neighboring neural progenitor cells. This reciprocal
regulation is particularly important for maintenance of neural
progenitor cells before formation of neurons
10.6 Oscillatory Versus Persistent Hes1
Expression
Hes1 expression oscillates in actively proliferating
neural progenitor cells, whereas it is persistent in
slowly proliferating boundary cells. The precise mech-
anism of how oscillatory versus persistent Hes1
expression is regulated remains to be determined.
10 Rhythmic Expression of Notch Signaling in Neural Progenitor Cells 141

It was found that Socs3 expression also oscillates

with a period of about 2 h in fibroblasts (Yoshiura
et al., 2007). Phosphorylated Stat3 (Stat3-P) induces
Socs3 expression, but Socs3 inhibits phosphorylation
of Stat3, forming a negative feedback loop. As a result,
formation of Stat3-P and expression of Socs3 oscil-
late in the opposite phase (Fig. 10.6) (Yoshiura et al.,
2007). Interestingly, inhibition of Stat3-Socs3 oscilla-
tions blocks Hes1 oscillation, suggesting that Stat3-
Socs3 signaling regulates oscillatory versus persistent
Hes1 expression, although the precise mechanism is
unknown (Yoshiura et al., 2007). It was previously
shown that formation of Stat3-P is inhibited in the
absence of Hes1 (Kamakura et al., 2004), suggest-
ing that Stat3-Socs3 oscillations and Hes1 oscillation
depend on each other (Fig. 10.6).
Another possible mechanism is Id, a HLH factor Fig. 10.6 The oscillator networks in neural progenitor cells.
Phosphorylated Stat3 (Stat3-P) induces Socs3 expression, but
that does not have a basic region. Id can form a het- Socs3 inhibits phosphorylation of Stat3, forming a negative feed-
erodimer with Hes1 through the HLH domains, but back loop. As a result, formation of Stat3-P and expression of
this heterodimer cannot bind to the DNA, because Socs3 oscillate in the opposite phase. It is likely that Stat3-Socs3
Id does not have a basic region. As a result, Id can oscillations and Hes1 oscillation depend on each other. Hes1
oscillation drives Ngn2 and Delta1 oscillations. These oscillators
inhibit Hes1 activity. Id is highly expressed in the are coupled and may regulate proliferation and differentiation of
dorsal midline regions of the neural tube, and it was neural progenitor cells
shown that Id prevents Hes1 from negative autoreg-
ulation, thereby persistently up-regulating Hes1 (Bai expression of only subsets of downstream genes such
et al., 2007). Thus, it is possible that Stat3-Socs3 sig- as Delta1, and persistent expression may be required
naling and Id may regulate oscillatory versus persistent for neuronal differentiation. Hes1 oscillation is also
Hes1 expression. important for efficient proliferation of neural progen-
itor cells, and cells expressing Hes1 persistently such
as boundary cells proliferate slowly.
10.7 Conclusions The adult brain also has many neural stem/
progenitor cells, and continuous neurogenesis is essen-
tial for the maintenance of the brain structure and
It was previously thought that only differentiating neu-
function (Imayoshi et al., 2008). It is known that adult
rons express activator-type bHLH genes and Notch
neural stem cells proliferate very slowly, raising the
ligands, thereby activating Notch signaling of neigh-
possibility that Hes1 could be persistently expressed
boring neural progenitor cells. However, it does not
by these cells. Induction of Hes1 oscillation could lead
explain how Notch signaling is activated in neural pro-
to more efficient expansion and neuronal differentia-
genitor cells before neuronal formation. Recent accu-
tion of adult neural stem cells. Further characterization
mulating evidence indicates that activator-type bHLH
of dynamics of gene expression will help understand
genes and Notch ligands are expressed in salt-and-
the molecular mechanism of how proliferation and dif-
pepper patterns by neural progenitor cells (Kageyama
ferentiation of embryonic and adult neural stem cells
et al., 2008). We have found that expression of the
are controlled.
activator-type bHLH gene Ngn2 and the Notch ligand
Delta1 oscillates in neural progenitor cells, suggesting Acknowledgments This work was supported by the Grants-in-
that these cells reciprocally activate Notch signaling aid from the Ministry of Education, Culture, Sports, Science
by Delta oscillation. Our results may also explain why and Technology of Japan, and the Uehara Memorial Foundation.
neural progenitor cells do not start neuronal differ- H.S. was supported by the twenty-first century Center of
Excellence Program of the Ministry of Education, Culture,
entiation at early developmental stages even though Sports, Science and Technology of Japan and Research
activator-type bHLH genes are expressed. Oscillatory Fellowships of the Japan Society for the Promotion of Science
expression of activator-type bHLH genes may induce for Young Scientists.
142
References
Alvarez-Buylla A, Garcia-Verdugo JM, and Tramontin AD
(2001) A unified hypothesis on the lineage of neural stem cells.
Nat Rev Neurosci 2:287293.
Anthony TE, Klein C, Fishell G, and Heintz N (2004) Radial
glia serve as neuronal progenitors in all regions of the central
nervous system. Neuron 41:881890.
Artavanis-Tsakonas S, Rand MD, and Lake RJ (1999) Notch sig-
naling: cell fate control and signal integration in development.
Science 284:770776.
Baek JH, Hatakeyama J, Sakamoto S, Ohtsuka T, and Kageyama
R (2006) Persistent and high levels of Hes1 expression regulate
boundary formation in the developing central nervous system.
Development 133:24672476.
Bai G, Sheng N, Xie Z, Bian W, Yokota Y, Benezra R, Kageyama
R, Guillemot F, and Jing N (2007) Id sustains Hes1 expres-
sion to inhibit precocious neurogenesis by releasing negative
autoregulation of Hes1. Dev Cell 13:283297.
Bertrand N, Castro DS, and Guillemot F (2002) Proneural genes
and the specification of neural cell types. Nat Rev Neurosci
3:517530.
Castella P, Sawai S, Nakao K, Wagner JA, and Caudy M (2000)
HES-1 repression of differentiation and proliferation in PC12
cells: role for the helix 3-helix 4 domain in transcription
repression. Mol Cell Biol 20:61706183.
Castro DS, Skowronska-Krawczyk D, Armant O, Donaldson
IJ, Parras C, Hunt C, Critchley JA, Nguyen L, Gossler A,
Gttgens B, Matter J-M, and Guillemot F (2006) Proneural
bHLH and Brn proteins coregulate a neurogenic program
through cooperative binding to a conserved DNA motif. Dev
Cell 11:831844.
Chen H, Thiagalingam A, Chopra H, Borges MW, Feder JN,
Nelkin BD, Baylin SB, and Ball DW (1997) Conservation
of the Drosophila lateral inhibition pathway in human lung
cancer: a hairy-related protein (HES-1) directly represses
achaete-scute homolog-1 expression. Proc Natl Acad Sci USA
94:53555360.
Chien C, Hsiao C-D, Jan LY, and Jan YN (1996) Neuronal
type information encoded in the basic-helix-loop-helix
domain of proneural genes. Proc Natl Acad Sci USA
93:1323913244.
Fisher AL, Ohsako S, and Caudy M (1996) The WRPW
motif of the Hairy-related basic helix-loop-helix repres-
sor proteins acts as a 4-amino-acid transcription repres-
sion and proteinprotein interaction domain. Mol Cell Biol
16:26702677.
Fode C, Ma Q, Casarosa S, Ang S-L, Anderson DJ, and
Guillemot F (2000) A role for neural determination genes in
specifying the dorsoventral identity of telencephalic neurons.
Genes Dev 14:6780.
Fujita S (2003) The discovery of the matrix cell, the identi-
fication of the multipotent neural stem cell and the devel-
opment of the central nervous system. Cell Struct Funct
28:205228.
Grbavec D and Stifani S (1996) Molecular interaction between
TLE1 and the carboxyl-terminal domain of HES-1 contain-
ing the WRPW motif. Biochem Biophys Res Commun 223:
701705.
H. Shimojo et al.
Hatakeyama J, Tomita K, Inoue T, and Kageyama R (2001)
Roles of homeobox and bHLH genes in specification of a
retinal cell type. Development 128:13131322.
Hatakeyama J, Bessho Y, Katoh K, Ookawara S, Fujioka M,
Guillemot F, and Kageyama R (2004) Hes genes regulate size,
shape and histogenesis of the nervous system by control of
the timing of neural stem cell differentiation. Development
131:55395550.
Heng JI, Nguyen L, Castro DS, Zimmer C, Wildner H,
Armant O, Skowronska-Krawczyk D, Bedogni F, Matter JM,
Hevner R, and Guillemot F (2008) Neurogenin 2 controls
cortical neuron migration through regulation of Rnd2. Nature
455:114118.
Hirata H, Tomita K, Bessho Y, and Kageyama R (2001)
Hes1 and Hes3 regulate maintenance of the isthmic orga-
nizer and development of the mid/hindbrain. EMBO J
20:44544466.
Hirata H, Yoshiura S, Ohtsuka T, Bessho Y, Harada T,
Yoshikawa K, and Kageyama R (2002) Oscillatory expression
of the bHLH factor Hes1 regulated by a negative feedback
loop. Science 298:840843.
Ishibashi M, Moriyoshi K, Sasai Y, Shiota K, Nakanishi
S, and Kageyama R (1994) Persistent expression of
helix-loop-helix factor HES-1 prevents mammalian neu-
ral differentiation in the central nervous system. EMBO J
13:17991805.
Imayoshi I, Sakamoto M, Ohtsuka T et al. (2008) Roles
of continuous neurogenesis in the structural and func-
tional integrity of the adult forebrain. Nat Neurosci
11:11531161.
Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, and Israel
A (1995) Signalling downstream of activated mammalian
Notch. Nature 377:355358.
Johnson JE, Birren SJ, Saito T, and Anderson DJ (1992) DNA
binding and transcriptional regulatory activities of mammalian
achaete-scute homologous (MASH) proteins revealed by inter-
action with a muscle-specific enhancer. Proc Natl Acad Sci
USA 89:35963600.
Kabos P, Kabosova A, and Neuman A (2002) Blocking HES1
expression initiates GABAergic differentiation and induces the
expression of p21(CIP1/WAF1) in human neural stem cells. J
Biol Chem 277:87638766.
Kageyama R, Ohtsuka T, Hatakeyama J, and Ohsawa R (2005)
Roles of bHLH genes in neural stem cell differentiation. Exp
Cell Res 306:343348.
Kageyama R, Ohtsuka T, and Kobayashi T (2007) The Hes gene
family: repressors and oscillators that orchestrate embryogen-
esis. Development 134:12431251.
Kageyama R, Ohtsuka T, Shimojo H, and Imayoshi I
(2008) Dynamic Notch signaling in neural progenitor cells
and a revised view of lateral inhibition. Nat Neurosci
11:12471251.
Kamakura S, Oishi K, Yoshimatsu T, Nakafuku M, Masuyama
N, and Gotoh Y (2004) Hes binding to STAT3 mediates
crosstalk between Notch and JAK-STAT signaling. Nat Cell
Biol 6:547554.
Malatesta P, Hartfuss E, and Gtz M (2000) Isolation of radial
glial cells by fluorescent-activated cell sorting reveals a neu-
ronal lineage. Development 127:52535263.
10 Rhythmic Expression of Notch Signaling in Neural Progenitor Cells
Masamizu Y, Ohtsuka T, Takashima Y, Nagahara H, Takenaka Y,
Yoshikawa K, Okamura H, and Kageyama R (2006) Real-time
imaging of the somite segmentation clock: revelation of unsta-
ble oscillators in the individual presomitic mesoderm cells.
Proc Natl Acad Sci USA 103:13131318.
Murata K, Hattori M, Hirai N, Shinozuka Y, Hirata H,
Kageyama R, Sakai T, and Minato N (2005) Hes1 directly con-
trols cell proliferation through the transcriptional repression of
p27Kip1 . Mol Cell Biol 25:42624271.
Nieto M, Schuurmans S, Britz O, and Guillemot F (2001) Neural
bHLH genes control the neuronal versus glial fate decision in
cortical progenitors. Neuron 29:401413.
Noctor SC, Flint AC, Weissmann TA, Dammerman RS,
and Kriegstein AR (2001) Neurons derived from radial
glial cells establish radial units in neocortex. Nature
109:714720.
Ohsawa R, Ohtsuka T, and Kageyama R (2005) Mash1 and
Math3 are required for development of branchiomotr neurons
and maintenance of neural progenitors. J Neurosci 25:5857
5865.
Ohtsuka T, Ishibashi M, Gradwohl G, Nakanishi S, Guillemot F,
and Kageyama R (1999) Hes1 and Hes5 as Notch effectors in
mammalian neuronal differentiation. EMBO J 18:21962207.
Ohtsuka T, Sakamoto M, Guillemot F, and Kageyama R (2001)
Roles of the basic helix-loop-helix genes Hes1 and Hes5 in
expansion of neural stem cells of the developing brain. J Biol
Chem 276:3046730474.
Paroush Z, Finley L JR, Kidd T, Wainwright SM, Ingham PW,
Brent R, and Ish-Horowictz D (1994) Groucho is required for
Drosophila neurogenesis, segmentation, and sex determination
and interacts directly with hairy-related bHLH proteins. Cell
79:805815.
Parras CM, Schuurmans C, Scardigli R, Kim J, Anderson DJ,
and Guillemot F (2002) Divergent functions of the proneu-
ral genes Mash1 and Ngn2 in the specification of neuronal
subtype identity. Genes Dev 16:324338.
143
Ross SE, Greenberg ME, and Stiles CD (2003) Basic
helix-loop-helix factors in cortical development. Neuron
39:1325.
Sasai Y, Kageyama R, Tagawa Y, Shigemoto R, and Nakanishi
S (1992) Two mammalian helix-loop-helix factors structurally
related to Drosophila hairy and Enhancer of split. Genes Dev
6:26202634.
Selkoe D and Kopan R (2003) Notch and presenilin: regulated
intramembrane proteolysis links development and degenera-
tion. Annu Rev Neurosci 26:565597.
Shimojo H, Ohtsuka T, and Kageyama R (2008) Oscillations
in notch signaling regulate maintenance of neural progenitors.
Neuron 58:5264.
Strm A, Arai N, Leers J, and Gustafsson J- (2000) The Hairy
and Enhancer of Split homologue-1 (HES-1) mediates the pro-
liferative effect of 17beta-estradiol on breast cancer cell lines.
Oncogene 19:59515953.
Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua
X, Fan G, and Greenberg ME (2001) Neurogenin promotes
neurogenesis and inhibits glial differentiation by independent
mechanisms. Cell 104:365376.
Takebayashi K, Sasai Y, Sakai Y, Watanabe T, Nakanishi S, and
Kageyama R (1994) Structure, chromosomal locus, and
promoter analysis of the gene encoding the mouse
helix-loop-helix factor HES-1: negative autoregula-
tion through the multiple N box elements. J Biol Chem
269:51505156.
Tomita K, Moriyoshi K, Nakanishi S, Guillemot F, and
Kageyama R (2000) Mammalian achaete-scute and
atonal homologs regulate neuronal versus glial fate
determination in the central nervous system. EMBO J
19:54605472.
Yoshiura S, Ohtsuka T, Takenaka Y, Nagahara H, Yoshikawa K,
and Kageyama R (2007) Ultradian oscillations of Stat, Smad,
and Hes1 expression in response to serum. Proc Natl Acad Sci
USA 104:1129211297.
Chapter 11

Neuron-Astroglial Interactions in Cell Fate Commitment

in the Central Nervous System

Joice Stipursky, Tnia Cristina Leite de Sampaio e Spohr, Luciana Ferreira Romo,
and Flvia Carvalho Alcantara Gomes

Contents actively involved in brain function than was formerly

thought. Recent evidence shows that astroglia, in addi-
11.1 Introduction. Astroglia: Old Cells, New tion to their previously known roles in neurotransmitter
Concepts . . . . . . . . . . . . . . . . . . 146 clearance, ion buffering, and neuronal trophic sup-
11.2 Astroglial Cells and Neurogenesis . . . . . . 147 port, are also involved in other functions, such as
11.2.1 Radial Glia Cells as Progenitor Cells . 147
synapse development and neurogenesis. In this review,
11.2.2 Potential Roles of Astrocytes
in Neurogenic Niches . . . . . . . . 149 we will focus on the role of astroglial cells, mainly
11.3 Role of Neuron-Glia Interactions in Astrocyte radial glia and astrocytes, in several events of nervous
Generation and Maturation . . . . . . . . . 152 system development such as cell fate commitment,
11.3.1 Neuron-Radial Glia Interactions:
neuronal and astrocyte maturation, and synapse forma-
Implications for Radial Glia Maintenance
and Astrocyte Generation . . . . . . 152 tion. We will argue that the functional architecture of
11.3.2 Role of Neuronal-Derived Molecules the brain depends on an intimate neuron-glia partner-
in Astrocyte Differentiation: Crosstalk ship. Finally, we will briefly discuss the emerging view
Between Growth Factors and
of astrocytes as essential actors in neurodegenerative
Neurotransmitters . . . . . . . . . . 156
11.4 Neuron-Astrocyte Interactions: Implications for diseases and neurological disorders, and how a better
Neuronal Differentiation and Synaptogenesis 158 understanding of glial development might open new
11.4.1 Neuron-Astrocyte Interactions avenues to develop therapeutic approaches to these
and Neuronal Differentiation . . . . . 159
pathologies.
11.4.2 Role for Glia in Synaptogenesis . . . . 161
11.5 Concluding Remarks . . . . . . . . . . . . 163
References . . . . . . . . . . . . . . . . . . . . 164 Keywords Astrocyte differentiation Neuronal
differentiation Neuron-astrocyte interactions Radial
glia Synaptogenesis
Abstract Until about 100 years ago, astroglial cells
were regarded solely as passive components of the Abbreviations
nervous system. During the past few years, however,
increasing knowledge of these cells has completely ALS Amyotrophic Lateral Sclerosis
changed this scenario: rather than mere inert brain AMPA -amino-3-hydroxy-5-methyl-4-
glue, astroglial cells are now considered active part- isoxazoleproprionic acid
ners of neurons, and they seem to be much more BLBP brain lipid-binding protein
BMP bone morphogenic protein
BrdU bromodeoxyuridine
CNTF ciliary neurotrophic factor
F.C.A. Gomes () CT-1 Cardiotrophin-1
Laboratrio de Neurobiologia Celular, Programa de Biologia E embryonic day
Celular e do Desenvolvimento, Instituto de Cincias
Biomdicas, Centro de Cincias da Sade, Universidade ECM extracellular matrix
Federal do Rio de Janeiro, Rio de Janeiro, Brazil EGF epidermal growth factor
e-mail: fgomes@anato.ufrj.br EGL external granular layer

H. Ulrich (ed.), Perspectives of Stem Cells, 145

DOI 10.1007/978-90-481-3375-8_11, Springer Science+Business Media B.V. 2010
146 J. Stipursky et al.

FGF fibroblast growth factor The above concept was developed by the German
-Gal -galactosidase pathologist Rudolf Virchow, who in the mid-nineteenth
GFAP glial fibrillary acidic protein century, by analyzing postmortem human tissues, pos-
GFP green fluorescent protein tulated that neuroglia was a connective tissue, acellular
GLAST astrocyte-specific glutamate-aspartate in nature (Virchow, 1846, quoted from Somjen, 1988).
transporter In 1851, the first neuroglial cells would be described by
GLT-1 glutamate transporter-1 Heinrich Mller, in the retina of several species (fish,
IGL internal granular layer amphibian, bird, and human): the radial cells, known
iGlu receptor ionotrophic glutamate receptor today as the Mller cells. Also in the mid-nineteenth
IL-6 interleukin-6 century, Otto Deiters provided illustrations of a cell
LGE lateral ganglionic eminence
type that resembles our modern notion of an astrocyte.
LIF leukemia inhibitor factor
Further contributions in the field of the cellular origin
LIFR leukemia inhibitor factor receptor beta
of glial cells resulted from the efforts of several histol-
LPA lysophosphatidic acid
ogists, in particular Camillo Golgi, Santiago Ramn y
MAPK mitogen-activated protein kinase
MGE medial ganglionic eminence Cajal and Po Del Ro Hortega (Somjen, 1988). Using
mGlu receptor metabotropic glutamate receptor a variety of microscopy techniques, they described a
NGF nerve growth factor wide diversity of glial cells in the brain. Although
NMDA N-methyl-d-aspartate observations of the intimate relationship between these
NMJ neuromuscular junction cells and blood vessels or nerve fibers might suggest a
NPC neural progenitor cell(s) functional role for glia, these cells were regarded as
NRG neuregulin(s) merely supportive and passive elements, completely
NS nervous system devoid of any role in brain function, for at least a
NSC neural stem cell(s) hundred years after their description. The past decade,
PI3-K phosphatidylinositol 3-K however, has been marked by a thorough revisitation of
PNS peripherical nervous system the role of glial cells in healthy brains, and especially
RA retinoic acid in brain disease.
RG radial glia Glial cells are classified into two main groups:
RGC retinal ganglion cells microglia, which is the subject of Chapter 12 of
SGZ subgranular zone this book; and macroglia, which are discussed here.
SVZ subventricular zone Macroglia are subdivided into four main specialized
T3 thyroid hormone cell types: ependymal cells, Schwann cells, oligo-
TGF- transforming growth factor- dendroglia, and astroglia. The last type includes the
TGF- transforming growth factor- astrocytes, marginal glia, radial glia in the developing
TGFRI transforming growth factor- brain and spinal cord, Bergmann cells in the cerebel-
receptor I
lar cortex, Mller cells in the retina, pituicytes in the
TGFRII transforming growth factor-
neurohypophysis, and tanycytes in the hypothalamus
receptor II
(Kettenmann and Ransom, 2005). Here we will focus
TSP thrombospondin(s)
on two subpopulations of astroglial cells: astrocytes
VZ ventricular zone
Wnt wingless and radial glia (RG) cells.
Evidence accumulating over the past decade has
revealed that neuron-astroglia interactions play key
roles in several events of brain development, such as
11.1 Introduction. Astroglia: Old Cells, the proliferation and differentiation of neuronal pre-
New Concepts cursors (Lim and Alvarez-Buylla, 1999; Song et al.,
2002; Krnyei et al., 2005; Lie et al., 2005; de
Sampaio e Spohr et al., 2008), neuronal migration
. . . this connective substance forms in the brain, in (Hatten, 2002), axonal guidance (Garcia-Abreu et al.,
the spinal cord, and in the higher sensory nerves a sort
of Nervenkitt (neuroglia), in which the nervous system
1995; Martinez and Gomes, 2002, 2005), synapse for-
elements are embedded Rudolf Virchow mation (Christopherson et al., 2005; Stevens et al.,
11 Neuron-Astroglial Interactions in Cell Fate Commitment
2007), and glial maturation (Gomes et al., 1999a;
de Sampaio e Spohr et al., 2002; Schmid et al.,
2003; Sousa et al., 2004; Barnab-Heider et al., 2005;
Stipursky and Gomes, 2007). Recently, RG cells and
astrocytes were described as neural stem cells in the
developing and adult brain, respectively (Noctor et al.,
2001; Alvarez-Buylla and Lim, 2004).
Astroglial cells are emerging as key mediators
of brain development, function, and plasticity, high-
lighting the critical need to better characterize the
mechanisms underlying their development and inter-
action with neurons. Although our understanding of
these cells has expanded dramatically during the past
decade, a great deal of mystery still surrounds the role
of astrocytes and RG cells in brain development and
injury.
In this chapter, we will discuss the conceptual shift
of the role of astroglia-neuron interactions in brain
development. We argue that astroglial cells should not
be viewed primarily as support cells, but rather as cells
that actively control the structural and functional orga-
nization of the developing and mature brain. We begin
by reviewing in vitro and in vivo studies that have
demonstrated a relatively novel attribute of RG cells
and astrocytes, as neural progenitor cells (NPC) in the
developing and adult nervous system, respectively. We
then discuss the role of neuron-astroglia interactions in
cell fate commitment, with a special focus on the tim-
ing of neurogenesis versus gliogenesis and astrocyte
maturation. Finally, we provide evidence of the role
of astrocytes in synapse development, and the conse-
quences of astroglia dysfunction for neurodegenerative
diseases and neurological disorders.
11.2 Astroglial Cells and Neurogenesis
One of the greatest challenges of neuroscience is to
understand how the widely diverse cell types of the
nervous system (NS) are generated. The vertebrate
NS originates as a flat sheet of neuroepithelial cells,
which gives rise to the great variety of glial and neu-
ronal types that form the adult brain (Kandel et al.,
2000). Shortly after the appearance of the first neurons,
neuroepithelial cells undergo a change in their charac-
teristics and acquire molecular and cytological features
typical of the astroglial lineage, as they give rise to the
RG cells (Malatesta et al., 2008). After their birth in
147
the ventricular surface of the neural tube, the neurons
must migrate to their final destination. The success of
their migration is partly dependent on the interaction
of postmitotic neurons and the system of RG fibers.
Although RG cells were originally described as guides
for neuronal migration, the prevailing view is that RG
constitute the main NPC for neurons and astrocytes
in most regions of the nervous system (Gtz et al.,
2002).
Until the mid-twentieth century, it was assumed
that no new neurons are generated after brain forma-
tion is completed. This idea, however, gave place to
the description of neurogenic areas in the adult brain
of different species. The recent identification of the
astroglial origin of these progenitor cells assigned a
new attribute to astrocytes, not previously discussed.
In this section, we will discuss the role of astroglia,
mainly RG cells and adult astrocytes, in neurogenesis,
both as progenitor cells themselves, and as a source of
trophic support in neurogenic areas.
11.2.1 Radial Glia Cells as Progenitor Cells
The radial glia cells were described in the mammal
fetal encephalon at the end of the nineteenth century by
Giuseppe Maggini (1888) (for review see Bentivoglio
and Mazzarello, 1999). These cells have a bipolar
morphology, with the cell body located in the ventric-
ular zone (VZ), and cell processes extending from the
ventricular through the pial surfaces.
Classical studies in several species, including
humans (Choi and Lapham, 1978; de Azevedo
et al., 2003), non-human primates (Schmechel and
Rakic, 1979), carnivores (Voigt, 1989), and rodents
(Takahashi, 1990), have suggested that RG cells trans-
form into astrocytes during cerebral cortical develop-
ment, as we will further address in Section 11.3.1 (for
review see Bentivoglio and Mazzarello, 1999). The
use of electron microscopy and immunohistochemistry
applied to primate embryos has confirmed the glial
phenotype of radial cells (for review see Rakic, 2003).
At the end of neurulation, the CNS is made up of
a pseudostratified epithelium, where radially arranged
bipolar cells span the entire thickness of the neural
tube. These neuroepithelial cells, like earlier progen-
itors, undergo a characteristic alternate movement of
the nucleus (interkinetic nuclear migration) between
148
the basal and the apical surface of the neural tube. This
migration is synchronized with the cell cycle, and these
cells undergo symmetrical cell division on the luminal
surface of the neural tube. From this stage, however,
an increasing number of cells begin to divide asym-
metrically, giving rise to another neuroepithelial cell
and to either a neuron, or, alternatively, to a progenitor
cell that will undergo mitosis at a significant distance
from the ventricular surface, the basal progenitors,
which will generate only neurons via symmetrical divi-
sion. However, shortly after the appearance of the
first neurons, neuroepithelial cells give rise to the RG
cells. Although RG cells maintain the morphology and
intermediate filament nestin expression similar to the
neuroepithelial cells, some of the features that define
the neuroepithelial-to-RG transition are the expres-
sion of genes that are typical of astrocytes, such as
the brain lipid-binding protein (BLBP), the astrocytic
glutamate-aspartate transporter (GLAST), the adhe-
sion molecule TN-C, the enzyme glutamine synthase,
the calcium-binding protein S100, the intermediate
filament vimentin, and, in some primates (but not in
rodents), the intermediate filament the glial fibrillary
acidic protein, GFAP. Also, glycogen granules begin
to accumulate in the RG cytoplasm. Notably, apart
from the peculiar shape, in many species no markers
are known that allow discrimination of RG from astro-
cytes. This is the case, for instance, in primates, where
the immunoreactivity for GFAP is shown by both cell
types; whereas in rodents it is limited to astrocytes
(Malatesta et al., 2008).
Initially described as only related to neuronal migra-
tion and cerebral cortex lamination (Hatten, 1999;
Rakic, 1971), RG cells are today well recognized for
their progenitor potential in all regions of the CNS
(Anthony et al., 2004). They are considered a major
intermediate between neural stem cells and cerebral
cortex neurons (Gtz and Barde, 2005). Several obser-
vations, both in vitro and in vivo, have shown that,
during neurogenesis, RG cells generate mostly neurons
(Malatesta et al., 2000; Noctor et al., 2001; Anthony
et al., 2004). The first direct observation of this event
came from the analysis of the differentiation of RG iso-
lated by fluorescence-activated cell sorting (Malatesta
et al., 2000). Cells were labeled by exploiting two inde-
pendent characteristics of RG: the ability to express
green fluorescent protein (GFP) under the control of
the human GFAP (hGFAP) promoter, and the pres-
ence of a contact with the basal membrane, allowing
J. Stipursky et al.
them to be stained with a lipophilic dye applied to
the pial surface (Malatesta et al., 2000). The analysis
demonstrated that the majority of RG cells generate
homogeneous clones, composed either of neurons or
of non-neuronal cells (glia and/or progenitor cells)
exclusively. The generation of neurons from RG in
the cerebral cortex was corroborated by video time-
lapse analyses showing the generation of a radial
unit by the asymmetric division of a single RG cell
(Noctor et al., 2001). Such radial units were com-
posed of the RG cell itself plus multiple postmitotic
neurons. Subsequently, in vivo lineage-tracing studies
based on transgenic-mice technology showed that RG
is the main source of postmitotic neurons in the cere-
bral cortex (Malatesta et al., 2003), and possibly in the
entire telencephalon (Anthony et al., 2004). Both stud-
ies were based on transgenic mouse lines where Cre-
recombinase expression was driven by RG-specific
promoters: the hGFAP- (Malatesta et al., 2003) and
the BLBP-promoter (Anthony et al., 2004). Although
both studies agreed that RG cells are responsible for
the generation of all projection neurons of the cerebral
cortex, discrepancies were found regarding the role of
RG in the generation of ventral telencephalic neurons
(Malatesta et al., 2008).
RG as well as their neurogenic potential appear to
be highly heterogeneous, both within and across differ-
ent brain regions (Kriegstein and Gtz, 2003). Cell-fate
mapping studies suggested that this diversity might
be associated with the expression of different tran-
scription factors such as Pax6, Olig2, and Gsh2 (Gtz
et al., 1998; Malatesta et al., 2003). In the dorsal telen-
cephalon, for example, RG cells contain the transcrip-
tion factor Pax6 (Gtz et al., 1998), whereas those from
the lateral ganglionic eminence (LGE) contain Gsh2,
and those in the medial ganglionic eminence (MGE)
contain Olig2 (Malatesta et al., 2003). All these tran-
scription factors have been implicated as potent cues in
patterning the developing neural tube (Sun et al., 2001;
Carney et al., 2008). Recent fate mapping of the entire
progeny of RG cells has demonstrated pronounced dif-
ferences in the ventral and dorsal telencephalon: while
cortical RG cells generate the vast majority of neu-
rons (more than 90%) in the cerebral cortex, RG in
the ventral telencephalon generates very few neurons
(Malatesta et al., 2003). Besides RG heterogeneity
within the brain, other RG-like cells also exist through-
out the CNS, such as the Mller glia in the retina and
the Bergmann glia in the cerebellum (Pinto and Gtz,
11 Neuron-Astroglial Interactions in Cell Fate Commitment
2007). These cells share some morphological and func-
tional features with cerebral cortical RG, such as their
radial morphology and role as neuronal guides.
During cerebellar development, Bergmann glial
cells support the migration of neurons from their birth-
place in the external granular layer (EGL) to their
final position in the internal granular layer (IGL)
(Nicholson and Altman, 1972). As cortical RG, they
express BLBP, GLAST, and vimentin. In contrast to
rodent cortical RG, Bergmann glia cells express GFAP.
Recently, it was observed that Bergmann glia express
stem cell markers of the Sox family of transcrip-
tion factors (Sox/Sox2) (Sottile et al., 2006); however,
a progenitor potential has not yet been reported for
these cells. In the retina, the Mller glia represents
the main glial cell type, which crosses the outer and
inner layers of the retina with a radial characteristic
morphology. This cell expresses cell markers such as
vimentin, GLAST, and the astrocyte-specific markers
GFAP and glutamine synthase (Okada et al., 1990;
Vardimon et al., 1993). In addition to their role in neu-
ronal migration, Mller glial cells were indicated as
the retina potential stem cells, implicated in generation
of new neurons after injury (Raymond and Hitchcock,
1997). Recent in vivo and in vitro works identified
stem-cell-like properties, including self-renewal and
multipotency, in mammalian Mller glia (Das et al.,
2006; Bernardos et al., 2007) and immortalized human
Mller cells (Lawrence et al., 2007).
Self-renewal and neuronal generation capacity are
key features that are lost during the transformation
of RG into astrocytes at the end of neurogenesis in
the mammalian brain. However, in lower vertebrates,
where neurogenesis persists in a rather widespread
fashion in the adult brain, RG with access to the
ventricle also persists into adulthood. Interestingly,
the persistence of RG may also be responsible for
the success of axonal regeneration in the CNS of
non-mammalian vertebrates, as they provide a good
substrate for axonal growth consistent with their nor-
mal role during development (for review see Pinto and
Gtz, 2007).
Taken together, the maintenance of RG cells or their
key aspects of embryonic development seems to be a
crucial feature to allow neuronal repair. Not only is the
persistence of RG cells in many non-mammalian ver-
tebrates highly correlated with the presence of stem
cells and adult neurogenesis, but in addition, neural
stem cells in the adult mammalian brain retain RG
149
features. As we will address in more detail in the fol-
lowing sections (11.2.2 and 11.3.1), identification of
the molecular cues that regulate RG identity might rep-
resent a crucial step in understanding neurogenesis in
both the developing and adult brain.
11.2.2 Potential Roles of Astrocytes
in Neurogenic Niches
A classical dogma of developmental neuroscience is
that, once neurons in the adult CNS are terminally
differentiated, they persist through the life of the organ-
ism, and are not replaced when they die. This idea,
however, began to change in the 1960s with the obser-
vation of thymidine-incorporating cells with neuronal
characteristics in the postnatal brain (Altman and Das,
1966). Later, a series of experiments in several dif-
ferent species, including birds, mice, and humans,
demonstrated ongoing neurogenesis in the adult verte-
brate brain (Goldman and Nottebohm, 1983; Galileo
et al., 1990; Alvarez-Buylla et al., 2001; Temple,
2001). This phenomenon occurs through the persis-
tence of niches that harbor neural stem cells (NSC)
and constitute a microenvironment that constantly
supports and regulates neurogenic activity (Alvarez-
Buylla and Lim, 2004). Surprisingly, evidence accu-
mulating in the last 10 years has supported an essential
role for glial cells in NSC adult niches. In this section,
we will discuss the role of astroglial cells in adult neu-
rogenesis, either by acting as primary precursors, or
by exerting influence on other cell types as supporting
cells.
Two germinal regions within the adult mammalian
brain have been shown to contain active neurogenesis
throughout life: the subventricular zone (SVZ) of the
lateral ventricles (Alvarez-Buylla and Lim, 2004), and
the subgranular zone (SGZ) of the dentate gyrus in the
hippocampus (Song et al., 2002). Neurogenesis out-
side these two regions appears to be extremely limited,
or nonexistent, in the intact adult mammalian CNS
(Alvarez-Buylla and Lim, 2004).
Strikingly, in both SVZ and SGZ, a subset of
astrocytes that is classically associated with support
functions in the brain was identified as the in vivo
primary precursors for adult neurogenesis (Doetsch
et al., 1999; Seri et al., 2001). These cells have been
defined as astrocytes based on their ultrastructural
150
features, markers that they express, and electrophysi-
ological properties. Supporting the idea of astrocytic
stem cells, SVZ and SGZ astrocytes remain labeled
with thymidine or bromodeoxyuridine (BrdU) after
long survival times, indicating their stem-cell property
(Doetsch et al., 1999; Seri et al., 2001). Further, they
are capable of repopulating the niche after the elimina-
tion of other cell types by the anti-mitotic compound
Ara-C (Doetsch et al., 1999). An additional support
for astrocytes as the NSC in SVZ and SGZ was pro-
vided by genetic ablation in vivo. Removal of GFAP-
positive cells eliminated the ability of the germinal
zone to reconstitute itself over time (Morshead et al.,
1994).
In addition to astrocytes, some workers have pre-
sented data suggesting that ependymocytes might also
represent neural stem cells (Johansson et al., 1999).
However, this view is not generally accepted, because
other reports have demonstrated that although ependy-
mal cells can proliferate in vitro, they do not generate
neurons (Chiasson et al., 1999), and do not proliferate
in vivo (Spassky et al., 2005).
The NSC of the SVZ, also referred to as Type
B cells, are embedded in a peculiar niche that also
contains rapid-cycling transit-amplifying progenitor
cells (Type C cells) and migrating neuroblasts (Type
A cells), which are guided towards the olfactory
bulb, where they become functional neurons (Doetsch,
2003). Type B cells have astroglial characteristics and
express GFAP, nestin, vimentin, GLAST, and Sox2,
but not S100 (another marker for astrocytes); accu-
mulate glycogen granules in their cytoplasm; and
are relatively quiescent (for review see Malatesta
et al., 2008). There is evidence that stem cells in
the hippocampus SGZ may correspond to the type
B cells that were described in the SVZ (Seri et al.,
2001).
Adult SVZ stem cells (Type B cells) have been
shown to be related to embryonic ventricular RG. The
conversion of RG into Type B cells involves retraction
of the RG basal processes, loss of the radial morphol-
ogy, and a slowing of the cell cycle (Merkle et al.,
2004). Supporting this morphological feature, RG and
Type B cells share molecular regulatory mechanisms,
such as expression of the neurogenic transcription fac-
tor Pax 6 and activation of the Notch signaling pathway
(Malatesta et al., 2008).
Astrocytes constitute the main source of trophic fac-
tors in CNS. These include members of the epidermal
J. Stipursky et al.
growth factor (EGF) family, transforming growth fac-
tor (TGF), and neuregulins (NRG); fibroblast growth
factor (FGF), nerve growth factor (NGF), and ciliary
neurotrophic factor (CNTF). By providing trophic sup-
port, astrocytes might affect several events of brain
development, such as neuronal precursor proliferation,
cell fate commitment, and synaptogenesis, as we will
address later in this chapter.
We demonstrated that EGF secreted by cultured
cerebellar astrocytes in response to thyroid hormone
(T3) induces neuronal cerebellar proliferation in vitro
(Gomes et al., 1999b). Later, we demonstrated that
cocultures of astrocytes and neurons potentiate this
effect, suggesting that neuron-astrocyte interaction is
a key element in cerebellar morphogenesis (Martinez
and Gomes, 2005).
Several growth factors and signaling pathways that
are present in astrocytes have a crucial role in adult
neurogenesis, suggesting that these cells might orches-
trate the proliferation in neurogenic niches. This is the
case of FGF, which is important for maintenance of
the NSC pool (Zheng et al., 2004); Notch1, which is a
crucial regulator of NSC maintenance and self-renewal
within the neurogenic SVZ niche (Alvarez-Buylla
and Lim, 2004); CNTF, which promotes self-renewal
of neural precursors in vitro by increasing Notch1
expression (Chojnacki et al., 2003); and transforming
growth factor- (TGF-), the endogenous ligand of the
EGF receptor, which controls the pool of progenitors
by modulating the proliferation of transit-amplifying
cells, the intermediate stage between the neural stem
cells and neuroblasts (Doetsch et al., 2002).
Astrocytes might also influence neuronal fate com-
mitment rather than precursor proliferation. Neuronal
differentiation from adult NPC in the neurogenic niche
proceeds partly because of the local presence of bone
morphogenic protein (BMP) antagonists. BMP was
shown to instruct adult NPC to adopt a glial fate (Lim
et al., 2000; Zheng et al., 2004). Ependymal cells in
the SVZ secrete Noggin (Lim et al., 2000), and astro-
cytes in the SGZ secrete neurogenesin-1 (Ueki et al.,
2003), both BMP antagonists, in the SVZ and SGZ
respectively. Another growth factor that was recently
identified as being produced by astrocytes, at least in
vitro, and induces NSC differentiation is retinoic acid
(RA). RA is one of the most powerful morphogenic
molecular regulators of neuronal cell-fate commit-
ment. Recently, it was demonstrated that RA can be
produced by astrocytes in vitro; however, in vivo it
11 Neuron-Astroglial Interactions in Cell Fate Commitment
was demonstrated that exposure to exogenous retinoids
or blocking RA signaling interferes with prolifera-
tion and differentiation within the neurogenic zones
of the mature brain, such as the higher vocal center
in songbirds, or the SVZ and hippocampus in mice.
Endogenous RA has recently been shown to be active
within the adult stem-cell niches, including the SVZ,
the rostral migratory stream, and the hippocampus. It
was also demonstrated that there are RA-responsive
astrocytes in the SVZ and a population of RA-activated
astroglial cells within the hippocampus of RA reporter
mice (Krniey et al., 2007).
Another astrocyte-derived signal that modulates
adult neurogenesis is the Wnt (wingless) pathway.
The Wnt family of secreted glycoproteins is known
to play crucial roles in regulating other somatic stem
cells in vivo (Lobo et al., 2007). Interestingly, Wnt3
is expressed by astrocytes of the adult hippocam-
pus (Lie et al., 2005). Overexpression of Wnt3 leads
to increased neuronal fate specification of adult hip-
pocampal NSC and increased proliferation of neurob-
lasts, while blockade of Wnt signaling results in reduc-
tions in the generation of new neurons both in vitro and
in vivo. These data point to Wnt signalling as a prin-
cipal regulator of adult hippocampal neurogenesis (Lie
et al., 2005). On the other hand, it has been shown that
the transforming growth factor beta (TGF-) signal-
ing pathway might also participate in the neurogenic
process in the adult hippocampal region (Wachs et al.,
2006). It has been shown that TGF-1, which is also
known to be secreted by astrocytes as we will discuss
further, promotes a decrease in the proliferation in the
hippocampus. These data are strengthened by a recent
paper showing that the expression of Noggin, a BMP
inhibitor, enhances neurogenesis in the hippocampus,
indicating that the TGF- family of proteins is crucial
for these events in the adult telencephalon (Bonaguidi
et al., 2008).
Within this scenario, a question arises: To what
extent is an astrocyte considered a cell at a termi-
nal stage of differentiation? In the last few years,
several studies have indicated that the astrocytes are
extremely plastic cells, in terms of their dedifferentia-
tion capacity. Since the 1990s, evidence has shown that
the embryonic encephalon contains soluble factors that
promote astrocyte differentiation, and has clarified the
discussion of astrocyte differentiation as a reversible
event (Hutter and Hatten, 1995). Growing evidence
from the past few years has indicated that the members
151
of the EGF family, such as TGF- and NRG, are key
elements in this event. Sharif and colleagues (2006)
reported that TGF- is an inducer of adult astrocyte
dedifferentiation in vitro. TGF- treatment induced
the appearance of RG phenotype features in astro-
cytes, such as expression of BLBP and RC2, radial
morphology, support of neuronal migration, and, more
surprisingly, neuron generation.
As we will discuss in Section 11.3.1 of this chapter,
Eva Antons group has shown that the NRG-ErbB2 sig-
naling is essential for RG maintenance (Schmid et al.,
2003). More recently, the same group has shown that,
in fact, the reinduction of the ErbB2 receptor expres-
sion in adult astrocytes promotes their dedifferentiation
into RG cells (Ghashghaei et al., 2007). Similarly, rein-
duction of the transcription factor Pax6, by Magdalena
Gtzs group, in postnatal astrocytes derived from
non-neurogenic regions enables these cells to generate
neurons (Berninger et al., 2007). Together, these data
open the possibility that manipulation of astrocyte dif-
ferentiation may represent a potential tool for neuronal
replacement in injury.
What are the differences between neurogenic and
non-neurogenic astrocytes? What makes the SVZ and
SGZ special in supporting the proliferation and neu-
ronal differentiation of multipotent neural progenitors?
Answers to these questions will lead to better under-
standing of adult neurogenesis in both healthy and
diseased individuals.
Recent evidence indicates that astrocytes of the
SVZ and SGZ are strongly influenced by their local
microenvironment. However, the environment alone
does not seem to be sufficient to induce non-germinal
astrocytes to behave as neural stem cells, suggest-
ing that specific cell-intrinsic differences between
germinal-center astrocytes and other astrocytes may
exist. The S100 protein known to be expressed by
many mature astrocytes is not expressed by most SVZ
astrocytes or by the radial astrocytes of the SGZ (Seri
et al., 2004; Raponi et al., 2007). The astrocytes in
the SVZ that express both S100 and GFAP gen-
erate significantly fewer neurospheres than do astro-
cytes that express GFAP alone, indicating that S100
expression denotes mature astrocytes that do not
act as stem cells (Raponi et al., 2007). Interestingly,
horizontal astrocytes of the SGZ divide and express
S100, and they are thought to function as local oligo-
dendroglial progenitors within the hilus (Seri et al.,
2004). It was also shown recently that the LeX-positive
152
GFAP-positive subpopulation of germinal astrocytes
expressing GFAP, contains the majority of multipo-
tent neural stem cells that are able to form multi-
potent neurospheres; whereas LeX-negative GFAP-
expressing astrocytes are unable to do this (Imura
et al., 2006). One important point is that astrocytes
derived from the cerebral cortex are LeX-negative and
non-neurogenic, suggesting the existence of significant
phenotypic and functional differences between these
two types of astrocytes (Ihrie and Alvarez-Buylla,
2007). In addition to S100 and LeX, other proteins
are also emerging as potential markers for germinal-
zone astrocytes, including brain-lipid binding protein,
nestin, and Sox2 (Filippov et al., 2003; Steiner et al.,
2006).
Therefore, it will become extremely important to
collect more information on the characteristics of
astrocytes in the neurogenic regions in normal or dis-
ease conditions. This will be a key to developing
means of effectively modulating neurogenesis in dis-
ease states. Understanding the molecular regulators
that normally control the fate of neural stem cells
and their progeny in the adult CNS may open new
avenues to develop alternative therapeutic approaches
to nervous-system injury.
11.3 Role of Neuron-Glia Interactions
in Astrocyte Generation
and Maturation
The Neuron Doctrine, which was created in the late
nineteenth century and which considered neurons as
the only cells with physiological relevance for NS
function, kept us in the dark concerning glial devel-
opment. While there is compelling evidence of the
effect of astrocyte factors on neurons, there is still a
lack of data on their effects on astrocytes. However,
although this subject has been less addressed, evidence
has accumulated that indicates that neurons are modu-
lators of astrocyte differentiation. In this section, we
will describe the emerging evidence for the role of
neuron-glia interactions in cell fate determination, and
the implications for the timing of neurogenesis and gli-
ogenesis. Further, we will discuss recent studies on the
role of neuronal-derived factors such as neurotransmit-
ters and growth factors in RG identity and astrocyte
maturation.
J. Stipursky et al.
11.3.1 Neuron-Radial Glia Interactions:
Implications for Radial Glia
Maintenance and Astrocyte
Generation
Development of the vertebrate CNS is achieved
through a common pool of precursor cells that sequen-
tially generate neurons and glial cells (Bayer and
Altman, 1991). In rodents, neurons are generated
from embryonic day (E) 12 to E18; astrocytes appear
around E18, with their numbers peaking in the neona-
tal period, and mature oligodendrocytes appear later.
Thus, multipotent precursors change their competence
over time to generate different cell types. The ability of
embryonic cortical precursors to make neurons when
cultured on embryonic cortical sections, but to make
astrocytes when cultured on postnatal cortical sections
(Morrow et al., 2001), suggests that the neurogenic-
to-gliogenic switch might depend on environmental
signals rather than only on an intrinsic developmental
program.
As discussed previously, one of the main neural
precursors of the nervous system is the RG cells.
Whereas in most vertebrates (fish, amphibians, rep-
tiles, birds) these cells persist into adulthood, in most
CNS regions of adult mammals they transform into
astrocytes (for review see Bentivoglio and Mazzarello,
1999). The most direct evidence for this transformation
was obtained in the cerebral cortex of ferrets by detec-
tion of fluorescent tracers in astrocytes after labeling
RG pial processes (Voigt, 1989). Later, this event was
demonstrated for several vertebrate species including
humans (for review see Bentivoglio and Mazzarello,
1999).
The most prevalent view is that RG-astrocyte trans-
formation is initiated by detachment of the ventricular
endfeet; the cell body moves upwards to a certain
depth in the cortex, maintaining its pial attachment.
After extending a few processes, the RG detaches
from the pia and progressively assumes the typical
astrocytic morphology (Voigt, 1989; Takahashi et al.,
1990). The RG-astrocyte transition is a hallmark of
the neurogenesis-to-gliogenesis switch in the cerebral
cortex. However, although RG-astrocyte transforma-
tion is well recognized, the molecular mechanisms
underlying this process are little understood.
The fact that gliogenesis coincides with the end of
neurogenesis, a period of generation of a large number
11 Neuron-Astroglial Interactions in Cell Fate Commitment
of neurons, suggests that neurons might signal for gli-
ogenesis. Further, the double function of RG during
CNS development, both as neuronal/astrocyte pro-
genitors and as a scaffolding supporting neuronal
migration (Campbell and Gtz, 2002) suggests that
there may be intimate links between the signaling
pathways that control RG development, neurogene-
sis/astrocytogenesis, and migration.
One of the first evidences of neuronal modula-
tion of the RG phenotype came in 1985, with the
demonstration that cultures of cerebellar neurons and
astrocytes induce a morphological, functional, and
molecular differentiation into RG (Hatten, 1985).
Later, the same group showed that expression of
RG cell identity is regulated in the mammalian fore-
brain by the availability of diffusible inducing signals
that are present in the embryonic brain (Hunter and
Hatten, 1995).
The idea that the neuron-RG interaction is a crit-
ical event for the establishment of RG identity and
morphology was further supported by several in vivo
and in vitro lines of evidence (Feng and Heintz,
1995; Anton et al., 1997; Soriano et al., 1997; Supr
et al., 2000). Ablation of Cajal-Retzius cells in the
cerebral cortex leads to a dramatic decrease in the
number of RG apical processes, inducing prema-
ture astrocyte differentiation (Supr et al., 2000).
Complementary transplantation experiments showed
that embryonic Cajal-Retzius cells grafted to cerebel-
lar tissue induce a rejuvenation of Bergmann astro-
cytes into an RG phenotype (Soriano et al., 1997).
Further compelling evidence has demonstrated that,
in addition to modulating RG morphology, neurons
also modulate the expression of RG molecular mark-
ers such as BLBP (Feng and Heintz, 1995; Patten et al.,
2003).
Although the radializing soluble factor has not
been identified, several molecules and signaling path-
ways activated by neurons have been indicated as
important for maintenance of RG identity, including
Notch, NRG-ErbB, Wnt, meteorin, and others (Zhou
et al., 2004; Nishino et al., 2004; Ever and Gaiano,
2005).
The Notch signaling pathway is best character-
ized as mediating cellcell signaling between adja-
cent cells. Both the ligands, members of the Delta
and Jagged families, are able to bind the membrane-
associated Notch molecule and activate the Notch
intracellular domain, which translocates to the nucleus
153
where it converts the CBF1 repressor complex into
an activator gene-transcription complex (Yoon and
Gaiano, 2005). The signaling pathway initiated by
Notch 1, which is present in NPC, promotes the RG
phenotype in the embryonic telencephalon (Gaiano
and Fishell, 2002) and the cerebellum (Eiraku et al.,
2005). This event implies cooperation between Notch
and other growth factors, such as FGF and NRG
(Patten et al., 2003; Schmid et al., 2003; Yoon et al.,
2004).
The NRG are a family of proteins containing an
EGF-like motif that activates membrane-associated
tyrosine kinases related to the EGF receptor (known as
ErbB-1). It has been reported that the NRG 1-ErbB2
signaling is essential to the establishment of RG in the
cerebellum (Patten et al., 2003). On the other hand, the
negative modulation of ErbB2 promotes its transfor-
mation into astrocytes in the cerebral cortex (Schmid
et al., 2003), and the introduction of the constitu-
tive active form of the ErbB2 receptor into mature
astrocytes promotes its dedifferentiation into RG cells
(Ghashghaei et al., 2007). Similarly, the ErbB4 recep-
tor is also involved in the regulation of the timing
of gliogenesis in the cerebral cortex, modulating the
transcription of glial genes during development (Sardi
et al., 2006).
The Wnt/catenin signaling pathway has been indi-
cated as inducing the progenitor phenotype in the
mammal brain. Wnts are secreted glycoprotein sig-
naling molecules that regulate many developmental
events in vertebrates and invertebrates. In mammals,
previous studies showed that Wnt signaling regu-
lates the development of the dorsal neural tube (Zhou
et al., 2004). It has been shown that in LRP6 mutant
mice, a co-receptor for the Wnt/catenin signaling
pathway shows several abnormalities in the produc-
tion of granular cells in the dentate gyrus of the
hippocampus and the formation of RG fibers (Zhou
et al., 2004). Moreover, it seems that the synergistic
interaction of Wnt and Notch confers neural stem-
cell properties on the retinal Mller glia (Das et al.,
2006).
During encephalic development, several molecules
interact synergistically or antagonistically to deter-
mine the correct time of cell generation. As mentioned
above, the onset of gliogenesis coincides with the
end of neurogenesis, suggesting a possible role for
the growing pool of newly generated neurons as a
source of soluble factors that promote gliogenesis. In
154
fact some studies indicate that cytokine signaling is
important, and the appropriate timing of astrogene-
sis can be mimicked in isolated clones of precursor
cells, which seems to argue that a cell-intrinsic mech-
anism regulates the onset of gliogenesis (Qian et al.,
2000). Because such clones contain both precursors
and newly born neurons, the hypothesis is that newly
born neurons secrete gliogenic factors that feed back
to instruct multipotent precursors to generate astro-
cytes. Within this idea, several studies have suggested
that neurons participate in glial differentiation in the
telencephalon.
Some of the best-studied soluble factors are the
leukemia inhibitor factors of the interleukin-6 (IL-6)
family, including CNTF, leukemia inhibitor fac-
tor (LIF), and Cardiotrophin-1 (CT-1) (Qian, 2000;
Barnab-Heider et al., 2005). These factors sig-
nal through heterodimerization of coreceptors LIFR
(leukemia inhibitor factor receptor beta) and gp130,
and activation of the intracellular Jak-STAT (for review
see Miller and Gauthier, 2007).
Several lines of evidence strongly support a role
for the Jak/STAT pathway in astrocytogenesis. First,
the STAT transcriptional factors have been shown to
cause direct transcriptional activation of two astro-
cytic genes, GFAP and S100, via STAT binding sites
within their promoters (Bonni et al., 1997; Nakashima
et al., 1999b; Miller and Gauthier, 2007). Further,
gp130 or LIFR deficiency causes profound impair-
ment in astrocyte generation, both in vitro (Ware
et al., 1995; Nakashima et al., 1999a) and in vivo
(Barnab-Heider et al., 2005). In the same context,
other groups have demonstrated that CNTF and LIF
are sufficient to induce astrogenesis, and that this
action requires LIFR and gp130 in vitro (Bonni et al.,
1997; Nakashima et al., 1999a, 1999b). In addition, it
has been shown that cortical neurons synthesize and
secrete the neurotrophic cytokine CT-1, which acti-
vates the gp130-Jak-STAT pathway and is essential for
the timed genesis of astrocytes in vitro and in vivo.
Through the use of cortical precursor cultures that
temporally mimic the in-vivo differentiation pattern,
it was demonstrated that CT-1 causes premature gli-
ogenesis. Moreover, the neonatal CT-1-deficient cor-
tex contains fewer astrocytes (Barnab-Heider et al.,
2005).
Recently, our group showed that cortical neurons
induce astrocyte differentiation from radial glia-like
cells by activating the TGF-1 pathway in vitro
J. Stipursky et al.
(Stipursky and Gomes, 2007). The TGF- superfamily
is constituted by multifunctional polypeptide mem-
bers, which perform critical functions in tissue repair
and development (Shi and Massague, 2003). In the
NS, TGF-1 has been implicated in organization of
the glial scar in response to injury and in several
neurodegenerative diseases (Zhu et al., 2002; Vivien
and Ali, 2006). Emerging evidence, however, has
suggested a key role for this factor in several NS
developmental processes such as cell adhesion, neu-
ronal migration and differentiation, synaptogenesis,
and blood-brain barrier formation (de Sampaio e Spohr
et al., 2002; Brionne et al., 2003; Miller 2003; Garcia
et al., 2004; Stipursky and Gomes, 2007; Feng and Ko,
2008).
TGF- signaling is mediated mainly by two ser-
ine threonine kinase receptors, transforming growth
factor-beta receptor I (TGFRI) and II (TGFRII), which
activate Smad 2/3 and Smad 4 transcription factors.
Phosphorylation and activation of these proteins is
followed by formation of the Smads 2/3-4 complex,
which translocates to the nucleus regulating transcrip-
tional responses to TGF- (Shi and Massagu, 2003).
We showed that embryonic cortical NPC cultures
enriched in RG-like cells, which are characterized by
their radial morphology and also by nestin and BLBP
expression, are direct targets of TGF-1, because these
cells express TGFRII. In that study we observed a
specific upregulation of glial markers, such as GFAP
and GLAST, and downregulation of RG markers,
such as nestin and BLBP, in the presence of TGF-
1 or a conditioned medium derived from neurons.
All of these events were followed by activation of
the SMADs2/3 proteins, with subsequent transloca-
tion to the nucleus. Inhibition of TGF-1 pathways by
the neutralizing antibody against this cytokine impairs
neuronal effects (Fig. 11.1, Stipursky and Gomes,
2007). These data and the observation that in the
adult rodent brain, TGF-1 seems to negatively mod-
ulate neurogenesis and stem-cell proliferation (Wachs
et al., 2006), together with the recent identification
of TGFs isoforms in the germinative layers of the
telencephalon and spinal cord, strongly support a role
for TGF- in the biology of RG cells (Mecha et al.,
2008).
Thus, during CNS development, several molecular
cues instruct neural precursors as to which differen-
tiation pathway, neuronal or glial, they should follow
(Fig. 11.2). Although several of these cues have been
11 Neuron-Astroglial Interactions in Cell Fate Commitment
Fig. 11.1 TGF-1 role in RG-astrocyte transformation. (a)
Treatment of E14.5 cerebral cortex-derived progenitors with
TGF-1 decreases the number of nestin/GLAST positive cells
(RG-like cells) and increases that of GFAP/GLAST positive
cells (Astrocytes). (b) RG cell in vitro shows a typical bipolar
morphology. (c) Schematic view of cerebral cortex develop-
ment. (1) RG cells show a bipolar morphology that spans
Fig. 11.2 Schematic model of neuron-RG interaction sig-
naling pathways. RG cell is a direct target of neuron-derived
radializing factors (Neuregulin (NRG), Delta/Notch, FGF) that
promote RG phenotype maintenance. However, growing evi-
dence suggests that neurons secrete astrocytogenic-inducing
growth factors (TGF-1, CT-1, ILs, CNTF). The balance
between these factors controls RG self-renewal (white curved
arrows) and its transformation into astrocytes (black curved
arrow)
155
the entire thickness of neural tube wall. During cerebral cor-
tex development, these cells generate neuroblasts that migrate
over radial fibers (migrating neurons); (2) Postmitotic neurons
form the cortical plate and differentiate terminally into layer-
specific neurons; (3) At the end of the neuronal migratory phase,
RG cells differentiate into astrocytes under the influence of
TGF-1
thoroughly investigated during the last few years, there
is still great interest in understanding how they interact.
In addition, there is a lack of knowledge concerning
what makes these progenitors lose their transient neu-
rogenic potential and differentiate towards a neuronal
or glial phenotype.
As mentioned before, it is known that early in devel-
opment, the signaling initiated by Neuregulins and its
receptor ErbBs, as well as Notch1 and FGF, for exam-
ple, play an important role in maintaining the undif-
ferentiated RG phenotype. On the other hand, later in
development the activation of the Jak-STAT pathway
by factors of the ILs family, as well as SMADs by
TGF-, promotes glial specification. Although these
are apparently temporally segregated events, in fact
the idea is that all of these pathways interact at a
key moment during CNS development, promoting the
switch of progenitor-neuronal-glial phases. It has been
shown that, apparently, the balance of these pathways,
transcription factors, as well as epigenetic silencing
gene mechanisms such as methylation and acetyla-
tion, control the correct timing of neuron and glial-
cell generation (for review see Miller and Gauthier,
2007).
156
11.3.2 Role of Neuronal-Derived
Molecules in Astrocyte
Differentiation: Crosstalk Between
Growth Factors and
Neurotransmitters
Neuron-astrocyte interactions in the synaptic cleft play
a pivotal role in synapse development. The intimate
relationship between astrocytes and synaptic termi-
nals in vivo (Ventura and Harris, 1999) suggests that
besides affecting synapse formation, astrocytes are
also potential targets for neuronal-derived molecules
such as neurotransmitters. This idea was further sup-
ported by the accumulating evidence that astrocytes
express a wide range of neurotransmitter receptors,
both in vitro as well as in vivo (Porter and Mccarthy,
1997; Volterra and Meldolesi, 2005). Synaptic trans-
mission is controlled by the astrocytic coverage of
synapses. Morphological adaptations in the astrocytes
are associated with changes in the peri-synaptic space
and in the localization of glutamate transporters, which
results in modified action of synaptically released glu-
tamate (Oliet et al., 2001). Glutamate clearance and,
as a consequence, glutamate concentration and diffu-
sion in the extracellular space, are associated with the
degree of astrocytic coverage of its neurons. Failure
of glutamate removal can lead to neuronal death due
to its well-known neurotoxic properties (Camacho and
Massieu, 2006).
It is now clear that astrocytes respond to a vari-
ety of synaptically released transmitters in addition
to glutamate. For example, noradrenaline (Duffy and
MacVicar, 1995), histamine, acetylcholine (Shelton
and McCarthy, 2000), ATP (Wang et al., 2000), and
GABA (Kang et al., 1998) can induce elevations of
astrocytic Ca2+ . Indeed, the list of transmitters that can
mobilize astrocytic Ca2+ is almost as long as the list of
molecules that activate neuronal receptors.
Astrocytes express high levels of GABAA recep-
tors (Tateishi et al., 2006), and differentiation of
immature astrocytes is at least partly mediated by
the inhibitory neurotransmitter GABA. In vitro and
in vivo studies have provided evidence that GABA
affects immature astrocytes by increasing both their
GFAP expression and their stellation (Mong et al.,
2002; Runquist and Alonso, 2003), strongly sug-
gesting a role for GABAergic signaling in astrocyte
differentiation.
J. Stipursky et al.
Two main subtypes of glutamate transporters have
been described in glia: GLAST, with expression pre-
dominating at early stages; and glutamate transporter-
1 (GLT-1), the expression of which progressively
increases with maturity. Recently, Regan and collab-
orators demonstrated a differential cellular expression
of glutamate transporters in the developing and mature
CNS, and also showed that glutamate signaling is
under local CNS region-specific modulation. These
results suggest the concept that astroglia is formed by
a heterogeneous population of cells that markedly vary
in their responsiveness to several agents such as neuro-
transmitters (Regan et al., 2007). Swanson and collab-
orators have reported that neurons can modulate astro-
cyte glutamate transporter expression and astrocyte
differentiation in vitro. In the absence of neurons, cor-
tical astrocytes maintain polygonal shapes and express
only the GLAST transporter. When co-cultured with a
neuronal layer, many of the astrocytes maintain a stel-
late shape and express GLT-1, suggesting that neurons
can modulate astrocyte differentiation (Swanson et al.,
1997). Although the nature of this neuronal signal
remains to be identified, reports have clearly demon-
strated that astrocyte differentiation is modulated by
neuronal soluble factors (Perego et al., 2000) or neuro-
transmitters (Runquist and Alonso, 2003; Romo et al.,
2008).
How can neurotransmitters affect the morphology
and differentiation of an astrocyte? The morphology
of these cells depends on the phosphorylation and
polymerization status of GFAP. It has been shown
that different neurotransmitters including glutamate
(Kommers et al., 1999, 2002), GABA (Runquist and
Alonso, 2003), and serotonin (Chang et al., 2005)
influence the phosphorylation of GFAP, and thus in
turn its degree of polymerization (Inagaki et al., 1994).
A secreted glial protein, S100, inhibits polymeriza-
tion of GFAP (Bianchi et al., 1995; Ziegler et al.,
1998) and may therefore regulate astrocyte morphol-
ogy in an autocrine fashion. There is evidence that
serotonin induces the release of S100 from astro-
cytes (Chang et al., 2005), suggesting a link between
neuronal (serotoninergic) activity, release of S100,
and astroglial morphology. Additional support for a
crosstalk between GFAP/astrocyte differentiation and
glutamate signaling emerged from the recent demon-
stration that expression of GFAP is essential to anchor
the glutamate transporter GLAST in the astrocyte
plasma membrane, thus enhancing GLAST-mediated
11 Neuron-Astroglial Interactions in Cell Fate Commitment
transport (Sullivan et al., 2007). This is consistent
with the observation that GFAP knockout mice exhibit
reduced glutamate clearance (Hughes et al., 2004), and
loss of GLT-1 leads to elevated glutamate levels and
subsequent neurodegeneration (Rothstein et al., 1996).
These studies suggest that changes in GFAP gene
expression and glutamate homeostasis might mutually
influence each other.
Glutamate is the major excitatory neurotrans-
mitter in the CNS, and its responses are mediated
by ionotropic (iGlu) and metabotropic glutamate
(mGlu) receptors. iGlu receptors are cation-specific
ion channels defined by a specific pharmacology in
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic
acid (AMPA), kainate, and N-methyl-d-aspartate
(NMDA) receptors. mGlu receptors are a family of
G-protein-coupled, seven transmembrane domain
receptors that exert a variety of effects on second
messenger systems and ion channels. They are divided
into eight different subtypes (mGlu18) and three
sub-groupings based on agonist pharmacology: Group
I (mGlu1 and mGlu5 receptors), Group II (mGlu2 and
mGlu3), and group III (mGlu4, mGlu6, mGlu7, and
mGlu8) (Pin and Duvoisin, 1995). mGlu receptors
modulate synaptic transmission, and are involved in
activity-dependent modification of synaptic transmis-
sion such as long-term potentiation and long-term
depression. Each receptor subtype exhibits a well-
defined expression pattern in several brain regions. In
neurons, mGlu1 and mGlu5 receptors are generally
found in postsynaptic densities and modulate post-
synaptic efficacy; whereas mGlu2, mGlu3, mGlu4,
mGlu7, and mGlu8 receptors are mainly (but not
exclusively) presynaptic and regulate neurotransmitter
release (Ferraguti and Shigemoto, 2006). mGlu recep-
tors are also found in glial cells, where their activation
exerts a variety of effects that are crucial for glial func-
tion and glial-neuronal interaction under physiological
and pathological conditions (Bruno et al., 2001; Flor
et al., 2002). The presence of mGlu receptors in glial
cells was initially inferred by a number of actions of
glutamate in cultured astrocytes, including the increase
in intracellular Ca2+ (Glaum et al., 1990). Both mGlu3
and mGlu5 receptors have been detected by RT-PCR
in hippocampal astrocytes acutely isolated from young
rats (Schools and Kimelberg, 1999), adult rats (Cai
et al., 2000), reactive astrocytes, human astrocytes, and
glioma cells (Aronica et al., 2000, 2003). Astrocytes
also express AMPA receptors (Zhou and Kimelberg,
157
2001), NMDA receptors (Verkhratsky and Kirchhoff,
2007), GABAA receptors (Tateishi et al., 2006), and
purinergic receptors (Abbracchio and Ceruti, 2006).
Metabotropic glutamate receptors have been con-
sidered to be potential targets for neuroprotective
drugs since their earliest characterization. Studies by
Nicolettis group demonstrated that neuroprotection
mediated by group-II mGlu receptors involves a novel
form of glial-neuronal interaction, which is promoted
by the activation of mGlu3 receptors present in astro-
cytes (Bruno et al., 1997; Battaglia et al., 1998). That
neuroprotection by glial mGlu3 receptors is mediated
by TGF-1 and TGF-2, which are released from
astrocytes and exert a potent neuroprotective activity in
in vitro and in vivo models of excitotoxic death (Bruno
et al., 1998). This activation of glial group-II mGlu
receptors enhances the de novo synthesis of TGF-1
through the activation of the mitogen-activated protein
(MAP) kinase and phosphatidylinositol (PI)-3-K path-
ways. Pharmacological inhibition of these two path-
ways prevents the neuroprotective activity of group-II
mGlu receptor agonists against excitotoxic neuronal
death (DOnofrio et al., 2001). Recently, Corti and col-
laborators used mGlu2/Glu3 receptor knockout mice
to examine whether these two receptor subtypes have
distinct roles in the processes of neurodegeneration
and neuroprotection. They demonstrated that neuro-
protection by the dual mGlu2/3 receptor is entirely
mediated by mGlu3 receptors (Corti et al., 2007). This
suggests the existence of a novel mechanism of neuro-
protection that is mediated by the secretion of TGF-
species from astrocytes in response to group-II mGlu
receptor activation. The importance of this mecha-
nism is strengthened by the evidence that TGF- pro-
tects against neuronal death induced by excitotoxins,
ischemia, or aggregates of -amyloid peptide (Copani
et al., 1995; Pratt and McPherson, 1997; Bruno et al.,
1998; Flanders et al., 1998; Buisson et al., 2003).
TGF-1 has several effects on astrocytes, includ-
ing inhibition of proliferation (Baghdassarian et al.,
1993; Krieglstein et al., 1998), change in morphol-
ogy (Flanders et al., 1993) and motility (Gagelin et al.,
1995), modulation of the cytoskeleton (Laping et al.,
1994), and the protein content of the extracellular
matrix (ECM) (Baghdassarian et al., 1993; Wyss-
Coray et al., 1995). More recently, the role of TGF-1
in ECM production was highlighted by the demon-
stration that TGF-1 knockout mice show reduced
expression of laminin (Brionne et al., 2003).
158
Fig. 11.3 Activation of GFAP gene by glutamate involves
the TGF-1/Smad pathway. Cerebral cortex astrocytes from
newborn transgenic mice were cultured alone (a, b); in the
presence of TGF-1 (10 ng/mL, (c, d)) or glutamate (100
M, (e, f)). (a, c and e) GFAP promoter-directed expression
of lac-Z was revealed by X-Gal (blue nuclei) prior to anti-
GFAP immunocytochemistry (gray staining). Immunolabeling
In order to gain insight into astrocyte differentiation
induced by neurons, we have focused on GFAP expres-
sion, which is the major component of the astrocytic
intermediate filaments (Eng et al., 1971; Bignami et al.,
1972). In the rodent CNS, astrocyte maturation is fol-
lowed by a replacement in the expression of vimentin
by GFAP (Dahl, 1981; Pixley and De Vellis, 1984). By
using transgenic mice bearing 2 kbp of the 5 flanking
region of the GFAP gene linked to the -galactosidase
(-Gal) reporter gene, we demonstrated that cortical
neurons activate the GFAP gene promoter, followed by
transgenic astrocyte differentiation in vitro. Addition
of a conditioned medium derived from cortical neurons
had a similar effect, suggesting that a soluble fac-
tor derived from neurons might be responsible for the
induction of the GFAP gene promoter (Gomes et al.,
1999a). Later, we identified TGF-1 as the major medi-
ator of this event (de Sampaio e Spohr et al., 2002).
Although both cell types, neurons and astrocytes, syn-
thesize and secrete this factor, addition of neurons to
astrocyte monolayers greatly increased TGF-1 syn-
thesis and secretion by astrocytes (de Sampaio e Spohr
et al., 2002; Sousa et al., 2004). More recently, we
demonstrated that glutamate activates the GFAP gene
promoter of cerebral cortical astrocytes through induc-
tion of the TGF-1 signaling pathway. We reported
that glutamate induced Smad 4 nuclear translocation,
followed by activation of the GFAP gene, in cortical
astrocytes. Both events were inhibited by addition of
J. Stipursky et al.
for Smad 4 revealed a cytoplasmic location in control cultures
(b) and a nuclear distribution in TGF-1 (c) and glutamate-
treated (d) cultures. Glutamate and TGF-1 increased -Gal
astrocyte number, followed by activation of the Smad pathway.
Scale bars correspond to 50 m
the mGlu2/3 receptor antagonist (Romo et al., 2008;
Fig. 11.3).
These data suggest a key role for TGF-1 as a
downstream mediator of the effect of glutamate and
neurons on the GFAP gene promoter. The associa-
tion of dysfunctional glial glutamate transporters and
receptors with several neurological disorders (Sheldon
and Robinson, 2007), together with the observation of
high levels of TGF-1 in several diseases where gluta-
mate metabolism dysfunctions are described (Tesseur
and Wyss-Coray, 2006), support the hypothesis that
glutamate/TGF-1 cross-talk might have a key role,
not only in development but in brain pathology.
11.4 Neuron-Astrocyte Interactions:
Implications for Neuronal
Differentiation and Synaptogenesis
It has been estimated that individual astrocytes in
the adult rodent brain ensheath and interact with as
many as 140,000 synapses (Bushong et al., 2002).
Traditionally, the function of astrocytes in synapses
has been associated with the regulation of ion concen-
trations, neurotransmitter clearance, and providing of
substrates for energy metabolism (Carmignoto, 2000).
Accumulated data from the last 7 years, however, sug-
gest that astrocytes are an integral part of synaptic
11 Neuron-Astroglial Interactions in Cell Fate Commitment
connections, either by (1) inducing neuronal differen-
tiation; or by (2) promoting synapse formation and
elimination; and even by (3) actively secreting neu-
romodulators or gliotransmitters. In this section, we
will focus on the role of glial cells in synaptic plastic-
ity, with special emphasis on items (1) and (2). First,
we will briefly discuss the role of astrocytes in neu-
ronal maturation, and axonal and dendritic growth,
essential steps for synapse establishment. Then, we
will present in vitro and in vivo evidence that astro-
cytes and Schwann cells can induce synaptogenesis,
and are involved in the maintenance of newly formed
synapses, in addition to ongoing structural support of
synapses.
11.4.1 Neuron-Astrocyte Interactions
and Neuronal Differentiation
During CNS development, neurons migrate, and as
soon as they reach their appropriate positions they
begin to extend axons in order to establish connections
with other neurons. This event is characterized by a
dramatic change in cell morphology that is of funda-
mental importance in brain development, as well as in
the regeneration of damaged nervous tissue (Guizzetti
et al., 2008).
Growing axons navigate toward their targets in
response to a variety of guidance signals in their sur-
rounding environment. These cues include diffusible
attractive or repellent molecules that are secreted by
the intermediate or final cellular targets of the axons.
Astrocytes are known as a potent source of cues that
influence neuronal fate and growth-cone navigation,
not only by secreting soluble factors, but also by
expressing extracellular matrix proteins (Garcia-Abreu
et al., 2000; de Sampaio e Spohr et al., 2008).
Recently, Kanemaru and co-workers (2007) sug-
gested a novel intracellular signaling mechanism by
which astrocytes mediate neurite outgrowth. These
authors showed that modulation of spontaneous Ca2+
oscillations in astrocytes leads to a reduction in the
expression of N-cadherin, which correlates with inhi-
bition of their neurite permissivity (Kanemaru et al.,
2007). Interestingly, it was reported that spontaneous
Ca2+ oscillations become less frequent with age (Parri
et al., 2001) and are lost in reactive astrocytes (Aguado
159
et al., 2002), which are not permissive for neurite out-
growth. Together, these events suggest that Ca2+ oscil-
lations in astrocytes might be an important mechanism
to control neuronal development and regeneration.
Another suggested mechanism by which astrocytes
are proposed to modulate neurite outgrowth is by their
pattern of expression of extracellular-matrix (ECM)
proteins such as laminin, fibronectin, and proteogly-
cans (Garcia-Abreu et al., 2000; Martinez and Gomes,
2002). Astrocytes have been recognized as the major
source of these ECM components, both in vivo and
in vitro (Garcia-Abreu et al., 2000). The pattern of
these ECM proteins on the astrocyte surface, which
is highly modulated by thyroid hormone, provides
directional cues for neurite outgrowth (Garcia-Abreu
et al., 2000; Martinez and Gomes, 2002). Our group
demonstrated in 2002 that EGF secreted by T3-treated
astrocytes induces EGL neurons to undergo differ-
entiation initiated by the outgrowth of neurites, and
this event is mediated by EGF modulation of laminin
and fibronectin astrocytic expression through MAPK
and PI3K pathways. We demonstrated that EGF could
have a binary role for EGF in cerebellar ontogene-
sis, directly on the proliferation of granular precursors,
and indirectly through ECM components in neurite
outgrowth.
However, the ability of astrocytes to induce neurite
outgrowth depends on the region where the astrocytes
originate. It was demonstrated that astrocytes derived
from different sections of the embryonic mouse mid-
brain show different permissivity to support neurite
outgrowth. This was related to differences in the
content of the ECM elements, laminin and sulfated
glycosaminoglycans (s-GAGs) in the astrocytes from
different sections: the punctuate pattern of laminin
expression was not permissive for neurite outgrowth
(Garcia-Abreu et al., 1995, 2000; Mendes et al., 2003).
Together, these differences support the concept that
astrocytes display heterogeneous specializations, with
reports of region-specific, species-specific, and func-
tionally distinct subpopulations. It is interesting that
astrocytes lose their permissivity to neurite outgrowth
with age (Bahr et al., 1995), indicating that this prop-
erty depends not only on the region of origin of
the astrocytes, but also on their developmental stage.
However, the underlying mechanism that controls this
property remains unclarified.
Thus, astroglial-derived soluble and membrane-
bound factors could promote neuronal fate and
160
arborization of neuronal precursors; however, the
molecules that may instruct the astrocytes to secrete
soluble factors or membrane-bound factors remain
unknown. Recently, we demonstrated that astrocytes
primed by lysophosphatidic acid (LPA) increase neu-
ronal differentiation, likely through a soluble factor,
and that this activity is dependent on activation of
defined LPA receptors expressed in astrocytes (de
Sampaio e Spohr et al., 2008). LPA is a lysophospho-
lipid that may regulate diverse biological processes;
however, little is known about its role in NS devel-
opment. Our group demonstrated that LPA-primed
astrocytes induce neuronal differentiation of neuronal
precursors. Analysis of neuronal morphology revealed
a marked increase in the number of processes per neu-
ron plated onto LPA-primed astrocytes, suggesting that
LPA-primed astrocytes may influence cell-fate specifi-
cation and neuronal maturation in vitro (de Sampaio e
Spohr et al., 2008) (Fig. 11.4).
One of the major specializations that neurons
undergo is that related to polarization: which branch
Fig. 11.4 LPA-primed astrocytes increase neuronal differ-
entiation. Cerebral cortical progenitors obtained from embry-
onic mice were cultured for 24 h on control (a) and LPA-primed
(b) astrocyte monolayers. Cells were immunostained for the
neuronal marker, -tubulin III. There was a significant enhance-
ment in the arborization and length of neurites of neurons plated
onto LPA-primed astrocytes. Scale bar corresponds to 30 m.
J. Stipursky et al.
is committed to become an axon or dendrites, which
are two morphologically and functionally distinct seg-
ments? Neuronal polarity is important for neuronal
specialization. The understanding of the mechanisms
and molecules that may modify axonal or dendritic
elongation or morphology during neuronal develop-
ment might yield some clues for a better understand-
ing of higher brain functions, such as learning and
memory. However, these mechanisms involved in the
creation and maintenance of neuronal polarity are still
the subject of study. What is known is that astrocytes
play important roles in this event: neuronal polarity
is regulated by several molecules, such as NGF and
BMP (a subclass of the TGF- superfamily), and these
molecules may be secreted by astrocytes (Prochiantz,
1995). It was also demonstrated that dendrite initi-
ation is regulated separately from that of the axon,
and that local, and thus region-specific, astrocyte-
derived factors are responsible for this phenomenon,
and this can be mediated by ECM (Chamak et al.,
1987).
(c) Schematic model of LPA effect on neurons mediated by
astrocytes. Extracellular LPA produced by postmitotic neurons
directly interacts with high-affinity LPA receptors present on
glial cells, which secrete a soluble factor in response to LPA.
Indirectly, LPA induces maturation of postmitotic neurons and
neuronal commitment of progenitors
11 Neuron-Astroglial Interactions in Cell Fate Commitment
Neuronal polarity defines the fine tuning of
responses to numerous types of signal inputs, inte-
grating and propagating them in the form of elec-
trical impulses over long distances to different tar-
gets. In addition to signal processing and propagation,
the axonal and dendritic surfaces also play house-
keeping roles such as receiving and releasing different
molecules from and into their environment (Bradke
and Dotti, 2000).
Astrocytes modify the number of synaptic con-
nections, by releasing a variety of factors that influ-
ence axonal and dendritic growth and thus the poten-
tial for synapse formation, such as laminins (Liesi
et al., 1983), neurotrophins (Althaus and Richter-
Landsberg, 2000), S100 (Nishiyama et al., 2002), and
the activity-dependent neurotrophic factor (Blondel
et al., 2000).
During development, inappropriate synapses must
be eliminated, and appropriate synaptic connections
must be maintained and strengthened. This dynamic
period of synaptic refinement coincides with the
appearance of astrocytes in the postnatal brain, and
recent evidence indicates a role for astrocyte-derived
signals in synapse development (Ullian et al., 2001;
Christopherson et al., 2005). Astrocyte contacts to
neurons may have different roles in the formation
of initial synapses, and the astrocytes may be able
to induce local structural and functional modifica-
tions of dendritic segments or individual synapses.
These contacts may also induce global maturation of
neurons and subsequently up-regulation of synapse
formation, promoting the global maturation of the
neuronal network. Recently, Nishida and Okabe
(2007) demonstrated that astrocytic contacts have
instructive roles in the subsequent stabilization and
maturation of immature dendritic protrusions, and that
these astrocytic contacts are selective: some dendritic
protrusions received astrocytic contacts and others
did not, indicating that the astrocytes are capable of
identifying the appropriate target.
All the lines of evidence discussed here increase
the support for the hypothesis that astrocytes play a
dynamic role in the maintenance of connections in
the nervous system proper, either by controlling neu-
ronal specification and polarization or by controlling
the numbers and establishment of the synapse con-
nections. Therefore, astrocytes, which were formerly
considered to be passive supporters of the chemical
synapse, have recently gained much recognition as
161
an integral and essential component of the tripartite
synapse in both the peripheral nervous system (PNS)
and the CNS (Feng and Ko, 2008), as we will better
address in the next section.
11.4.2 Role for Glia in Synaptogenesis
In the CNS, astrocytes envelop the pre- and postsy-
naptic neuronal components. This cellular architecture
yields the concept of the tripartite synapse, consti-
tuted by neurons and peri-synaptic glial cells, which
is regarded today as a third integral component of
synapses (Araque et al., 1999). Peri-synaptic glia have
been observed in several central and peripheral NS,
including astrocytes in the cerebral cortex (Ventura
and Harris, 1999), Bergmann glia in the cerebellum
(Grosche et al., 2002), Mller cells in the retina,
and Schwann cells at the neuromuscular junction
(Robitaille, 1998).
The first evidence of the role of astrocytes in
synaptogenesis came from Ben Barres group at the
end of the twentieth century. By using glia-free cul-
tures of purified retinal ganglion cells (RGC), Pfrieger
and Barres (1997) found that astrocytes and oligo-
dendrocytes strongly enhanced spontaneous synaptic
activity and the reliability of synaptic transmission.
These effects were attributed to an increase in the effi-
cacy of existing synapses (Pfrieger and Barres, 1997).
They found that astrocytes increased the total num-
ber of synapses on each neuron sevenfold. Moreover,
astrocytes were required to maintain these synapses,
because most synapses that had formed in the presence
of astrocytes were quickly lost when these cells were
removed (Ngler et al., 2001; Ullian et al., 2001, 2004).
Later, the idea that glial cells induce synapse forma-
tion was validated, in the central and PNC, by a number
of studies in different neuronal cell lines (murine and
human) (Mauch et al., 2001; Ngler et al., 2001; Ullian
et al., 2001), and neuromuscular junction (NMJ) neu-
rons (Ullian et al., 2004). More recently, Johnson and
collaborators demonstrated that synaptic transmission
in human embryonic stem cell (hES cell) derived
neuronal cultures is enhanced by exogenous astrocytes
(Johnson et al., 2007). These data suggest that glia-
induced synaptogenesis might be a general mechanism
that is used by organisms ranging from lower inverte-
brates, as recently shown for C. elegans (Bacaj et al.,
2008), to humans.
162
If there is compelling evidence for the role of
astrocytes in synaptogenesis, these findings raise a
vital question of how glial cells enhance synaptoge-
nesis and regulate synaptic transmission. In vitro or
in vivo studies have revealed a variety of putative
synaptogenic secreted and contact glia-derived fac-
tors. These include glutamate (Parpura and Haydon,
2000), cholesterol (Mauch et al., 2001; Goritz et al.,
2005), the extracellular matrix protein thrombospondin
(Christopherson et al., 2005), TGF-1 (Feng and Ko,
2008), adenosine triphosphate (Koizumi et al., 2003),
the neuromodulator D-serine (Yang et al., 2003), and
the glial protein S100 (Nishiyama et al., 2002).
It is conceivable that astrocytes secrete other fac-
tors, not yet identified, with synapse-promoting activ-
ity (Parpura and Haydon, 2000; Yang et al., 2003;
Elmariah et al., 2005).
Recent studies showed that cultured neurons from
the mammalian CNS require glia-derived cholesterol
to form numerous and efficient synapses. Neurons
appear to produce enough cholesterol to survive,
to differentiate axons and dendrites, and to form a
few inefficient synapses. The massive formation of
synapses, however, may require additional choles-
terol, which must be delivered by astrocytes via
ApoE-containing lipoproteins (Mauch et al., 2001;
Pfrieger, 2003; Goritz et al., 2005). So far, how-
ever, it remains unclear how cholesterol enhances the
formation and function of synapses. Goritz and col-
laborators demonstrated that dendrite differentiation is
the rate-limiting step for glia-induced synaptogenesis
in RGC, and that this process requires cholesterol. In
addition, they demonstrated that cholesterol directly
enhances presynaptic differentiation, supports contin-
uous synapse development, and is essential for the
stability of evoked synaptic transmission (Goritz et al.,
2005).
Christopherson and collaborators identified the
ECM proteins thrombospondins (TSPs), TSP-1-2,
as the synaptogenic astrocyte-derived molecule
(Christopherson et al., 2005). Addition of TSP-1
or TSP-2 to cultured RGC caused a large increase
in the number of synapses, similar to that induced
by astrocytes. Immunodepletion of TSP-2 from
astrocyte-conditioned medium reduced the number
of induced synapses to control levels. Moreover, the
TSP-1 and TSP-2 functions are essential for pro-
moting synaptogenesis: TSP-1/TSP-2 double-mutant
mice exhibit a dramatic reduction in the number
J. Stipursky et al.
of synapses formed during postnatal stages. TSP-1
and -2 are both expressed in the developing brain
during the peak period of synaptogenesis, but are
shut off by adulthood. These findings suggest that
immature astrocytes provide a developmental window
during which synaptogenesis can occur, by producing
a permissive environment through the secretion
of TSP (Allen and Barres, 2005; Christopherson
et al., 2005).
Similar glia-enhanced synaptogenesis has also been
observed at PNS synapses (Feng and Ko, 2008). Feng
and Ko demonstrated that TGF-1 is a Schwann
cell-derived molecule that promotes synaptogene-
sis at nerve-muscle contacts in vitro. This event
is mainly due to induction of expression of neu-
ronal agrin, a large proteoglycan, which leads to
a global increase in the formation of acetylcholine
receptor clusters on muscle cells. This expression
induced by TGF-1 acts directly on presynaptic neu-
rons rather than through muscle cells (Feng and Ko,
2008).
During development, neurons acquire competence
to receive synapses. Two distinct mechanisms are
essential to the success of this process: dendrite devel-
opment, as discussed in the previous section; and
synaptic receptivity. Although it is generally assumed
that neurons are generated with the ability to both
form and receive synapses, Fletcher et al. (1994) found
that embryonic hippocampal neurons are capable of
forming but not receiving synapses. Recently, it was
shown that astrocyte contact induces the acquisition
of competence for synaptic receptivity (Barker et al.,
2008).
The formation of mature neural circuits requires
the selective elimination of inappropriate synaptic con-
nections. Several lines of evidence suggest that astro-
cytes may play a key role in this process, either by
activating specific signaling pathways, such as the
integrin-dependent pathway (Hama et al., 2004), or by
physically eliminating the synapses, through release of
proteases, protease inhibitors (Nelson et al., 1995), and
components of the complement cascade (Stevens et al.,
2007).
Murai and coworkers revealed an influence of
ephrin signaling on the regulation of spine morphology
in mouse hippocampal pyramidal cells in situ (Murai
et al., 2003). They showed that the interaction between
ephrin-A3, located on astrocytic processes, and its
EphA4 receptor on dendritic spines leads to retraction
11 Neuron-Astroglial Interactions in Cell Fate Commitment
of spines. This raises the question of whether contact
between astrocytic processes and dendritic spines dur-
ing development is responsible for the elimination of
synapses.
Stevens and collaborators showed that a protein
in the classical complement cascade is expressed by
postnatal neurons in response to immature astrocytes,
and is located on synapses throughout the postna-
tal CNS and retina. They identified an unexpected
role for astrocytes and the classical complement cas-
cade in mediating CNS synapse elimination in the
retinogeniculate pathway. The finding that the classical
complement cascade helps to mediate developmental
CNS synapse elimination adds to the growing evidence
that components of the immune system contribute
to brain development and function (Stevens et al.,
2007).
This evidence, together with the emerging char-
acterization of gliotransmitters (Bezzi and Volterra,
2001) and several neurotransmitters and receptors in
astrocytes (which are not within the scope of this chap-
ter), argues that astrocytes are active partners of neu-
rons, not only in synapse development but in function
as well.
11.5 Concluding Remarks
The relative number of astrocytes, expressed both as
a proportion of the total brain cell number and as a
ratio to the neuronal number, increases dramatically
with phylogeny and brain complexity (Nedergaard
et al., 2003). It has been proposed that human cor-
tical evolution is accompanied by increasing com-
plexity in the form and function of astrocytes, which
reflects an expansion of their functional roles in synap-
tic modulation and cortical circuitry. This is exem-
plified by the observation that each domain of a
single rodent astrocyte covers approximately 90,000
synapses, whereas in the human cortex each astrocyte
supports and modulates the function of roughly two
million synapses. Further, in addition to the increase
in number and complexity of astrocytes in evolution,
the growing association of glia in synapses, which
may be related to NS evolution, is even more striking.
Although one cannot completely attribute the intellec-
tual capacity of humans to astrocyte complexity, the
loss of astrocytic domains has been associated with
163
disturbances of neuronal function that are found in
several neurological disorders such as epilepsy and
schizophrenia.
One key issue in developmental neurobiology is to
understand how the brain orchestrates the differentia-
tion of various cell types. A range of epigenetic signals
are involved in neural-fate potential, lineage specifica-
tion, and cellular differentiation in the CNS and PNS.
The identification of RG and astrocytes as neural stem
cells in the developing and mature nervous system,
respectively, shed light on the importance of identify-
ing the molecules that control astroglia differentiation.
Although several molecules involved in this event have
already been identified, we are only at the threshold
of understanding fully the nature and consequences
of these newly discovered astroglial functions. The
scenario is still more rudimentary concerning brain
pathology.
Emerging evidence indicates that glial cells are
essential elements in the establishment of several
neurological disorders, such as epilepsy, schizophre-
nia, and depression; and neurodegenerative diseases,
such as Alzheimers, Parkinsons, and Huntingtons
diseases and Amyotrophic Lateral Sclerosis (ALS)
(for review see Seifert et al., 2006; Barres, 2008).
Whether glial dysfunction is the primary deficit or
a consequence of neuronal damage, remains to be
investigated in each pathology. The question is even
more complex when we consider that astroglia are not
unique, but a heterogeneous group of cells that express
different growth factors and neurotransmitter recep-
tors, molecular markers, different stem-cell potentials,
and synaptogenic properties. This evidence leads to
specific questions such as What is the difference
between neurogenic and non-neurogenic astrocytes?
or Why do some synapses not require astrocyte sig-
nals? or What signals might reprogram astrocytes
to a RG phenotype in the adult brain? or Which
pathways lead to astrocyte activation or dysfunction?
Answers to these questions would certainly provide
a better understanding of glial biology and neuron-
glia interaction during brain development and aging.
Further, the key roles of glial cells in CNS pathol-
ogy, together with the accumulating evidence that
RG cells and astrocytes (and/or neurons) are con-
vertible cell types, open the possibility that manipu-
lation of glia differentiation signaling pathways may
be a promising therapeutic target for brain insults
(Fig. 11.5).
164
Fig. 11.5 Schematic view of neuron-astroglial interactions.
Radial glia cells and astrocytes are involved in brain develop-
ment, either acting as neuronal progenitors (1) or secreting sol-
uble and ECM molecules that modulate neuronal fate commit-
ment, neuronal differentiation, and synaptogenesis (2). Deficits
in astrocyte functions play a key role in the establishment of
Acknowledgment For financial support: FAPERJ, CNPq,
CAPES (Brazil).
References
Abbracchio MP, Ceruti S (2006) Roles of P2 receptors in glial
cells: focus on astrocytes. Purinergic Signal 4:595604.
Aguado F, Espinosa-Parrilla JF, Carmona MA, Soriano E (2002)
Neuronal activity regulates correlated network properties
of spontaneous calcium transients in astrocytes in situ. J
Neurosci 22:94309444.
Allen NJ, Barres BA (2005) Signaling between glia and neurons:
focus on synaptic plasticity. Curr Opin Neurobiol 5:542548.
Althaus HH, Richter-Landsberg C (2000) Glial cells as targets
and producers of neurotrophins. Int Rev Cytol 197:203277.
Altman J, Das GD (1966) Autoradiographic and histological
studies of postnatal neurogenesis. I. A longitudinal investi-
gation of the kinetics, migration and transformation of cells
incorporating tritiated thymidine in neonate rats, with special
reference to postnatal neurogenesis in some brain regions. J
Comp Neurol 126:337389.
Alvarez-Buylla A, Garca-Verdugo JM, Tramontin AD (2001) A
unified hypothesis on the lineage of neural stem cells. Nat
Rev Neurosci 2:287293.
Alvarez-Buylla A, Lim DA (2004) For the long run: maintaining
germinal niches in the adult brain. Neuron 41:683686.
J. Stipursky et al.
several neurodegenerative diseases or neurological disorders (3).
Manipulation of signaling pathways that modulate astrocyte
commitment lineage (black and gray dashed lines); this func-
tion might be crucial to use glial cells as potential therapeutic
targets
Anthony TE, Klein C, Fishell G, Heintz N (2004) Radial glia
serves as neuronal progenitors in all regions of the central
nervous system. Neuron 25:881890.
Anton ES, Marchionni MA, Lee KF, Rakic P (1997) Role of
GGF/neuregulin signaling in interactions between migrating
neurons and radial glia in the developing cerebral cortex.
Development 124:35013510.
Araque A, Parpura V, Sanzgiri RP, Haydon PG (1999) Tripartite
synapses: glia, the unacknowledged partner. Trends Neurosci
5:208215.
Aronica E, Gorter JA, Ijlst-Keizers H, Rozemuller AJ, Yankaya
B, Leenstra S, Troost D (2003) Expression and functional
role of mGluR3 and mGluR5 in human astrocytes and glioma
cells: opposite regulation of glutamate transporter proteins.
Eur J Neurosci 10:21062118.
Aronica E, van Vliet EA, Mayboroda OA, Troost D, da Silva FH,
Gorter JA (2000) Upregulation of metabotropic glutamate
receptor subtype mGluR3 and mGluR5 in reactive astro-
cytes in a rat model of mesial temporal lobe epilepsy. Eur
J Neurosci 7:23332344.
Bacaj T, Tevlin M, Lu Y, Shaham S (2008) Glia are essential for
sensory organ function in C. elegans. Science 322:744747.
Baghdassarian D, Toru-Delbauffe D, Gavaret JM, Pierre M
(1993) Effects of transforming growth factor-beta 1 on the
extracellular matrix and cytoskeleton of cultured astrocytes.
Glia 7:193202.
Bahr M, Przyrembel C, Bastmeyer M (1995) Astrocytes from
adult rat optic nerves are nonpermissive for regenerating
retinal ganglion cell axons. Exp Neurol 131:211220.
11 Neuron-Astroglial Interactions in Cell Fate Commitment
Barker AJ, Koch SM, Reed J, Barres BA, Ullian EM (2008)
Developmental control of synaptic receptivity. J Neurosci
33:81508160.
Barnab-Heider F, Wasylnka JA, Fernandes KJ, Porsche C,
Sendtner M, Kaplan DR, Miller FD (2005) Evidence that
embryonic neurons regulate the onset of cortical gliogenesis
via cardiotrophin-1. Neuron 48:253265.
Barres B (2008) The mystery and magic of glia: a perspective on
their roles in health and disease. Neuron 60:430440.
Battaglia G, Bruno V, Ngomba RT, Di Grezia R, Copani
A, Nicoletti F (1998) Selective activation of group-II
metabotropic glutamate receptors is protective against exci-
totoxic neuronal death. Eur J Pharmacol 356:271274.
Bayer SA, Altman J (1991) Development of the endopiriform
nucleus and the claustrum in the rat brain. Neuroscience
45:391412.
Bentivoglio M, Mazzarello P (1999) The history of radial glia.
Brain Res Bull 49:305315.
Bernardos RL, Barthel LK, Meyers JR, Raymond PA (2007)
Late-stage neuronal progenitors in the retina are radial
Mller glia that function as retinal stem cells. J Neurosci
26:70287040.
Berninger B, Costa MR, Koch U, Schroeder T, Sutor B, Grothe
B, Gtz M (2007) Functional properties of neurons derived
from in vitro reprogrammed postnatal astroglia. J Neurosci
32:86548664.
Bezzi P, Volterra A (2001) A neuron-glia signalling network in
the active brain. Curr Opin Neurobiol 11:387394.
Bianchi R, Garbuglia M, Verzini M, Giambanco I, Spreca A,
Donato R (1995) S-100 protein and annexin II2-p11(2)
(calpactin I) act in concert to regulate the state of assem-
bly of GFAP intermediate filaments. Biochem Biophys Res
Commun 208:910918.
Bignami A, Eng LF, Dahl D, Uyeda CT (1972) Localization of
glial fibrillary acidic protein in astrocyte by immunofluores-
cence. Brain Res 43:429435.
Blondel O, Collin C, McCarran WJ, Zhu S, Zamostiano
R, Gozes I, Brenneman DE, McKay RD (2000) A glia-
derived signal regulating neuronal differentiation. J Neurosci
20:80128020.
Bonaguidi MA, Peng CY, McGuire T, Falciglia G, Gobeske KT,
Czeisler C, Kessler JA (2008) Noggin expands neural stem
cells in the adult hippocampus. J Neurosci 37:91949204.
Bonni A, Sun Y, Nadal-Vicens M, Bhatt A, Frank DA,
Rozovsky I, Stahl N, Yancopoulos GD, Greenberg ME
(1997) Regulation of gliogenesis in the central nervous
system by the JAK-STAT signaling pathway. Science 278:
477483.
Bradke F, Dotti CG (2000) Establishment of neuronal polar-
ity: lessons from cultured hippocampal neurons. Cur Opin
Neurobiol 10:574581.
Brionne TC, Tesseur I, Masliah E, Wyss-Coray T (2003) Loss
of TGF-beta1 leads to increased neuronal cell death and
microgliosis in mouse brain. Neuron 40:11331145.
Bruno V, Battaglia G, Casabona G, Copani A, Caciagli F,
Nicoletti F (1998) Neuroprotection by glial metabotropic
glutamate receptors is mediated by transforming growth
factor-beta. J Neurosci 18:95949600.
Bruno V, Battaglia G, Copani A, DOnofrio M, Di Iorio P,
De Blasi A, Melchiorri D, Flor PJ, Nicoletti F (2001)
Metabotropic glutamate receptor subtypes as targets for
165
neuroprotective drugs. J Cereb Blood Flow Metab 9:
10131033.
Bruno V, Sureda FX, Storto M, Casabona G, Caruso A, Knopfel
T, Kuhn R, Nicoletti F (1997) The neuroprotective activity
of group-II metabotropic glutamate receptors requires new
protein synthesis and involves a glial-neuronal signaling. J
Neurosci 17:18911897.
Buisson A, Lesne S, Docagne F, Ali C, Nicole O, MacKenzie
ET, Vivien D (2003) Transforming growth factor-beta and
ischemic brain injury. Cell Mol Neurobiol 23:539550.
Bushong EA, Martone ME, Jones YZ, Ellisman MH (2002)
Protoplasmic astrocytes in CA1 stratum radiatum occupy
separate anatomical domains. J Neurosci 1:183192.
Cai Z, Schools GP, Kimelberg HK (2000) Metabotropic gluta-
mate receptors in acutely isolated hippocampal astrocytes:
developmental changes of mGluR5 mRNA and functional
expression. Glia 1:7080.
Camacho A, Massieu L (2006) Role of glutamate transporters in
the clearance and release of glutamate during ischemia and
its relation to neuronal death. Arch Med Res 1:1118.
Campbell K, Gtz M (2002) Radial glia: multi-purpose cells for
vertebrate brain development. Trends Neurosci 25:235238.
Carmignoto G (2000) Reciprocal communication systems
between astrocytes and neurones. Prog Neurobiol 6:
561581.
Carney RS, Cocas LA, Hirata T, Mansfield K, Corbin JG (2008)
Differential regulation of telencephalic pallial-subpallial
boundary patterning by Pax6 and Gsh2. Cereb Cortex
19:745759.
Chamak B, Fellous A, Glowinski J, Prochiantz A (1987) MAP2
expression and neuritic outgrowth and branching are coregu-
lated through region-specific neuro-astroglial interactions. J
Neurosci 7:31633170.
Chang MS, Ariah LM, Marks A, Azmitia EC (2005) Chronic
gliosis induced by loss of S-100B: knockout mice have
enhanced GFAP-immunoreactivity but blunted response to a
serotonin challenge. Brain Res 1031:19.
Chiasson BJ, Tropepe V, Morshead CM, van der Kooy D (1999)
Adult mammalian forebrain ependymal and subependymal
cells demonstrate proliferative potential, but only subependy-
mal cells have neural stem cell characteristics. J Neurosci
19:44624471.
Choi BH, Lapham LW (1978) Radial glia in the human fetal
cerebrum: a combined Golgi, immunofluorescent and elec-
tron microscopic study. Brain Res 148:295311.
Chojnacki A, Shimazaki T, Gregg C, Weinmaster G, Weiss S
(2003) Glycoprotein 130 signaling regulates Notch1 expres-
sion and activation in the self-renewal of mammalian fore-
brain neural stem cells. J Neurosci 23:17301741.
Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell
JW, Agah A, Lawler J, Mosher DF, Bornstein P, Barres BA
(2005) Thrombospondins are astrocyte-secreted proteins that
promote CNS synaptogenesis. Cell 120:421433.
Copani A, Bruno V, Battaglia G, Leanza G, Pellitteri R, Russo
A, Stanzani S, Nicoletti F (1995) Activation of metabotropic
glutamate receptors protects cultured neurons against apop-
tosis induced by beta-amyloid peptide. Mol Pharmacol
47:890897.
Corti C, Battaglia G, Molinaro G, Riozzi B, Pittaluga A, Corsi
M, Mugnaini M, Nicoletti F, Bruno V (2007) The use of
knock-out mice unravels distinct roles for mGlu2 and mGlu3
166
metabotropic glutamate receptors in mechanisms of neurode-
generation/neuroprotection. J Neurosci 27:82978308.
Dahl D (1981) The vimentin-GFA protein transition in rat neu-
roglia cytoskeleton occurs at the time of myelination. J
Neurosci Res 6:741748.
Das AV, Mallya KB, Zhao X, Ahmad F, Bhattacharya S,
Thoreson WB, Hegde GV, Ahmad I (2006) Neural stem cell
properties of Mller glia in the mammalian retina: regulation
by Notch and Wnt signaling. Dev Biol 299:283302.
Davis AA, Temple S (2000) Timing of CNS cell generation: a
programmed sequence of neuron and glial cell production
from isolated murine cortical stem cells. Neuron 28:6980.
de Azevedo LC, Fallet C, Moura-Neto V, Daumas-Duport C,
Hedin-Pereira C, Lent R (2003) Cortical radial glial cells
in human fetuses: depth-correlated transformation into astro-
cytes. J Neurobiol 55:288298.
de Sampaio e Spohr TCL, Martinez R, da Silva EF, Moura
Neto V, Gomes FC (2002) Neuro-glia interactions effects on
GFAP gene: a novel role for transforming growth factor beta
1. Eur J Neurosci 16:20592069.
de Sousa OV, Romo L, Neto VM, Gomes FC (2004) Glial fibril-
lary acidic protein gene promoter is differently modulated by
transforming growth factor-beta 1 in astrocytes from distinct
brain regions. Eur J Neurosci 19:17211730.
Doetsch F (2003) A niche for adult neural stem cells. Curr Opin
Genet Dev 13:543550.
Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-
Buylla A (1999) Subventricular zone astrocytes are neural
stem cells in the adult mammalian brain. Cell 97:703716.
Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-
Buylla A (2002) EGF converts transit-amplifying neurogenic
precursors in the adult brain into multipotent stem cells.
Neuron 36:10211034.
Duffy S, MacVicar BA (1995) Adrenergic calcium signaling in
astrocyte networks within the hippocampal slice. J Neurosci
15:55355550.
DOnofrio M, Cuomo L, Battaglia G, Ngomba RT, Storto M,
Kingston AE, Orzi F, De Blasi A, Di Iorio P, Nicoletti F,
Bruno V (2001) Neuroprotection mediated by glial group-
II metabotropic glutamate receptors requires the activation
of the MAP kinase and the phosphatidylinositol-3-kinase
pathways. J Neurochem 78:435445.
Eiraku M, Tohgo A, Ono K, Kaneko M, Fujishima K, Hirano T,
Kengaku M (2005) DNER acts as a neuron-specific Notch
ligand during Bergmann glial development. Nat Neurosci
8:873880.
Elmariah SB, Oh EJ, Hughes EG, Balice-Gordon RJ (2005)
Astrocytes regulate inhibitory synapse formation via Trk-
mediated modulation of postsynaptic GABAA receptors. J
Neurosci 25:36383650.
Eng LF, Vanderhaeghen JJ, Bignami A, Gerste B (1971) An
acidic protein isolated from fibrous astrocytes. Brain Res
28:351354.
Ever L, Gaiano N (2005) Radial glial progenitors: neurogenesis
and signaling. Curr Opin Neurobiol 1:2933.
Feng L, Heintz N (1995) Differentiating neurons activate tran-
scription of the brain lipid-binding protein gene in radial
glia through a novel regulatory element. Development 121:
17191730.
Feng Z, Ko CP (2008) Schwann cells promote synaptogenesis at
the neuromuscular junction via transforming growth factor-
beta1. J Neurosci 39:95999609.
J. Stipursky et al.
Ferraguti F, Shigemoto R (2006) Metabotropic glutamate recep-
tors. Cell Tissue Res 326:483504.
Filippov V, Kronenberg G, Pivneva T, Reuter K, Steiner B,
Wang LP, Yamaguchi M, Kettenmann H, Kempermann G
(2003) Subpopulation of nestin-expressing progenitor cells
in the adult murine hippocampus shows electrophysiologi-
cal and morphological characteristics of astrocytes. Mol Cell
Neurosci 23:373382.
Flanders KC, Ldecke G, Renzig J, Hamm C, Cissel DS,
Unsicker K (1993) Effect of TGF-s and bFGF on astroglial
cell growth and gene expression in vitro. Mol Cell Neurosci
4:406417.
Flanders KC, Ren RF, Lippa CF (1998) Transforming growth
factor-betas in neurodegenerative disease. Prog Neurobiol
54:7185.
Fletcher TL, De Camilli P, Banker G (1994) Synaptogenesis
in hippocampal cultures: evidence indicating that axons and
dendrites become competent to form synapses at different
stages of neuronal development. J Neurosci 11:66956706.
Flor PJ, Battaglia G, Nicoletti F, Gasparini F, Bruno V (2002)
Neuroprotective activity of metabotropic glutamate receptor
ligands. Adv Exp Med Biol 513:197223.
Gagelin C, Pierre M, Gavaret JM, Toru-Delbauffe D (1995)
Rapid TGF beta 1 effects on actin cytoskeleton of astro-
cytes: comparison with other factors and implications for cell
motility. Glia 13:283293.
Gaiano N, Fishell G (2002) The role of notch in promoting glial
and neural stem cell fates. Annu Rev Neurosci 25:471490.
Galileo DS, Gray GE, Owens GC, Majors J, Sanes JR (1990)
Neurons and glia arise from a common progenitor in chicken
optic tectum: demonstration with two retroviruses and cell
type-specific antibodies. Proc Natl Acad Sci USA 87:
458462.
Garcia CM, Darland DC, Massingham LJ, DAmore PA (2004)
Endothelial cell-astrocyte interactions and TGF beta are
required for induction of blood-neural barrier properties. Dev
Brain Res 152:2538.
Garcia-Abreu J, Mendes FA, Onofre GR, Freitas MS, Silva LCF,
Moura Neto V, Cavalcante L (2000) Contribution of heparin
sulfate to the non-permissive role of the midline glia to the
growth of midbrain neurites. Glia 29:260272.
Garcia-Abreu J, Moura-Neto V, Carvalho SL, Cavalcante LA
(1995) Regionally specific properties of midbrain glia. I.
Interactions with midbrain neurons. J Neurosci Res 40:
417477.
Ghashghaei HT, Weimer JM, Schmid RS, Yokota Y, McCarthy
KD, Popko B, Anton ES (2007) Reinduction of ErbB2 in
astrocytes promotes radial glial progenitor identity in adult
cerebral cortex. Genes Dev 21:32583271.
Glaum SR, Holzwarth JA, Miller RJ (1990) Glutamate receptors
activate Ca2+ mobilization and Ca2+ influx into astrocytes.
Proc Natl Acad Sci USA 87:34543458.
Goldman SA, Nottebohm F (1983) Neuronal production, migra-
tion, and differentiation in a vocal control nucleus of
the adult female canary brain. Proc Natl Acad Sci USA
80:23902394.
Gomes FCA, Garcia-Abreu J, Galou M, Paulin D, Moura Neto
V (1999a) Neurons induce GFAP gene promoter of cultured
astrocytes from transgenic mice. Glia 26:97108.
Gomes FCA, Maia CG, Menezes JRL, Moura Neto V (1999b)
Cerebellar astrocytes treated by thyroid hormone modulate
neuronal proliferation. Glia 25:247255.
11 Neuron-Astroglial Interactions in Cell Fate Commitment
Goritz C, Mauch D, Pfrieger FW (2005) Multiple mechanisms
mediate cholesterol-induced synaptogenesis in a CNS neu-
ron. Mol Cell Neurosci 29:190201.
Grosche J, Kettenmann H, Reichenbach A (2002) Bergmann
glial cells form distinct morphological structures to interact
with cerebellar neurons. J Neurosci Res 2:138149.
Guizzetti M, Moore N, Giordano G, Costa LG (2008)
Modulation of neuritogenesis by astrocyte muscarinic recep-
tors. J Biol Chem 283:3188431897.
Gtz M, Barde YA (2005) Radial glial cells defined and major
intermediates between embryonic stem cells and CNS neu-
rons. Neuron 46:369372.
Gtz M, Hartfuss E, Malatesta R (2002) Radial glia as neuronal
precursors: a new perspective on the correlation of morphol-
ogy and lineage restriction in the developing cerebral cortex
of mice. Dev Brain Res Bull 57:777788.
Gtz M, Stoykova A, Gruss P (1998) Pax6 controls radial glia
differentiation in the cerebral cortex. Neuron 21:10311044.
Hama H, Hara C, Yamaguchi K, Miyawaki A (2004) PKC sig-
naling mediates global enhancement of excitatory synapto-
genesis in neurons triggered by local contact with astrocytes.
Neuron 41:405415.
Hatten ME (1985) Neuronal regulation of astroglial morphology
and proliferation in vitro. J Cell Biol 2:384396.
Hatten ME (1999) Central nervous system neuronal migration.
Annu Rev Neurosci 22:511539.
Hatten ME (2002) New directions in neuronal migration.
Science 297:16601663.
Hughes EG, Maguire JL, McMinn MT, Scholz RE, Sutherland
ML (2004) Loss of glial fibrillary acidic protein results
in decreased glutamate transport and inhibition of PKA-
induced EAAT2 cell surface trafficking. Mol Brain Res
124:114123.
Hunter KE, Hatten ME (1995) Radial glial cell transformation
to astrocytes is bidirectional: regulation by a diffusible fac-
tor in embryonic forebrain. Proc Natl Acad Sci USA 92:
20612065.
Ihrie RA, Alvarez-Buylla A (2007) Cells in the astroglial lineage
are neural stem cells. Cell Tissue Res 331:179191.
Imura T, Nakano I, Kornblum HI, Sofroniew MV (2006)
Phenotypic and functional heterogeneity of GFAP-
expressing cells in vitro: differential expression of
LeX/CD15 by GFAP-expressing multipotent neural stem
cells and non-neurogenic astrocytes. Glia 53:277293.
Inagaki M, Nakamura Y, Takeda M, Nishimura T, Inagaki N
(1994) Glial fibrillary acidic protein: dynamic property and
regulation by phosphorylation. Brain Pathol 4:239243.
Johansson CB, Svensson M, Wallstedt L, Janson AM, Frisen J
(1999) Neural stem cells in the adult human brain. Exp Cell
Res 253:733736.
Johnson MA, Weick JP, Pearce RA, Zhang SC (2007) Functional
neural development from human embryonic stem cells:
accelerated synaptic activity via astrocyte coculture. J
Neurosci 12:30693077.
Kandel ER, Schwartz JH, Jessell TM (2000) Principles of
Neural Science, 4th ed. McGraw-Hill, New York.
Kanemaru K, Okubo Y, Hirose K, Iino M (2007) Regulation
of neurite growth by spontaneous Ca2+ oscillations in astro-
cytes. J Neurosci 27:89578966.
Kang J, Jiang L, Goldman SA, Nedergaard M (1998) Astrocyte-
mediated potentiation of inhibitory synaptic transmission.
Nature Neurosci 1:683692.
167
Kettenmann H, Ransom BR (2005) Neuroglia, 2nd ed. Oxford
University Press, USA.
Koizumi S, Fujishita K, Tsuda M, Shigemoto-Mogami Y, Inoue
K (2003) Dynamic inhibition of excitatory synaptic transmis-
sion by astrocyte-derived ATP in hippocampal cultures. Proc
Natl Acad Sci USA 19:1102311028.
Kommers T, Rodnight R, Boeck C, Vendite D, Oliveira D, Horn
J, Oppelt D, Wofchuk S (2002) Phosphorylation of glial fib-
rillary acidic protein is stimulated by glutamate via NMDA
receptors in cortical microslices and in mixed neuronal/glial
cell cultures prepared from the cerebellum. Dev Brain Res
137:139148.
Kommers T, Rodnight R, Oppelt D, Oliveira D, Wofchuk S
(1999) The mGluR stimulating GFAP phosphorylation in
immature hippocampal slices has some properties of a group
II receptor. Neuroreport 10:21192123.
Krieglstein K, Henheik P, Farkas L, Jaszai J, Galter D, Krohn
K, Unsicker K (1998) Glial cell line-derived neurotrophic
factor requires transforming growth factor- for exerting its
full neurotrophic potential on peripheral and CNS neurons. J
Neurosci 18:98229834.
Kriegstein AR, Gtz M (2003) Radial glia diversity: a matter of
cell fate. Glia 43:3743.
Krnyei Z, Gcza E, Rhl R, Orsolits B, Vrs E, Szab B,
Vgovits B, Madarsz E (2007) Astroglia-derived retinoic
acid is a key factor in glia-induced neurogenesis. FASEB J
21:114.
Krnyei Z, Szlvik V, Szab B, Gcza E, Czirk A, Madarsz
E (2005) Humoral and contact interactions in astroglia/stem
cell co-cultures in the course of glia-induced neurogenesis.
Glia 49:430444.
Laping NJ, Morgan TE, Nichols NR, Rozovsky I, Young-Chan
CS, Zarow C, Finch CE (1994) Transforming growth factor-
beta 1 induces neuronal and astrocyte genes: tubulin alpha
1, glial fibrillary acidic protein and clusterin. Neuroscience
58:563572.
Lawrence JM, Singhal S, Bhatia B, Keegan DJ, Reh TA,
Luthert PJ, Khaw PT, Limb GA (2007) MIO-M1 cells and
similar muller glial cell lines derived from adult human
retina exhibit neural stem cell characteristics. Stem Cells
25:20332043.
Lie DC, Colamarino SA, Song H-J, Dsir L, Mira H, Consiglio
A, Lein ES, Jessberger S, Lansford H, Dearie AR, Gage FH
(2005) Wnt signalling regulates adult hippocampal neuroge-
nesis. Nature 437:13701375.
Liesi P, Dahl D, Vaheri A (1983) Laminin is produced by early
rat astrocytes in primary culture. J Cell Biol 96:920924.
Lim DA, Alvarez-Buylla A (1999) Interaction between astro-
cytes and adult subventricular zone precursors stimulates
neurogenesis. Proc Natl Acad Sci USA 96:75267531.
Lim DA, Tramontin AD, Trevejo JM, Herrera DG, Garcia-
Verdugo JM, Alvarez-Buylla A (2000) Noggin antagonizes
BMP signaling to create a niche for adult neurogenesis.
Neuron 28:713726.
Lobo NA, Shimono Y, Qian D, Clarke MF (2007) The biology
of cancer stem cells. Annu Rev Cell Dev Biol 23:675699.
Malatesta P, Appolloni I, Calzolari F (2008) Radial glia and
neural stem cells. Cell Tissue Res 331:165178.
Malatesta P, Hack MA, Hartfuss E, Kettenmann H, Klinkert
W, Kirchhoff F, Gtz M (2003) Neuronal or glial
progeny: regional differences in radial glia fate. Neuron
37:751764.
168
Malatesta P, Hartfuss E, Gtz M (2000) Isolation of radial glial
cells by fluorescent-activated cell sorting reveals a neuronal
lineage. Development 127:52535263.
Martinez R, Gomes FC (2002) Neuritogenesis induced by
thyroid hormone-treated astrocytes is mediated by epi-
dermal growth factor/mitogen-activated protein kinase-
phosphatidylinositol 3-kinase pathways and involves mod-
ulation of extracellular matrix proteins. J Biol Chem
51:4931149318.
Martinez R, Gomes FC (2005) Proliferation of cerebellar neu-
rons induced by astrocytes treated with thyroid hormone is
mediated by a cooperation between cell contact and solu-
ble factors and involves the epidermal growth factor-protein
kinase a pathway. J Neurosci Res 3:341349.
Mauch DH, Nagler K, Schumacher S, Goritz C, Muller EC, Otto
A, Pfrieger FW (2001) CNS synaptogenesis promoted by glia
derived cholesterol. Science 294:13541357.
Mecha M, Rabadn MA, Pea-Melin A, Valencia M, Mondjar
T, Blanco MJ (2008) Expression of TGF-betas in the embry-
onic nervous system: analysis of interbalance between iso-
forms. Dev Dyn 237:17091717.
Mendes FA, Onofre GR, Silva LCF, Cavalcante LA, Garcia-
Abreu J (2003) Concentration-dependent actions of glial
chondroitin sulfate on the neuritic growth of midbrain neu-
rons. Dev Brain Res 142:111119.
Merkle FT, Tramontin AD, Garcia-Verdugo JM, Alvarez-Buylla
A (2004) Radial glia give rise to adult neural stem cells
in the subventricular zone. Proc Natl Acad Sci USA 101:
1752817535.
Miller MW (2003) Expression of transforming growth factor-
beta in developing rat cerebral cortex: effects of prenatal
exposure to ethanol. J Comp Neurol 460:410424.
Miller FD, Gauthier AS (2007) Timing is everything: making
neurons versus glia in the developing cortex. Neuron 54:
357369.
Mong JA, Nuez JL, McCarthy MM (2002) GABA mediates
steroid-induced astrocyte differentiation in the neonatal rat
hypothalamus. J Neuroendocrinol 14:4555.
Morrow T, Song MR, Ghosh A (2001) Sequential specification
of neurons and glia by developmentally regulated extracellu-
lar factors. Development 128:35853594.
Morshead CM, Reynolds BA, Craig CG, McBurney MW,
Staines WA, Morassutti D, Weiss S, van der Kooy D (1994)
Neural stem cells in the adult mammalian forebrain: a
relatively quiescent subpopulation of subependymal cells.
Neuron 13:10711082.
Murai KK, Nguyen LN, Irie F, Yamaguchi Y, Pasquale EB
(2003) Control of hippocampal dendritic spine morphol-
ogy through ephrin-A3/EphA4 signaling. Nat Neurosci 6:
153160.
Nakashima K, Nakashima K, Wiese S, Yanagisawa M, Arakawa
H, Kimura N, Hisatsune T, Yoshida K, Kishimoto T, Sendtner
M, Taga T (1999a) Developmental requirement of gp130 sig-
naling in neuronal survival and astrocyte differentiation. J
Neurosci 19:54295434.
Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune
T, Kawabata M, Miyazono K, Taga T (1999b) Synergistic
signaling in fetal brain by STAT3-Smad1 complex bridged
by p300. Science 284:479482.
Nedergaard M, Ransom B, Goldman SA (2003) New roles for
astrocytes: redefining the functional architecture of the brain.
Trends Neurosci 26:523530.
J. Stipursky et al.
Nelson PG, Fields RD, Liu Y (1995) Neural activity, neuron-
glia relationships, and synapse development. Perspect Dev
Neurobiol 2:399407.
Nicholson JL, Altman J (1972) The effects of early hypo- and
hyperthyroidism on the development of rat cerebellar cor-
tex. I. Cell proliferation and differentiation. Brain Res 44:
1323.
Nishida H, Okabe S (2007) Direct astrocytic contacts regulate
local maturation of dendritic spines. J Neurosci 2:331340.
Nishino J, Yamashita K, Hashiguchi H, Fujii H, Shimazaki T,
Hamada H (2004) Meteorin: a secreted protein that regu-
lates glial cell differentiation and promotes axonal extension.
EMBO J 23:19982008.
Nishiyama H, Knopfel T, Endo S, Itohara S (2002) Glial pro-
tein S100 modulates long-term neuronal synaptic plasticity.
Proc Natl Acad Sci USA 99:40374042.
Noctor SC, Flint AC, Weissman TA, Dammerman RS,
Kriegstein AR (2001) Neurons derived from radial glial cells
establish radial units in neocortex. Nature 409:714720.
Ngler K, Mauch DH, Pfrieger FW (2001) Glia-derived sig-
nals induce synapse formation in neurones of the rat central
nervous system. J Physiol 533:665679.
Okada M, Matsumura M, Ogino N, Honda Y (1990) Mller
cells in detached human retina express glial fibrillary acidic
protein and vimentin. Graefes Arch Clin Exp Ophthalmol
228:467474.
Oliet SH, Piet R, Poulain DA (2001) Control of glutamate clear-
ance and synaptic efficacy by glial coverage of neurons.
Science 5518:923926.
Parpura V, Haydon PG (2000) Physiological astrocytic cal-
cium levels stimulate glutamate release to modulate adjacent
neurons. Proc Natl Acad Sci USA 15:86298634.
Parri HR, Gould TM, Crunelli V (2001) Spontaneous astrocytic
Ca2+ oscillations in situ drive NMDAR mediated neuronal
excitation. Nature Neurosci 4:803812.
Patten BA, Peyrin JM, Weinmaster G, Corfas G (2003)
Sequential signaling through Notch1 and erbB recep-
tors mediates radial glia differentiation. J Neurosci
23:61326140.
Perego C, Vanoni C, Bossi M, Massari S, Basudev H, Longhi R,
Pietrini G (2000) The GLT-1 and GLAST glutamate trans-
porters are expressed on morphologically distinct astrocytes
and regulated by neuronal activity in primary hippocampal
cocultures. J Neurochem 3:10761084.
Pfrieger FW (2003) Role of cholesterol in synapse formation and
function. Biochim Biophys Acta 2:271280.
Pfrieger FW, Barres BA (1997) Synaptic efficacy enhanced by
glial cells in vitro. Science 277:16841687.
Pin JP, Duvoisin R (1995) The metabotropic glutamate recep-
tors: structure and functions. Neuropharmacology 34:126.
Pinto L, Gtz M (2007) Radial glial cell heterogeneity the
source of diverse progeny in the CNS. Prog Neurobiol
83:223.
Pixley SKR, De Vellis J (1984) Transition between immature
radial glia and mature astrocytes studied with a monoclonal
antibody to vimentin. Dev Brain Res 15:201209.
Porter JT, McCarthy KD (1997) Astrocytic neurotrans-
mitter receptors in situ and in vivo. Prog Neurobiol
51:439455.
Pratt BM, McPherson JM (1997) TGF-beta in the central ner-
vous system: potential roles in ischemic injury and neurode-
generative diseases. Cytokine Growth Factor Rev 8:267292.
11 Neuron-Astroglial Interactions in Cell Fate Commitment
Prochiantz A (1995) Neuronal polarity: giving neurons heads
and tails. Neuron 15:743746.
Qian X, Shen Q, Goderie SK, He W, Capela A, Davis
AA, Temple S (2000) Timing of CNS cell generation:
a programmed sequence of neuron and glial cell pro-
duction from isolated murine cortical stem cells. Neuron
28:6980.
Rakic P (1971) Guidance of neurons migrating to the fetal
monkey neocortex. Brain Res 33:471476.
Rakic P (2003) Developmental and evolutionary adaptations of
cortical radial glia. Cereb Cortex 13:541549.
Raponi E, Agenes F, Delphin C, Assard N, Baudier J,
Legraverend C, Deloulme JC (2007) S100 expression
defines a state in which GFAP-expressing cells lose their
neural stem cell potential and acquire a more mature devel-
opmental stage. Glia 55:165177.
Raymond PA, Hitchcock PF (1997) Retinal regeneration: com-
mon principles but a diversity of mechanisms. Adv Neurol
72:171184.
Regan MR, Huang YH, Kim YS, Dykes-Hoberg MI, Jin L,
Watkins AM, Bergles DE, Rothstein JD (2007) Variations in
promoter activity reveal a differential expression and physi-
ology of glutamate transporters by glia in the developing and
mature CNS. J Neurosci 25:66076619.
Robitaille R (1998) Modulation of synaptic efficacy and synaptic
depression by glial cells at the frog neuromuscular junction.
Neuron 4:847855.
Romo LF, de Sousa OV, Neto VM, Gomes FC (2008)
Glutamate activates GFAP gene promoter from cultured
astrocytes through TGF-beta1 pathways. J Neurochem
2:746756.
Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L,
Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP,
Welty DF (1996) Knockout of glutamate transporters reveals
a major role for astroglial transport in excitotoxicity and
clearance of glutamate. Neuron 16:675686.
Runquist M, Alonso G (2003) Gabaergic signaling mediates
the morphological organization of astrocytes in the adult rat
forebrain. Glia 41:137151.
Sardi SP, Murtie J, Koirala S, Patten BA, Corfas G (2006)
Presenilin-dependent ErbB4 nuclear signaling regulates the
timing of astrogenesis in the developing brain. Cell 127:
185197.
Schmechel DE, Rakic P (1979) A Golgi study of radial glial
cells in developing monkey telencephalon: morphogene-
sis and transformation into astrocytes. Anat Embryol 156:
115152.
Schmid RS, Mcgrath B, Berechid BE, Boyles B, Marchionni M,
Sestan N, Anton ES (2003) Neuregulin 1-erbB2 signaling is
required for the establishment of radial glia and their trans-
formation into astrocytes in cerebral cortex. Proc Natl Acad
Sci USA 7:42514256.
Schools GP, Kimelberg HK (1999) mGluR3 and mGluR5 are
the predominant metabotropic glutamate receptor mRNAs
expressed in hippocampal astrocytes acutely isolated from
young rats. J Neurosci Res 58:533543.
Seifert G, Schilling K, Steinhauser C (2006) Astrocyte dysfunc-
tion in neurological disorders: a molecular perspective. Nat
Rev Neurosci 7:194206.
Seri B, Garca-Verdugo JM, Collado-Morente L, McEwen BS,
Alvarez-Buylla A (2004) Cell types, lineage, and architecture
169
of the germinal zone in the adult dentate gyrus. J Comp
Neurol 478:359378.
Seri B, Garca-Verdugo JM, McEwen BS, Alvarez-
Buylla A (2001) Astrocytes give rise to new neurons
in the adult mammalian hippocampus. J Neurosci
21:71537160.
Sharif A, Legendre P, Prvot V, Allet C, Romao L, Studler J-
M, Chneiweiss H, Junier M-P (2006) Transforming growth
factor promotes sequential conversion of mature astro-
cytes into neural progenitors and stem cells. Oncogene 26:
26952706.
Sheldon AL, Robinson MB (2007) The role of gluta-
mate transporters in neurodegenerative diseases and poten-
tial opportunities for intervention. Neurochem Int 51:
333355.
Shelton MK, McCarthy KD (2000) Hippocampal astrocytes
exhibit Ca2+ -elevating muscarinic cholinergic and histamin-
ergic receptors in situ. J Neurochem 74:555563.
Shi Y, Massagu J (2003) Mechanisms of TGF-beta signaling
from cell membrane to the nucleus. Cell 11:685700.
Somjen GG (1988) Nervenkitt: notes on the history of the
concept of neuroglia. Glia 1:29.
Song HJ, Stevens CF, Gage FH (2002) Astroglia induce neuro-
genesis from adult neural stem cells. Nature 417:3944.
Soriano E, Alvarado-Mallart RM, Dumesnil N, Del Ro J,
Sotelo C (1997) Cajal-Retzius cells regulate the radial glia
phenotype in the adult and developing cerebellum and alter
granule cell migration. Neuron 18:563577.
Sottile V, Li M, Scotting PJ (2006) Stem cell marker expression
in the Bergmann glia population of the adult mouse brain.
Brain Res 1099:817.
Spassky N, Merkle FT, Flames N, Tramontin AD, Garca-
Verdugo JM, Alvarez-Buylla A (2005) Adult ependymal
cells are postmitotic and are derived from radial glial cells
during embryogenesis. J Neurosci 25:1018.
de Sampaio e Spohr TCL, Choi JW, Gardell SE, Herr DR,
Rehen SK, Gomes FC, Chun J (2008) Lysophosphatidic acid
receptor-dependent secondary effects via astrocytes promote
neuronal differentiation. J Biol Chem 283:74707479.
Steiner B, Klempin F, Wang L, Kott M, Kettenmann H,
Kempermann G (2006) Type-2 cells as link between glial
and neuronal lineagein adult hippocampal neurogenesis. Glia
54:805814.
Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson
KS, Nouri N, Micheva KD, Mehalow AK, Huberman
AD, Stafford B, Sher A, Litke AM, Lambris JD, Smith
SJ, John SW, Barres BA (2007) The classical comple-
ment cascade mediates CNS synapse elimination. Cell
6:11641178.
Stipursky J, Gomes FC (2007) TGF-beta1/SMAD signaling
induces astrocyte fate commitment in vitro: implications for
radial glia development. Glia 55:10231033.
Sullivan SM, Lee A, Bjorkman ST, Miller SM, Sullivan RK,
Poronnik P, Colditz PB, Pow DV (2007) Cytoskeletal
anchoring of GLAST determines susceptibility to brain
damage: an identified role for GFAP. J Biol Chem
282:2941429423.
Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua
X, Fan G, Greenberg ME (2001) Neurogenin promotes neu-
rogenesis and inhibits glial differentiation by independent
mechanisms. Cell 104:365376.
170
Supr H, Del Ro JA, Martnez A, Prez-Sust P, Soriano E Wachs FP, Winner B, Couillard-Despres S, Schiller T, Aigner
(2000) Disruption of neuronal migration and radial glia in
the developing cerebral cortex following ablation of Cajal-
Retzius cells. Cereb Cortex 10:602613.
Swanson RA, Liu J, Miller JW, Rothstein JD, Farrell K, Stein
BA, Longuemare MC (1997) Neuronal regulation of gluta-
mate transporter subtype expression in astrocytes. J Neurosci
3:932940.
Takahashi T, Misson JP, Caviness VS Jr (1990) Glial process
elongation and branching in the developing murine neo-
cortex: a qualitative and quantitative immunohistochemical
analysis. J Comp Neurol 302:1528.
Tateishi N, Shimoda T, Manako J, Katsumata S, Shinagawa
R, Ohno H (2006) Relevance of astrocytic activation
to reductions of astrocytic GABAA receptors. Brain Res
1:7991.
Temple S (2001) The development of neural stem cells. Nature
414:112117.
Tesseur I, Wyss-Coray T (2006) A role for TGF-beta signaling
in neurodegeneration: evidence from genetically engineered
models. Curr Alzheimer Res 3:505513.
Ueki T, Tanaka M, Yamashita K, Mikawa S, Qiu Z, Maragakis
NJ, Hevner RF, Miura N, Sugimura H, Sato K (2003)
A novel secretory factor, Neurogenesin-1, provides neuro-
genic environmental cues for neural stem cells in the adult
hippocampus. J Neurosci 23:1173211740.
Ullian EM, Christopherson KS, Barres BA (2004) Role for glia
in synaptogenesis. Glia 47:209216.
Ullian EM, Sapperstein SK, Christopherson KS, Barres BA
(2001) Control of synapse number by glia. Science 291:
657661.
Vardimon L, Ben-Dror I, Havazelet N, Fox LE (1993) Molecular
control of glutamine synthetase expression in the developing
retina tissue. Dev Dyn 196:276282.
Ventura R, Harris KM (1999) Three-dimensional relationships
between hippocampal synapses and astrocytes. J Neurosci
16:68976906.
Verkhratsky A, Kirchhoff F (2007) NMDA receptors in glia.
Neuroscientist 1:2837.
Vivien D, Ali C (2006) Transforming growth factor-beta sig-
nalling in brain disorders. Cytokine Growth Factor Rev
17:121128.
Voigt T (1989) Development of glial cells in the cere-
bral wall of ferrets: direct tracing of their transforma-
tion from radial glia into astrocytes. J Comp Neurol
289:7488.
Volterra A, Meldolesi J (2005) Astrocytes, from brain glue to
communication elements: the revolution continues. Nat Rev
Neurosci 6:626640.
J. Stipursky et al.
R, Winkler J, Bogdahn U, Aigner L (2006) Transforming
growth factor-beta1 is a negative modulator of adult
neurogenesis. J Neuropathol Exp Neurol 65:358370.
Wang Z, Haydon PG, Yeung ES (2000) Direct observation of
calcium-independent intercellular ATP signaling in astro-
cytes. Analyt Chem 72:20012007.
Ware CB, Horowitz MC, Renshaw BR, Hunt JS, Liggitt D,
Koblar SA, Gliniak BC, McKenna HJ, Papayannopoulou
T, Thoma B (1995) Targeted disruption of the low-affinity
leukemia inhibitory factor receptor gene causes placental,
skeletal, neural and metabolic defects and results in perinatal
death. Development 121:12831299.
Wyss-Coray T, Feng L, Masliah E, Ruppe MD, Lee HS, Toggas
SM, Rockenstein EM, Mucke L (1995) Increased central ner-
vous system production of extracellular matrix components
and development of hydrocephalus in transgenic mice over-
expressing transforming growth factor-beta1. Am J Pathol
147:5367.
Yang Y, Ge W, Chen Y, Zhang Z, Shen W, Wu C, Poo M, Duan S
(2003) Contribution of astrocytes to hippocampal long-term
potentiation through release of D-serine. Proc Natl Acad Sci
USA 100:1519415199.
Yoon K, Gaiano N (2005) Notch signaling in the mammalian
central nervous system: insights from mouse mutants. Nat
Neurosci 8:709715.
Yoon K, Nery S, Rutlin ML, Radtke F, Fishell G, Gaiano N
(2004) Fibroblast growth factor receptor signaling promotes
radial glial identity and interacts with Notch1 signaling in
telencephalic progenitors. J Neurosci 24:94979506.
Zheng W, Nowakowski RS, Vaccarino FM (2004) Fibroblast
growth factor 2 is required for maintaining the neural stem
cell pool in the mouse brain subventricular zone. Dev
Neurosci 26:181196.
Zhou M, Kimelberg HK (2001) Freshly isolated hippocampal
CA1 astrocytes comprise two populations differing in gluta-
mate transporter and AMPA receptor expression. J Neurosci
21:79017908.
Zhou CJ, Zhao C, Pleasure SJ (2004) Wnt signaling mutants
have decreased dentate granule cell production and radial
glial scaffolding abnormalities. J Neurosci 24:121126.
Zhu Y, Yang GY, Ahlemeyer B, Pang L, Che XM, Culmsee C,
Klumpp S, Krieglstein J (2002) Transforming growth factor-
beta 1 increases bad phosphorylation and protects neurons
against damage. J Neurosci 22:38983909.
Ziegler DR, Innocente CE, Leal RB, Rodnight R, Gonalves CA
(1998) The S100 protein inhibits phosphorylation of GFAP
and vimentin in a cytoskeletal fraction from immature rat
hippocampus. Neurochem Res 23:12591263.
Chapter 12
The Origin of Microglia and the Development of the Brain
Flavia R. S. Lima, Anna Carolina C. da Fonseca, Giselle P. Faria, Luiz Gustavo F. Dubois,
Trcia R. Alves, Jane Faria, and Vivaldo Moura Neto
Contents
12.1 Microglia: Origin and Development . . . . . 172
12.1.1 Origin of Microglia . . . . . . . . . . 173
12.1.2 Invasion of the CNS by Microglial
Precursors During Development . . . . . 174
12.1.3 Expansion of Microglial Population within
CNS . . . . . . . . . . . . . . . . . 175
12.1.4 Microglial Development and Thyroid
Hormones . . . . . . . . . . . . . . 177
12.1.5 Adult CNS: Ramified Microglia . . . . . 178
12.2 Microglia and Regressive Processes During Brain
Development: Phagocytosis and Neurotoxic
Factors . . . . . . . . . . . . . . . . . . . 179
12.3 Microglial Secreted Neurotrophic Factors: Role
in Neural Development . . . . . . . . . . . . 181
12.3.1 Microglia and Neural Progenitor Cells . . 182
12.4 The Future . . . . . . . . . . . . . . . . . 183
References . . . . . . . . . . . . . . . . . . . . 184
Abstract We discuss the origin and development
of microglial cells and their influence on neural
development. Unlike astrocytes, oligodendrocytes, and
neurons, which are derived from neuroectoderm,
microglial cells originate from mesoderm. Microglial
hematopoietic precursors enter the developing CNS
from the bloodstream, ventricles, and meninges. In
the brain, these cells migrate and proliferate, as
ameboid microglia, and become distributed through-
out the nervous parenchyma. Ameboid microglial
cells differentiate into ramified microglia when they
reach their definitive location. The factors that con-
trol the invasion of the nervous parenchyma, migration,
F.R.S. Lima ()
Programa de Anatomia, Instituto de Cincias Biomdicas,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
e-mail: flima@anato.ufrj.br
H. Ulrich (ed.), Perspectives of Stem Cells,
DOI 10.1007/978-90-481-3375-8_12, Springer Science+Business Media B.V. 2010
proliferation, and differentiation of microglial cells are
not completely known. These important events may
depend on environmental factors such as soluble or
cell-surface-bound molecules and components of the
extracellular matrix. In the developing CNS, microglial
cells are involved in clearing cell debris and withdraw-
ing transitory or misdirected axons, and presumably
support neurogenesis, cell survival, and neurite growth.
In the adult brain, activated microglia occur mostly
in response to neuronal injuries, when, they destroy
invading microorganisms, remove harmful debris, and
promote tissue repair, as well as partaking in the
immune response by secreting cytokines, facilitat-
ing the return to homeostasis. Microglial activation
is an important event in the defense of the nervous
parenchyma against infectious, neurodegenerative, and
inflammatory diseases. The majority of data in the
literature describes microglial cells in a neuropatho-
logical context. Little is known of the development
of these cells in the nervous parenchyma and their
effective role in neurogenesis.
Keywords Microglia Development Progenitor
Growth factor Neurogenesis
Abbreviations
b-FGF basic fibroblast growth factor
CSF colony stimulating factor
GDNF glial-derived neurotrophic factor
GFAP glial fibrillary acidic protein
GM granulocyte macrophage
gw gestational weeks
HGF hepatocyte growth factor
HPC hippocampal progenitor cell(s)
ICAM-1 intercellular adhesion molecule-1
171
172 F.R.S. Lima et al.

IFN- interferon gamma These latter cell types can also appear in the adult brain
IL interleukin in pathological conditions.
iNOS inducible NO synthase Macrophages correspond to the terminal stages of
LFA-1 lymphocyte function-associated antigen-1 mononuclear phagocyte lineage differentiation. In gen-
LIF leukemia inhibitory factor eral, macrophages do not constitute a homogeneous
LPS lypopolysaccharide population, but rather are heterogeneous and have spe-
MAP2 microtubule-associated protein 2 cific structure and functions depending on the tissue.
MAPK mitogen-activated protein kinase In the CNS, macrophages can be present in different
MHC II class II major histocompatibility complex forms, according to their location, the stage of devel-
MIP macrophage inflammatory protein opment, and tissue integrity (Perry et al., 1994; Streit,
NGF nerve growth factor
2001). Microglial cells are considered a subpopula-
NSC neural stem cell(s)
tion of intraparenchymal cells, which are distinguished
NT neurotrophin
from phagocytes located in the meninges and in the
OPC oligodendrocyte precursor cell(s)
coroid plexus stroma, or from macrophages situated in
PAF platelet-activating factor
PI3K phosphoinositol-3-kinase the cerebral capillary walls (Fig. 12.1).
PKC protein kinase C In the immature CNS, microglial cells show a het-
PS phosphatidylserine erogeneous morphology, either a dilated cell body
RCA-1 Ricinus communed agglutinin-1 tending to several forms for developing pseudopods
ROS reactive-oxygen species and filopods, or a rounded cell body without cytoplas-
TGF 1 transforming growth factor 1 matic expansions. These variations may be associated
TH thyroid hormones with the origin and cerebral location of these cells
TNF tumor necrosis factor (Perry and Gordon, 1988; Streit, 2001). From the
ultrastructural viewpoint, ameboid microglia have a
cytoplasm rich in organelles, a developed Golgi appa-
ratus, with vacuoles and lysosomes in abundance, indi-
cating intense activity of synthesis and phagocytosis
12.1 Microglia: Origin and Development (Murabe and Sano, 1982). The expression of non-
specific esterases, acid phosphatases, and 5nucleoti-
Together with astrocytes and oligodendrocytes, dases activity by ameboid microglia also distinguish
microglial cells comprise the principal glial popu- them from the ramified microglia (Ling, 1981; Hanish
lations in the CNS. Microglial cells were initially and Kettenmann, 2007).
described by Del Ro Hortega in 1932; however, only There are two extreme microglial phenotypes: ram-
in the 1980s did they begin to receive more attention ified microglia, which are at first not very active func-
and study. Using silver-carbonate stains in nervous- tionally; and ameboid microglia, which are involved
tissue preparations, Del Ro Hortega (1932) described in CNS growth and repair. However, the situation is
cells constituted by a small cell body and numerous
ramified cell process. Because of their morphology,
this cell type was denominated microglia, in contrast
to macroglia (astrocytes and oligodendrocytes).
In the cerebral parenchyma, microglial cells are
distinguished from the other cell types by their mor-
phology, and are recognized by the expression of
specific mononuclear phagocyte markers. According
to the maturation stage of the CNS or to nerve-tissue
integrity, these cells can acquire different phenotypes.
In the absence of injury, the ramified microglial cells Fig. 12.1 Purified microglia cultures from neonatal mice
cortex. Cells stained by Isolectin B4 derived from Griffonia sim-
are the typical microglia of the adult nervous tissue. plicifolia conjugated with FITC. Nuclei stained by DAPI. Left
In the immature CNS, brain macrophages, also known panel, bar 50 m. Right panel, bar 10 m. Photomicrographs
as ameboid microglial cells, accumulate transitorily. taken by Anna Carolina Fonseca (UFRJ)
12 The Origin of Microglia and the Development of the Brain
more complex than it seems. It has been suggested that
intermediate microglial phenotypes exist, modifying
morphology and function during brain development
in particular, and also after injury. These intermediate
microglial cell forms appear during differentiation pro-
cesses or activation of the microglia (Perry et al., 1994;
Kreutzberg, 1996; Streit, 2001; Schwartz et al., 2006).
12.1.1 Origin of Microglia
The origin of microglia has been discussed for many
years. However, current information indicates that
they derive from mesenchymal precursor infiltration.
During brain development, precursors generated in
the bone marrow invade the nervous parenchyma and
differentiate into microglial cells (Ling and Wong,
1993; Cuadros and Navascus, 1998; Kaur et al., 2001;
Chan et al., 2007). The first studies that supported
this hypothesis were done in the 1980s. Attempting
to show that microglial cells derive from monocytic
lineages, Ling and cols. (1980) injected monocytes
labeled with carbon into the bloodstream of new-
born rats. Subsequently, they detected the appearance
of microglial cells containing carbon particles in the
CNS, particularly in the corpus callosum, in the first
days of postnatal life. Using antibodies against the
F4/80 antigen, a highly specific marker for mononu-
clear phagocytes in rodents, immunohistochemical
studies revealed positive labeling for circulating mono-
cyte invaders in the nervous tissue in embryonic stages,
ameboid microglia in the prenatal period, and rami-
fied microglia in the adult brain (Hume et al., 1983;
Perry et al., 1985). Later, Giulian et al. (1995) demon-
strated that, in vitro, monocytes isolated from the
newborn brain give rise to cells with microglial fea-
tures. However, these cells do not appear in cultures
from monocytes derived from other sources. In this
context, it was also reported that a cellular subpopula-
tion derived from bone marrow showed the same ionic
channel model as the microglial cells (Banati et al.,
1991; Chan et al., 2007). These pioneering studies sug-
gested that bone marrow produces specific precursors
for microglia, which are different from those that will
originate macrophages for other tissues.
However, microglial cells appear within the CNS
before brain vascularization (Ashwell, 1991; Sorokin
et al., 1992; Cuadros et al., 1993; Wang et al., 1996)
173
and before monocytes are produced in hemopoietic
tissues (Hurley and Streit, 1996; Navascus et al.,
2000). Thus, it is possible that not all microglial cells
originate from circulating monocytes during develop-
ment.
In the adult CNS, discrimination between infiltrat-
ing mononuclear cell and activated resident microglia
is difficult, as well as their corresponding tissue of
origin or precursor cell lineages, mostly due to the
lack of cell surface specific markers to distinguish
these cells (reviewed by Chan et al., 2007). Expression
of the myeloid-specific transcription factor PU.1 by
microglia (Walton et al., 2000) indicates a possible
myeloid origin, but not necessarily from blood mono-
cyte progenitors (Asheuer et al., 2004; Hess et al.,
2004; Vallieres and Sawchenko, 2003; Vitry et al.,
2003).
Monocytes ingress into the brain and seem to be
the direct circulating precursors of postnatal microglia
only under defined conditions, such as in whole-body
irradiated chimeric mice (Mildner et al., 2007) or
during post inflammatory period of bacterial menin-
gitis (Djukic et al., 2006). Despite having a strong
potential for proliferation and self-renewal in the adult
CNS, microglial proliferation normally occurs at very
low levels, increasing significantly after CNS injury
(Graeber et al., 1988; Ajami et al., 2007).
Recently, new concepts about the origin of
microglia during development were proposed in a
review paper (Chan et al., 2007). The authors pro-
posed that the CNS invasion by microglial precursors
occurs in two waves (Fig. 12.2). The first occurs during
fetal development, and cells are derived mainly from
extravascular progenitors (of myeloid/mesenchymal
but not monocytic origin). The second population
invades the brain during perinatal and postnatal devel-
opment. These cells are derived from circulating pro-
genitors (probably monocytes), and give rise to so-
called ameboid microglia (Rezaie and Male, 2002;
Simard and Rivest, 2004; Rezaie et al., 2005; Tsuchiya
et al., 2005; Chan et al., 2007).
In accordance with this idea, Razaie and cols.
(2005) reported that during human brain develop-
ment, the cerebral wall of the human telencephalon
is colonized by at least two putative populations
of cells that originate microglia mainly during the
second trimester of gestation. These subpopulations
can be discriminated based on differences in pheno-
type, morphological characteristics, and distribution
174 F.R.S. Lima et al.

Fig. 12.2 Simplified

scheme of the theory
of mesodermal/myeloid
origin for microglia during
development. Hematopoietic
stem cells give rise to the
lymphoid and myeloid
blood-cell lineages.
A committed myeloid
precursor may give rise to
monocyte precursors or to
granulocyte precursors, and
both may originate microglia.
Adapted from Chan et al.
(2007)

patterns within the developing nervous system. The
first progenitor population is RCA-1(++; Ricinus com-
muned agglutinin-1), CD68(), class II major histo-
compatibility complex [MHCII()], underlies the mes-
enchyme and meninges, and resides in the subplate.
These cells differentiate early within the subplate.
The second progenitor population is CD68(++), RCA-
1(+) and MHCII(), and adopts the typical ameboid
macrophage-like forms. These progenitor cells reside
in the deep marginal layer and at the boundary between
the cortical plate and the subplate at 1417 gestational
weeks (gw), where they also appear to differentiate and
progressively populate the upper part of this region by
24 gw. At this time, microglia can be found all over
the developing human fetal telencephalon, and differ-
entiation into ramified forms characteristic of adult
microglial cells begins also be seen, probably consti-
tuting a merger of the two populations of progenitor
cells (Rezaie et al., 2005).
Microglial progenitors undergo homing to the ner-
vous system, so they could be usefull tools for ther-
apeutic purposes (Dobrenis, 1998; Cucchiarini et al.,
2003; Watanabe et al., 2002; Chan et al., 2007).
Upon genetic manipulation, microglia progenitors
could locally deliver specific gene products for the
treatment of several disorders, such as metachromatic
leukodystrophy, gliomas and stroke (Chan et al., 2007).
Nevertheless, genetic or cell therapy will only be pos-
sible when tissue of origin or cell lineage of resident
microglia can be precisely identified.
12.1.2 Invasion of the CNS by Microglial
Precursors During Development
Considering that microglial precursors originate out-
side the nervous parenchyma, studies have demon-
strated, in rodents, that these cells appear in the
12 The Origin of Microglia and the Development of the Brain
nervous parenchyma especially at the end of embryo-
genesis and in the first days of postnatal life, although
their presence is already observed from the 12th day
of embryonic life (E12; Perry et al., 1985; Ashwell,
1991; Milligan et al., 1991; Navascus et al., 2000;
Chan et al., 2007). In humans, precursors invade the
nervous tissue during the first two trimesters of ges-
tation (Billiards et al., 2006; Monier et al., 2007).
The entry of microglial precursors during this period,
added to the proliferation of these cells within the
CNS, must provide enough precursors to produce the
mature microglial population, because the turnover of
microglia in the adult brain is slow (Lawson et al.,
1992; Navascus et al., 2000).
Three invasion routes have been proposed. The first
route is from the bloodstream. In this route, blood cir-
culating cells may enter the CNS, adhering to vessel
walls and gaining access to the nervous parenchyma
through a mechanism similar to that described for the
invasion of other tissues by macrophages and mono-
cytes (Cramer, 1992). The LFA-1/ICAM-1 (lympho-
cyte function-associated antigen-1/lymphocytes/ inter-
cellular adhesion molecule-1) adhesion system, which
is involved in the entry of leukocytes into the injured
brain parenchyma, must also be involved in this mech-
anism (Akiyama et al., 1994; Cuadros and Navascus,
1998). Because the blood-brain barrier is formed later
in development, many microglial precursors use this
route to invade the CNS and to reach their final location
without needing to migrate alone for long distances.
However, microglial cells also appear in brain regions
that are not vascularized at some stage of devel-
opment, such as in the mammal retina, where they
appear before this region is vascularized (Schnitzer,
1989; Diaz-Araya et al., 1995; Chen et al., 2002).
Therefore other routes should be considered, such as
the entry of microglial precursors from the ventricles.
The idea that microglial precursors may reach the ner-
vous parenchyma from the ventricles is based on the
presence of monocyte and macrophage lineages in the
ventricular lumen (Jordan and Thomas, 1988; Monier
et al., 2007). Moreover, microglial cells apparently
cross the ventricular surface during brain development
(Cuadros et al., 1994; Cuadros and Navascus, 1998).
The third route that has been documented is from
the meninges. This entry route of microglial precur-
sors was first proposed by Del Ro-Hortega (1932).
Studies by later investigators supported this idea, and
from studies using electron microscopic images, they
175
suggested that microglial precursors cross the pial bor-
der to reach the nervous parenchyma (Boya et al.,
1991; Navascus et al., 1996; Dalmau et al., 1997;
Monier et al., 2007). Navascus group reported that
most microglial cells in the optic tectum and cerebel-
lum seem to derive from precursors that have entered
the nervous parenchyma by this route (Cuadros et al.,
1994; 1997; Navascus et al., 2000).
It appears that the microglial precursors can use all
three of these invasion routes. It has been suggested
that these cells enter the nervous tissue via different
pathways, and generate different microglial subpop-
ulations (Provis et al., 1996; Navascus et al., 2000;
Chan et al., 2007).
12.1.3 Expansion of Microglial
Population within CNS
12.1.3.1 Proliferation
The growth of the microglial population does not result
exclusively from infiltration of the mesodermal pre-
cursors. Different studies have shown that microglial
cells proliferate actively during brain development. In
rodents, the number of microglia detected in the cor-
pus callosum grows significantly between the 5th day
and 2nd week of postnatal life. The high proliferation
capacity of these cells is attested by a high rate of
incorporation of tritiated thymidine, which reaches a
peak of almost 80% of labeled cells on the 12th post-
natal day (Imamoto and Leblond, 1978; Mallat et al.,
1997; Dalmau et al., 2003).
In vitro studies have characterized certain Colony
Stimulating Factors (CSFs) as mitogenes for microglia
(Suzumura et al., 1990; Elkabes et al., 1996), such
as colony-stimulating factors-1 (CSF-1), granulo-
cyte macrophage (GM)-CSF and interleukin (IL)-3
(Giulian and Ingeman, 1988; Hao et al., 1990; Lee
et al., 1993; Imai and Kohsaka, 2002). In particu-
lar, CSF-1 specifically stimulates mononuclear phago-
cyte production, and it is expressed in several tissues,
including the brain.
Mitogenic effects of vitamin E, glial-derived neu-
rotrophic factor (GDNF), transforming growth fac-
tor (TGF) 1, and neurotrophin 3 (NT-3) were also
observed in microglial cells in vitro (Elkabes et al.,
1996; Salimi et al., 2003; Flanary and Streit, 2006).
176
In vivo, a decrease in the number of microglial cells in
the subventricular zone was seen in knockout mice for
NT-3 and its trkC receptor (Kahn et al., 1999).
12.1.3.2 Migration
Navascus group has been studying the microglial
migration in some regions of the quail brain, such
as the optic tectum (Cuadros et al., 1994), the cere-
bellum (Cuadros et al., 1997), and, mostly, the retina
(Navascus et al., 1995; Marn-Teva et al., 1999). They
established a model according to which the migration
in these regions occurs in two different steps. During
the first step, termed tangential migration, microglial
precursors move together from their entry point within
the nervous parenchyma, traveling over long distances
on oriented substrates. These substrates (axon fasci-
cles) may act as highways, leading the precursors into
the interior of the nervous parenchyma. In the sec-
ond stage, termed radial migration, microglial cells
change direction and move towards the pial surface,
reaching all parts of the nervous parenchyma (Monier
et al., 2007). Moreover, analyses during the retina
development suggest that microglial cells migrate
along the radial processes of Mller cells (Navascus
et al., 1996). Thus, microglial precursors leave high-
ways and enter secondary roads to reach their final
destination within the nervous parenchyma (Cuadros
and Navascus, 1998, 2001). In vitro morphological
and immunocytochemical analyses indicated the pos-
sibility that microglia may alternate migration and
proliferation cycles, contributing to the expansion of
the microglial population during retina development
(Marn-Teva et al., 1999; Navascus et al., 2000).
The migration model described above refers to
regions of the immature quail brain, which has a lam-
inar organization. Although it is not yet clear whether
this model applies to all regions of the quail brain
or whether similar steps occur in other species, some
studies in immature CNS of mammals have indicated
this possibility (Perry et al., 1985; Milligan et al., 1991;
Monier et al., 2007).
Ameboid microglia as well as astrocytes produce
cytokines that are involved in invasion and migration
of microglial cells, such as TGF, CSF-1, and the
extracellular matrix protein thrombospondin 1 (Wang
et al., 1988; Constam et al., 1992; Mansfield and
Suchard, 1994; Bartholdi and Schwab, 1997; Salimi
F.R.S. Lima et al.
et al., 2003; Rozenfeld et al., 2005). Indeed, analyses
using recombinant CSF-1 on microglial cultures have
shown that this factor, which is secreted essentially by
astrocytes, may induce microglial migration in vitro
(Calvo et al., 1998). Recently, it was demonstrated that
treatment with ATP increased the migration of cultured
rat microglia by about 4-fold, and that prostaglandin E
(PGE)-2 effectively reduced this effect (Nagano et al.,
2008).
12.1.3.3 Differentiation
The macrophage phenotype of microglia disappears
progressively during CNS maturation. In the rat cor-
pus callosum, this alteration is observed from the 2nd
week of postnatal life (Imamoto and Leblond, 1978;
Dalmau et al., 2003). Part of these ameboid microglia
that are present in the embryonic brain may degen-
erate, as occurs with activated microglia in the adult
brain (Gehrmann and Banati, 1995; Jones et al., 1997).
However, many of them develop narrow processes,
becoming progressively ramified. This microglial dif-
ferentiation idea has been suggested by several workers
(Perry and Gordon, 1988; Ling and Wong, 1993; Davis
et al., 1994; Cuadros and Navascues, 2001), and it was
first demonstrated in situ with different markers, such
as tritiated thymidine (Imamoto and Leblond, 1978),
carbon (Ling et al., 1980), and rhodamine isothyocy-
anate (Leong and Ling, 1992). This morphological
transformation is accompanied by loss of the phago-
cytic function (Kaur and Ling, 1991), decreased
expression of some macrophagic markers such as CR3
and CMH I, and the disappearance of antigens such
as ED1 in the rat (Ling et al., 1991). Retinoic acid
can induce loss of phagocytic activity, favoring dif-
ferentiation in vitro (Giulian and Baker, 1986). Many
immunohistochemical features that are present in ame-
boid microglia but are lost in ramified microglia,
later reappear in the activated microglia (Streit and
Kreutzberg, 1988; Davis et al., 1994; Hanisch and
Kettenmann, 2007). The distribution of microtubules
can also change in cultured microglial cells. Ramified
microglia show more stable microtubules, with a
higher rate of acetylation and detyrosination than in
ameboid microglia (Ilschner and Brandt, 1996). It has
been suggested that the microglial cell variability in
different regions of the brain depends on environmen-
tal factors (Lawson et al., 1990; Sievers et al., 1995;
12 The Origin of Microglia and the Development of the Brain
Streit, 2001; Schwartz et al., 2006). Microglial cells
in brain regions that are exposed to plasma proteins
are less ramified than are microglia in regions where
the blood-brain barrier is complete (Perry and Gordon,
1991). In the immature CNS, environmental alterations
as well as specific properties of the CNS at each devel-
opmental stage and in each region might influence
microglial differentiation.
Astrocytes appear to play an important role in deter-
mining the mature microglial phenotype, since the
co-culturing of microglial cells with astrocytes con-
sistently yields high numbers of ramified microglia
(Tanaka and Maeda, 1996; Navascus et al., 2000).
Astrocytes are the main source of CSF-1 in the CNS,
and they can produce this factor in culture constitu-
tively (Thry et al., 1990; Lee et al., 1993). In addition,
ameboid microglia stimulated by LPS can also pro-
duce CSF-1 (Thry et al., 1992; Lee et al., 1993). The
spontaneous growth of microglial cells in astrocyte
monolayers is blocked by the addition of anti-CSF-1
antibodies to the cultures (Thry and Mallat, 1993).
Several studies have demonstrated that CSF-1 prevents
apoptosis of cultured ameboid microglia (Tomozawa
et al., 1996), and stimulates their proliferation (Giulian
and Ingeman, 1988; Hao et al., 1990; Lee et al., 1993;
Imai and Kohsaka, 2002) as well as the growth of cell
processes, transforming these macrophagic cells into
cells with a morphology that is reminiscent of rami-
fied microglia (Sawada et al., 1990). Through analyses
using neutralizing antibodies for TGF, M. Mallats
group demonstrated that neurons secrete TGF, which
potentiates the microglial proliferation promoted by
CSF-1 (Dobbertin et al., 1997).
Op (osteopetrosis) mice have a recessive muta-
tion in the region of the gene that codes for CSF-1.
Consequently, these animals do not produce biologi-
cally active CSF-1 (Raivich et al., 1994; Berezovskaya
et al., 1995). Immunohistochemical studies have
shown that the microglial density decreased 47% in
the corpus callosum, 37% in the parietal cortex, and
34% in the frontal cortex in op mice. Moreover, in
the frontal cortical region, microglial cells are smaller
and have less-developed cell processes, in compari-
son to normal animals (Wegiel et al., 1998; Kondo
et al., 2007). These studies suggest that CSF-1 plays
an important role in the maturation of the microglia in
vivo.
Treatment with fibronectin, an extracellular matrix
protein secreted by different cells and particularly by
177
astrocytes, can also promote microglial transformation
to the ramified phenotype. This process can be
reversed in vitro by another extracellular matrix pro-
tein, laminin, suggesting that these two molecules
must be involved in the regulation of the microglial
differentiation in the CNS (Chamak and Mallat, 1991).
12.1.4 Microglial Development and
Thyroid Hormones
Many questions regarding microglial recruitment,
expansion, and differentiation still remain without
answers. The endocrine regulation of the microglial
development is not well understood. Thyroid hor-
mones (THs) are involved in the regulation of impor-
tant events that occur during the development of the
CNS (Lima et al., 1997; Gomes et al., 2001; Trentin,
2006). The postnatal development of rat microglia is
marked by a large increase in the number of microglial
cells and the growth of their ramified processes. Our
group, in collaboration with Mallats team, demon-
strated that THs play a crucial role in the development
of microglia (Lima et al., 2001; Mallat et al., 2002).
Microglial processes were markedly less abundant in
hypothyroid rat pups than in age-matched normal ani-
mals, from post-natal day 4 up to the end of the third
postnatal week of life. A delay in the extension of pro-
cesses and a decrease in the density of microglial cell
bodies in the developing cortex of normal and hypothy-
roid animals were responsible for these differences.
Conversely, neonatal rat hyperthyroidism accelerated
the extension of microglial processes and increased
the density of cortical microglial cell bodies above
physiological levels, during the first postnatal week
of life (Fig. 12.3). Consistent with the effects of THs
observed in vivo, thyroid hormone (T3) favored the
survival of cultured purified microglial cells and the
growth of their processes (Lima et al., 2001).
Proliferation, migration, and differentiation of
microglia are closely regulated by intracellular
signaling cascades. Protein kinase C (PKC) and
phosphoinositol-3-kinase (PI3K) mediate the prolifer-
ation and differentiation of rat microglia from organ-
otypic cortex brain sections (Zassler et al., 2003).
Using a human microglial cell line (C13NJ), Martin
et al. (2003) have shown in a wound-healing model
and a chemotaxis assay that microglial migration can
178 F.R.S. Lima et al.

Fig. 12.3 Microglial cells in the parietal cortex of hypothy- processes. a, b: Arrows point to microglial cells, which are
roid (a), euthyroid (b), and hyperthyroid (c) rats treated with shown at higher magnification in the insets. Microglial cell bod-
TH at postnatal day 4. Peroxidase staining with isolectin B4 ies and processes appear more numerous from a to c. Bar, 100
reveals blood vessels ( arrowheads ) and branched microglial m. Figure from Lima et al. (2001)

be induced by the neuropeptide neurotensin via a
mechanism that is dependent on both the PI3K and
mitogen-activated protein kinase (MAPK) pathways.
12.1.5 Adult CNS: Ramified Microglia
In the absence of injury, the ramified phenotype of
microglia is typical in the adult vertebrate CNS. In
the mouse, microglial cells comprise 512% of the
total of CNS cells. Although they are present in all
mature brain regions, their density varies among areas
of the nervous parenchyma. Ramified microglial cells
are more numerous in the gray matter than in the
white matter. The hippocampus, olfactory bulb, and
basal ganglia are regions with higher microglial den-
sity, whereas the cortex, thalamus, and hypothalamus
have moderate microglial density, and the cerebellum
and brainstem have lower microglial density (Perry
and Gordon, 1988; Lawson et al., 1990). In the retina
and certain cortical regions, studies have revealed that
microglial cells are arranged to form a cellular layer.
In this layer, each cell covers with its ramifications a
certain territory, which other microglial cells do not
appear to invade (Hume et al., 1983; Schnitzer, 1989).
In the adult brain, the role of ramified microglia,
also termed resting microglia, is still unclear. Although
these cells have certain macrophagic markers, such
as the Fc fragment receptors of immunoglobulins or
the CR3 receptors, they do not show properties of

Fig. 12.4 Schematic

representation of
(1) molecules involved with
the regulation of the
microglial development,
(2) phagocytosis and
microglial neurotoxic
factors related with
regressive processes during
brain development,
(3) neurotrophic factors
secreted by microglia that
can be participating
of the neural development
and, (4) microglia as
possible multipotential stem
cells. The majority of studies
related with microglia
and development was
performed in vitro and
deserves further investigation
12 The Origin of Microglia and the Development of the Brain
phagocytosis, and moreover appear to be in a qui-
escent state. However, ramified microglial cells are
highly sensitive to environmental conditions and can
transform rapidly into activated cells, in the case of dis-
order in the homeostasis of nervous tissue. Therefore,
they may have an important role in microglial survival,
constituting an endogenous macrophage reservoir of
the CNS (Perry and Gordon, 1988). For this reason,
Hanisch and Kettenmann (2007) suggested that rest-
ing microglia should be renamed surveying microglia
in the mature brain.
Microglial cells develop and react according
to changes in their environment (Fig. 12.4). The
CNS constitutes a particular protected environment,
where macrophage recruitment tends to be maximally
limited, in comparison to those observed in other tis-
sues, where inflammatory reactions are much more
marked (Perry et al., 1993). A better understanding of
the interactions between microglia and their microen-
vironment in the CNS will benefit the comprehension
of the fundamental mechanisms for macrophagic regu-
lation in vivo, as well as neuropathologies that are still
incurable (Perry et al., 1993; Streit, 2001; Hanisch and
Kettenmann, 2007).
12.2 Microglia and Regressive Processes
During Brain Development:
Phagocytosis and Neurotoxic
Factors
As described here, microglial cells exhibit an ame-
boid phenotype that is typical of macrophages in the
developing brain (Vilhardt, 2005). These cells have a
wide spectrum of important biological responses dur-
ing brain development: migration, proliferation, nitric-
oxide (NO) production and respiratory burst, phagocy-
tosis, antigen presentation, and secretion of diffusible
factors (Garden and Moller, 2006). Some of these are
discussed here in more detail.
Neuronal or axonal degeneration induces microglial
proliferation. Cytokines such as IL-1, IL-4, inter-
feron gamma (IFN-), CSF-1 (Kim and de Vellis,
2005) and GM-CSF (Suh et al., 2005), in addi-
tion to neurotrophic factors such as brain-derived
neurotrophic factor (BDNF) and NT-3 (Elkabes
et al., 1996), strongly promote this proliferation. In
179
addition to proliferation, when they are confronted
with this kind of insult, microglial cells may also pro-
duce and release toxic reactive-oxygen species (ROS)
into the neural environment, leading to oxidative injury
(Garden and Moller, 2006). Cultured rodent microglial
cells express inducible NO synthase (iNOS) (Kim and
de Vellis, 2005) and NADPH oxidase (Sankarapandi
et al., 1998), which are enzymes that generate NO and
produce superoxide anions and hydrogen peroxide,
respectively (Garden and Moller, 2006). For exam-
ple, it has been demonstrated that NO induces the
death of rat cerebral cortical neurons through apopto-
sis, with the involvement of activated caspase-3 (Wang
et al., 2003). Another example is that microglial cells
recognize mouse Purkinje cells expressing activated
caspase-3, engulf these cells, and produce high levels
of superoxide ions, promoting neuronal death during
brain development (Marin-Teva et al., 2004).
During the development of the CNS, microglial
cells participate in the formation of the complex net-
work of connections that are present in the adult brain.
In fact, these cells are capable of phagocytosing tran-
sient or aberrant axons, apoptotic cells, and cellular
debris (Ashwell, 1990; Ferrer et al., 1990; Innocenti
et al., 1983). For example, it has been demonstrated
in a rodent model that neurohypophysial microglial
cells engulf axonal terminations without commitment
of neuronal viability, suggesting that microglial cells
play a role in the remodeling of terminal arborizations
of neurosecretory neurons and in processing or degrad-
ing hormones and peptides that they contain (Pow
et al., 1989). Moreover, microglial cells engulf apop-
totic retinal cells during development. Through con-
focal microscopy, Egensperger et al. (1996) observed
the presence of cells in the retina whose nuclei were
TUNEL- (marker for apoptotic nucleus) negative, but
the cytoplasm was TUNEL-positive. This observation
strongly suggests that these cells are microglial cells,
because their cytoplasmic labeling probably results
from the ingestion of the fragmenting DNA of a dying
retinal cell. The importance of the role of microglia
in phagocytosing apoptotic cells is that this process
eliminates dead cells safely by preventing leakage
of potentially cytotoxic or antigenic substances from
dying cells and by suppressing the proinflammatory
effects of engulfing phagocytes (Savill et al., 2002),
leaving the CNS undamaged.
Among the signals for recognition of these cellular
elements are the externalization of phosphatidylserine
180
(PS) on the surface of apoptotic cells, and the interac-
tions with the microglial vitronectin receptor and the
CD36 scavenger receptor (Mallat et al., 2005; Stolzing
and Grune, 2004; Witting et al., 2000). More specifi-
cally, it has been demonstrated that in a co-culture of
induced PC12 apoptotic cells with rat microglial cells,
blockage of the CD36 scavenger receptor using spe-
cific antibodies results in a significant reduction of the
degradation of apoptotic bodies by microglia (Stolzing
and Grune, 2004). Moreover, in a similar model utiliz-
ing a co-culture of induced cerebellar apoptotic cells
with cerebral cortical microglial cells, the presence of
ligands that potentially interfere with the PS recep-
tor and the vitronectin receptor diminishes the ratio of
engulfment of apoptotic bodies by microglia (Witting
et al., 2000).
Still in the context of phagocytosis, it has recently
been demonstrated that microglial cells have the capac-
ity to not only phagocyte apoptotic cell bodies, but
also to promote cell death by engulfment, during
the normal CNS development (Mallat et al., 2005;
Marin-Teva et al., 2004). For example, as previ-
ously mentioned, microglial cells induce the death
of developing Purkinje cells by engulfing cerebel-
lar neurons expressing activated caspase-3 (Marin-
Teva et al., 2004). Thus, in addition to phagocytosing
cells undergoing apoptosis, pathological proteins, and
microbes (Garden and Moller, 2006), microglial cells
are also actively involved in promoting the elimination
of developing neurons in the healthy brain (Vilhardt,
2005).
In response to specific extracellular signals, a sub-
set of microglia cells may function as an antigen-
presenting cell (APC) to T cells. For this, they up-
regulate the expression of dendritic cell markers such
as MHC-II and CD11c, co-stimulatory molecules such
as B7.1 and B7.2, and the cathepsin protease required
for generation of antigen peptides (Aloisi et al., 2000).
There are four major classes of molecules that
are responsible for the communication signals from
microglia to CNS cells and invading leukocytes, espe-
cially during brain injury; however, many of them also
appear to be present in brain development, as dis-
cussed below. The molecular classes are cytokines,
chemokines, trophic factors, and small molecule medi-
ators of inflammation.
Cytokines are small proteins that regulate the
growth, survival, differentiation, and activities of cells.
Interleukins are included in this class. During brain
F.R.S. Lima et al.
injury, microglial cells express and release IL-1/,
IL-3, IL-6, IL-8, IL-12, IL-15, tumor necrosis factor
(TNF) , and interferons (IFNs) as anti-inflammatory
cytokines such as IL-4, IL-10, IL-13, and TGF
(Hanisch, 2002; Kim and de Vellis, 2005). In this situa-
tion, microglia also express receptors for most of these
cytokines, resulting in autocrine feedback loops and
efficient interaction with astrocytes that are crucial for
the homeostasis of the CNS (Hanisch, 2002; Rozenfeld
et al., 2003, 2005; Garden and Moller, 2006).
The role of the well-known proinflammatory
cytokines IL-1, IL-6, and TNF is not always detri-
mental to the CNS. For example, many hippocam-
pal progenitor cells (HPC) die shortly after birth.
Investigating what cell or molecule could cause this
death, Cacci et al. (2005) demonstrated that the addi-
tion of TNF to the medium of a cultured HPC
line shortly after the cells stopped division promotes
apoptosis of these cells. Once the microglial cells syn-
thesized and released this factor, they cultured the
HPC line with conditioned medium from a microglial
cell line and found a similar effect, which suggests
that, during development, microglial TNF seems
to control the number of HPC by promoting the
death of these cells (Cacci et al., 2005). Still during
development, it has been demonstrated in vitro that
microglial cells synthesize and secrete IL-1, which, in
turn, stimulates proliferation of cerebral cortical astro-
cytes. Because the primitive astrocytes that are found
in radial patterns provide routes along which neurons
migrate from germinal zones to cortical regions of the
CNS (Bignami and Dahl, 1974; Schmechel and Rakic,
1979; Levitt and Rakic, 1980), and, at later periods
of brain development, astrocytes secrete extracellular
matrix constituents, such as laminin, which promote
selective axonal growth (Letourneau, 1975; Lander
et al., 1982; Hopkins et al., 1985; Garcia-Abreu et al.,
1995; Lima et al., 2007), this proliferation induced by
microglial IL-1 may help to direct pathways of axonal
outgrowth or to stabilize newly formed circuits, play-
ing an important role in the maturation of the CNS
(Giulian et al., 1988).
Chemokines are a class of chemoattractant
molecules that act through G-protein-coupled recep-
tors. Cultured microglia that are exposed to microbes,
cytokines, or pathological proteins express and secrete
chemokines such as macrophage inflammatory protein
(MIP)-2, CCL2, CCL3, CCL4, CCL5, CXCL1,
CXCL8, CXCL10, and fractalkine (Hanisch, 2002).
12 The Origin of Microglia and the Development of the Brain
Microglia also express receptors for many of the
chemokines, suggesting that one important function of
chemokine secretion is to attract additional microglial
cells to the site of the insult (Garden and Moller,
2006).
Studies of the role of chemokines in the CNS have
been increasingly active in recent years, and appear to
be very promising. For example, it has been demon-
strated that fractalkine secreted from adjacent neurons,
microglial cells, or astrocytes promotes the activation
of neuronal survival pathways in hippocampal neu-
rons expressing the receptor for fractalkine (CX3 CR1)
(Meucci et al., 2000).
Trophic factors that promote neuronal survival are
frequently synthesized in and secreted from microglia.
Microglial cells, in vitro and in vivo, synthesize classi-
cal neurotrophins such as nerve growth factor (NGF),
BDNF, NT-3, basic fibroblast growth factor (b-FGF),
and GDNF (Elkabes et al., 1996; Honda et al., 1999;
Presta et al., 1995).
The role of trophic factors may be indirectly bene-
ficial. For example, it has been demonstrated in vivo
that, during development, microglial NGF promotes
the death of retinal neurons that specifically express the
neurotrophin receptor p75 (Frade and Barde, 1998). It
has also been observed that both the volume occupied
by the area of cell death and the density of its pyknotic
fragments undergo considerable variation during the
development of the retina, and this is thought to occur
in order to create space to accommodate the incoming
axons of the retinal ganglion cells, converging toward
the optic disk, for the correct formation of the optic
nerve, showing that microglia has an essential role in
this well-regulated process (Cuadros and Rios, 1988;
Martin-Partido et al., 1988).
Small-molecule lipid mediators of inflammation
such as arachidonic acid, prostaglandins D2 , E2,
and F2 , platelet-activating factor (PAF), tromboxane
B2, and leukotriene B4 (Minghetti and Levi, 1998)
also play a role in the communication signals from
microglia to CNS cells and invading leukocytes.
During brain injury, microglial cells secrete large
amounts of prostaglandins and express several recep-
tors for them (Minghetti and Levi, 1998). Besides
mediating inflammation, prostaglandins may function
as neuroprotector molecules (Rozenfeld et al., 2003).
It has been demonstrated that prostaglandins amelio-
rate the glutamate-induced toxicity of cortical neurons
(Akaike et al., 1994; Cazevieille et al., 1994).
181
As discussed here, microglial cells aim to promote
the correct ontogenesis and protection of the CNS.
Even events that might seem harmful, such as the
promotion of neuronal death or the release of proin-
flammatory cytokines, are beneficial at some point,
proving the fundamental role of microglial cells in the
development of the healthy brain.
12.3 Microglial Secreted Neurotrophic
Factors: Role in Neural
Development
The majority of studies describe microglia in a patho-
logical context. There is still little information about
these cells and their effective participation in the devel-
opment of cells of the nervous system.
In the developing brain, microglial cells take part
in many morpho- and histogenetic events, helping to
establish the complex network of connections that
is present in the adult brain. Several studies suggest
that microglial cells participate in neuritogenesis and
guidance of axons during development (Nagata et al.,
1993; Chamak et al., 1994; Navascus et al., 2000;
Streit, 2001; Rochefort et al., 2002). In fact, Stolz and
cols. (1991) have shown that ameboid microglial cells
guide the neurite growth of motoneurons in culture.
These cells are able to transform CNS regions into
permissive substrates for axonal growth (David et al.,
1990; Rabchevsky and Strait, 1997; Rochefort et al.,
2002; Batchelor et al., 2002). Moreover, microglial
cells increase myelination and modulate neuronal sur-
vival and synaptogenesis (Hamilton and Rome, 1994;
Shin et al., 2004; Bessis et al., 2007). In addition, they
stimulate CNS vascularization, and also promote astro-
cyte differentiation and astrocyte proliferation, both
during development and after injury, favoring the for-
mation of glial scars (Giulian et al., 1988; Navascus
et al., 2000; Checchin et al., 2006; Nakanishi et al.,
2007). Trophic factors secreted by microglia partic-
ipate in these events (Mallat and Chamak, 1994;
Nakajima et al., 2007). In the developing CNS, IL-
1, which is involved in astrocyte proliferation, is only
detected in the brain after the appearance of the ame-
boid microglia (Giulian et al., 1988). In fact, in vitro
microglial cells synthesize many interleukins, such
as IL-1, IL-4, and IL-6, besides TNF, in response
to several stimuli (Giulian et al., 1988; Frei et al.,
182
1989; Htier et al., 1990, Park et al., 2005; Nakanishi
et al., 2007). Additionally, microglial cells secrete in
culture, spontaneously, FGF2 (Shimojo et al., 1991),
GDNF (Matsushita et al., 2008), NGF after stimula-
tion with LPS (Mallat et al., 1989), NT-3 (Elkabes
et al., 1996; Nakajima et al., 2001), and BDNF (Bessis
et al., 2007), as well as others. All these factors are
involved in the regulation of growth of the CNS. In par-
ticular, the role of microglia in neuronal network for-
mation has been specifically studied. Microglial cells
isolated from the developing rat brain spontaneously
secrete factors that stimulate neurite growth in differ-
ent regions of the CNS (Nagata et al., 1993; Chamak
et al., 1994; Streit, 2001). One of these microglial
factors, thrombospondin, was identified both in vitro
and in vivo (Chamak et al., 1994, 1995). Recently,
Nakajima and cols. (2007) have demonstrated that neu-
rons stimulate microglia to secrete NGF and enhance
the microglial secretion of other trophic factors such as
GDNF, FGF2, neurotrophin 4/5 (NT-4/5), TGF1, IL-
3, and IL-10. These factors induced neuronal survival
and maturation in culture (Nakajima et al., 2007).
12.3.1 Microglia and Neural Progenitor
Cells
Another important event that has been widely debated
in recent years, regarding the role of microglial cells
during CNS development, is their involvement in regu-
lating neural induction and determining the fate of neu-
ral progenitors (Fig. 12.4). The differentiation and pro-
liferation of neural stem cells (NSC) are regulated by
a combination of their intrinsic properties (e.g., tran-
scription factors, epigenetic factors) and cell-extrinsic
properties from the microenvironment around NSC
(e.g., cytokines, growth factors, and cell-cell contact).
Recently, there has been great interest in clarifying the
mechanism of the influence of the microenvironment
on NSC, especially cell-cell contact between NSC and
other nearby types of cells (Zhu et al., 2008). It has
been reported that astrocytes promote the differenti-
ation of NSC, suggesting the importance of cell-cell
interactions between glial cells and NSC. However, the
contribution of microglia to the modulation of neuro-
genesis is still unclear. Both pro- and anti-neurogenic
effects have been demonstrated (Walton et al., 2006;
Cacci et al., 2008; Ideguchi et al., 2008).
F.R.S. Lima et al.
NSC of the subventricular neuraxis have the abil-
ity to self-renew and form multipotent neurospheres in
vitro. This property to generate committed neuroblasts
is progressively lost with continued culture. Walton
and cols. (2006) showed that when NSC were co-
cultured with microglial cells or with their conditioned
medium, neurogenesis was greatly expanded, indicat-
ing that microglia secrete factors that are essential for
neurogenesis. In fact, it was reported that microglial
cells can interact with tenascin-R and secrete NGF,
BDNF, and TGF, promoting proliferation of NSC and
differentiation into neurons but not oligodendrocytes
(Liao et al., 2008). Cacci and cols. (2008) also demon-
strated in microglia that the induction of the proin-
flammatory cytokines IL-1, IL-1, IL-6, and TNF
was sharply reduced after chronic lypopolysaccharide
(LPS) stimulation, compared with acute LPS stimu-
lation (24 h). Conversely, the production of the anti-
inflammatory cytokine IL-10 and the immunomodula-
tory PGE2 was still increased after chronic LPS expo-
sure. Acutely activated microglia reduced survival of
neural precursor cells, prevented neuronal differentia-
tion, and strongly increased glial differentiation, likely
through the release of proinflammatory cytokines;
whereas chronically activated microglia were permis-
sive to neuronal differentiation and cell survival, and
still supported glial differentiation. These events indi-
cate the complexity of the microglial activation process
during neural differentiation (Cacci et al., 2008).
Evidence suggests that microglia have the capacity
to influence the differentiation, not only of embryonic
neural precursors, but also of adult neural precursors
toward a neuronal phenotype (Aarum et al., 2003;
Butovsky et al., 2006; Ziv et al., 2006). Indeed, Battista
and cols. (2006), using adrenalectomized animals,
have reported that activated microglia may secrete
TGF, promoting neurogenesis in adult NSC from the
dentate gyrus of the hippocampus.
Microglia are also implicated in the regulation of
gliogenesis, in both the developing and adult brain.
In vitro studies have shown that microglia induce
oligodendrocyte differentiation with involvement of
the transcription factor NF-kappa B (Nicholas et al.,
2001). Additionally, Golli proteins are expressed in
both the nervous and immune systems. LPS-treated
microglia, but not LPS-treated astrocytes, secrete these
proteins and induce proliferation of oligodendrocyte
precursor cells (OPC) from the cortical subventricular
12 The Origin of Microglia and the Development of the Brain
zone (Filipovi and Zecevi, 2005). Lalive and cols.
(2005) have also described TGF as a factor involved
in oligodendrocyte differentiation. They suggested
that, in the mature CNS after injury, TGF may play
a pivotal role in remyelination by inducing microglia
to release HGF (hepatocyte growth factor), which is
both a chemotactic and differentiation factor for OPC
(Lalive et al., 2005).
The promotion of astrocyte differentiation of neural
progenitor cells by microglia has also been investi-
gated. Using NSC and microglial cultures obtained
from rat embryonic day 16 of the subventricular zone
and rat postnatal day 1 of the cortex, respectively,
Nakanishi and cols (2007) showed that the microglial-
conditioned medium had no significant effect on the
proliferation of NSC. However, this medium increased
the percentage of cells that were positive for GFAP,
a marker of astrocytes, during differentiation. In addi-
tion, Nakanishi and cols (2007) reported that at least
IL-6 and LIF (leukemia inhibitory factor) released
by activated microglia promote astrocytic differentia-
tion of NSC via the activation of the JAK/STAT and
mitogen-activated protein kinase (MAPK) pathways.
Another study, using co-cultures of microglia-NSC,
demonstrated that ramified microglia promote astrogli-
ogenesis of NSC by activating the STAT3 function and
via the notch and sox9 signaling pathways (Zhu et al.,
2008).
Although microglial cells have a mesodermal ori-
gin, recent studies have suggested a novel role for
microglia as multipotential stem cells to give rise
to neurons, astrocytes, or oligodendrocytes. Tanakas
group demonstrated that rat microglial cells express
nestin, A2B5, and O4 antigens, which are markers
of OPC. When microglial cells were cultured in the
presence of 10% fetal bovine serum for 3 days, fol-
lowed by culture in the presence of 70% serum for 2
days, they became highly proliferative and dediffer-
entiated. Then, dedifferentiated cells were transferred
into serum-free medium, and a substantial number
of the cells rapidly turned into long-process-bearing
cells, which expressed microtubule-associated pro-
tein 2, synapsin I, neurofilament proteins, glial fib-
rillary acidic protein (GFAP), or galactocerebroside
(Yokovama et al., 2004, 2006). In accordance with
these data, Sugimotos group has recently shown that
microglia are multipotential stem cells that give rise to
microtubule-associated protein (MAP) 2 -positive and
183
GFAP-positive cells. They also investigated the possi-
bility of microglia to differentiate into functional neu-
rons. Electrophysiological studies demonstrated that
microglia-derived MAP2-positive cells have a sim-
ilar pattern to cultured cortical neurons, suggesting
that these cells possess properties of actual functional
neurons (Matsuda et al., 2008). Moreover, western
blot and immunocytochemical analyses revealed that
activation of bone morphogenetic protein (BMP) sig-
naling through Smad and Id2 proteins is one of the
molecular pathways involved in the generation of
microglia-derived MAP2-positive and GFAP-positive
cells (Niidome et al., 2008). These reports are very
recent, and need to be confirmed. We cannot discard
the possibility that microglia are really multipotential
stem cells, since they originate from hematopoietic
precursors. However there is still little information
available on this issue, which deserves further inves-
tigation.
12.4 The Future
In this chapter, we have exploited the data on
microglial development looking to the interactions
with parenchyma. The majority of data in the litera-
ture describes microglial cells in a neuropathological
context. Little is known about the development of these
cells in the nervous parenchyma and their effective role
in neurogenesis. The interactions of microglia with
others glial cells and neurons contribute importantly
to the harmony of the brain. These interactions are
based on exchange of secreted factors as ILs, growth
factors or the noticed chemokines. However these
exchanges are a very complex system of cell interac-
tions, as it is suggested in the Fig. 12.4. Microglial
cells are considered a subpopulation of intraparenchy-
mal cells, which are distinguished from phagocytes
located in the meninges and in the coroid plexus
stroma, or from macrophages situated in the cerebral
capillary walls. More recently, attention is done to neu-
ral progenitor cells and its implications on therapy.
Microglial progenitors undergo homing to the nervous
system, could they be usefull tools for therapeutic pur-
poses? Upon genetic manipulation, microglia progen-
itors could locally deliver specific gene products for
the treatment of several disorders, such as metachro-
matic leukodystrophy, gliomas and stroke, as well as
184
comment by Chan et al., 2007. Nevertheless, genetic or
cell therapy will only be possible when tissue of origin
or cell lineage of resident microglia can be precisely
identified.
A better understanding of the interactions between
microglia and their microenvironment in the CNS
will benefit the comprehension of the fundamental
mechanisms for macrophagic regulation in vivo, devel-
opment, as well as neuropathologies that are still
incurable.
References
Aarum J, Sandberg K, Haeberlein SL, Persson MA (2003)
Migration and differentiation of neural precursor cells can
be directed by microglia. Proc Natl Acad Sci USA 100:
1598315988.
Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM (2007)
Local self-renewal can sustain CNS microglia mainte-
nance and function throughout adult life. Nat Neurosci 10:
15381543.
Akaike A, Kaneko S, Tamura Y, Nakata N, Shiomi H, Ushikubi
F, Narumiya S (1994) Prostaglandin E2 protects cultured
cortical neurons against N-methyl-D-aspartate receptor-
mediated glutamate cytotoxicity. Brain Res 663:237243.
Akiyama H, Tooyama I, Kondo H, Ikeda K, Kimura H, McGreer
EG, McGreer PL (1994) Early response of brain resident
microglia to kainic acid-induced hippocampal lesions. Brain
Res 635:257268.
Aloisi F, Serafini B, Adorini L (2000) Glia-T cell dialogue.
J Neuroimmunol 107:111117.
Asheuer M, Pflumio F, Benhamida S, Dubart-Kupperschmitt
A, Fouquet F, Imai Y, Aubourg P, Cartier N (2004)
Human CD34+ cells differentiate into microglia and express
recombinat therapeutic protein. Proc Natl Acad Sci USA
101:35573562.
Ashwell K (1990) Microglia and cell death in the developing
mouse cerebellum. Brain Res Dev Brain Res 55:219230.
Ashwell K (1991) The distribution of microglia and cell death in
the fetal rat forebrain. Dev Brain Res 58:112.
Banati RB, Rothe G, Valet G, Kreutzberg GW (1991)
Respiratory burst in the ameboid microglia: a flow cytometric
study on cultured rat microglia. Neuropathol Appl Neurobiol
17:223230.
Bartholdi D, Schwab ME (1997) Expression of pro-
inflammatory cytokine and chemokine mRNA upon
experimental spinal cord injury in mouse: an in situ
hybridization study. Eur J Neurosci 9:14221438.
Batchelor PE, Porritt MJ, Martinello P, Parish CL, Liberatore
GT, Donnan GA, Howells DW (2002) Macrophages and
microglia produce local trophic gradients that stimulate
axonal sprouting toward but not beyond the wound edge. Mol
Cell Neurosci 21:436453.
Battista D, Ferrari CC, Gage FH, Pitossi FJ (2006) Neurogenic
niche modulation by activated microglia: transforming
F.R.S. Lima et al.
growth factor beta increases neurogenesis in the adult dentate
gyrus. Eur J Neurosci 23:8393.
Berezovskaya O, Maysinger D, Fedoroff S (1995) The
hematopoetic cytokine colony stimulating factor in the CNS:
Congenital absence of CSF-1 in mice results in abnormal
microglial response and increased neuron vulnerability to
injury. Int J Devl Neurosci 13:285299.
Bessis A, Bchade C, Bernard D, Roumier A (2007) Microglial
control of neuronal death and synaptic properties. Glia
55:233238.
Bignami A, Dahl D (1974) Astrocyte-specific protein and radial
glia in the cerebral cortex of newborn rat. Nature 252:
5556.
Billiards SS, Haynes RL, Folkerth RD, Trachtenberg FL, Liu
LG, Volpe JJ, Kinney HC (2006) Development of microglia
in the cerebral white matter of the human fetus and infant.
J Comp Neurol 497:199208.
Boya J, Calvo JL, Carbonell AL, Borregon A (1991) A lectin
histochemistry study on the development of rat microglial
cells. J Anat 175: 229236.
Butovsky O, Ziv Y, Schwartz A, Landa G, Talpalar AE, Pluchino
S, Martino G, Schwartz M (2006) Microglia activated by
IL-4 or IFN-gamma differentially induce neurogenesis and
oligodendrogenesis from adult stem/progenitor cells. Mol
Cell Neurosci 31:149160.
Cacci E, Ajmone-Cat MA, Anelli T, Biagioni S, Minghetti L
(2008) In vitro neuronal and glial differentiation from embry-
onic or adult neural precursor cells are differently affected by
chronic or acute activation of microglia. Glia 56:412425.
Cacci E, Claasen JH, Kokaia Z (2005) Microglia-derived tumor
necrosis factor-alpha exaggerates death of newborn hip-
pocampal progenitor cells in vitro. J Neurosci Res 80:
789797.
Calvo CF, Dobbertin A, Gelman M, Glowinski J, Mallat M
(1998) Identification of CSF-1 as a ameboid microglia migra-
tory activity produced by astrocytes. Glia 24:180186.
Cazevieille C, Muller A, Meynier F, Dutrait N, Bonne C (1994)
Protection by prostaglandins from glutamate toxicity in cor-
tical neurons. Neurochem Int 24:395398.
Chamak B, Dobbertin A, Mallat M (1995) Immunochemical
detection of thrombospondin in microglia in the developing
rat brain. Neuroscience 69:177187.
Chamak B, Mallat M (1991) Fibronectin and laminin regulate
the in vitro differentiation of microglial cells. Neuroscience
45:513527.
Chamak B, Morandi V, Mallat M (1994) Brain macrophages
stimulate neurite growth and regeneration by secreting
thrombospondin. J Neuros Res 38:221233.
Chan WY, Kohsaka S, Razaie P (2007) The origin and cell
lineage of microglia new concepts. Brain Res Rev 53:
344354.
Checchin D, Sennlaub F, Levavasseur E, Leduc M, Chemtob
S (2006) Potential role of microglia in retinal blood vessel
formation. Invest Ophthalmol Vis Sci 47:35953602.
Chen L, Yang P, Kijlstra A (2002) Distribution, markers,
and functions of retinal microglia. Ocul Immunol Inflamm
10:2739.
Constam DB, Philipp J, Malipiero UV, Ten-Dijke P, Schachner
M, Fontana A (1992) Differential expression of TGF1,
-2, -3 by glioblastoma cells, astrocytes and microglia.
J Immunol 148:14041410.
12 The Origin of Microglia and the Development of the Brain
Cramer EB (1992) Cell biology of phagocyte migration from
the bone marrow, out of the bloodstream, and across organ
epithelia. In: Inflammation: Basic Principles and Clinical
Correlates, 2nd ed., (Gallin JI, Goldstein IM and Snyderman
R, eds.), pp. 341351. Raven Press, New York.
Cuadros MA, Martin C, Coltey P, Almendros A, Navascues
J (1993) First appearance, distribution and origin pf
macrophages in the early development of the avian central
nervous system. J Comp Neurol 330:113129.
Cuadros MA, Moujahid A, Quesada A, Navascus J (1994)
Development of microglia in the quail optic tectum. J Comp
Neurol 348:207224.
Cuadros MA, Navascus J (1998) The origin and differentita-
tion of microglial cells during development. Prog Neurobiol
56:173189.
Cuadros MA, Navascus J (2001) Early origin and coloniza-
tion of the developing central nervous system by microglial
precursors. Prog Brain Res 132:5159.
Cuadros MA, Rios A (1988) Spatial and temporal correla-
tion between early nerve fiber growth and neuroepithelial
cell death in the chick embryo retina. Anat Embryol 178:
543551.
Cuadros MA, Rodriguez-Ruiz J, Calvente R, Almendros A,
Marin-Teva JL, Navascus J (1997) Microglia development
in the quail cerebellum. J Comp Neurol 389:390401.
Cucchiarini M, Ren XL, Perides G, Terwilliger EF (2003)
Selective gene expression in brain microglia mediated via
adeno-associated virus type2 and type 5 vectors. Gene Ther
10:657667.
Dalmau I, Finsen B, Tonder N, Zimmer J, Gonzalez
B, Castellano B (1997) Development of microglia in
the prenatal rat hippocampus. J Comp Neurol 377:
7084.
Dalmau I, Vela JM, Gonzlez B, Finsen B, Castellano B (2003)
Dynamics of microglia in the developing rat brain. J Comp
Neurol 458:144157.
David S, Bouchard C, Tsatas O, Giftochristos N (1990)
Macrophages can modify the nonpermissive nature of the
adult central nervous system. Neuron 5:463469.
Davis EJ, Foster TD, Thomas WE (1994) Cellular forms and
functions of brain microglia: Brain Res Bull 34:7378.
Del Rio Hortega P (1932) Microglia. Section X. In: Cytology
and Cellular Pathology of Nervous System (Penfield W ed.),
pp. 481534. Paul B. Hoeber, New York.
Diaz-Araya CM, Provis JM, Penfold PL, Billson FA (1995)
Development of microglia topography in human retina
J Comp Neurol 363:5368.
Djukic M, Mildner A, Schmidt H, Czesnik D, Bruck W, Priller
J, Nau R, Prinz M (2006) Circulating monocytes engraft
in the brain, differentiate into microglia and contribute to
the pathology following meningitis in mice. Brain 129:
23942403.
Dobbertin A, Schmid P, Gelman M, Glowinski J, Mallat
M (1997) Neurons promote macrophage proliferation by
producing transforming growth factor-beta2. J Neurosci
17:53055315.
Dobrenis K (1998) Microglia in cell culture and in transplan-
tation therapy for central nervous system disease. Methods
16:320344.
Egensperger R, Maslim J, Bisti S, Hollander H, Stone J (1996)
Fate of DNA from retinal cells dying during development:
185
uptake by microglia and macroglia (Muller cells). Brain Res
Dev Brain Res 97:18.
Elkabes ST, Di Cicco-Bloom EM, Black IB (1996) Brain
microglia/macrophages express neurotrophins that selec-
tively regulate microglial proliferation and function.
J Neurosci 16:25082521.
Ferrer I, Bernet E, Soriano E, del Rio T, Fonseca M (1990)
Naturally occurring cell death in the cerebral cortex of the
rat and removal of dead cells by transitory phagocytes.
Neuroscience 39:451458.
Filipovic R, Zecevic N (2005) Interaction between microglia and
oligodendrocyte cell progenitors involves Golli proteins. Ann
N Y Acad Sci 1048:166174.
Flanary BE, Streit WJ (2006) Alpha-tocopherol (vitamin E)
induces rapid, nonsustained proliferation in cultured rat
microglia. Glia 53:669674.
Frade JM, Barde YA (1998) Microglia-derived nerve growth
factor causes cell death in the developing retina. Neuron
20:3541.
Frei K, Malipiero UV, Leist TP, Zinkernagel RM, Schwab ME,
Fontana A (1989) On the cellular source and function of
interleukin 6 produced in the central nervous system in viral
diseases. Eur J Immunol 19:689694.
Garcia-Abreu J, Cavalcante LA, Moura Neto V (1995)
Differential patterns of laminin expression in lateral and
medial midbrain glia. Neuroreport 6:761764.
Garden GA, Moller T (2006) Microglia biology in health and
disease. J Neuroimmune Pharmacol 1:127137.
Gehrmann J, Banati RB (1995) Microglial turnover in the injured
CNS: activated microglia undergo delayed DNA fragmen-
tation following peripheral nerve injury. J Neuropathol Exp
Morphol 54:680688.
Giulian D, Baker TJ (1986) Characterization of ameboid
microglia isolated from developing mammalian brain.
J Neurosci 6:21632178.
Giulian D, Haverkamp LJ, Li J, Karshin WL, Yu J, Tom D, Li X,
Kirkpatrick JB (1995) Senile plaques stimulate microglia to
release a neurotoxin found in Alzheimer brain. Neurochem
Int 27:119137.
Giulian D, Ingeman GE (1988a) Colony-stimulating fac-
tors as promoters of ameboid microglia. J Neurosci 8:
47074717.
Giulian D, Young DG, Woodward J, Brown DC, Lachman LB
(1988b) Interleukin-1 is an astroglial growth factor in the
developing brain. J Neurosci 8:709714.
Gomes FA, Lima FRS, Trentin AG and Moura Neto V (2001)
Thyroid hormone role on nervous system morphogenesis.
Progr Brain Res (Rev), 132:4150.
Graeber MB, Tetzlaff W, Streit WJ, Kreutzberg GW (1988)
Microglial cells but not astrocytes undergo mitosis following
facial nerve axotomy. Neurosci Lett 85:317321.
Hamilton SP, Rome LH (1994) Stimulation of in vitro myelin
synthesis by microglia. Glia 11:326335.
Hanisch UK (2002) Microglia as a source and target of
cytokines. Glia 40:140155.
Hanisch UK, Kettenmann H (2007) Microglia: active sensor and
versatile effector cells in the normal and pathologic brain.
Nat Neurosci 10:13871394.
Hao C, Guilbert LJ, Fedoroff S (1990) Production of colony-
stimulating factor-1 (CSF-1) by mouse astroglia in vitro.
J Neurosci Res 27:314323.
186
Hess DC, Abe T, Hill WD, Studdard AM, Carothers J, Masuya
M, Fleming PA, Drake CJ, Ogawa M (2004) Hematopoietic
origin of microglial and perivascular cells in brain. Exp
Neurol 186:134144.
Honda S, Nakajima K, Nakamura Y, Imai Y, Kohsaka S (1999)
Rat primary cultured microglia express glial cell line-derived
neurotrophic factor receptors. Neurosci Lett 275:203206.
Hopkins JM, Ford-Holevinski TS, McCoy JP, Agranoff BW
(1985) Laminin and optic nerve regeneration in the goldfish.
J Neurosci 5:30303038.
Hume DA, Perry H, Gordon S (1983) Immunohistochemical
localization of a macrophage-specific antigen in developing
mouse retina: phagocytosis of dying neurons and differen-
tiation of microglial cells to form a regular array in the
plexiform layers. J Cell Biol 97:253257.
Hurley SD, Streit WJ (1996) Microglia and the mononuclear
phagocytic system. In: Topical Issues in Microglia Research
(Ling EA, Tan CK and Tan CBC, eds.), pp. 119. Singapore
Neuroscience Association, Singapore.
Htier E, Ayala J, Bousseau A, Denfle P, Prochiantz A (1990)
Amoeboid microglial cells and not astrocytes synthesize
TNF in Swiss mouse brain cell cultures. Eur J Neurosci
2:762768.
Ideguchi M, Shinoyama M, Gomi M, Hayashi H, Hashimoto
N, Takahashi J (2008) Immune or inflammatory response by
the host brain suppresses neuronal differentiation of trans-
planted ES cell-derived neural precursor cells. J Neurosci
Res 86:19361943.
Ilschner S, Brandt R (1996) The transition of microglia to a ram-
ified phenotype is associated with the formation of stable
acetylated and detyrosinated microtubules. Glia 18:129140.
Imai Y, Kohsaka S (2002) Intracellular signaling in M-
CSF-induced microglia activation: role of Iba1. Glia 40:
164174.
Imamoto K, Leblond CP (1978) Radioautographic investigation
of gliogenesis in the corpus callosum of young rats. J Comp
Neurol 180:139164.
Innocenti GM, Clarke S, Koppel H (1983) Transitory
macrophages in the white matter of the developing visual
cortex. II. Development and relations with axonal pathways.
Brain Res 313:5566.
Jones LL, Banati RB, Graeber MB, Bonfanti L, Raivich G,
Kreutzberg GW (1997) Population control of microglia: does
apoptosis play a role? J Neurocytol 26:755770.
Jordan FL, Thomas WE (1988) Brain macrophage: question of
origin and interrelationship. Brain Res Rev 13:164178.
Kahn MA, Kumar S, Liebl D, Chang R, Parada LF, De Vellis
J (1999) Mice lacking NT-3, and its receptor TrkC, exhibit
profound deficiencies in CNS glial cells. Glia 26:153165.
Kaur C, Hao AJ, Wu CH, Ling EA (2001) Origin of microglia.
Microsc Res Tech 54:29.
Kaur C, Ling EA (1991) A study of the transformation of
amoeboid microglia cells into microglia labeled with the
isolectin Griffonia simplicifolia in postnatal rats. Acta Anat
142:118125.
Kim SU, de Vellis J (2005) Microglia in health and disease.
J Neurosci Res 81:302313.
Kondo Y, Lemere CA, Seabrook TJ (2007) Osteopetrotic (op/op)
mice have reduced microglia, no Abeta deposition, and
no changes in dopaminergic neurons. J Neuroinflammation
4:3141.
F.R.S. Lima et al.
Kreutzberg G (1996) Microglia: a sensor for pathological events
in the CNS. Trends Neurosci 19:312319.
Lalive PH, Paglinawan R, Biollaz G, Kappos EA, Leone DP,
Malipiero U, Relvas JB, Moransard M, Suter T, Fontana A
(2005) TGF-beta-treated microglia induce oligodendrocyte
precursor cell chemotaxis through the HGF-c-Met pathway.
Eur J Immunol 35:727737.
Lander AD, Fujii DK, Gospodarowicz D, Reichardt LF (1982)
Characterization of a factor that promotes neurite outgrowth:
evidence linking activity to a heparan sulfate proteoglycan.
J Cell Biol 94:574585.
Lawson LJ, Perry VH, Dri P, Gordon S (1990) Heterogeneity in
the distribution and morphology of microglia in the normal
adult mouse brain. Neurosci 39:151170.
Lawson LJ, Perry VH, Gordon S (1992) Turnover of resident
microglia in the normal adult mouse brain. Neurosci 48:
405415.
Lee SC, Liu W, Roth P, Dickson DW, Berman JW, Brosnan
CF (1993) Macrophage colony stimulating factor in fetal
astrocytes and microglia: differential regulation by citokines
and lipopolysaccharide and modulation of class II MHC on
microglia. J Immunol 150:594604.
Leong SK, Ling EA (1992) Amoeboid and ramified microglia:
their interrelationship and response to brain injury. Glia
7:3947.
Letourneau PC (1975) Possible roles for cell-to-substratum
adhesion in neuronal morphogenesis. Dev Biol 44:7791.
Levitt P, Rakic P (1980) Immunoperoxidase localization of glial
fibrillary acidic protein in radial glial cells and astrocytes
of the developing rhesus monkey brain. J Comp Neurol
193:815840.
Liao H, Huang W, Niu R, Sun L, Zhang L (2008) Cross-talk
between the epidermal growth factor-like repeats/fibronectin
6-8 repeats domains of Tenascin-R and microglia modulates
neural stem/progenitor cell proliferation and differentiation.
J Neurosci Res 86:2734.
Lima FR, Arantes CP, Muras AG, Nomizo R, Brentani RR,
Martins VR (2007) Cellular prion protein expression in
astrocytes modulates neuronal survival and differentiation.
J Neurochem 103:21642176.
Lima FRS, Gervais A, Colin C, Izambar M, Moura Neto V,
Mallat M (2001) Regulation of microglial development: a
novel role for thyroid hormone. J Neurosci 21:20282038.
Lima FRS, Trentin AG, Rosenthal D, Chagas C, Moura Neto
V (1997) Thyroid hormone induces protein secretion and
morphological changes in astroglial cells with an increase
in expression of glial fibrillary acidic protein. J Endocrinol
154:167175.
Ling EA (1981) The origin and nature of microglia. In: Advances
in Cell Neurobiology. Vol. 2 (Fedoroff S and Hertz L, eds.),
pp. 3382. Academic Press. London.
Ling EA, Kaur C, Wong WC (1991) Expression of major his-
tocompatibility complex and leukocyte common antigens in
amoeboid microglia in postnatal rats. J Anat 177:117126.
Ling EA, Penney D, Leblond CP (1980) Use of carbon labeling
to demonstrate the role of blood monocytes as precursors of
the ameboid cells present in the corpus callosum of postnatal
rats. J Comp Neur 193:631657.
Ling EA, Wong WC (1993) The origin and nature of rami-
fied and amoeboid microglia: a historical review and current
concepts. Glia 7:918.
12 The Origin of Microglia and the Development of the Brain
Mallat M, Calvo CF, Dobbertin A (1997) Migration and pro-
liferation of mononuclear phagocytes in the central nervous
system. Adv Exp Med Biol 429:99108.
Mallat M, Chamak B (1994) Brain macrophage: neurotoxic or
neurotrophic effector cells? J Leukoc Biol 56:416422.
Mallat M, Houlagatte R, Brachet P, Prochiantz A (1989)
Lipopolysaccharide-stimulated rat brain macrophages
release NGF in vitro. Dev Biol 133:309311.
Mallat M, Lima FRS, Gervais A, Colin C, Moura Neto V (2002)
New insights in the role of thyroid hormone in the CNS: the
microglial track. Mol Psychiatry 7:78.
Mallat M, Marin-Teva JL, Cheret C (2005) Phagocytosis in the
developing CNS: more than clearing the corpses. Curr Opin
Neurobiol 15:101107.
Mansfield PJ, Suchard SJ (1994) Thrombospondin promotes
chemotaxis and haptotaxis of peripheral blood monocytes.
J Immunol 153:42194229.
Marin-Teva JL, Dusart I, Colin C, Gervais A, van Rooijen N,
Mallat M (2004) Microglia promote the death of developing
Purkinje cells. Neuron 41:535547.
Martin S, Vincent JP, Mazella J (2003) Involvement of the neu-
rotensin receptor-3 in the neurotensin-induced migration of
human microglia. J Neurosci 23:11981205.
Martin-Partido G, Rodriguez-Gallardo L, Alvarez IS, Navascues
J (1988) Cell death in the ventral region of the neural retina
during the early development of the chick embryo eye. Anat
Rec 222:272281.
Marn-Teva JL, Almendros A, Calvente R, Cuadros MA,
Navascus J (1999) Proliferation of actively migrating ame-
boid microglia in the developing quail retina. Anat Embryol
200:289300.
Matsuda S, Niidome T, Nonaka H, Goto Y, Fujimura K, Kato
M, Nakanishi M, Akaike A, Kihara T, Sugimoto H (2008)
Microtubule-associated protein 2-positive cells derived from
microglia possess properties of functional neurons. Biochem
Biophys Res Commun 368:971976.
Matsushita Y, Nakajima K, Tohyama Y, Kurihara T, Kohsaka
S (2008) Activation of microglia by endotoxin suppresses
the secretion of glial cell line-derived neurotrophic fac-
tor (GDNF) through the action of protein kinase C
alpha (PKCalpha) and mitogen-activated protein kinases
(MAPKS). J Neurosci Res 86:19591971.
Meucci O, Fatatis A, Simen AA, Miller RJ (2000) Expression of
CX3CR1 chemokine receptors on neurons and their role in
neuronal survival. Proc Natl Acad Sci USA 97:80758080.
Mildner A, Schmid H, Nitsche M, Merkler D, Hanisch UK,
Mack M, Heikenwalder M, Bruck W, Priller J, Prinz
M (2007) Microglia in the adult brain arise from Ly-
6Chi CCR2+ monocytes only under defined host conditions.
Nature Neurosci 12:15441553.
Milligan CE, Cunningham J, Levitt P (1991) Differential
immunochemical markers reveal the normal distribution of
brain macrophages and microglia in the developing rat brain.
J Comp Neurol 314:125135.
Minghetti L, Levi G (1998) Microglia as effector cells in brain
damage and repair: focus on prostanoids and nitric oxide.
Prog Neurobiol 54:99125.
Monier A, Adle-Biassette H, Delezoide AL, Evrard P, Gressens
P, Verney C (2007) Entry and distribution of microglial cells
in human embryonic and fetal cerebral cortex. J Neuropathol
Exp Neurol 66:372382.
187
Murabe Y, Sano Y (1982) Microglial cells in the cerebral cortex
of the rat, with special reference to their possible involvement
in synaptic function. Cell Tissue Res 223:493506.
Nagano T, Kimura SH, Takemura M (2008) Prostaglandin E2
reduces extracellular ATP-induced migration in cultured rat
microglia. Brain Res 1221:15.
Nagata K, Nakajima K, Kohsaka S (1993) Plasminogen pro-
motes the development of rat mesencephalic dopaminergic
neurons in vitro. Dev Brain Res 75:3137.
Nakajima K, Honda S, Tohyama Y, Imai Y, Kohsaka S, Kurihara
T (2001) Neurotrophin secretion from cultured microglia.
J Neurosci Res 65:322331.
Nakajima K, Tohyama Y, Maeda S, Kohsaka S, Kurihara T
(2007) Neuronal regulation by which microglia enhance
the production of neurotrophic factors for GABAergic, cat-
echolaminergic, and cholinergic neurons. Neurochem Int
50:807820.
Nakanishi M, Niidome T, Matsuda S, Akaike A, Kihara T,
Sugimoto H (2007) Microglia-derived interleukin-6 and
leukaemia inhibitory factor promote astrocytic differentia-
tion of neural stem/progenitor cells. Eur J Neurosci 25:
649658.
Navascus J, Calvente R, Marn-Teva JL, Cuadros MA (2000)
Entry, dispersion and differentiation of microglia in the
developing central nervous system. An Acad Bras C 72:
91102.
Navascus J, Cuadros MA, Almendros A (1996) Development
of microglia: evidences from studies in the avian central
nervous system. In: Topical Issues in Microglia Research
(Ling EA, Tan CK and Tan CBC, eds.), pp. 4364. Singapore
Neuroscience Association, Singapore.
Navascus J, Moujahid A, Almendros A, Marin-Teva JL,
Cuadros J (1995) Origin of microglia in the quail retina:
central-to-peripheral and vitreal-to-scleral migration of
microglial precursors during development. J Comp Neurol
354:209228.
Nicholas RS, Wing MG, Compston A (2001) Nonactivated
microglia promote oligodendrocyte precursor survival and
maturation through the transcription factor NF-kappa B. Eur
J Neurosci13:959967.
Niidome T, Matsuda S, Nonaka H, Akaike A, Kihara T,
Sugimoto H (2008) A molecular pathway involved in
the generation of microtubule-associated protein 2-positive
cells from microglia. Biochem Biophys Res Commun 370:
184188.
Park KW, Lee DY, Joe EH, Kim SU, Jin BK (2005)
Neuroprotective role of microglia expressing interleukin-4.
J Neurosci Res 81:397402.
Perry VH, Anderson PB, Gordon S (1993) Macrophages and
inflammation in the central nervous system. Trends Neurosci
16:268273.
Perry VH, Gordon S (1988) Macrophages and microglia in the
nervous system. Trends Neurosci 11:273277.
Perry VH, Gordon S (1991) Macrophages and the nervous
system. Int Rev Cytol 125:203244.
Perry VH, Hume DA, Gordon S (1985) Immunohistochemical
localization of macrophages and microglia in the adult and
developing mouse brain. Neurosci 15:313326.
Perry VH, Lawson LJ, Reid DM (1994) Biology of the mononu-
clear phagocyte system of the central nervous system and
HIV infection. J Leukoc Biol 56:399406.
188
Pow DV, Perry VH, Morris JF, Gordon S (1989) Microglia in
the neurohypophysis associate with and endocytose terminal
portions of neurosecretory neurons. Neurosci 33:567578.
Presta M, Urbinati C, Dellera P, Lauro GM, Sogos V, Balaci L,
Ennas MG, Gremo F (1995) Expression of basic fibroblast
growth factor and its receptors in human fetal microglia cells.
Int J Dev Neurosci 13:2939.
Provis JM, Diaz CM, Penfold PL (1996) Microglia in human
retina: a heterogenous population with distinct ontogenies.
Persp Dev Neurobiol 3:213222.
Rabchevsky AV, Streit WJ (1997) Grafting of cultured microglial
cells into the lesioned spinal cord of adult rats enhances
neurite outgrowth. J Neurosci Res 47:3448.
Raivich G, Gehrmann J, Kreutzberg GW (1994) Inhibition of
posttraumatic microglial proliferation in a genetic model
of macrophage colony-stimulating factor deficiency in the
mouse. Eur J Neurosci 6:16151618.
Rezaie P, Dean A, Male D, Ulfig N (2005) Microglia in the cere-
bral wall of the human telencephalon at second trimester.
Cereb Cortex 15:938949.
Rezaie P, Male D (2002) Mesoglia and microglia a historical
review of the concept of mononuclear phagocytes within the
central nervous system. J Hist Neurosci 11:325374.
Rochefort N, Quenechdu N, Watroba L, Mallat M, Giaume C,
Milleret C (2002) Microglia and astrocytes may participate
in the shaping of visual callosal projections during postnatal
development. J Physiol Paris 96:183192.
Rozenfeld C, Martinez R, Figueiredo RT, Bozza MT, Lima
FR, Pires AL, Silva PM, Bonomo A, Lannes-Vieira J, De
Souza W, Moura-Neto V (2003) Soluble factors released by
Toxoplasma gondii-infected astrocytes down-modulate nitric
oxide production by gamma interferon-activated microglia
and prevent neuronal degeneration. Infect Immun 71:
20472057.
Rozenfeld C, Martinez R, Seabra S, Santanna C, Gonalves JG,
Bozza M, Moura-Neto V, De Souza W (2005) Toxoplasma
gondii prevents neuron degeneration by interferon-gamma-
activated microglia in a mechanism involving inhibition of
inducible nitric oxide synthase and transforming growth
factor-beta1 production by infected microglia. Am J Pathol
167:10211031.
Salimi K, Moser KV, Marksteiner J, Reindl M, Humpel C (2003)
GDNF and TGF-beta1 promote cell survival in serum-free
cultures of primary rat microglia. Cell Tissue Res 312:
135139.
Sankarapandi S, Zweier JL, Mukherjee G, Quinn MT, Huso
DL (1998) Measurement and characterization of superox-
ide generation in microglial cells: evidence for an NADPH
oxidase-dependent pathway. Arch Biochem Biophys 353:
312321.
Savill J, Dransfield I, Gregory C, Haslett C (2002) A blast
from the past: clearance of apoptotic cells regulates immune
responses. Nat Rev Immunol 2:965975.
Sawada M, Suzumura A, Yamamoto H, Marunouchi T (1990)
Activation and proliferation of the isolated microglia by
CSF-1 and possible involvement of protein kinase C. Brain
Res 509:119124.
Schmechel DE, Rakic P (1979) A Golgi study of radial glial
cells in developing monkey telencephalon: morphogene-
sis and transformation into astrocytes. Anat Embryol 156:
115152.
F.R.S. Lima et al.
Schnitzer J (1989) Enzyme-histochemical demonstration of
microglial cells in the adult and postnatal rabbit retina.
J Comp Neurol 282:249263.
Schwartz M, Butovsky O, Brck W, Hanisch UK (2006)
Microglial phenotype: is the commitment reversible? Trends
Neurosci 29:6874.
Shimojo M, Nakajima K, Takei N, Hamanoue M, Kohsaka
S (1991) Production of basic fibroblast growth factor in
cultured rat brain microglia. Neurosci Lett 123:229231.
Shin WH, Lee DY, Park KW, Kim SU, Yang MS, Joe EH,
Jin BK (2004) Microglia expressing interleukin-13 undergo
cell death and contribute to neuronal survival in vivo. Glia
46:142152.
Sievers J, Bamberger C, Debus OM, Lucius R (1995)
Regeneration in the optic nerve of adult rats: influences
of cultured astrocytes and optic nerve grafts of different
ontogenetic stages. J Neurocytol 10:783793.
Simard AR, Rivest S (2004) Bone marrow stem cells have
the ability to populate the entire central nervous system
into fully differentiated parenchymal microglia. FASEB
J 18:9981000.
Sorokin SJ, Hoyt RF, Blunt DG, McNelly NA (1992)
Macrophage development: II. Early ontogeny of macrophage
populations in brain, liver, and lungs of rat embryos as
revealed by a lectin marker. Anat Record 232:527550.
Stolz B, Erulkar S, Kuffler DP (1991) Macrophages direct pro-
cess elongation from adult frog motoneurons in culture. Proc
R Soc Lond B 244:227231.
Stolzing A, Grune T (2004) Neuronal apoptotic bodies: phago-
cytosis and degradation by primary microglial cells. FASEB
J 18:743745.
Streit WJ (2001) Microglia and macrophages in the developing
CNS. Neurotoxicol 22:619624.
Streit WJ, Kreutzberg GW (1988) Response of endogenous glial
cells to motor neuron degeneration induced by toxic ricin.
J Comp Neurol 268:248263.
Suh HS, Kim MO, Lee SC (2005) Inhibition of granulocyte-
macrophage colony-stimulating factor signaling and
microglial proliferation by anti-CD45RO: role of Hck
tyrosine kinase and phosphatidylinositol 3-kinase/Akt.
J Immunol 174:27122719.
Suzumura A, Sawada M, Yamamoto H, Marunouchi T (1990)
Effects of colony stimulating factors on isolated microglia in
vitro. J Neuroimmunol 30:237244.
Tanaka J, Maeda N (1996) Microglia ramification requires
nondiffusible factors derived from astrocytes. Exp Neurol
137:367375.
Thry C, Htier E, Evrard C, Mallat M (1990) Expression of
macrophage colony-stimulating factor gene in the mouse
brain during development. J Neurosci Res 26:129133.
Thry C, Mallat M (1993). Influences of interleukin 1 and
tumor necrosis factor-a on the growth of microglial cells in
primary cultures of mouse cerebral cortex: involvement of
colony-stimulating factor 1. Neurosci Lett 150:195199.
Thry C, Stanley ER, Mallat M (1992) Interleukin 1 and
tumor necrosis factor-a stimulate the production of colony-
stimulating factor 1 by murine astrocytes. J Neurochem
59:11831186.
Tomozawa Y, Inoue T, Takahashi M, Adachi M, Satoh M
(1996) Apoptosis of cultured microglia by the deprivation of
macrophage. Neurosci Res 25:715.
12 The Origin of Microglia and the Development of the Brain
Trentin AG. (2006) Thyroid hormone and astrocyte morphogen-
esis. J Endocrinol 189:189197.
Tsuchiya T, Park KC, Toyonaga S, Yamada SM, Nakabayashi
H, Nakai E, Ikawa N, Furuya M, Tominaga A, Shimizu K
(2005) Characterization of microglia induced from mouse
embryonic stem cells and their migration into the brain
parenchyma. J Neuroimmunol 160:210218.
Vallieres L, Sawchenko PE (2003) Bone marrow-derived cells
that populate the adult mouse brain preserve their hematopoi-
etic identity. J Neurosci 23:51975207.
Vilhardt F (2005) Microglia: phagocyte and glia cell. Int
J Biochem Cell Biol 37:1721.
Vitry S, Bertrand JY, Cumano A, Dubois-Dalcq M (2003)
Primordial hematopoietic stem cells generate microglia but
not myelin-forming cells in a neural environment. J Neurosci
23:1072410731.
Walton MR, Gibbons H, MasGibbo GA, Sirimanne E, Saura
J, Gluckman PD, Dragunow M (2000) PU.1 expression in
microglia. J Neuroimmunol 104:109115.
Walton NM, Sutter BM, Laywell ED, Levkoff LH, Kearns
SM, Marshall GP 2nd; Scheffler B, Steindler DA (2006)
Microglia instruct subventricular zone neurogenesis. Glia
54:815825.
Wang JM, Chen ZG, Colella S, Bonilla MA, Welte K, Bordignon
C, Mantovani A (1988) Chemotactic activity of recombi-
nant human granulocyte colony-stimulating factor. Blood
72:14561460.
Wang JY, Shum AY, Ho YJ (2003) Oxidative neurotoxicity
in rat cerebral cortex neurons: synergistic effects of H2 O2
and NO on apoptosis involving activation of p38 mitogen-
activated protein kinase and caspase-3. J Neurosci Res
72:508519.
Wang CC, Wu CG, Shieh JY, Wen CY, Ling EA (1996)
Immunohistochemical study of ameboid microglial cells in
fetal rat brain. J Anat 189:567574.
189
Watanabe R, Takase-Yoden S, Fukumitsu H, Nakajima K (2002)
Cell transplantation to the brain with microglia labeled by
neuropathogenic retroviral vector system. Cell Transplant
11:471473.
Wegiel J, Wisniewski HM, Dziewiatkowski J, Tarnawski M,
Kozielski R, Trenknert E, Wiktor-Jedrzejczak W (1998)
Reduced number and altered morphology of microglial cells
in colony stimulating factor-1-deficient osteopetrotic op/op
mice. Brain Res 804:135139.
Witting A, Muller P, Herrmann A, Kettenmann H, Nolte
C (2000) Phagocytic clearance of apoptotic neurons by
Microglia/Brain macrophages in vitro: involvement of lectin-
, integrin-, and phosphatidylserine-mediated recognition.
J Neurochem 75:10601070.
Yokoyama A, Sakamoto A, Kameda K, Imai Y, Tanaka J
(2006) NG2 proteoglycan-expressing microglia as multipo-
tent neural progenitors in normal and pathologic brains. Glia
53:754768.
Yokoyama A, Yang L, Itoh S, Mori K, Tanaka J (2004)
Microglia, a potential source of neurons, astrocytes and
oligodendrocytes. Glia 45:96104.
Zassler B, Schermer C, Humpel C (2003) Protein kinase C
and phosphoinositol-3-kinase mediate differentiation or pro-
liferation of slice-derived rat microglia Pharmacology 67:
211215.
Zhu P, Hata R, Cao F, Gu F, Hanakawa Y, Hashimoto
K, Sakanaka M (2008) Ramified microglial cells pro-
mote astrogliogenesis and maintenance of neural stem
cells through activation of Stat3 function. FASEB J 22:
38663877.
Ziv Y, Avidan H, Pluchino S, Martino G, Schwartz M
(2006) Synergy between immune cells and adult neu-
ral stem/progenitor cells promotes functional recovery
from spinal cord injury. Proc Natl Acad Sci USA 103:
1317413179.
Chapter 13

Tissue Biology of Proliferation and Cell Death Among

Retinal Progenitor Cells

Rafael Linden, Rodrigo A.P. Martins, Mariana S. Silveira, Helena L. Borges, Alfred
Sholl-Franco, Lucianne Fragel-Madeira, and Ana Carolina Dudenhoeffer-Carneiro

Contents Abstract The retina is a complex network of various

13.1 Introduction . . . . . . . . . . . . . . . . . 192
13.1.1 Retinal Progenitor Cells . . . . . . . . 193
13.1.2 Cell Proliferation in the Retina: On-the-fly
Restriction of Phenotype . . . . . . . . 194
13.1.3 Retinal Tissue and Microenvironment
Around Progenitor Cells . . . . . . . . 194
13.2 The Cell Cycle Among Retinal Progenitor Cells 195
13.2.1 Morphology of Retinal Progenitor Cells . 195
13.2.2 Interkinetic Nuclear Migration and the Cell
Cycle in the Developing Retina . . . . . 196
13.2.3 The Cell Cycle Machinery in Retinal
Progenitor Cells . . . . . . . . . . . . 197
13.2.4 Checkpoint Control of the Cell Cycle . . 198
13.3 Control of Retinal Progenitor Cell Proliferation
by Growth Factors and Cytokines . . . . . . 199
13.3.1 Growth Factors . . . . . . . . . . . . 199
13.3.2 Interleukins . . . . . . . . . . . . . . 200
13.3.3 Neurotrophins . . . . . . . . . . . . . 200
13.3.4 Hedgehog, Notch and Wnt . . . . . . . 201
13.3.5 Platelet Activating Factor . . . . . . . . 202
13.4 Control of the Retinal Cell Cycle
by Neurotransmitters and Neuromodulators . 203
13.4.1 Classical Neurotransmitters . . . . . . . 203
13.4.2 Neuropeptides . . . . . . . . . . . . . 209
13.5 Signal Transduction in the Extrinsic Control of
the Retinal Cell Cycle . . . . . . . . . . . . 210
13.6 Death and Survival of Retinal Progenitor Cells 212
13.6.1 Mechanisms of Cell Death . . . . . . . 212
13.6.2 Sensitivity to Cell Death Within the Retinal
Cell Cycle . . . . . . . . . . . . . . 215
13.6.3 Molecular Mechanisms of Cell Death
Among Retinal Progenitor Cells . . . . . 216
13.7 Conclusion and Future Directions . . . . . . 217
References . . . . . . . . . . . . . . . . . . . . 218
R. Linden ()
Instituto de Biofsica, Universidade Federal do Rio de Janeiro,
Cidade Universitaria, Rio de Janeiro, Brazil
e-mail: rlinden@biof.ufrj.br
molecularly and neurochemically distinct cell types.
These heterogeneous and highly interactive neurons
and glia are stratified in rather precisely organized
layers, and often distributed in regular arrays, sur-
rounded by rich, laminated extracellular matrix, as well
as a dual vascular system. All components of retinal
tissue affect the proliferation and survival of retinal
progenitor cells through a combination of tightly reg-
ulated genetic and microenvironmental mechanisms.
The latter are provided for by a variety of extrin-
sic modulators, including neurotrophins, interleukins,
neurotransmitters and neuropeptides, acting through
numerous types of plasma membrane receptors, and
cross-talking intracellular signaling pathways. This
chapter reviews some of the major determinants of reti-
nal cell population dynamics, which are a pre-requisite
for the design of tissue engineering applied to retinal
degenerations.
Keywords Cell cycle Growth factors
Neurotransmitters Programmed cell death Stem
cells
Abbreviations
5-HT 5-hydroxytryptamine, serotonin
AC adenylyl cyclase
ACh acetylcholine
AIF apoptosis-inducing factor
AMPA -amino-3-hydroxy-5-methyl-4-
isoxazole propionic acid
Apaf-1 apoptotic protease-activating factor-1
ATM ataxia and teleangiectasia mutated
ATP adenosine triphosphate
ATR ATM and Rad3-related

H. Ulrich (ed.), Perspectives of Stem Cells, 191

DOI 10.1007/978-90-481-3375-8_13, Springer Science+Business Media B.V. 2010
192 R. Linden et al.

Bcl-2 B-cell lymphoma protein-2 OLO olomoucine

BDNF brain-derived growth factor PACAP Pituitary Adenylyl Cyclase-activating
BMP bone morphogenetic protein Polypeptide
BrdU bromo-deoxyuridine PAF platelet activating factor
CAK CDK activating kinase PCD programmed cell death
cAMP cyclic adenosine-3 -5 -monophosphate PDTC pyrrolidinedithiocarbamate
CDC cell division cycle PI3K phosphoinositide-3-kinase
CDK cyclin-dependent protein kinase PKA cAMP-activated protein kinase
CE cilliary epithelium PKC protein kinase C
CK2 casein kinase 2 PLC phospholipase C
Cl- chloride ion PNMT phenylethanolamine N-methyltransferase
CMZ cilliary marginal zone Rb retinoblastoma protein
CNS central nervous system RPC retinal progenitor cell(s)
CNTF ciliary neurotrophic factor Shh sonic hedgehog
CREB cAMP Response Element Binding protein STAT signal transducer and activator
DDC DOPA decarboxylase of transcription
DFO deferoxamine TGF transforming growth factor
Dhh desert hedgehog TH tyrosine hydroxylase
DNA-PK DNA protein kinase TrK tyrosine kinase receptor
DOPA dihydroxyphenylalanine twhh tiggy-winkle hedgehog
DSB double strand break Wnt wingless-int oncogene
EGF epidermal growth factor
ERK extracellular signal-related kinase
FasL Fas ligand
FGF fibroblast growth factor
GABA gamma amino butiric acid 13.1 Introduction
GAD glutamic acid decarboxylase
GCL ganglion cell layer Recent discoveries concerning stem cells in both the
GTP guanosine triphosphate developing and adult nervous system, and current
Gy gray experimental assays of cell therapies have spread a
Hh hedgehog proteins wave of excitement concerning the possibility of devel-
IFN interferon oping restorative treatments for degenerative diseases
IGF insulin-related growth factor of the nervous system (Muotri and Gage, 2006). The
Ihh Indian hedgehog case of the retina includes both retinal dystrophies
IL interleukin (Vugler et al., 2007; MacLaren and Pearson, 2007), as
INL inner nuclear layer well as glaucomatous neurodegeneration (Bull et al.,
INM interkinetic nuclear migration
2008).
IP3 inositol triphosphate
As a necessary step for the establishment of new
IR ionizing radiation
technologies of regenerative neurology and neuro-
mAChR muscarinic acetylcholine receptor(s)
ophthalmology, a thorough understanding of develop-
MAPK mitogen activated kinase
MG132 carbobenzoxyl-leucinyl-leucinyl-leucinal mental mechanisms depends heavily upon unraveling
mGluR metabotropic glutamate receptor the basic biology of stem/progenitor cells. A large
MIMO mimosine body of work has been directed at the behavior of stem
MOP-R opioid receptor cells, mostly in vitro, and a number of both genetic and
nAChR nicotinic acetylcholine receptor(s) epigenetic events involved in either the maintenance
NBL neuroblastic layer or the exit from an undifferentiated state have been
NMDA N-methyl-D-aspartate uncovered in recent years (Yeo et al., 2008; Lunyak
NO nitrous oxide and Rosenfeld, 2008).
NPY neuropeptide Y Nonetheless, stem/progenitor cells reside among
NT neurotrophin a complex environment, and just like any other
ODC ornitine decarboxylase issue concerning developmental biology, would greatly
13 Tissue Biology of Proliferation and Cell Death
benefit from studying their dependence on the struc-
ture, cellular and extracellular matrix components,
cellcell communication and signaling systems in the
context of the tissue (e.g. Raff, 1992; Linden, 2000;
Rutishauer, 2008). The present chapter aims at review-
ing some of the cellular and molecular interactions, and
elements of their signal transduction pathways, that
affect two of the major events of the life cycle of retinal
progenitor cells, namely the cell cycle and consequent
proliferation rates, and the sensitivity to programmed
cell death.
13.1.1 Retinal Progenitor Cells
The mammalian neural retina is derived from retinal
progenitor cells (RPC) of neuroectodermal origin, ini-
tially found in the protrusive optic vesicles on both
sides of the neural tube. RPC proliferate repeatedly,
and daughter cells eventually exit the cell cycle and
differentiate into several dozen types, which form reg-
ular mosaics distributed in alternating layers of cell
bodies and synaptic contacts (Galli-Resta et al., 2008).
Therein, individual retinal cell types can be identified
by their position, morphology, biochemical properties
and function (Adler, 1986; Kolb et al., 1992; Pourcho,
1996; Kolb, 1997; Jeon et al., 1998; Cayouette et al.,
2006).
Mature retinal cells are grouped in 6 distinct classes
of neurons ganglion cells, amacrine (inclusive of
interplexiform) cells, horizontal cells, bipolar cells,
cone photoreceptors and rod photoreceptors and one
type of glia, the Mller cell (Livesey and Cepko,
2001). These cell classes are produced according to an
evolutionary conserved birth order (Finlay, 2008), in
which ganglion cells, cone photoreceptors, horizontal
cells and roughly half of the amacrine cells are gener-
ated first during embryogenesis, followed by bipolar
cells, Mller glia and the remaining amacrine cells.
Rod photoreceptors are generated throughout retinal
development (Young, 1985a,b; Wallace, 2008).
Similar to other layered structures of the CNS,
the RPC are located in a pseudostratified proliferative
epithelium (Fig. 13.1, see Section 2.3), analogous to
the ventricular zone of the developing brain (Miyata,
2008), and here referred to as the neuroblastic layer
(NBL). The majority of RPC cell division occur at the
apical tip of the NBL (Baye and Link, 2008), although
193
A
GCL
INL
NBL
M G1 S G2 M G1
B C
Fig. 13.1 Retinal progenitor cells and interkinetic nuclear
migration in the newborn rat retina. (a) Drawing of a cross-
section of the neural retina from a newborn rat, showing the
differentiating cell body and plexiform layers found at birth in
this species. Basal (inner) is up, apical (outer) is down. Round
grey profiles depict differentiating ganglion cells in the gan-
glion cell layer (GCL), amacrine cells in the inner nuclear layer
(INL), and the inner plexiform layer (horizontal lines) between
GCL and INL. Occasional differentiating photoreceptors are
shown at the lower rim, and horizontal cells in the middle of
the neuroblastic layer (NBL). The white profiles straddling the
retinal thickness represent proliferating cells undergoing interki-
netic nuclear migration at successive phases of the cell cycle
(M-G1-S-G2-M, from left to right), amidst a population of
unsynchronized proliferating cells (light grey-lined profiles in
the background). (b, c). The S-phase (b) and M-phase (c) strata
of the NBL in the newborn rat. Both photomicrographs are lim-
ited to the NBL (bracket in a), and show immunohistochemical
staining of bromo-deoxy-uridine (BrdU) injected 30 min before
fixation (b), and of phosphorylated Histone H3 (c), markers of
DNA synthesis and of the G2/M transition, respectively. The
pigment epithelium was removed when retinae were explanted
(Mod from Carneiro et al., 2008)
some mitotic figures have been observed within the
inner nuclear layer of the developing vertebrate retina,
which give rise exclusively to horizontal cells that
remain in the same laminar location as their mother
cell (Rapaport et al., 1985; Rapaport and Vietri, 1991;
Godinho et al., 2007; Rompani and Cepko, 2008).
Neurogenesis in the mammalian retina ceases at
the end of the developmental period and is essentially
absent in the adult. Exception to this rule is the reac-
tive proliferation of Mller glial cells after retinal
injury, which may be followed by neuronal differen-
tiation, thus indicating potential neurogenic properties
194
of reactive Mller cells in adult mammalian retina
(Fischer and Reh, 2001; Ooto et al., 2004; Bringmann
et al., 2006; Osakada et al., 2007; Florian et al., 2008).
In addition, retinal cell differentiation has also been
reportedly derived in vitro from quiescent progeni-
tor cells located in both the ciliary body and iris
pigment epithelium (Tropepe et al., 2000; Ohta et
al., 2008), which would suggest similarities with the
ciliary marginal zone and retinal pigment epithelium
of teleost fish, amphibia and birds (Kubota et al., 2002;
Reh and Fischer, 2006). Nevertheless, recent studies
suggest that the ciliary epithelium (CE) of mammals
does not contain retinal stem cells, since all prolifer-
ating cells of CE-derived spheres display features of
differentiated pigmented epithelial cells. In addition,
these pigmented proliferating cells from either human
or mouse CE-derived spheres failed to fully differ-
entiate into retinal cell types, which was interpreted
as the activation of an incomplete transdifferentiation
program (Cicero et al., 2009).
13.1.2 Cell Proliferation in the Retina:
On-the-fly Restriction
of Phenotype
In the developing nervous system, multipotent progen-
itor cells may undergo either symmetric or asymmetric
cell division. Symmetrical divisions may give rise
either to multipotent daughter cells that reenter the
cell cycle, or to a pair of postmitotic cells. Following
asymmetrical divisions the daughter cells have distinct
fates: Usually one cell reenters the cell cycle, while
the other differentiates into a post-mitotic cell (Chenn
and McConnell, 1995; Mione et al., 1997; Yoshikawa,
2000; Cayouette and Raff, 2003; Roegiers and Jan,
2004; Cayouette et al., 2006).
Lineage analysis has shown that RPC are multi-
potent and individual RPC can give rise to a variety
of combinations of retinal neurons and glia (Price et
al., 1987; Turner et al., 1990). However, RPC are
somewhat heterogeneous (Lillien, 1998), and during
retinal development, these cells undergo progressive
changes in their competence to generate distinct cell
types (Cepko et al., 1996), such that RPC that exit the
cell cycle early in development give rise to the early
born cell classes, whereas RPC born later are restricted
R. Linden et al.
to the generation of the so-called late born cell
classes.
The mechanisms underlying progressive lineage
restriction in RPC are still poorly understood, but
evidence has accumulated that both intrinsic factors,
as well as responses to extrinsic factors regulate the
behavior of RPC, to ensure that appropriate numbers of
the various retinal cell types are generated throughout
retinal development (Reh and Levine, 1998; Cepko,
1999; Marquardt and Gruss, 2002; Marquardt, 2003;
Donovan and Dyer, 2005; Cayouette et al., 2006).
13.1.3 Retinal Tissue
and Microenvironment Around
Progenitor Cells
Notwithstanding the remarkable intrinsic factors that
guide both RPC proliferation and phenotypic deter-
mination, their sensitivity to extrinsic control raises
fundamental questions as to the role of cellcell inter-
actions upon the behavior of RPC. Within the develop-
ing retina, similar to other areas of the central nervous
system, RPC thrive amidst a rich, varied, and histo-
typically structured environment (Fig. 13.1). It is of
particularly importance that the morphology of prolif-
erating retinal cells, which span the full depth of the
retina, allows for a changing set of partners to inter-
act with successive generations of RPC, as postmitotic
cells progressively populate the retinal layers during
development.
Several studies have shown that changes in the time
course and spatial patterns of expression or distribu-
tion of growth factors and cytokines are critical for the
control of not only the proliferative state and the spec-
ification of cell fate in the retina (Gaur et al., 1992;
Zhao and Barnstable, 1996; Zhao and Lemke, 1998;
McFarlane et al., 1998; von Bartheld, 1998; Jaynes
and Turner, 2000; Patel and McFarlane, 2000; Dyer
and Cepko, 2001; Das et al., 2006; Silva et al., 2008),
but also of the survival of early proliferating neural
progenitor cells (de la Rosa and de Pablo, 2000).
Both the pigment epithelium and the differentiating
retinal cells themselves provide a continuously chang-
ing source of a large number of distinct cytokines
(Holtkamp et al., 2001), as well as neurotransmitters
and neuromodulators (Linden et al., 2005; Martins and
13 Tissue Biology of Proliferation and Cell Death
Pearson, 2007). Adding to these, adhesion molecules
(Sharma and Johnson, 2000), and extracelular matrix
components (Inatani and Tanihara, 2002; Erlich et al.,
2003) present within the developing retinal tissue, may
interact with responsive RPC. Vascular development,
together with the presence of immune cells in the
developing retina (Linden et al., 1986) contribute an
additional set of extracellular factors that may affect
the developing RPC.
Independent of the coupling between proliferation
and cell differentiation, the rate of cell proliferation
per se (Levine and Green, 2004), together with pro-
grammed cell death (Linden et al., 1999; de la Rosa
and de Pablo, 2000) rank among the major determi-
nants of the correct wiring of the retina. Therefore, the
understanding of RPC to a level allowing their eventual
manipulation in therapeutic context requires a thor-
ough analysis of the factors that control the retinal cell
cycle and the sensitivity of retinal progenitor cells to
programmed cell death.
13.2 The Cell Cycle Among Retinal
Progenitor Cells
13.2.1 Morphology of Retinal
Progenitor Cells
The neural retina is derived from bilateral evaginations
of the anterior neural plate, which form the bilayered
optic cup, the inner layer of which contains the multi-
potent RPC. Retinal volume and eye size are primarily
determined by the number of times each progenitor
cell divides before its final division to generate two
postmitotic cells. In mammals and birds, proliferation
of RPC ceases before retinal histogenesis is complete
(Young, 1985a; Cepko et al., 1996; Dhomen et al.,
2006). Retinal cell differentiation obeys the inner-to-
outer and central-to-periphery gradients, beginning in
the inner layer of the central optic cup and progressing
concentrically in a wave-like fashion until it reaches
the peripheral edges of the retina (Rapaport and Stone,
1983; Young, 1985b; Beazley et al., 1987; Prada et al.,
1991; Marquardt and Gruss, 2002).
Retinal progenitor cells are bipolar in shape, bear-
ing one process that extends towards the apical surface
195
of the epithelium and another process that terminates
at the basal surface, show typical epithelial features
and are highly polarized along their apicalbasal axis
(Turner et al., 1990; Gtz and Huttner, 2005; Godinho
et al., 2007). The proliferative NBL appears to be strat-
ified because the nuclei of RPC are distributed along
the apical-basal axis, and migrate up and down during
the cell cycle (see below).
During cell division, most RPC maintain both their
basal and apical attachments throughout the cell cycle.
After division, one daughter cell inherits the basal
process and its sibling cell initiates a new one. The
inheritor, which can be either a neuron or a progen-
itor cell, uses the elongated process for translocation
and positioning (Saito et al., 2003; Baye and Link,
2008). Previous findings using DiI-labeled cerebral
wall slices, also demonstrated that the basal process of
each cortical progenitor cell is maintained throughout
the M phase, and is inherited by one of its daughter
cells (Miyata et al., 2001, 2002). Interestingly, how-
ever, and differing from the developing cerebral cortex,
retinal progenitors either do not, or only partially
develop radial glial-cell features during neurogenesis,
suggesting that the migration of early generated neu-
rons is guided by the basal process (Turner et al., 1990;
Kriegstein and Gtz, 2003; Gtz and Huttner, 2005;
Kriegstein et al., 2006). However, it was additionally
shown that a small number of committed precursor
cells lose their attachment to the apical surface and give
rise exclusively to two horizontal cells (Godinho et al.,
2007).
The apicalbasal polarity of RPC is an important
basis for their symmetric versus asymmetric division
(Fig. 13.2). Some studies indicate that through the con-
trol of the orientation of the cleavage plane during cell
division, progenitors may differentially distribute cell
fate-determining factors to daughter cells, thereby pro-
ducing equal or unequal outcomes (Cepko et al., 1996;
Gtz and Huttner, 2005; Cayouette et al., 2006). In
vivo imaging of retrovirally labeled progenitor cells
in the rat retina showed that cleavage planes that are
either parallel to the apical surface or oriented verti-
cally have distinct outcomes, as to whether the daugh-
ter cells will adopt either the same or distinct fates
(Chenn and McConnell, 1995; Cayouette and Raff,
2003; Cayouette et al., 2006; Godinho et al., 2007).
Thereby, controlling the orientation of RPC division
could help establishing distinct lineages of RPC.
196
basal
either...
apical
G1 S G2
Fig. 13.2 Symmetrical and asymmetrical divisions in the
retinal neuroepithelium. Mitotic cells can divide either hor-
izontally or vertically at the apical margin of the neuroblastic
layer, close to the retinal pigment epithelium (RPE), Depending
13.2.2 Interkinetic Nuclear Migration
and the Cell Cycle in the
Developing Retina
In 1935, Sauer correlated the shape and histological
appearance of the nuclei of proliferating cells in the
ventricular layer of the cerebral cortex of vertebrate
embryos, with their distance to the lumen of the neu-
ral tube, and established the concept of interkinetic
nuclear migration (INM). This event has so far been
described only in the nervous system, and is char-
acterized by intracellular movement of the nuclei of
the neuroblasts, as these cells pass through successive
phases of the cell cycle (Sauer, 1935).
The nuclei originated in each mitotic division (M-
phase) in the apical margin of the ventricular zone
migrate towards the basal margin, where DNA repli-
cation (S-phase) takes place. Once DNA synthesis
is completed, the nucleus migrates back to the api-
cal margin, where cell division occurs. The G1 and
G2 phases occur along the nuclear migration pathway
(Figs. 13.1, and 13.2). Thus, cell cycle phases corre-
late with nucleus position during interkinetic migration
(Sauer, 1935; Sidman, 1961; Fujita, 1962; Rapaport et
al., 1984; Young, 1985a; Jacobson, 1991; Takahashi et
al., 1996; Saito et al., 2003; Gtz and Huttner, 2005;
Cayouette, 2007).
INM was confirmed by experimental observations
in which treatment with mitotic inhibitors resulted in
arrested metaphase cells only at the apical surface and
3 H-thymidine pulse-labeling experiments showed dif-
ferential labeling of progenitor cell nuclei throughout
R. Linden et al.
ILM
or...
RPE
M G1 M G1
on the plane of cell division, daughter cells can either remain in
the cell cycle and proceed through the G1 phase with nuclear
migration anchored to the inner limiting membrane (ILM), or
leave the cell cycle and differentiate
the thickness of the ventricular layer (Sauer and
Chittenden, 1959; Sauer and Walker, 1959; Langman
et al., 1966; Baye and Link, 2008 for review).
In the developing retina, similar to the ventricu-
lar zone of other parts of the central nervous sys-
tem, the nuclei of cell progenitors migrate back and
forth through the full thickness of the NBL in syn-
chrony with the cell cycle (Sidman, 1961; Fujita, 1962;
Young, 1985b; Rapaport et al., 1984). DNA is dupli-
cated in the innermost margin, while mitosis occurs
in the outer rim of the NBL (Fig. 13.1, Fujita, 1962;
Sidman, 1961).
The total duration of the cell cycle in the retina
increases as development proceeds, in part due to
lengthening of the S phase, which comprises at least
half of the cell cycle during postnatal development of
the rat retina. Therefore, roughly 50% of the asyn-
chronous population of proliferating cells are in S
phase at any time in the newborn rat retina, and
these cells can be identified by a single injection of
a nucleotide analogue, such as Bromo-deoxyuridine
(BrdU) (Alexiades and Cepko, 1996; Rehen et al.,
1999). Immediately after incorporating the marker
nucleotide, the labeled nuclei of cells in S phase are
found within the basal (inner) half to two-thirds of the
NBL in the newborn rat retina (Fig. 13.1).
Hayes and Nowakowski, using DNA labeling tech-
niques, correlated the distance and velocity of move-
ment of nuclei to the phases of the cell cycle, and
observed that during G1 the nuclei move for various
distances and velocities, reaching the inner half of the
ventricular zone where they distribute along various
levels. Upon entering S phase, the nuclei stop moving
13 Tissue Biology of Proliferation and Cell Death
and remain still until completion of DNA synthesis.
The passage through G2 is fast, and the nuclei soon
reach the ventricular surface, where they stay during
mitosis (Hayes and Nowakowski, 2000).
It is likely that positioning of intracellular nuclei
during INM is regulated by extracellular signals
present along different strata of the proliferative
layer, associated with transitions of cell cycle phases.
Disruption of retinal cell cycle by genetic alterations
or pharmacological manipulations accordingly alters
INM (Pearson et al., 2005b; Willer et al., 2005).
Moreover, when cell cycle progression is partially or
completely inhibited, INM is either slowed or stopped
(Ueno et al., 2006; L. Fragel-Madeira, unpublished
results). An elegant study recently showed that an
apical-basal gradient of notch signaling regulates the
interplay of INM and the exit from the cell cycle (Del
Bene et al., 2008). Notwithstanding, the causal rela-
tionships between nuclear migration and progression
of the cell cycle are yet to be uncovered. Indeed, the
inextricable link between the INM and the succession
of phases of the cell cycle still defies investigators in
this field.
13.2.3 The Cell Cycle Machinery in Retinal
Progenitor Cells
The molecular mechanisms involved in the control
of the cell cycle have been usually viewed as evo-
lutionarily conserved. However, cloning of the genes
that encode components of the cell-cycle machinery
revealed multiple, closely related family members for
almost all major regulators, and current evidence sup-
ports the notion that related family members play
highly specialized roles during development, which
may vary among distinct species (Dyer and Cepko,
2001).
Progression through the distinct phases of the cell
cycle depends upon the activity of a family of ser-
ine/threonine kinases, called CDK (cyclin-dependent
kinases). Cyclins are regulatory proteins that bind and
activate the kinase activity of the CDK. In general, the
CDKs are constitutive, while the expression of cyclins
changes during the different phases of the cell cycle
(Sherr, 1993). The formation of specific cyclin/CDK
complexes, and the consequent kinase activity, is fun-
damental for progression through the distinct phases
197
of the cell cycle (Gran and Reddy, 1995; Stewart et
al., 2003). Therefore, both the transcriptional control
of cyclin mRNA and the cyclic degradation cyclin
proteins are necessary to allow exit from mitosis and
re-entry into a new proliferative cycle (Page and Hieter,
1997; Stewart et al., 2003).
Distinct combinations of cyclins and CDK regulate
the transition between different phases of the cell cycle
by directly regulating the phosphorylation state of mul-
tiple target proteins (Sanchez and Dynlacht, 2005 for
review). Following the completion of cell division, the
decision to exit the cell cycle and turn into a post-
mitotic cell, or to re-enter in S-phase, depends on the
state of phosphorylation of the retinoblastoma (Rb)
family proteins. Distinct cyclin/CDK holoenzymes can
phosphorylate the Rb and/or its family members (p107
and p130) (Sherr, 1996; Weinberg, 1995). Ample evi-
dence support a model of sequential phosphorylation
of the Rb proteins by distinct cyclin/CDK complexes.
The three D-type cyclins (D1, D2 and D3), which are
expressed in a tissue-specific manner, associate with
either one of two CDK (CDK4 and CDK6). Cyclin
D/CDK complexes are believed to act early during
G1, while the cyclin E/CDK2 complex is believed to
be the main Rb/p107/p130 kinase later in G1. The
phosphorylation of the Rb family proteins leads to
their dissociation from the transcription factor E2F.
When dissociated from Rb proteins, E2Fs regulate
the transcription of genes required for the transition
and progression through S-phase. Conversely, if Rb is
unphosphorylated, the cell will exit the cell cycle and
become post-mitotic (Morgan, 1997).
Lack of Rb causes ectopic mitoses and increased
cell death in the mouse retina at E16.5 (Maandag et
al., 1994), and the loss of both Rb and p107 leads to
severe retinal dysplasia and the formation of tumors
(Robanus-Maandag et al., 1998, Zhang et al., 2004).
Studies of the regulation of retinal development by
Rb proteins have demonstrated a crucial role of these
in the control of RPC proliferation (MacPherson et
al., 2004; Zhang et al., 2004). Importantly, Rb fam-
ily members are not interchangeable in the developing
retina, therefore, when Rb gene is inactivated in the
developing mouse retina, a compensatory increase in
p107 expression was observed. In addition, Rb and
p130 are redundantly expressed in a subset of reti-
nal cells (Donovan et al., 2006). The development of
tumors in response to the inactivation of Rb family
genes, as well as the redundancy and compensation
198
described above, emphasize the importance of the Rb
pathway for control of the cell cycle during retinal
development (MacPherson et al., 2004; Zhang et al.,
2004).
Rb family proteins bind and regulate various mem-
bers of the E2F transcription factor family. E2F1-
3 are believed to function as transcriptional activa-
tors, while E2F4-5 act as repressors (for reviews:
Cam and Dynlacht, 2003; Korenjak and Brehm,
2005). Expression of E2F transcription factors in the
developing retina has been previously demonstrated.
Specifically, both E2F1 and E2F2 are expressed in the
NBL, while expression of E2F3-4 may be confined
to postmitotic cells of the developing retina (Dagnino
et al., 1997). Ectopic expression of E2F1 driven by a
photoreceptor-specific promoter induces the prolifera-
tion of differentiating photoreceptors, indicating that
E2F1 activity must be downregulated to ensure cell
cycle exit (Lin et al., 2001). Further evidence for the
regulation of RPC proliferation by E2Fs was shown
in a knock-in mouse. Developing retinas expressing a
mutant Rb protein incapable of binding E2F1-3, dis-
played similar defects as described for Rb-deficient
retinas, including ectopic proliferation of RPC. These
findings reinforce the importance of the Rb/E2F path-
way in the regulation of the RPC cell cycle (Sun et al.,
2006).
In addition to the association to the cyclins, the
activity of CDK complexes is also regulated in
other ways. Two families of cyclin-dependent kinase
inhibitors (CKIs) have been identified that can inhibit
the activity of the CDK (Sherr and Roberts, 1995;
Vidal and Koff, 2000; Stewart et al., 2003, Harper and
Brooks, 2005). The INK family (p15Ink4a , p16Ink4b ,
p18Ink4c and p19Ink4d ) is believed to bind prefer-
entially cyclin-DCDK complexes and lead to dis-
ruption of the holoenzyme subunits. Cip/Kip CKIs
(p21Cip1 , p27Kip1 and p57Kip2 ) can form stable com-
plexes with either cyclin-DCDK or cyclin-ECDK.
In addition, several transcriptional mechanisms, as
well as post-translational regulation (e.g., phospho-
rylation and ubiquitination) of cyclins, CDKs and
CKIs have been reported. For example, CAKs (Cdk
Activating Kinases) are known to phosphorylate Thr-
160, which promotes structural changes and increases
CDK2 kinase activity by several orders of magnitude.
Similarly, CDKs are inhibited by phosphorylation by
the WEE1 family of kinases, which may be counter-
acted by the action of CDC25 phosphatases, which
dephosphorylate WEE1 sites (Thr-14, Tyr-15) leading
R. Linden et al.
to CDK activation (Kaldis, 1999; Abbas and Dutta,
2006 for reviews).
Retinal cells appear to be critically dependent on
both cyclin D1 and p27Kip1 , as shown in transgenic
mice. The retinae of cyclin D1-knockout mice show
reduced cell number (Sicinski et al., 1995), lower rates
of proliferation and an unique pattern of photorecep-
tor cell death (Ma et al., 1998), while p27Kip1 -deficient
animals show disturbances in retinal organization with
an increased cell number which is consistent with
a requirement for p27Kip1 in RPC cell cycle exit
(Nakayama et al., 1996). The p27Kip1 protein was
reportedly upregulated during late G2 or early G1 in
most cells that leave the cell cycle at any stage of devel-
opment (Dyer and Cepko, 2001). In turn, p57Kip2 is
expressed in only a subset of progenitor cells that leave
the cell cycle during a brief period of retinal develop-
ment in mouse embryos. RPC that upregulate p57Kip2
differ from those that upregulate p27Kip1 , and these
proteins are upregulated at distinct times during the
cell cycle (Dyer and Cepko, 2001). Little is known
about the role of CAK, WEE or CDC25 in the regu-
lation of progenitor cell cycle in developing vertebrate
retina.
Cyclin D1 was shown to be genetically upstream of
cyclin E and p27Kip1 function. Nonetheless, prolifera-
tion still occurs in the cyclin D1-null retina, which may
be due to either or both persistent expression of cyclin
D3 or to a D-cyclin-independent mechanism. This sug-
gests that multiple mechanisms may exist to support at
least a basal level of G1 progression in RPC (Levine
and Green, 2004).
In the retina, p57Kip2 is both necessary and suffi-
cient for appropriate cell cycle exit. However, p57Kip2
is not expressed in RPC exiting the cell cycle dur-
ing the postnatal period. RPC deficient in p19Ink4d
also proliferate beyond the normal period, although the
phenotype of p19Ink4d -null retinas is less severe than
that of p27Kip1 knockout (Levine and Green, 2004).
The available data, therefore, indicate that certain
regulators of the cell cycle, such as cyclin D family
members and several CKIs, have major roles in the
control of the proliferation of RPC.
13.2.4 Checkpoint Control
of the Cell Cycle
Together with the cell cycle progression machinery,
cells are equipped with checkpoints that guarantee the
13 Tissue Biology of Proliferation and Cell Death
correct ordering of cell cycle events. Cell cycle check-
points are mechanisms capable to arrest or block phase
transition until the recognition of appropriate sig-
nals necessary to cell cycle progression (Hartwell and
Weinert, 1989). Checkpoints are composed of cellu-
lar surveillance and signaling pathways that coordinate
DNA repair with chromosome metabolism and cell-
cycle transitions (Branzei and Foiani, 2006; Bartek and
Lukas, 2007).
For example, the genome is under constant threat
of damage from both exogenous agents and intrinsic
mechanical events that may damage DNA, including
the unwinding of chromatin during the normal cell
cycle (Branzei and Foiani, 2008 for review). DNA
lesions activate checkpoint pathways that regulate spe-
cific DNA-repair mechanisms in the various phases
of the cell cycle, and result in a reversible delay of
cell cycle progression (Walworth, 2000; Sampath and
Plunkett, 2001; Bartek and Lukas, 2001; Borges et al.,
2004).
Post-translational modifications, such as phospho-
rylation, ubiquitylation and sumoylation, are crucial
for the regulation of the checkpoint machinery, thereby
regulating important cell cycle events (Gutierrez and
Ronai, 2006). These post-translational modifications
may affect the recruitment of repair proteins to dam-
aged DNA, or direct the repair machinery towards a
certain type of lesion, often to allow repair in a specific
cell-cycle phase.
The checkpoint proteins are recruited to DNA
lesions, transducer complexes transmit and amplify the
signals to downstream targets such as the DNA-repair
apparatus and the cell-cycle machinery (Branzei and
Foiani, 2006). The transmission of the signal or acti-
vation of these targets is often achieved by phosphory-
lation events that affect either the transcription level
or activity of repair genes, and modulate cell-cycle
transitions.
There are three major checkpoints, depending on
cell cycle phase and all related to DNA damage (Bartek
and Lukas, 2001; Cortez, 2003; Xiao et al., 2003).
G1 checkpoint results in that DNA synthesis will not
occur if the replication machinery is not ready; intra-S
checkpoint monitors DNA integrity during its dupli-
cation, and G2/M checkpoint verifies DNA damage
before cell enters M-phase (Walworth, 2000; Sampath
and Plunkett, 2001; Bartek and Lukas, 2001).
Central components of the checkpoint machinery
are the phosphoinositide 3-kinase related kinases ATM
199
(ataxia-telangiectasia mutated), ATR (ATM and Rad3-
related) and DNA protein kinase (Bartek and Lukas,
2007). ATM and ATR kinases respond to distinct types
of DNA damage. ATM mainly responds to double-
strand breaks (DSBs), whereas ATR is activated by S
phase damage such as single stranded (ss)DNA and
stalled forks (Shilloh, 2003).
When ATM and ATR are recruited to sites of
damage, they target various substrates, including
checkpoint kinase-2 (CHK2) and CHK1, respectively
(Abraham, 2001; Matsuoka et al., 2007 for review).
ATM or ATR phosphorylate and activates CHK1/2,
which phosphorylate, for example, distinct members
of CDC25 phosphatase family depending of cell cycle
phase. This phosphorylation reduces CDC25 activ-
ity and prevents removal of inhibitory phosphates
of cyclin-CDK complexes required for cell cycle
progression (Branzei and Foiani, 2008). DSB resec-
tion and activation of ATR requires both ATM and
cyclin-dependent kinase (CDK) activity (Jazayeri et
al., 2006), whereas in other circumstances, ATR may
be required for activation of ATM (Burdak-Rothkamm
et al., 2008).
Notwithstanding the classical responses to DNA
damage, the possibility cannot be discarded that other
pathways of activation of checkpoint kinases such as
ATM/ATR, or CHK1/CHK2 may exist, and may be
subject to extrinsic control in proliferating cells.
13.3 Control of Retinal Progenitor Cell
Proliferation by Growth Factors
and Cytokines
13.3.1 Growth Factors
Experiments in vitro showed that acid fibroblast
growth factor (aFGF, or FGF-1), basic FGF (bFGF, or
FGF-2), insulin, or insulin-like growth factors (IGF-1
and IGF-2) promote cell proliferation in rat (E15-E18),
chicken (E-6), and teleost fish (Carassius auratus,
315 cm in standard) retina (Frade et al., 1996a;
Hernandez-Sanchez et al., 1995; Lillien and Cepko,
1992; Otteson et al., 2002). The dependence on stage
of development observed for these proliferative effects
may reflect important stage-dependent factors associ-
ated with cell responsiveness. Other studies showed an
200
effect of epidermal growth factor (EGF) upon prolif-
eration, but not self-renewal of embryonic (E18.5) and
early postnatal (P1) mouse RPC (Ahmad et al., 2004;
James et al., 2004), although mouse adult retinal cells
proliferate and self-renew in response to both FGF-
2 and EGF (Tropepe et al., 2000), depending on the
presence of laminin (Otaegi et al., 2007).
Basic fibroblastic growth factor has important roles
in the CNS (Hicks, 1998), inclusive of RPC. FGF-
2 is abundantly expressed in both embryonic and
adult RPC, and regulates retinal growth, organiza-
tion, and differentiation (Hicks, 1998). The prolifer-
ation of rodent retinal cells was stimulated by bFGF
(Guilarducci-Ferraz et al., 2008; Hicks and Courtois,
1992) in monolayer cultures, coupled with a sig-
nificant increase in the number of photoreceptors
(Hicks and Courtois, 1992). Both the proliferation and
differentiation induced by bFGF depend on an uncom-
mitted stage of retinal precursors, as well as of the
presence of laminin (Otaegi et al., 2007).
It has also been shown that earlier RPC express
lower levels of EGF receptors (EGF-R) than late pro-
genitors (Lillien and Wancio, 1998), suggesting that
EGF contributes to the timing of progenitor cell dif-
ferentiation (Lillien and Wancio, 1998). These data
are consistent with the previous observation of age-
dependent differences in mitotic responses to tumor
growth factor alfa (TGF), and TGF-3 in vitro
(Anchan and Reh, 1995; Lillien and Cepko, 1992;
Lillien and Wancio, 1998). Immature RPC were main-
tained in vitro as long as 6 months in serum free
medium with both bFGF and EGF (Akagi et al.,
2003), and could be stimulated to form neurospheres
(Liljekvist-Soltic et al., 2008). Both EGF and bFGF
were shown to establish Mller cell spheres (Das et al.,
2006). Either withdrawal of both factors or the addi-
tion of TGF were optimal to establish changes in the
proliferative state of RPC at the time of birth in rats
(Lillien, 1995; Lillien and Cepko, 1992).
Intraocular injections of bFGF, EGF, or insulin
stimulated the proliferation of nonpigmented cells in
distinct regions of the ciliary body. This led to dif-
ferentiation of some of these cells into various types
of retinal neurons, in a regional specific way, which
indicates differential responsiveness to growth fac-
tors (Fischer and Reh, 2003). The same properties
were observed in some retinal pigmented cells in mice
(Ahmad et al., 2000; Tropepe et al., 2000). This effect
can be obtained by either insulin or EGF, as well as
R. Linden et al.
by the co-administration of insulin with EGF or bFGF
(Fischer et al., 2002; Fischer and Reh, 2003), indicat-
ing that these growth factors have similar effects during
development of the retina. Basic FGF, EGF, and TGF
stimulate the proliferation of embryonic RPC (Anchan
and Reh, 1995; Anchan et al., 1991; Lillien and Cepko,
1992), and induce the differentiation of neurofilament-
expressing retinal ganglion cells in embryonic chick
retina in vivo (McCabe et al., 1999) as well as at
the retinal margin of the retina of postnatal chickens
(Fischer et al., 2002).
IGF-1 is expressed at high levels in the teleost
retina, and was shown to induce RPC to stop prolif-
eration and produce new rod photoreceptors (Otteson
et al., 2002). Corroborating these data, the blockade of
IGF receptors reduced the number of dividing rod pro-
genitor cells in the outer nuclear layer (Zygar et al.,
2005).
13.3.2 Interleukins
Cytokines and chemokines have often been recognized
as relevant players in neuroimmune interactions (Bauer
et al., 2007; Schafers and Sorkin, 2008; Reale et al.,
2008; Fontaine et al., 2008; Zhang et al., 2008), but lit-
tle is known of their roles in the control of the cell cycle
of neural progenitor cells (Vela et al., 2002; Vallieres
et al., 2002; Wang et al., 2007; Nakanishi et al., 2007;
Kang and Kang, 2008).
We have, however, recently shown that interleukin
(IL)-4 has an anti-proliferative effect upon RPC of
newborn mice, an effect mediated by activation of
protein kinase A. This effect was associated with
blockade of the transition from G1 to S phase, since
IL-4 enhanced expression of p27Kip1 and decreased the
expression of cyclin D1 (Fig. 13.3, Silva et al., 2008).
13.3.3 Neurotrophins
Brain-derived neurotrophic factor (BDNF) also
increased the number of neuronal progeny from
the RPC population at the expense of Mller glia
(Ahmed et al., 1995). Reports indicated that Mller
glia express both the high affinity TrkB and the low
affinity p75 receptors for neurotrophins (Garcia et
al., 2003; Oku et al., 2002), and that the loss of p75
13 Tissue Biology of Proliferation and Cell Death
A B
Fig. 13.3 Modulation of the retinal cell cycle by interleukin-
4. (a) Incubation of retinal explants from neonatal rats with IL-4
reduces the incorporation of tritiated thymide, and indication
of partial inhibition of the cell cycle. An inhibitor of protein
is implicated in the progression of retinoblastoma
(Dimaras et al., 2006). The influence of treatment with
BDNF and erythropoietin on the differentiation and
on de-differentiation of mammalian Mller glia into
RPC was analyzed in vitro (Nickerson et al., 2008).
Treatment with BDNF induced Mller glia prolifera-
tion, RPC phenotype up-regulation, and development
of sphere-like colonies, similar to treatment with both
EGF and bFGF (Das et al., 2006).
Neurotrophin-3 (NT-3) regulates neural prolifera-
tion, differentiation, and survival in the nervous system
(Chalazonitis, 2004; Frade et al., 1999). In the retina,
the expression and localization of both NT-3 and its
high affinity receptor TrkC were characterized at early
developmental stages, when RPC predominate (Das et
al., 2000). During retinal development, the disruption
of NT-3 signaling by overexpression of a truncated
TrkC isoform produced a significant reduction in the
numbers of all retinal cell types, as well as decreased
cell proliferation, suggesting that NT-3 targets RPC
rather than differentiated cells (Das et al., 2000).
Similar to other growth factors, the inhibition of RPC
proliferation is not complete, consistent with in vitro
studies showing that RPC change their responsiveness
to mitogenic signals in a spatial and temporal man-
ner during development, and that growth factors may
act in combination (Anchan and Reh, 1995; Anchan
et al., 1991; Fischer et al., 2002; Fischer and Reh,
2003; Lillien and Cepko, 1992). At later stages, NT-3
201
C
kinase A (H-89) prevents the effect of IL-4. (b, c) IL-4 induces
a decrease in content of cyclin D1 and an increase in the CDK
inhibitor p27Kip1 , which are likely to underlie the blockade of
the cell cycle (Mod. from Silva et al., 2008)
produced by retinal amacrine and RGC acts on central
retinal targets (von Bartheld et al., 1996). The mecha-
nisms of control of the cell cycle by NT-3 were studied
in oligodendroglial progenitor cells. NT-3 regulates
entry into S-phase through the expression of c-myc
and cdc2 (Kumar et al., 1998). Consequently, blockade
of NT-3 signaling, by either antibodies or overexpres-
sion of truncated TrkC (Bovolenta et al., 1996; Das et
al., 2000), inhibits the transition to S phase, and thus
lengthens the cell cycle.
13.3.4 Hedgehog, Notch and Wnt
The first member of the hedgehog (Hh) family was
described in Drosophila, but now highly conserved Hh
proteins have been identified in various organisms. The
mammalian homologues are: Sonic hedgehog (Shh),
Indian hedgehog (Ihh) and Desert hedgehog (Dhh);
and in zebrafish besides sonic hedgehog (shh), tiggy-
winkle hedgehog (twhh) was also described. These
signaling molecules are well known to regulate various
phases of the development of different tissues in a vari-
ety of organisms and are also related to the arousal of
tumors (Ingham and McMahon, 2001; Agathocleous et
al., 2007; Amato et al., 2004; Burke and Basler, 1996).
In the retina, hedgehog proteins (Hh) are required
for progression of the neurogenic wave, RPC
202
proliferation, photoreceptor differentiation, and RGC
axon growth (Russell, 2003). Hh acts as a mitogen in
the developing mammalian retina (Jensen and Wallace,
1997; Levine et al., 1997). It is expressed in mouse
retina by cells in the ganglion cell layer and inner
nuclear layer, and produces an increased number of
rod photoreceptors, amacrine cells, and Mller glial
cells (Jensen and Wallace, 1997). In fact, it was pro-
posed that Hh in the retina accelerates both the G1
and G2 phases of the cell cycle, and then pushes these
rapidly dividing cells out of the cell cycle prematurely
(Agathocleous et al., 2007), such that quiescent RPC
are converted into fast-cycling, transient amplifying
progenitors that are closer to both cell cycle exit and
differentiation, similar to that observed in other tissues
(Amato et al., 2004; Ingham and McMahon, 2001).
Mutations in the Hh and bone morphogenetic
protein-4 (BMP4) signaling pathways can give rise to
severe developmental defects, including anophthalmia-
microphthalmia (AM), retinal dystrophy, myopia,
brain anomalies, and polydactyly (Bakrania et al.,
2008). Using in situ hybridization in human embryos,
expression of BMP4 was shown in the optic vesicle,
developing retina and lens, pituitary region, and dig-
its. Reduced Hh signaling from the ventral forebrain is
likely to be the cause of cyclopia (Chan et al., 2007;
Muller et al., 2000). Moreover, it was demonstrated
that knockdown of Hh expression slows or arrests rod
and cone photoreceptor differentiation (Stenkamp et
al., 2000). Thus, Hh seems to be required for prolif-
eration and differentiation of several cell classes in the
retina.
Activation of the Notch-Delta pathway may also be
important for the maintenance of the undifferentiated
state in RPC. These cells from both rodent embryo
(E14 and E18) and adult ciliary margin express Notch,
whereas the expression of its ligand Delta increases
from E14 to adult (Ahmad et al., 2004; Bao and Cepko,
1997), particularly in the outer neuroblastic layer, sim-
ilar to the distribution of STAT3 (Shao-Min Zhang et
al., 2005). Moreover, it was shown that conditional
removal of Notch1 early in development led to a reduc-
tion in the size of the retina due to a decrease in
the number of progenitor cells and premature neu-
rogenesis. Additionally, ablation of Notch1 in early
progenitor cells led to enhanced cone photoreceptor
production, and ablation of Notch1 at later points led
to an almost exclusive production of rod photorecep-
tor cells. Altogether, these data suggest that Notch1
R. Linden et al.
not only maintains the progenitor state, but is required
to inhibit the photoreceptor fate (Jadhav et al., 2006;
Yaron et al., 2006). Jadhav and colleagues (2006))
also showed that prolonged Notch activity in progeni-
tor cells maintains cells in the progenitor state without
perturbing temporal identity, promoting early progen-
itor characteristics early in development and late pro-
genitor characteristics later in development. However,
eventually, constitutive Notch activation led these cells
to acquire characteristics of glial and stem cells. And
reactivating the Notch pathway in newly postmitotic
retinal cells promoted mature glial cell formation in a
subset of cells.
The role of Wnt in the regulation of the prolifer-
ation of progenitors in the marginal ciliary zone has
also been discussed (Kubo et al., 2003; Kubo and
Nakagawa, 2008). In addition, activation the Wnt-
Frizzled signaling pathway leads to neurosphere for-
mation of adult ciliary margin stem cells (Ahmad et
al., 2004). In adult mammalian retina, the presence of
injury could activate quiescent properties of RPC in
Mller cells in a process that involves activation of
both Wnt and Notch (Das et al., 2006).
13.3.5 Platelet Activating Factor
Platelet-activating factor (PAF) is the trivial
name for the 1-O-alcyl-2-acetyl-sn-glycero-3-
phosphorylcholine phospholipid, which elicits a wide
range of physiological responses in a variety of cell
types, such as proliferation, differentiation, inflamma-
tion and allergy (Izumi and Shimizu, 1995). Almost
all signaling pathways triggered by PAF are mediated
by a G protein-coupled receptor and tyrosine kinases
(Dhar and Shukla, 1991, 1994; Thurston et al., 1993;
Zhu and Shukla, 1993; Honda et al., 1994; Kuruvilla
et al., 1994; Liu et al., 1994).
PAF induced proliferation in human bone marrow,
fibroblasts and cell lines such as endometrial adenocar-
cinome (HEC1A) and immature erythrocytes (K562)
(Ishii and Shimizu, 2000). Tumor cell lines both syn-
thesize PAF and express its membrane receptor, thus
producing an autocrine proliferative effect together
with a paracrine effect in endothelial cells, which pro-
motes angiogenesis required for tumor establishment
and development (Dupuis et al., 1997; Bussolati et al.,
2000; Denizot et al., 2001; Montruchio et al., 2000).
13 Tissue Biology of Proliferation and Cell Death
PAF was shown to accumulate in the retina in
response to injury, such as ischemic proliferative
retinopathy or diabetic retinopathy, and was impli-
cated in vincristine-induced experimental retinopathy
and other retinal impairments (Hardy et al., 2005; de
La Cruz et al., 1998; Doly et al., 1995; Cluzel et al.,
1995). It was also shown both that acetylcholine and
dopamine promote the production of PAF in immature
cells of the embryonic chick retina (Bussolino et al.,
1988, 1989).
Both a PAF-like lipid and PAF surface receptor
were found in the developing rat retina, and treatment
with exogenous PAF partially inhibited interkinetic
nuclear migration of proliferating cells pulse-labeled
with BrdU, whereas both the total number of prolif-
erating cells and the incorporation of nucleotide were
preserved. The reduced number of cell nuclei that
reached the outer stratum following treatment with
PAF had a heterochromatic pattern of labeling for
BrdU, suggesting incorporation at the end of S phase,
at which time the dividing cells may have escaped
a period of sensitivity to the lipid. The velocity of
interkinetic nuclear migration in cells that passed the
S/G2 transition was, however, the same in either PAF-
treated or control retinal tissue. The results strongly
suggest the induction by PAF of an S/G2 checkpoint,
resulting in blockade of interkinetic nuclear migration
in the developing retina. The finding that Mller cells
produce abundant PAF suggest that glial cells may con-
trol the cell cycle through lipid mediators, inclusive of
PAF (Fragel-Madeira et al., manuscript in preparation).
13.4 Control of the Retinal Cell Cycle
by Neurotransmitters
and Neuromodulators
13.4.1 Classical Neurotransmitters
13.4.1.1 Acetylcholine
Markers of cholinergic activity (e.g. choline acetyl
transferase and acetylcholine esterase) are expressed
in the neuroblastic layer of the chick retina (Layer,
1991), and evidence have also been reported of the
expression of the cholinergic system around birthdate
in mouse and ferret (Feller et al., 1996). Starburst
amacrine cells, which are born during the first few
203
days of neurogenesis (Prada et al., 1991), are the pre-
dominant subpopulation of retinal cholinergic cells
(Baughman and Bader, 1977), in addition to immature
horizontal cells that may transiently release ACh prior
to synaptogenesis (Kim et al., 1998, 2000).
Acetylcholine (Ach) signals both ligand-gated
ionotropic receptors and metabotropic G protein-
coupled receptors, respectively known as nico-
tinic (nAChR) and muscarinic receptors (mAChR).
Activated nicotinic receptors allow sodium influx,
while activated mAChR lead either to the release
of Ca2+ from IP3 -sensitive intracellular stores or to
the production of cAMP by adenylyl cyclase (AC)
(Albuquerque et al., 1995; van Koppen and Kaiser,
2003). Both nAChR and mAChR are expressed in the
developing retina of various species. In chick retina,
nAChR mRNA is present by E4.5 (Hamassaki-Britto
et al., 1994), but both the expression and functional
studies performed in mammals indicate that expres-
sion of nicotinic receptors may be restricted to dif-
ferentiating cells (Wong, 1995; Pearson et al., 2002).
Muscarinic AChRs are present in both the avian and
mammalian retina at the start of neurogenesis (Wong,
1995; McKinnon and Nathanson, 1995; Pearson et al.,
2002). Imaging studies have shown that, as early as E3
in the chick and E20 in the rabbit, the activation of
functional mAChR in retinal progenitor cells induces
changes in the concentration of intracellular calcium
([Ca2+ ]i ) (Yamashita and Fukuda, 1993; Yamashita
et al., 1994; Wong, 1995; Pearson et al., 2002).
Regulation of cell proliferation by ACh through the
activation of mAChR has been reported in a num-
ber of biological systems. Some studies suggested that
muscarinic agonists induce cell proliferation in cul-
tured glial cells (Cohen et al., 1996, Guizzetti et al.,
1996, 1998). However, the activation of mAChR in the
developing chick retina negatively regulates the pro-
liferation of progenitor cells, by increasing the time
necessary for a progenitor cell to divide during mitosis
(Pearson et al., 2002). Regulation of RPC proliferation
by endogenous cholinergic activity was also observed
in cultures of immature rat retina. While muscarinic
antagonists led to a small increase in cell proliferation,
agonists reduced DNA synthesis and cell proliferation,
corroborating previous evidence of a negative regula-
tion of cellular proliferation after activation of mAChR
(dos Santos et al., 2003).
Interestingly, exposure to either muscarinic ago-
nists or antagonists leads to smaller or larger eyes,
204
respectively, in agreement with reported changes in the
number of proliferating cells (Pearson et al., 2002).
Also consistent with the demonstrated roles of ACh in
the regulation of RPC proliferation through mAChR,
the proportion of progenitor cells responding to mus-
carinic agonists, which is maximal during the peak
of neurogenesis, declines rapidly, and is maintained
only in differentiating neurons of the inner retina
(Yamashita et al., 1994; Wong, 1995; Pearson et al.,
2002). Although a growing number of studies demon-
strate a role of muscarinic signaling in retinal develop-
ment, it is still hard to distinguish between effects on
either cell proliferation or cell differentiation, since the
molecular mechanisms that underlie the effects of ACh
in the regulation of retinal progenitor cell proliferation
are not completely understood.
13.4.1.2 Glutamate
In adult retinas, the excitatory neurotransmitter glu-
tamate is the primary neurotransmitter of rod pho-
toreceptors, bipolar cells and ganglion cells, which
comprise the vertical visual pathway (Thoreson and
Witkovsky, 1999; Yang, 2004; Shen et al., 2006).
Glutamate is found in several areas of the developing
CNS, including the retina (Fletcher and Kalloniatis,
1997; Miranda-Contreras et al., 1998, 1999, 2000),
where glutamate was detected by immunohistochem-
istry in cells of the neuroblastic layer in both rat
and rabbit. Immunoreactivity was first detected around
birth in the rat, and during late embryonic development
(E20) in rabbit retinas (Fletcher and Kalloniatis, 1997;
Pow et al., 1994). It is possible that progenitor cells
may release glutamate into the retinal environment, but
since glutamate can be synthesized via several bio-
chemical pathways, it is not trivial to determine the
precise source of this neurotransmitter.
Glutamate signals through both ionotropic and
metabotropic receptors. Ionotropic receptors are clas-
sified in either NMDA receptors, permeable to Na+ ,
K+ and Ca2+ , or AMPA/Kainate receptors, perme-
able to Na+ and K+ , but with low permeability to
Ca2+ . However, an edited version of the subunit
GluR2 confers Ca2+ permeability to AMPA recep-
tors (Murphy and Miller, 1989; Pruss et al., 1991;
Burnashev et al., 1992), and this Ca2+ -permeable
AMPA receptor is prevalent during early development
R. Linden et al.
(Pellegrini-Giampietro et al., 1992). Metabotropic glu-
tamate receptors (mGluR) are grouped in three fami-
lies differentially coupled to G-proteins: mGluR1 and
mGluR5 receptors are linked to the IP3 /Ca2+ signal-
ing cascade (Abe et al., 1992; Aramori and Nakanishi,
1992), while other classes of metabotropic receptors
inhibit the production of cAMP (Prezeau et al., 1994).
Pioneering studies in the embryonic rabbit retina
demonstrated the expression of functional glutamate
receptors during retinal development (Wong, 1995),
which was later confirmed in other species (Allcorn et
al., 1996; Grunder et al., 2000; Martins et al., 2006).
Wong showed that activation of glutamate receptors
leads to an increase in [Ca2+ ]i in cells located in
the neuroblastic layer, possibly retinal progenitors
or recent post-mitotic cells. Interestingly, NMDA-
induced responses were restricted to differentiating
cells (Wong, 1995). In the chick retina, expression
of both AMPA/kainate subunits and the NR1 subunit
of the NMDA receptor was reported as early as E5,
mostly in immature ganglion cells (Silveira dos Santos
and Hamassaki-Britto, 2001). Imaging analysis con-
firmed the expression studies, by showing that the
induction of calcium influx by glutamate was restricted
to post-mitotic cells (Pearson et al., 2002). In the rat
retina, the NR1 subunit of the NMDA receptor was
first detected in ganglion cells at E21 and strong label-
ing was detected in ganglion, amacrine and horizontal
cells by P3 (Grunder et al., 2000). Other NMDA recep-
tor subunits (NR2A, NR2B and NR3A) have been
detected with similar patterns (Hartveit et al., 1994;
Sucher et al., 2003). All AMPA/ Kainate receptor sub-
units (GluR1, 2, 3 and 4) are expressed in ganglion and
amacrine cells by late embryonic development (E20)
in the rat retina (Grunder et al., 2000). Expression of
NMDA receptor subunit NR1 and AMPA receptor sub-
units GluR1 and GluR5 was also reported in retinal
progenitor cells, as verified by co-labeling in cells that
incorporated 3 [H]-thymidine, in both embryonic and
postnatal mouse retina (Martins et al., 2006).
Activation of glutamatergic receptors has been
shown to regulate cell proliferation in various areas
of the developing CNS. Interestingly, both stimulatory
and inhibitory effects of glutamate receptor activa-
tion upon proliferation of neuronal progenitor cells
were demonstrated. While in the cerebral cortex, glu-
tamate has an anti-proliferative effect through the
AMPA/kainate receptors (LoTurco et al., 1995; Haydar
et al., 2000), in developing striatum, activation of
13 Tissue Biology of Proliferation and Cell Death
NMDA receptor induced proliferation of progenitor
cells (Luk et al., 2003).
Glutamatergic regulation of proliferation of retinal
progenitor cells was studied in various species. In the
developing chick retina, activation of glutamate recep-
tors did not change the time retinal progenitors spent
to complete mitosis, and no change in the eye size
was observed after in vivo modulation of glutamatergic
signaling (Pearson et al., 2002). An anti-proliferative
role for glutamate upon mouse retinal progenitor cells
was recently described. It was shown that retinal pro-
genitor cells express AMPA/Kainate receptor subunits,
and that activation of these receptors induced cell
cycle exit with no effect upon cell death. Lineage
analysis studies after pharmacological blockade of
AMPA/kainate receptors, and overexpression of domi-
nant negative isoforms of GluR1 receptor, suggested a
cell-autonomous role for glutamatergic signaling in the
regulation of proliferation of progenitor cells during
development of the mouse retina (Fig. 13.4, Martins et
al., 2006). However, it cannot be ruled out that activa-
tion of glutamate receptors may regulate the expression
of other anti-proliferative extrinsic factors. The molec-
ular mechanisms underlying glutamate-induced cell
cycle exit were also examined in this study. Even
though no difference in the expression of various cell
cycle regulators, such as cyclin D1, p27Kip1 , p57Kip2 ,
A C
IRE S nuclear LacZ
LTR LTR
% clones
B 30
1
2
4 3
5 20
1 6
7
8
9
Fig. 13.4 Modulation of neurotransmitter signaling and
its effects upon clonal expansion of retinal progenitor cells
(RPC). (a) Retinal explants were infected with a replication-
incompetent retrovirus NIN-E, that encodes nuclear-localized
-galactosidase. (b) After 10 days in culture, E14.5 mouse
retinal explants were stained with X-gal and sectioned. The
clonal expansion of RPC after the modulation of glutamater-
gic signaling was determined by quantifying the number
of clones and the number of cells in each clone. (c, d)
205
CDK2/4 was observed, a decrease in the kinase activ-
ity of CDK2 was demonstrated after activation of
glutamate receptors (Martins et al., 2006). In agree-
ment with previous studies reporting induction of cell
cycle exit by cell-extrinsic factors (Nagahara et al.,
1999), a decrease in the phosphorylation of CDK2
threonine 160 was observed. These results indicate
a post-transcriptional mechanism for the glutamate-
induced cell cycle exit.
Modulation of cell proliferation in response to
glutamate-induced neuronal injury has been demon-
strated in adult retina (Dyer and Cepko, 2000; Fischer
and Reh, 2001). These studies demonstrated the induc-
tion of proliferation of Mller and microglia cells, in
response to in vivo overstimulation of glutamatergic
receptors in the chick and mouse eyes. Expression
of glutamatergic receptors was previously reported in
Mller glial cells (Grunder et al., 2000) and recent
studies suggest that direct activation of these recep-
tors by sub toxic concentrations of glutamate would
directly trigger cell proliferation of Mller glial cells
(Takeda et al., 2008). However, it cannot be ruled out
that glial cell proliferation in response to excitotoxic
insults in the adult retina could be a consequence of
secondary events that may take place in retinal tissue
after neuronal injury.
50 untreated D 50 untreated
glutamate NBQX
40 40
p=7x10
% clones
30
20
10 10
0 0
1 2 3 >4 1 2 3 >4
clone size (cells/clone) clone size (cells/clone)
Graphs show the proportion of clones containing the indicated
number of cells. Pharmacological activation (c) or blockade
(d) of glutamate receptors had opposite effects upon RPC
clonal expansion. Treatment with the AMPA/Kainate recep-
tor antagonist NBQX (100 M) decreased the proportion of
one-cell clones. Consistently, the proportion of clones con-
taining four or more cells was increased after inhibition of
non-NMDA receptor signaling (Mod from Martins et al.,
2006)
206
13.4.1.3 GABA and Glycine
The classical pathway of synthesis of GABA is through
decarboxylation of glutamate by the enzyme glutamic
acid decarboxylase (GAD). However, an alternative
pathway of GABA synthesis was demonstrated, in
which ornithine decarboxylase (ODC) is the limiting
synthesizing enzyme and putrescine is used as a sub-
strate (Yamasaki et al., 1999). In the developing chick
retina, GABA uptake and immunostaining for GABA-
positive neuroblast-like cells have been reported as
early as E6 (Frederick, 1987; Hokoc et al., 1990),
which confirmed the presence of this neurotransmitter
in the embryonic retinal environment. Elegant stud-
ies of thymidine incorporation revealed that GAD-
expressing neurons (amacrine and horizontal cells),
first detected around E10 (Hokoc et al., 1990), are pre-
dominantly born between E6 and E9 (da Costa Calaza
et al., 2000). In rats, GABA is detected in the devel-
oping retinal tissue around birth (Versaux-Botteri et
al., 1989; Fletcher and Kalloniatis, 1997). Although
the precise onset of GAD immunoreactivity in the
developing rat retina is controversial (see: Yamasaki
et al., 1999 and Dkhissi et al., 2001), the presence of
GABA in the retinal environment before the detection
of GAD-immunopositive interneurons in the chick,
and most likely in the rat retina, indicates that the
ODC-mediated pathway of GABA synthesis plays an
important role in vertebrate retinal development.
GABA signals through both ionotropic and
metabotropic receptors. Ligand-gated ion channels
include GABAA and GABAC , while GABAB receptor
is a G-protein coupled metabotropic receptor (see
Rudolph et al., 2001; Yang, 2004). GABA is widely
accepted as an inhibitory neurotransmitter, because
in the mature nervous system, activation of GABAA
receptors leads to an influx of chloride ions (Cl- )
through the receptor channel. However, during devel-
opment, GABA acts as an excitatory neurotransmitter
(see Ben-Ari, 2002 for review). A delay in the matura-
tion of the Cl- extrusion system relative to Cl- uptake
mechanisms results in a developmental window in
which the concentration of [Cl- ]i is higher than that
found in the extracellular environment (Nishi et al.,
1974; Zhang et al., 1991). During this period, GABAA
receptor activation and the consequent increase in Cl
conductance and Cl efflux result in an increase in cell
excitability. On the other hand, activation of GABAB
receptors inhibits cAMP production. This receptor
R. Linden et al.
is also indirectly coupled to Ca2+ and K+ channels
(North et al., 1987; Hill, 1985)
Expression of functional GABA receptors in the
developing retina has been extensively reported in
a number of species (Huang and Redburn, 1996;
Mitchell and Redburn, 1996; Mitchell et al., 1999;
Greka et al., 2000; Catsicas and Mobbs, 2001; Wu
and Cutting, 2001; Pearson et al., 2002). However, it
is still not clear if these receptors are expressed in
retinal progenitor cells or in cells that have already
left the cell cycle. In the mouse retina, mRNA for
the GABAA receptor subunit 6 was detected in reti-
nal progenitor cells during embryonic development,
but the expression of a functional receptor in these
cells was not determined (Young and Cepko, 2004).
In the chick retina, it has been suggested that GABA-
evoked Ca2+ response is largely restricted either to
immature neurons in the ganglion cell layer (GCL) and
inner nuclear layer (INL) or to migrating post-mitotic
cells, rather than progenitor cells (Catsicas and Mobbs,
2001; Pearson et al., 2002).
Glycine receptors are ligand-gated channels that
form both homo- and heteropentamers comprised of
multiple combinations of alpha (1-4) and beta ()
subunits. Activation of glycine receptors in the mature
nervous system leads to Cl- influx. However, similar to
GABA, activation of glycinergic receptors also induces
membrane Ca2+ influx in the developing nervous sys-
tem. In addition to glycine, other amino acids, such as
-alanine, L-alanine, L-serine, proline and taurine can
also activate glycinergic receptors.
Expression of glycine receptors in developing
retina has been shown in both rat and mouse.
Immunostaining for glycine receptors appears as dif-
fuse staining in the neuroblastic layer during early
postnatal development of the rat retina. However, this
observation does not directly demonstrate expression
of glycine receptors in retinal progenitor cells. Later in
development, glycine receptor expression is restricted
to the INL (Sassoe-Pognetto and Wassle, 1997).In the
mouse, transcripts for the alpha2 subunit (GlyR2)
were first detected at birth in the outer half of the
neuroblastic layer (Young and Cepko, 2004).
A pioneering study by Altshuler and colleagues
demonstrated that taurine was the factor within retinal-
conditioned medium responsible for inducing rod
photoreceptor fate (Altshuler et al., 1993). Taurine
is present at high levels in developing nervous
system (Sturman, 1988; Lombardini, 1991) and may
13 Tissue Biology of Proliferation and Cell Death
signal either by entering the cells through a trans-
porter or through the activation of glycine or GABA
receptors (for review, see: Tapaz, 2004). Recently,
Young and Cepko reported that endogenous taurine
present within developing retinal tissue activates both
GABAergic and glycinergic pathways in developing
mouse retina (Young and Cepko, 2004). In addition,
this study demonstrated that pharmacological block-
ade of either glycine or GABA receptors prevented the
rod-promoting effect of taurine.
However, the regulation of retinal progenitor cell
proliferation by GABA and glycine during develop-
ment appears to differ between species. Lineage anal-
ysis experiments in which glycine receptor GlyR2
was overexpressed indicated that glycinergic signaling
induces retinal progenitor cells to exit the cell cycle
(Young and Cepko, 2004). In contrast, in the devel-
oping chick retina GABA receptor activation would
not directly affect retinal progenitor cell proliferation.
Even though endogenous activation of GABA recep-
tors appears to be responsible for generating sponta-
neous [Ca2+ ]i transients in cells located at the NBL
of the embryonic chick retina (Pearson et al., 2002),
GABA-responding cells were predominantly interpha-
sic cells that expressed the early neuronal marker, -
III-tubulin (Tuj1), suggesting that, in the chick, GABA
only signals postmitotic cells. In addition, GABA had
little effect on the time taken by retinal progenitors of
the developing chick retina to progress through mitosis
(Pearson et al., 2002).
13.4.1.4 Adrenergics
The expression of adrenergic neurotransmitters and
receptors was previously reported in both the devel-
oping and the adult retina (Hadjiconstantinou et al.,
1984; Zarbin et al., 1986; Shelke et al., 1997; Lograno
et al., 2000; Woldemussie et al., 2007). In addition,
the activity of the epinephrine biosynthetic enzyme,
phenylethanolamine N-methyltransferase (PNMT)
(Cohen, 1987), as well as the activation of specific
signaling pathways by norepinephrine have been
reported during retinal development (Kubrusly et al.,
2007). However, no study has as yet demonstrated
either the expression of functional adrenergic recep-
tors in retinal progenitor cells or the regulation of
proliferation of progenitor cells by manipulation of
adrenergic signaling in developing retinal tissue.
207
13.4.1.5 Dopamine
Dopamine is expressed by a subpopulation of amacrine
cells, but is also present at very early stages in
the developing retinal environment (Martin-Martinelli
et al., 1989). The identification of dopamine-producing
cells may be made by the detection of the limiting
enzyme for its synthesis, tyrosine hydroxylase (TH).
In the rat, TH immunoreactivity was detected in late
embryonic to early postnatal development (E19-P2)
(Nguyen-Legros et al., 1983; Wu and Cepko, 1993),
by P3 in the rabbit (Osborne et al., 1984; Casini and
Brecha, 1992) and in the chick at the equivalent period
of development (E12) (Gardino et al., 1993). In addi-
tion, it was recently demonstrated that dopamine may
be produced via a TH-independent pathway. In the
chick retina, the enzyme DOPA decarboxylase (DDC),
which was first detected as early as E8, is able to pro-
duce dopamine from L-DOPA before TH expression
is detected. Only in E18, DDC was clearly identi-
fiable in dopaminergic amacrine cells. On the other
hand, around E8, expression of DDC was found in
the immature outer plexiform layer (Kubrusly et al.,
2003). Recently, it was reported that DDC is expressed
in the neuroblastic layer and INL of P0 rat retinas
(Kralj-Hans et al., 2006), suggesting that retinal pro-
genitors and/or post-mitotic cells may be a source of
dopamine synthesized from L-DOPA. It is, neverthe-
less, not yet known if this TH-independent pathway for
dopamine synthesis is present at more immature stages
of mammalian retinal development.
Dopamine acts through the activation of specific
G-protein coupled receptors, classified in two sub-
families, D1-like (receptors D1 and D5) and D2-
like (D2, D3 and D4). The former are coupled to
protein Gs , while the latter are usually coupled to
Go or Gi proteins. The distribution of D1 receptor
(D1R) immunoreactivity was investigated during post-
natal development of the rat retina and this receptor
was found already at birth (Koulen, 1999). Kralj-
Hans and colleagues (2006) confirmed the pattern
observed around P0, and also detected D1R expres-
sion in the neuroblastic layer, which suggests that
it may be expressed in either progenitors or recent
post-mitotic cells. D2R in rat retinas is mainly asso-
ciated with extrasynaptic membranes of dopaminergic
cells, as well as in processes of non-dopaminergic
amacrine cells. Derouiche and Asan (1999) also sug-
gested that D4R is the D2-like receptor expressed in
photoreceptors.
208
Some studies have already suggested a role for
dopamine in the regulation of cell proliferation in
the retinal tissue. It was reported that, in albino rats,
dopamine increased the time retinal progenitor cells
take to complete mitosis, and reduced the number of
progenitors arriving in the proliferative zone, whereas
the same effects were not observed in pigmented reti-
nas (Kralj-Hans et al., 2006). The interpretation of
these findings may be either that dopaminergic signal-
ing in developing retina regulates cell-cycle length, as
suggested by the authors, or that the reduction in the
number of proliferating cells at the proliferative zone
may also be due to regulation of migration of retinal
progenitor cells.
In another study, in vivo administration of L-DOPA
was shown to regulate cell division and the orienta-
tion of cleavage in developing rat retina (Tibber et al.,
2006). L-DOPA is believed to act through its conver-
sion to dopamine by DDC. Although it was suggested
that L-DOPA may have neurotransmitter activity inde-
pendent of its conversion, L-DOPA-specific receptors
have not been found. For example, treatment with L-
DOPA in vitro decreased the number of mitotic figures
in embryonic rat retinas (Ilia and Jeffery, 1999) and
it was not determined if this effect was dependent on
dopamine synthesis.
These studies suggest an antiproliferative role for
dopamine in developing rat retina. Testing how the
modulation of endogenous dopaminergic signaling
regulates the proliferation of retinal progenitor cells
will be important to confirm the developmental role
of this neurotransmitter. An alternative proposal is that
some extracellular factors may otherwise specifically
regulate cell-cycle length, but not cell-cycle exit, as
was shown for Shh in the developing Xenopus retina
(Locker et al., 2006).
13.4.1.6 Serotonin
Serotonin (5-hydroxytryptamine, 5-HT) acts through
the activation of 5-HT receptor subtypes. At least thir-
teen different receptor subtypes, grouped into seven
families (5HT1- 5HT7), have now been described
(Hoyer et al., 2002).
The expression of 5-HT1b was detected by in situ
hybridization in both developing and adult mouse
retina (Upton et al., 1999), apparently restricted to
the ganglion cell layer. Transcripts for both 5-HT1a
R. Linden et al.
and 5-HT7 were found in adult rat retina (Pootanakit
and Brunken, 2000). In adult rabbit retina, mRNA for
5-HT1a, 5-HT2a, 5-HT3a, 5-HT3b and 5-HT7 were
detected, and immunohistochemical assays demon-
strated protein expression of 5-HT2a and 5-HT3
(Pootanakit et al., 1999; Pootanakit and Brunken,
2001).
Conversely, De Lucchini et al. (2003) reported that,
in developing Xenopus retina, 5-HT2B and 5-HT2C
receptors are expressed at, comparatively, much earlier
stages in both the proliferative zone (stage 40) and in
the ciliary margin zone (stage 45), the latter of which
is a region at the anterior margin of the retina in some
vertebrates, where it is believed new neurons are gen-
erate throughout life. Both transcripts were expressed
in BrdU incorporating (i.e., proliferating) cells, and
expression of 5-HT2B was maintained in post-mitotic
cells located in the INL layer (De Lucchini et al.,
2003).
Evidence for the role of serotonin upon the pro-
liferation of retinal progenitor cells was obtained in
Xenopus. In this study, a combination of pharmaco-
logical and genetic manipulations was used to demon-
strate that signaling through 5-HT2B receptors ontrols
both cell proliferation and cell death in developing
Xenopus retina, with impact in morphogenesis of the
eye (De Lucchini et al., 2005). Overexpression of 5-
HT2B, followed by treatment with serotonin, increased
the number of mitotic profiles. Conversely, pharma-
cological inhibition with ritanserin and morpholinos
targeting 5-HT2B receptors resulted in smaller eyes
and disorganization of retinal cytoarchitecture. In addi-
tion, these morpholinos reduced the expression of
cyclin D1. These results strongly suggest a mitogenic
role for serotonin in developing Xenopus retina. Data
are still lacking to determine whether cyclin D1 is
required for the mitogenic effects of serotonin in vivo,
and whether this role for serotonin is conserved among
species.
13.4.1.7 ATP
Both neurons and glia are potential sources of extracel-
lular ATP. In the adult CNS, ATP is released together
with several other neurotransmitters such as ACh, glu-
tamate and GABA, and it is reasonable to suggest
that the same may happen in the developing retina. To
date, ATP release has been demonstrated in cultures of
13 Tissue Biology of Proliferation and Cell Death
isolated amacrine cells (Santos et al., 1999), Mller
glia (Newman, 2001) and embryonic chick RPE
(Pearson et al., 2005).
ATP and its related nucleotides activate both
ionotropic and metabotropic receptors. P2X receptors
are ligand-gated ion channels, permeable to Na+ , K+
and Ca2+ and P2Y receptors are G-protein coupled
receptors, predominantly coupled to the IP3 /Ca2+ cas-
cade, although some isoforms are linked with adenylyl
cyclase/cAMP. ATP receptors are among the earli-
est functional receptors (Laasberg, 1990), but due to
limited availability of research tools, such as specific
antibodies, the precise onset of the multiple purinergic
receptor subunits during development of the vertebrate
retina is still unclear. Imaging studies provided evi-
dence of the expression of functional ATP receptors
in the embryonic chick retina. ATP-induced increase
in [Ca2+ ]i through the activation of P2Y receptors was
the largest at early developmental stages (E3), and
was predominantly observed in retinal progenitor cells,
rather than immature neurons (Pearson et al., 2002,
2005; Sakaki et al., 1996; Sugioka et al., 1996).
13.4.1.8 Adenosine
G-protein-coupled receptors (GPCRs) for adenosine
were initially classified as A1 and A2, which inhibit
and stimulate cAMP accumulation, respectively. Later
these receptors were subclassified, and A2-adenosine
receptors were termed A2A, to designate the recep-
tors that required low concentrations of adenosine and
adenosine analogues for the stimulation of Adenylyl
Cyclase. A second subtype was named A2B, which
also signals via Gs . The two other receptors, called A1-
and A3-adenosine receptors, interact with pertussis-
toxin-sensitive G proteins of the Gi and Go family
(Zezula and Freissmuth, 2008).
Both A1 and A2 adenosine receptors are present
since early stages of chick retina development (Paes-
de-Carvalho, 2002). In immature mammalian retina it
was shown the expression of A2a receptors in ganglion
cells and in the INL during retinal development in dogs
(Taomoto et al., 2000). The expression of purinergic
neurotransmitters early in retinal development raised
the possibility that they might modulate proliferation
and/or differentiation.
However, the potential roles of adenosine in the
control of proliferation have not been fully described.
209
Sanches et al. (2002) found that adenosine had no
effect on the proliferation of embryonic chick retinal
cells in culture, although an earlier report described a
small reduction in [3 H]-thymidine incorporation, sug-
gesting an inhibitory effect on proliferation, in chick
retinal explant cultures (Sugioka et al., 1999b).
On the other hand evidence accumulates on the
effects of adenosine in cell survival (Fredholm, 1997;
Dunwiddie and Masino, 2001), and it may have simi-
lar actions in the developing retina. For instance, when
purified cultures of immature retinal neurons are re-
fed with fresh medium they display an intense cell
death, an effect that can be greatly reduced when the
cultures are pre-incubated with adenosine (reviewed in
Paes-de-Carvalho, 2002).
13.4.2 Neuropeptides
Various neuropeptides and/or their receptors have
already been described in the developing reti-
nal tissue of mammals, such as substance P
and other tachykinins, somatostatin, neuropeptide
Y, corticotrophin releasing factor and related pep-
tides, angiotensin, opioid peptides, pituitary adenylyl
cyclase-activating polypeptide and vasoactive intesti-
nal peptide (see for review Bagnoli et al., 2003).
However, evidence is scarce concerning the role of
neuropeptides in the control of proliferation in retinal
progenitor cells.
Several opioid peptides were detected in the mam-
malian retina. Early studies detected the expression
of [Met5 ]-enkephalin, preproenkephalin mRNA and
[Met5 ]-enkephalin binding sites in the NBL and in
the GCL of early postnatal as well as embryonic
rat retinas (Isayama et al., 1991, 1995), and pre-
proenkephalin mRNA was found in the INL of adult
rat retinas (Isayama et al., 1996). Moreover, opioid
receptors (MOP-R) were detected in ganglion cells of
rats and monkeys, and receptors for enkephalins were
described in guinea pig and developing human reti-
nas (Wamsley et al., 1981; Altschuler et al., 1982;
Yew et al., 1991; Jotwani et al., 1994). In addition,
Makman and Dvorkin (1997) also showed binding for
nociceptin (orphanin FQ) in the retina of adult rats.
Limited data suggested a role for [Met5 ]-enkephalin
in the modulation of cell proliferation (Isayama et al.,
1991). [Met5 ]-enkephalin presented a subtle effect of
210
a 10% reduction in the labeling index, defined by
the number of [3 H]-thymidine positive cells 4 h after
injection in postnatal day 1 (P1) rats. Although the
co-injection of the antagonists naloxone or naltrexone
both reverted the effect of [Met5 ]-enkephalin, only nal-
trexone induced an increase in the labeling index when
injected alone (Isayama et al., 1991).
Neuropeptide Y (NPY) was also shown as a poten-
tial modulator of the proliferation of retinal progen-
itors in rat retinal cultures (lvaro et al., 2008).
Immunoreactivity for NPY was detected in amacrine
and displaced amacrine cells in mammalian retinas. In
addition, in some species, such as cat and human, NPY
was also found in ganglion cells (Oh et al., 2002). In
the retina of the rat, NPY-positive cells appear in the
GCL at late prenatal stages, and a transient increase in
the expression of this peptide was found close to the
time of eye opening (Ferriero and Sagar, 1989).
Alvaro and collaborators (2007) showed that NPY
and NPY receptors were expressed in differentiated
cells in retinal cultures from P3-P5 rats, but the expres-
sion pattern of NPY earlier in retinal development is
not available. The same group has recently suggested
a role for NPY in the stimulation of proliferation,
which involved the participation of nitric oxide, in
cultures of retinal cells from the same developmen-
tal stage mentioned above (Alvaro et al., 2008). In
this study, treatment of dissociated cell cultures for
48 h with NPY increased in almost 65% the num-
ber of BrdU+ -nestin+ positive cells. This prolifera-
tive effect was reverted by NO synthase as well as
soluble guanylyl cyclase inhibitors, and it was sug-
gested to depend on ERK1/2 activation (Alvaro et al.,
2008).
An anti-mitogenic role was identified for pituitary
adenylyl cyclase-activating polypeptide (PACAP) in
the neonatal rat retina (Njaine et al., 2009). In this
study, we found that mRNA for PACAP precursor is
present in rat retina from embryonic day 19 (E19),
while mRNA for the PACAP-specific PAC1 recep-
tor was detected from E16 (Njaine et al., 2009).
Previously, reports of expression of PACAP were
restricted to adult rat retinas in the ganglion cell, inner
plexiform and inner nuclear layers (Seki et al., 1998),
and in fetal human retinas at 1218 weeks of gestation
(Olianas et al., 1997). On the other hand, expres-
sion of receptors for PACAP was reported in ganglion
and amacrine cells, innerplexiform layer, outer nuclear
layer and outer plexiform layer in adult rat retinas (Seki
R. Linden et al.
et al., 1997, 1998), as well as in all layers in early
postnatal rat retina (Silveira et al., 2002).
Interestingly, it has been shown that glucagon-
expressing neurons in the retina may have a role in
the regulation of the proliferation of progenitor cells
in the CMZ of the postnatal chicken eye (Fischer
et al., 2005). Glucagon is a neuropeptide highly con-
served among species that belong to the same family of
PACAP, and was reportedly expressed by a subpopula-
tion of amacrine cells in the chicken retina (Tornqvist
et al., 1981; Kuwayama et al., 1982; Tornqvist and
Ehinger, 1983; Ekman and Tornqvist, 1985), although
its roles are not precisely known. It was suggested that
glucagon antagonizes the role of insulin or insulin-like
growth factor in the regulation of the proliferation of
retinal progenitors in the CMZ, to control de addition
of new cells to the edge of the retina (Fischer et al.,
2005).
13.5 Signal Transduction in the Extrinsic
Control of the Retinal Cell Cycle
Extracellular mediators, including neurotransmitters,
activate a number of second messengers and signal-
ing pathways that may act upon the retinal cell cycle
(Martins and Pearson, 2008 for review). For the most
part, however, little is known of signal transduction
triggered by extracellular modulators of the cell cycle
in the nervous system.
Transients of intracellular calcium are required for
progression through several steps in the proliferative
cell cycle including the G1/S-phase transition, S phase,
entry into mitosis and key points within mitosis includ-
ing the metaphaseanaphase transition and induction
of cytokinesis (Short et al., 1993; Berridge, 1995;
Santella et al., 1998; Whitaker and Larman, 2001).
For exemple, P2Y receptor stimulation leads to the
activation of the IP3 /Ca2+ second messenger cascade
(Sugioka et al., 1999a; Pearson et al., 2002; Sanches
et al., 2002). This leads to the activation of phospho-
lipase C (PLC), activation of PKC and stimulation of
the ERK/MAPK cascade (Sanches et al., 2002; Nunes
et al., 2007). The ERK/MAPK pathway is usually
associated with stimulation of cell-cycle progression,
primarily by relieving cell-cycle blockades at the step
between G1 and S-phase (Sherr, 1996). Noteworthy,
13 Tissue Biology of Proliferation and Cell Death
evidence was gathered that factors released by
postmitotic cells may interfere with the sensitivity of
the P2 receptors (Frana et al., 2007), which suggests
tissue constraints upon the regulation of the cell cycle
of retinal progenitor cells by purinergic receptors.
A number of extracellular mediators act via either
stimulatory or inhibitory G-protein-coupled receptors
to modulate adenylyl cyclase and the conversion of
ATP to cAMP. An important downstream effector
of cAMP is protein kinase A (PKA), which phos-
phorylates a wide array of target proteins that reg-
ulate diverse cellular functions. For example, phos-
phorylation of the transcription factor CREB (cAMP
Response Element Binding protein) regulates the
expression of various genes involved in cell survival
and synaptic plasticity (Tao et al., 1998; Patterson
et al., 2001). Recent data suggest that neurotrans-
mitters may act via cAMP to regulate the expres-
sion of cell cycle molecules in cultured mouse stem
cells. Thus, dopamine receptor activation led to a
decrease in DNA synthesis and reduced expression of
cyclins and CDK (Lee et al., 2006). Activation of the
cAMP/PKA pathway inhibited the cell cycle exit of
zebrafish retinoblasts (Masai et al., 2005). Forskolin
treatment, which increases the levels of cAMP, com-
pletely inhibits the generation of new post-mitotic cells
in the neural retina. Thus, in developing zebrafish
retina activation of PKA causes retinal progenitor cells
to remain in the cell cycle, although the molecular
mechanisms that underlie this effect are not clear. One
possibility is that PKA inhibits the expression of the
CDK inhibitor p27Kip1 . Alternatively, a major PKA
substrate, CREB, binds to the promoter and may reg-
ulate the expression of cell-cycle regulators such as
cyclin D1 (Lonze and Ginty, 2002). In contrast with
the data observed in zebrafish, cAMP/PKA antago-
nizes the proliferative role of Shh in other areas of the
nervous system in mammals (Waschek et al., 2006).
In addition, PKA-mediated regulation of WEE kinases
and cdc25 phosphatase activities was also reported (for
reviews, see Shibuya, 2003; Han and Conti, 2006). We
have also found that activation of PKA is involved in
the anti-proliferative effect of interleukin-4 upon the
developing retina, associated with increased expres-
sion of p27kip1 and reduced cyclin D1 (Silva et al.,
2008).
Another important intracellular signaling path-
way activated by extracellular mitogens involves the
enzyme PI3-kinase (PI3K). Downstream targets of
211
PI3K include protein kinases, such as PDKs, Akt and
S6 kinase, and regulators of small GTPases (Rodgers
and Theibert, 2002; Cantrell, 2001; Wymann and
Pirola, 1998 for reviews). Recently, it was reported that
ATP, acting via P2YR, might promote the prolifera-
tion of neural stem cells through the PI3K-dependent
pathway (Ryu et al., 2003; Heo and Han, 2006).
The expression of signal transducers and activators
of transcription (STATs) were modulated specifically
by some growth factors, and STAT3 was activated in a
restricted spatial pattern in the presence of aFGF, cil-
iary neurotrophic factor (CNTF), leukemia inhibitory
factor (LIF), and interferon- (IFN-), but not by
bFGF, EGF, IFN-, and retinoic acid (RA) (Shao-
Min Zhang et al., 2005). Since activated STAT3 co-
localized with incorporation of BrdU, it is likely that
STAT3 is directly involved in maintaining RPC in
mammalian retina. In fact, it was shown that STAT3
acts in the transition from G1 to S phase of the cell
cycle, through up-regulation of cyclins D2, D3 and
A, and cdc25A, with simultaneous down-regulation of
p21 and p27Kip1 (Fukada et al., 1998), which agrees
with studies that indicated a pivotal role for STAT3
in the maintenance of the undifferentiated state and
pluripotency of embryonic stem cells in the CNS
(Bauer and Patterson, 2006; Niwa et al., 1998).
Regarding the effects of PAF upon the retinal cell
cycle, the inhibition of either p38 or p42/44 MAPKs,
but not of PKC, prevented the inhibition of interkinetic
nuclear migration by PAF. PAF also induced activa-
tion of Chk1, which is likely responsible for the cell
cycle arrest at the S/G2 transition, because a pharma-
cological inhibitor of Chk1 prevented the effect of PAF
(Fragel-Madeira et al., unpublished).
Finally, other protein kinases have been implicated
in the cell cycle or in interkinetic nuclear migration,
although upstream pathways have not been identified.
Thus, in the developing rat retina, MAP kinases of
the p38 family were shown to regulate the metaphase-
anaphase transition within M-phase (Campos et al.,
2002), whereas CK2 was implicated in various phases
of the retinal cell cycle, including motor aspects of
interkinetic nuclear migration (Fig. 13.5, Carneiro et
al., 2008).
Therefore, several signal transduction pathways
have already been implicated in the control of the reti-
nal cell cycle by neuroactive substances. Nonetheless,
the definition of the relevant pathways is still fragmen-
tary.
212
A B
C 125
3H-TDR INCORPORATION (% control)
100
75
50
25
CTR 1 3
TBB (M)
Fig. 13.5 Role of CK2 in the retinal cell cycle. (a, b)
Distribution of BrdU-labeled nuclei within the NBL (see Fig.
13.1), at 3 h of incubation of retinal explants made 30 min
after intraperitoneal injection of the nucleotide analogue, show-
ing normal interkinetic nuclear migration (a), and its blockade
by 10 M 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor
of CK2 activity (b). (c) TBB also inhibits incorporation of
tritiated thymidine into retinal tissue, indicating that CK2 activ-
ity is required in S-phase. AraC = cytosine-arabinofuranoside.
Control experiments showed that the blockade of nuclear migra-
tion is not a consequence of the blockade of DNA synthesis
(Mod. from Carneiro et al., 2008)
13.6 Death and Survival of Retinal
Progenitor Cells
Extensive work has been reported on naturally-
occurring cell death during development of the nervous
system. It is well established that programmed cell
death occurs not only among differentiating neurons
forming synaptic connections, but also among neural
progenitor cells (de la Rosa and de Pablo, 2000; Lossi
and Merighi, 2003; Buss et al., 2006).
Notwithstanding several earlier studies that demon-
strated both the occurrence and extrinsic control of
retinal progenitor cell death (Silver and Robb, 1979;
Cuadros and Rios, 1988; Martin-Partido et al., 1988;
Frade et al., 1996b, 1997; Diaz et al., 1999, 2000),
only recently has evidence appeared on intracellular
R. Linden et al.
mechanisms that modulate cell death among early neu-
ral progenitors. It has been shown that developing
photoreceptors undergo progressive changes in mech-
anisms of programmed cell death, associated with the
transition from the proliferative state to the stage of
phenotypic differentiation. Thus, in the developing
rat retina, although the degeneration of both photore-
ceptors and proliferating/immediate postmitotic cells
induced by various agents depended on protein syn-
thesis, an increase of intracellular cyclic AMP blocked
degeneration of postmitotic, but not of proliferat-
ing photoreceptor precursors (Chiarini et al., 2003).
Recently, it was shown that neuroprotection provided
by insulin in the chick retina is distinctly mediated
by either the MAP kinase or the PI3-kinase signal-
ing pathways in either proliferating or postmitotic
differentiating cells (Chavarria et al., 2007).
10 AraC On the other hand, despite the reports that the den-
sity of pyknotic profiles in the neuroblastic layer of the
chick retina, prior to all but the genesis of ganglion
cells, is of the same order of magnitude as for post-
mitotic ganglion cells (de la Rosa and de Pablo, 2000;
Chavarria et al., 2007), it is as difficult to estimate the
true rates of cell death among proliferating neural pro-
genitor cells as it is for postmitotic neurons (Linden
and Reese, 2006). One of the major reasons for the
lack of reliable estimates of the absolute magnitude
of developmental cell death is the multiplicity of cell
death programs available to the developing cells (Fig.
13.6, Guimares and Linden, 2004), which cast doubts
on the estimates usually made on the basis of counting
pyknotic profiles in sections of the developing nervous
system (Linden and Reese, 2006).
Nonetheless, some evidence is available of factors
that affect the sensitivity of RPC to programmed cell
death depending on the phase of the cell cycle. Both the
major cell death programs and evidence for differential
sensitivity to the induction of cell death among distinct
phases of the cell cycle will be reviewed in this section.
13.6.1 Mechanisms of Cell Death
13.6.1.1 Apoptosis
Apoptosis, or programmed cell death type I, has been
extensively studied due to its importance for the devel-
opment of multicellular organisms, homeostasis of
13 Tissue Biology of Proliferation and Cell Death 213

PCD Type III (Necrosis)

Loss of membrane integrity
PCD Type II (Autophagy) Cell swelling
Degradation of organeles Organelle swelling
PCD Type I (Apoptosis)
No change in cell size No DNA laddering
Cell shrinkage
No DNA laddering
Blebbing
Partial chromatin condensation
Chromatin condensation
Numerous autophagic vesicles
DNA laddering
Nuclear fragmentation
Membrane integrity maintained

Fig. 13.6 Major types of programmed cell death. Expanding
the classical distinction between apoptosis, as programmed cell
death, and necrosis, as accidental cell death (Kerr et al., 1972),
current studies support at least 3 major types of programmed
cell death (Guimares and Linden, 2004), all of which have been
detected in the nervous system, and are likely to affect neural
progenitor cells in specific circumstances. Cornification has been
recognized as a fourth major type of programmed cell death by a
nomenclature committee (Kroemer et al., 2008), but is exclusive
of upper epidermal layers and unlikely to affect progenitor cells
in any tissue

adult tissues, as well as human diseases. It is acknowl-
edged as part of a natural process to remove damaged
cells during metazoan development, and to sculp-
ture tissues and organs, a good example of which is
the formation of digits (Chen and Zhao, 1998; Kerr,
2002; Borges et al., 2008, Domingos and Steller, 2007;
Degterev and Yuan, 2008). Besides physiological con-
ditions, apoptosis is one of the pathways by which
damaged cells are eliminated in pathological circum-
stances, or following genotoxic stress such as in cancer
treatment.
The term apoptosis was coined by Kerr in 1972 to
define an active, organized cell death process involv-
ing membrane blebbing, shrinkage of the cytoplasm
and nucleus, which generate pyknotic features. Then,
apoptotic cells disintegrate into apoptotic bodies sur-
rounded by an intact cell membrane that are engulfed
by surrounded cells and macrophages. Apoptosis is
often accompanied by biochemical markers such as
DNA fragmentation, mitochondrial membrane perme-
abilization (MMP), and activation of aspartate-specific
cystein-dependent proteases called caspases (Kerr,
2002; DSa et al., 2003; Kumar, 2007).
Apoptosis has, nonetheless, been more recently
recognized as a cell death process that could
occurs through caspase-dependent or independent
pathways. DNA condensation and fragmentation can
occur by a caspase-independent pathway. The best-
studied mechanism include the nuclear translocation of
Apoptosis-Inducing Factor (AIF), a flavoprotein local-
ized in the mitochondrial intermembrane space that is
conserved in eukaryotic cells from yeasts to humans
(Lorenzo et al., 1999; Cande et al., 2002). Cleavage
and release of AIF from mitochondria is regulated by
calpain I. Calpains are cystein proteases that are acti-
vated by autoprocessing due to intracellular calcium
increase. Calpain I and II (micro- and milli-calpain) are
expressed in the retina and are activated by micromolar
and millimolar calcium concentrations, respectively.
Activation of calpain has been associated with cell
death in retinal degeneration models (Marigo, 2007).
The caspase-dependent machinery was revealed
first from studies of developmentally programmed cell
death in the nematode C. elegans (Domingos and
Steller, 2007; Kumar, 2007) and is evolutionarily con-
served (Liang et al., 2008; Low et al., 2008; Low
214
and Yang, 2008). Mice lacking either caspase-3 or
caspase-9 show an excess of cells in the develop-
ing nervous system, as a consequence of decreased
rates of apoptosis among immature post-mitotic neu-
rons and progenitor cells. In addition, mice deficient
in molecules that are important for caspase 9 activa-
tion, such as cytochrome c or Apaf-1, also show an
excess of cells in nervous tissue (Kuida et al., 1996;
Cecconi et al., 1998; Hakem et al., 1998; Kuida et al.,
1998; Yoshida et al., 1998; Kuan et al., 2000; Hao
et al., 2005). These data support an important role of
caspases at the core of the cell death pathways in the
nervous system.
How caspases are activated will depend on the apop-
totic stimuli. In summary, if cell death is triggered by
extrinsic factors, such as tumor necrosis factor (TNF)
or Fas ligand (FasL), transmembrane receptors will
activate caspase 8, which will activate downstream cas-
pase 3, to execute cell death (Jin and El-Deiry, 2005;
Kim, 2005). If apoptosis is triggered by an intrinsic
damage, such as DNA damage, caspase activation will
usually depend on activation of p53 (Hurley and Bunz,
2007; Lavin and Kozlov, 2007; Borges et al., 2008).
The tumor suppressor p53 is a downstream target of
the DNA damage-signaling network, which involves
apical kinases such as ataxia-telangiectasia mutated
(ATM), and downstream kinases such as Chk2. ATM
and Chk2 phosphorylate p53, respectively at serines 15
and 20, leading to its stabilization and activation of its
transcriptional function (Hurley and Bunz, 2007; Lavin
and Kozlov, 2007).
Among p53 induced genes, the pro-apoptotic mem-
bers of the B-cell lymphoma protein-2 (Bcl-2) family,
such as Puma, Noxa, Bax and Bak, are major players
to induce cell death. Puma (Yu and Zhang, 2003; Roos
and Kaina, 2006) and Noxa (Roos and Kaina, 2006)
will activate Bax or Bak to cause cytochrome c release
from the mitochondria (Abu-Qare and Abou-Donia,
2001). Cytochrome c binds to and stimulates apop-
totic protease-activating factor-1 (Apaf-1) to assemble
the apoptosome, leading to the activation of caspase
9 (Hajra and Liu, 2004; Riedl and Salvesen, 2007).
Active caspase 9 will then activate caspase 3 in a
feedback loop to increase caspase activation, and to
cleave cellular substrates that will lead to morpho-
logical and biochemical characteristics of apoptosis
(Enari et al., 1998; Sahara et al., 1999). Moreover, cas-
pases cleave DNA repair enzymes and transcription
R. Linden et al.
regulators, including the retinoblastoma protein (Rb)
(Chau et al., 2002), which are substrates involved in the
regulation of apoptosis. For example, the developing
ganglion cell layer that expresses a mutant RB, resis-
tant to caspase cleavage, shows less cell death induced
by axonal damage (Chau et al., 2002). Retinal progen-
itors, however, do not depend on RB cleavage for cell
death induced by ionizing radiation (Chau et al., 2002).
Inhibition or deletion of caspases will delay or block
cell death, showing the central role of caspases on the
apoptotic process. Nevertheless, cells will often find an
alternative way to die by other mechanism (Guimares
and Linden, 2004).
13.6.1.2 Autophagy
Autophagy is an evolutionarily conserved, homeo-
static process that allows for lysosomal-dependent
degradation of long-lived proteins and organelles
(Tasdemir et al., 2008). Under either starvation or
stress, autophagy provides a cell protective func-
tion. However, it can also induce cell death if over-
stimulated (Guimares and Linden, 2004; Galluzzi
et al., 2008). Strategies for discriminating cell death
despite autophagic attempts at adaptation from cell
death through autophagy are a current focus of discus-
sion (Tasdemir et al., 2008).
Autophagic cell death is characterized by mas-
sive accumulation of large autophagic vacuoles called
autophagosomes, derived from part of the endoplas-
mic reticulum. The contents of autophagosomes are
degraded by lysosomal enzymes after autophagosomes
fuse with lysosomes (Guimares and Linden, 2004;
Kim, 2005; Degterev and Yuan, 2008; Galluzzi et
al., 2008; Tasdemir et al., 2008). In addition to the
presence of the double-membrane vesicular organelles,
autophagic cell death can show partial chromatin con-
densation, but neither cell shrinkage nor DNA ladder-
ing are observed (Henriquez et al., 2008).
Autophagic and apoptotic processes are intercon-
nected in several ways. For example, molecules in
the apoptotic pathways are also related to autophagy,
i.e. the p53 protein, as stated above. Another exam-
ple is the autophagy-specific gene Bcn-1, which is
identified as a novel member of the pro-apoptotic
BH3-only family of Bcl-2 proteins (Henriquez et al.,
2008). Moreover, inducers of apoptosis can induce
13 Tissue Biology of Proliferation and Cell Death
autophagy and vice-versa (Ciechomska et al., 2008)
and the blockade of one type of cell death may
increase the other (Ciechomska et al., 2008). For exam-
ple, the large-scale loss of differentiating cells, which
occurs during normal neural development and requires
classical apoptotic proteins (caspases and APF-1),
can undergo caspase-independent cell death involv-
ing autophagy when apoptosis is impaired (Oppenheim
et al., 2008). On the other hand, inhibition of
autophagy sensitizes cells to caspase-dependent apop-
tosis (Tasdemir et al., 2008).
13.6.1.3 Necrosis
13.6.2 Sensitivity to Cell Death Within
A typical necrotic cell is swollen, with enlarged
organelles and no DNA laddering is present. Moreover,
necrotic cells lose membrane integrity, which cause
spillage of intracellular contents and triggers inflam-
mation. In tissue culture and in the absence of phago-
cytosis, apoptotic cells become secondarily necrotic,
which sometimes obscures studies of cell death.
Besides morphological criteria, however, there is no
clear biochemical hallmark of necrotic cell death leav-
ing characterization of negative markers for apoptosis
and autophagy (Krysko et al., 2008).
Overwhelming physical (membrane damage, DNA
damage, loss of ion homeostasis) or chemical (tox-
icity, ATP depletion) stress, incompatible with cell
survival, causes necrosis. Consistent with this view,
necrotic cell death is frequently detected under patho-
logical conditions. For example, at the core of ischemic
areas, necrotic figures are found, whereas apoptotic
features predominate at peripheral regions. In addi-
tion to pathological conditions, necrosis occurs also
during development and represents a third type of
programmed cell death (Type III).
Cell death has historically been subdivided into
regulated (apoptosis) and unregulated mechanisms
(necrosis) (Borges et al., 2008; Degterev and Yuan,
2008; Henriquez et al., 2008). Recent findings,
however, indicate that necrosis not only can be
accidental but also programmed (Guimares and
Linden, 2004; Degterev and Yuan, 2008; Henriquez
et al., 2008). Indeed, when both apoptosis and
autophagy are blocked, a necrotic form of cell death
ensues. For example, the simultaneous treatment with
pan-caspase-3 inhibitor and the autophagy inhibitor
215
3-methyladenine (3-MA) increase light-induced
necrosis of photoreceptors (Kunchithapautham and
Rohrer, 2007).
These results changed the view of necrosis as
a passive form of degeneration and suggest that
it represent a backup pathway (Henriquez et al.,
2008). Mechanisms of programmed necrosis, also
known as necroptosis, are currently under investigation
(Degterev et al., 2005, Degterev and Yuan, 2008; Li et
al., 2008), but details remain to be elucidated.
the Retinal Cell Cycle
Sensitivity to cell death among proliferating cells
has been investigated in several models using ioniz-
ing radiation (IR). This stimulus will mostly induce
caspase-dependent apoptosis, which can be identified
in vivo by the detection of TUNEL-positive condensed
profiles. The use of ionizing radiation has additional
advantages for the study of intrinsic sensitivity to DNA
damage during the cell cycle, since this physical agent
penetrates tissue and even organisms independent on
the permeability of biologic membranes.
In several cell lines, the highest radioresistant stage
of the cell cycle is found in middle to late S phase
(Cheong et al., 1994; Biade et al., 1997). In the devel-
oping retina, IR induced two waves of cell death in
the NBL, and these were related with distinct phases
of the cell cycle. Approximately 60% of the apoptotic
profiles found at 6 hours after irradiation were neu-
roblasts, the nuclei of which are preferentially located
at the outermost region of the proliferative zone. This
corresponds to the expected location of the G2/M
transition (Fujita, 1962), which is usually one of the
most radiosensitive phases of the cell cycle (Cheong
et al., 1994). However, 40% of the early apoptotic
cells were young post-mitotic cells, since they were
neither labeled with proliferation nor with cellular dif-
ferentiation markers (Borges and Linden, 1999). The
second wave of apoptosis was observed 24 hr after
IR, close to the inner margin of the NBL, where the
nuclei of cells in S phase are located. These two waves
are likely independent, since an increase in lipid per-
oxidation was detected at 6 hr, but not at 24 hr after
216
-IRRADIATION
EARLY APOPTOSIS (6h)
-40% POST-MITOTIC CELLS
-60% PCNA+ CELLS (out of S-phase)
lipid peroxidadion
rescued by PDTC
Fig. 13.7 Two waves of programmed cell death induced
by gamma irradiation of retinal progenitor cells in S-phase.
An early wave of apoptosis kills both undifferentiated post-
mitotic cells and proliferating neuroblasts which were not in
S-phase at the time of irradiation, and depends on an increase
in lipid peroxidation. A later wave of apoptosis kills neuroblasts
IR. Accordingly, the antioxidant pyrrolidinedithiocar-
bamate (PDTC) prevented the first, but not the second
wave of apoptosis (Fig. 13.7, Borges and Linden,
1999). Therefore, the studies of IR-induced cell death
in the developing retina of rodents demonstrated that
the dying retinal cells could be grouped in at least
two populations that exhibit spatially and temporally
distinct apoptotic responses, which are influenced by
developmental stage as well as by the phase of the
cell cycle (Borges and Linden, 1999; Borges et al.,
2004).
R. Linden et al.
LATE APOPTOSIS (24h)
NEUROBLASTS IN S-PHASE
no increased lipid peroxidation
not rescued by PDTC
that are in S-phase cells at the time of irradiation, either when
cells re-enter a second S-phase after irradiation, or following
a prolonged intra-S checkpoint, and does not depend on lipid
peroxidation. Both waves of apoptosis require ATM and phos-
phorylation of p53 in serine-18 (Mod from Borges and Linden,
1999 and Borges et al., 2004, 2008).
13.6.3 Molecular Mechanisms of Cell
Death Among Retinal Progenitor
Cells
Proliferating cells are preferential targets of several
cytotoxic stimuli, among which blockers of cell cycle
kinases and proteasome inhibitors. Pharmacological
blockers of cyclin-dependent kinases (CDK), such as
deferoxamine (DFO), mimosine (MIMO), and olo-
moucine (OLO) induce cell death in retinal explants.
13 Tissue Biology of Proliferation and Cell Death
These cell cycle inhibitors induced cell death in the
retinal neuroblastic layer (NBL), where DFO and
MIMO killed only proliferating cells, whereas OLO
affected both proliferating and undifferentiated post-
mitotic cells (Rehen et al., 2002). The inhibitor of
proteasome function carbobenzoxyl-leucinyl-leucinyl-
leucinal (MG132) induced cell death in a subset of
cells within the neuroblastic layer of the retinal tissue.
MG132-sensitive population included both proliferat-
ing cells, most likely in their last round of cell division,
and post-mitotic undifferentiated cells. These results
show that both retinal progenitors and retinal cells in
the transition from the cell cycle to the differentiated
state are particularly sensitive to cell death induced by
either proteasome or CDK inhibitors, thus defining a
window of development with particular mechanism of
cell death (Neves et al., 2001; Rehen et al., 2002).
Irradiation induces a complex death response in the
developing retinal tissue both in vivo and in vitro,
involving progenitors at distinct phases of the cell
cycle and stages of differentiation. Although these
waves showed different sensitivity to lipid peroxida-
tion (Borges and Linden, 1999), they share similar
mechanism of execution of apoptosis regarding the
DNA damage-activated network. Genetic studies have
shown that the ATM-p53 pathway is necessary for
both waves of programmed cell death (Borges et al.,
2004). Genes of the ATM-p53 pathway, however, con-
tributed differently, depending on the dose of IR used.
For instance, while the lack of p53 resulted in com-
plete resistance to both low and high doses (2 Gy and
14 Gy), deficiency of Atm conferred only partial resis-
tance, suggesting that other pathways converge upon
p53 to induce cell death. The defect of apoptosis in
p53 knockout mice is comparable to Puma-/- mice,
suggesting that p53-dependent cell death is mainly
meditated thought activation of Puma (Yu and Zhang,
2003).
ATM can contribute to activation of p53 both
directly and indirectly, through activation of the Chk2
protein kinase. Knockout mice for Chk2 are defec-
tive in apoptosis induced by 5-8 Gy of IR in all areas
of the developing CNS examined (Takai et al., 2002).
Thus, similar to p53, Chk2 is required for IR-induced
apoptosis.
The importance of several posttranslational mod-
ifications in p53 was investigated in vivo by tak-
ing advantage of knock-in mutations. For instance,
knock-in mice p53-mutated at serine 18 (the direct
217
phosphorylation site of ATM in mice, comparable to
serine 15 in humans), showed a response to IR similar
to Atm mutant mice (Borges et al., 2004). This result
suggests that major contribution of ATM to cell death
is through direct activation of p53. Moreover, a lysine
residue in mouse p53, Lys317, is also acetylated in
vivo after DNA damage, and a knock-in mutation at
this site indicated that acetylation negatively regulates
p53-induced apoptosis in the developing retina (Chao
et al., 2006). Therefore, posttranslational modifications
of p53 can result in either positive or negative regula-
tion of apoptosis. These findings further illustrate the
complex regulation of the DNA damage response.
Interestingly, despite caspase activation, in RPC
no impairment of DNA fragmentation following ion-
izing radiation was observed in the presence of a
pan-caspase inhibitor, indicating p53-dependent, but
caspase-independent pathways of cell death (Herzog
et al., 2007).
In conclusion, cells in either distinct stage of dif-
ferentiation or distinct phases of the cell cycle show
diverse sensitivity to the same death stimulus. This
selective sensitivity either reflects upon the timing of
cellular demise, or defines a window of development
with selective sensitivity to specific mechanisms of cell
death. In addition, it is now clear that cell death can
occur with a variety of morphologies and mechanisms,
even when cells are exposed to the same stimulus. An
extensive crosstalk does exist among the 3 major types
of programmed cell death.
13.7 Conclusion and Future Directions
The data above show that the rate of prolifera-
tion of progenitor cells in the developing retina is
deeply affected by a variety of signals from neigh-
boring cells. Some of these signals modulate specif-
ically certain cell cycle transitions, and a few com-
ponents of signal transduction have been identified.
Nevertheless, most details are still missing as to
how the cell cycle is regulated by the barrage of
incoming signals produced by differentiated and pro-
liferating cells in their vicinity. In turn, since the
phase of the cell cycle also affects the sensitiv-
ity to cell death, both the capacity to proliferate
as well as the survival of stem/progenitor cells are
likely to depend on the tissue environment, including
218
both the identity and the activity of the various cell
types.
Understanding the life cycle and the determinants
of cell proliferation, differentiation and survival of
stem/progenitor cells is required for their successful
application in novel therapeutic procedures. Further
studies of the tissue biology of stem/progenitor cells
are thus warranted, to provide the basic knowledge
necessary for the safe development of cell therapies for
retinal, as well as other neural degenerations.
References
Abbas T, Dutta A (2006) CDK2-activating kinase (CAK): more
questions than answers. Cell Cycle 5:11231124.
Abe T, Sugihara H, Nawa H, Shigemoto R, Mizuno N,
Nakanishi S (1992) Molecular characterization of a novel
metabotropic glutamate receptor mGluR5 coupled to inos-
itol phosphate/Ca2+ signal transduction. J Biol Chem
267:1336113368.
Abraham RT (2001) Cell cycle checkpoint signaling through the
ATM and ATR kinases. Genes Dev 15:21772196.
Abu-Qare AW, Abou-Donia MB (2001) Biomarkers of apop-
tosis: release of cytochrome c, activation of caspase-3,
induction of 8-hydroxy-2 -deoxyguanosine, increased 3-
nitrotyrosine, and alteration of p53 gene. J Toxicol Environ
Health B Crit Rev 4:313332.
Adler R (1986) Developmental predetermination of the struc-
tural and molecular polarization of photoreceptor cells. Dev
Biol 117:520527.
Agathocleous M, Locker M, Harris WA, Perron M (2007) A gen-
eral role of hedgehog in the regulation of proliferation. Cell
Cycle 6:156159.
Ahmad I, Das AV, James J, Bhattacharya S, Zhao X (2004)
Neural stem cells in the mammalian eye: types and regula-
tion. Semin Cell Dev Biol 15:5362.
Ahmad I, Tang L, Pham H (2000) Identification of neural pro-
genitors in the adult mammalian eye. Biochem Biophys Res
Commun 270:517521.
Ahmed S, Reynolds BA, Weiss S (1995) BDNF enhances the
differentiation but not the survival of CNS stem cell-derived
neuronal precursors. J Neurosci 15:57655778.
Akagi T, Haruta M, Akita J, Nishida A, Honda Y, Takahashi M
(2003) Different characteristics of rat retinal progenitor cells
from different culture periods. Neurosci Lett 341:213216.
Albuquerque EX, Pereira EF, Castro NG, Alkondon M,
Reinhardt S, Schroder H, Maelicke A (1995) Nicotinic recep-
tor function in the mammalian central nervous system. Ann
N Y Acad Sci 757:4872.
Alexiades MR, Cepko CL (1996) Quantitative analysis of pro-
liferation and cell cycle length during development of the rat
retina. Dev Dyn 205:293307.
Allcorn S, Catsicas M, Mobbs P (1996) Developmental expres-
sion and self-regulation of Ca2+ entry via AMPA/KA recep-
tors in the embryonic chick retina. Eur J Neurosci 8:
24992510.
R. Linden et al.
Altschuler RA, Mosinger JL, Hoffman DW, Parakkal MH
(1982) Immunocytochemical localization of enkephalin-like
immunoreactivity in the retina of the guinea pig. Proc Natl
Acad Sci U S A 79:23982400.
Altshuler D, Lo Turco JJ, Rush J, Cepko C (1993) Taurine pro-
motes the differentiation of a vertebrate retinal cell type in
vitro. Development 119:13171328.
Alvaro AR, Martins J, Arajo IM, Rosmaninho-Salgado J,
Ambrsio AF, Cavadas C (2008) Neuropeptide Y stimulates
retinal neural cell proliferation involvement of nitric oxide.
J Neurochem 105:25012510.
Alvaro AR, Rosmaninho-Salgado J, Santiago AR, Martins J,
Aveleira C, Santos PF, Pereira T, Gouveia D, Carvalho AL,
Grouzmann E, Ambrsio AF, Cavadas C (2007) NPY in rat
retina is present in neurons, in endothelial cells and also in
microglial and Mller cells. Neurochem Int 50:757763.
Amato MA, Boy S, Perron M (2004) Hedgehog signaling in ver-
tebrate eye development: a growing puzzle. Cell Mol Life Sci
61:899910.
Anchan RM, Reh TA (1995) Transforming growth factor-beta-
3 is mitogenic for rat retinal progenitor cells in vitro.
J Neurobiol 28:133145.
Anchan RM, Reh TA, Angello J, Balliet A, Walker M (1991)
EGF and TGF-alpha stimulate retinal neuroepithelial cell
proliferation in vitro. Neuron 6:923936.
Aramori I, Nakanishi S (1992) Signal transduction and pharma-
cological characteristics of a metabotropic glutamate recep-
tor, mGluR1, in transfected CHO cells. Neuron 8:757765.
Bagnoli P, Dal Monte M, Casini G (2003) Expression of neu-
ropeptides and their receptors in the developing retina of
mammals. Histol Histopathol 18:12191242.
Bakrania P, Efthymiou M, Klein JC, Salt A, Bunyan DJ, Wyatt
A, Ponting CP, Martin A, Williams S, Lindley V, Gilmore
J, Restori M, Robson AG, Neveu MM, Holder GE, Collin
JR, Robinson DO, Farndon P, Johansen-Berg H, Gerrelli D,
Ragge NK (2008) Mutations in BMP4 cause eye, brain, and
digit developmental anomalies: overlap between the BMP4
and hedgehog signaling pathways. Am J Hum Genet 82:
304319.
Bao ZZ, Cepko CL (1997) The expression and function of
Notch pathway genes in the developing rat eye. J Neurosci
17:14251434.
Bartek J, Lukas J (2001) Mammalian G1 and S-phase check-
points in response to DNA damage. Curr Opin Cell Biol
13:738747.
Bartek J, Lukas J (2007) DNA damage checkpoints: from
initiation to recovery or adaptation. Curr Opin Cell Biol
19:238245.
Bauer S, Kerr BJ, Patterson PH (2007) The neuropoietic
cytokine family in development, plasticity, disease and
injury. Nat Rev Neurosci 8:221232.
Bauer S, Patterson PH (2006) Leukemia inhibitory factor pro-
motes neural stem cell self-renewal in the adult brain.
J Neurosci 26:1208912099.
Baughman RW, Bader CR (1977) Biochemical characterization
and cellular localization of the cholinergic system in the
chicken retina. Brain Res 138:469485.
Baye LM, Link BA (2008) Nuclear migration during retinal
development. Brain Res 1192:2936.
Beazley LD, Perry VH, Baker B, Darby JE (1987) An inves-
tigation into the role of ganglion cells in the regulation of
13 Tissue Biology of Proliferation and Cell Death
division and death of other retinal cells. Dev Brain Res
33:169184.
Ben-Ari Y (2002) Excitatory actions of gaba during develop-
ment: the nature of the nurture. Nat Rev Neurosci 3(9):
72839.
Berridge MJ (1995) Calcium signalling and cell proliferation.
Bioessays 17:491500.
Biade S, Stobbe CC, Chapman JD (1997) The intrinsic radiosen-
sitivity of some human tumor cells throughout their cell
cycles. Radiat Res 147:416421.
Borges HL, Chao C, Xu Y, Linden R, Wang JY (2004)
Radiation-induced apoptosis in developing mouse retina
exhibits dose-dependent requirement for ATM phosphoryla-
tion of p53. Cell Death Differ 11:494502.
Borges HL, Linden R (1999) Gamma irradiation leads to two
waves of apoptosis in distinct cell populations of the retina
of newborn rats. J Cell Sci 112:43154324.
Borges HL, Linden R, Wang JY (2008) DNA damage-induced
cell death: lessons from the central nervous system. Cell Res
18:1726.
Bovolenta P, Frade JM, Marti E, Rodriguez-Pena MA, Barde
YA, Rodriguez-Tebar A (1996) Neurotrophin-3 antibod-
ies disrupt the normal development of the chick retina.
J Neurosci 16:44024410.
Branzei D, Foiani M (2006) The Rad53 signal transduction
pathway: replication fork stabilization, DNA repair, and
adaptation. Exp Cell Res 312:26542659.
Branzei D, Foiani M (2008) Regulation of DNA repair through-
out the cell cycle. Nat Rev Mol Cell Biol 9:297308.
Bringmann A, Pannicke T, Grosche J, Francke M, Wiedemann P,
Skatchkov SN, Osborne NN, Reichenbach A (2006) Mller
cells in the healthy and diseased retina. Prog Retin Eye Res
25:397424.
Bull ND, Johnson TV, Martin KR (2008) Stem cells
for neuroprotection in glaucoma. Prog Brain Res 173:
511519.
Burdak-Rothkamm S, Rothkamm K, Prise KM (2008) ATM acts
downstream of ATR in the DNA damage response signaling
of bystander cells. Cancer Res 68:70597065.
Burke R, Basler K (1996) Hedgehog-dependent patterning in the
Drosophila eye can occur in the absence of Dpp signaling.
Dev Biol 179:360368.
Burnashev N, Monyer H, Seeburg PH, Sakmann B (1992)
Divalent ion permeability of AMPA receptor channels is
dominated by the edited form of a single subunit. Neuron
8:189198.
Buss RR, Sun W, Oppenheim RW (2006) Adaptive roles of
programmed cell death during nervous system development.
Annu Rev Neurosci 29:135.
Bussolati B, Biancone L, Cassoni P, Russo S, Rola-Pleszczynski
M, Montrucchio G, Camussi G (2000) PAF produced by
human breast cancer cellspromotes migration and prolif-
eration of tumor cells and neo-angiogenesis. Am J Pathol
157:17131725.
Bussolino F, Pescarmona GP, Camussi G, Gremo F (1988)
Acetylcholine and dopamine promote the production of
Platelet-activating factor in immature cells of chick embry-
onic retina. J Neurochem 51:17551759.
Bussolino F, Torelli S, Stefanini E, Gremo F (1989) Platelet-
activating factor production occurs through stimulation of
cholinergic and dopaminergic receptors in the chick retina.
J Lipid Mediat 1:283288.
219
Cam H, Dynlacht BD (2003) Emerging roles for E2F: beyond
the G1/S transition and DNA replication. Cancer Cell 3:
311316.
Campos CB, Bdard PA, Linden R (2002) Activation of p38
mitogen-activated protein kinase during normal mitosis in
the developing retina. Neuroscience 112:583591.
Cande C, Cecconi F, Dessen P, Kroemer G (2002) Apoptosis-
inducing factor (AIF): key to the conserved caspase-
independent pathways of cell death? J Cell Sci 115:
47274734.
Cantrell DA (2001) Phosphoinositide 3-kinase signalling path-
ways. J Cell Sci 114:14391445.
Carneiro AC, Fragel-Madeira L, Silva-Neto MA, Linden R
(2008) A role for CK2 upon interkinetic nuclear migration
in the cell cycle of retinal progenitor cells. Dev Neurobiol
68:620631.
Casini G, Brecha NC (1992) Postnatal development of tyro-
sine hydroxylase immunoreactive amacrine cells in the rabbit
retina: I. Morphological characterization. J Comp Neurol
326:283301.
Catsicas M, Mobbs P (2001) GABAb receptors regulate chick
retinal calcium waves. J Neurosci 21:897910.
Cayouette M (2007) Expanding neuronal layers by the local
division of committed precursors. Neuron 56:575577.
Cayouette M, Poggi L, Harris WA (2006) Lineage in the verte-
brate retina. Trends Neurosci 29:563570.
Cayouette M, Raff M (2003) The orientation of cell division
influences cell-fate choice in the developing mammalian
retina. Development 130:23292339.
Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA, Gruss P
(1998) Apaf1 (CED-4 homolog) regulates programmed cell
death in mammalian development. Cell 94:727737.
Cepko CL (1999) The roles of intrinsic and extrinsic cues and
bHLH genes in the determination of retinal cell fates. Curr
Opin Neurobiol 9:3746.
Cepko CL, Austin CP, Yang X, Alexiades M, Ezzeddine D
(1996) Cell fate determination in the vertebrate retina. Proc
Natl Acad Sci U S A 93:589595.
Chalazonitis A (2004) Neurotrophin-3 in the development of the
enteric nervous system. Prog Brain Res 146:243263.
Chan A, Lakshminrusimha S, Heffner R, Gonzalez-Fernandez F
(2007) Histogenesis of retinal dysplasia in trisomy 13. Diagn
Pathol 2:4849.
Chao C, Wu Z, Mazur SJ, Borges H, Rossi M, Lin T, Wang
JY, Anderson CW, Appella E, Xu Y (2006) Acetylation of
mouse p53 at lysine 317 negatively regulates p53 apop-
totic activities after DNA damage. Mol Cell Biol 26:
68596869.
Chau BN, Borges HL, Chen TT, Masselli A, Hunton
IC, Wang JY (2002) Signal-dependent protection from
apoptosis in mice expressing caspase-resistant Rb. Nat Cell
Biol 4: 757765.
Chavarra T, Valenciano AI, Mayordomo R, Egea J, Comella JX,
Hallbk F, de Pablo F, de la Rosa EJ (2007) Differential,
age-dependent MEK-ERK and PI3K-Akt activation by
insulin acting as a survival factor during embryonic retinal
development. Dev Neurobiol 67:17771788.
Chen Y, Zhao X (1998) Shaping limbs by apoptosis. J Exp Zool
282:691702.
Chenn A, McConnell SK (1995) Cleavage orientation and
the asymmetric inheritance of Notch1 immunoreactivity in
mammalian neurogenesis. Cell 82:631641.
220
Cheong N, Wang X, Wang Y, Iliakis G (1994) Loss of S-phase-
dependent radioresistance in irs-1 cells exposed to X-rays.
Mutat Res 314:7785.
Chiarini LB, Leal-Ferreira ML, de Freitas FG, Linden R (2003)
Changing sensitivity to cell death during development of
retinal photoreceptors. J Neurosci Res 74:875883.
Cicero SA, Johnson D, Reyntjens S, Frase S, Connell S, Chow
LM, Baker SJ, Sorrentino BP, Dyer MA (2009) cells previ-
ously identified as retinal stem cells are pigmented ciliary
epithelial cells. Proc. Natl Acad Sci USA. 106(16):6685
6690.
Ciechomska IA, Goemans CG, Tolkovsky AM (2008) Molecular
links between autophagy and apoptosis. Methods Mol Biol
445:175193.
Cluzel J, Doly M, Bazan NG, Bonhomme B, Braquet P (1995)
Inhibition of platelet-activating factor-induced retinal impair-
ments by cholera and pertussis toxins. Ophthalmic Res
27:153157.
Cohen J (1987) Postnatal development of phenylethanolamine-
N-methyltransferase activity of rat retina. Neurosci Lett
83:138142.
Cohen RI, Molina-Holgado E, Almazan G (1996) Carbachol
stimulates c-fos expression and proliferation in oligodendro-
cyte progenitors. Brain Res Mol Brain Res 43:193201.
Cortez D (2003) Caffeine inhibits checkpoint responses with-
out inhibiting the ataxia-telangiectasia-mutated (ATM) and
ATM- and Rad3-related (ATR) protein kinases. J Biol Chem
278:3713937145.
Cuadros MA, Ros A (1988) Spatial and temporal correla-
tion between early nerve fiber growth and neuroepithelial
cell death in the chick embryo retina. Anat Embryol 178:
543551.
da Costa Calaza K, Hokoc JN, Gardino PF (2000) Neurogenesis
of GABAergic cells in the chick retina. Int J Dev Neurosci
18:721726.
Dagnino L, Fry CJ, Bartley SM, Farnham P, Gallie BL, Phillips
RA (1997) Expression patterns of the E2F family of tran-
scription factors during mouse nervous system development.
Mech Dev 66:1325.
Das AV, Mallya KB, Zhao X, Ahmad F, Bhattacharya S,
Thoreson WB, Hegde GV, Ahmad I (2006) Neural stem cell
properties of Mller glia in the mammalian retina: regulation
by Notch and Wnt signaling. Dev Biol 299:283302.
Das I, Sparrow JR, Lin MI, Shih E, Mikawa T, Hempstead BL
(2000) Trk C signaling is required for retinal progenitor cell
proliferation. J Neurosci 20:28872895.
de la Cruz JP, Moreno A, Ruiz-Ruiz MI, Garcia-Campos J,
Sanchez de la Cuesta F (1998) Effect of WEB 2086-
BS, an antagonist of platelet-activating factor receptors, on
retinal vascularity in diabetic rats. Eur J Pharmacol 360:
3742.
de la Rosa EJ, de Pablo F (2000) Cell death in early neural devel-
opment: beyond the neurotrophic theory. Trends Neurosci
23:454458.
De Lucchini S, Ori M, Cremisi F, Nardini M, Nardi I (2005)
5-HT2B-mediated serotonin signaling is required for eye
morphogenesis in Xenopus. Mol Cell Neurosci 29:299 312.
De Lucchini S, Ori M, Nardini M, Marracci S, Nardi I (2003)
Expression of 5-HT2B and 5-HT2C receptor genes is associ-
ated with proliferative regions of Xenopus developing brain
and eye. Mol Brain Res 115:196201.
R. Linden et al.
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima
N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005)
Chemical inhibitor of nonapoptotic cell death with thera-
peutic potential for ischemic brain injury. Nat Chem Biol
1:112119.
Degterev A, Yuan J (2008) Expansion and evolution of cell death
programmes. Nat Rev Mol Cell Biol 9:378390.
Del Bene F, Wehman AM, Link BA, Baier H (2008) Regulation
of neurogenesis by interkinetic nuclear migration through an
apical-basal notch gradient. Cell 134:10551065.
Denizot Y, Desplat V, Drouet M, Bertin F, Melloni B (2001)
Is there a role of platelet-activating factor in human lung
cancer? Lung Cancer 33:195202.
Derouiche A, Asan E (1999) The dopamine D2 receptor sub-
family in rat retina: ultrastructural immunogold and in situ
hybridization studies. Eur J Neurosci 11:13911402.
Dhar A, Shukla SD (1991) Involvement of pp60c-src in
platelet-activating factor-stimulated platelets. Evidence for
translocation from cytosol to membrane. J Biol Chem 266:
1879718801.
Dhar A, Shukla SD (1994) Electrotransjection of pp60v-src
monoclonal antibody inhibits activation of phospholipase C
in platelets. A new mechanism for platelet-activating factor
responses. J Biol Chem 269:91239127.
Dhomen NS, Balaggan KS, Pearson RA, Bainbridge JW, Levine
ED, Ali RR, Sowden JC (2006) Absence of chx10 causes
neural progenitors to persist in the adult retina. Invest
Ophthalmol Vis Sci 47:386396.
Dimaras H, Coburn B, Pajovic S, Gallie BL (2006) Loss of
p75 neurotrophin receptor expression accompanies malig-
nant progression to human and murine retinoblastoma. Mol
Carcinog 45:333343.
Dkhissi O, Julien JF, Wasowicz M, Dalil-Thiney N, Nguyen-
Legros J, Versaux-Botteri C (2001) Differential expression
of GAD(65) and GAD(67) during the development of the rat
retina. Brain Res 919:242249.
Doly M, Cluzel J, Bonhomme B, Millerin M, Braquet P (1995)
Protective effect of a specific PAF antagonist on vincristine-
induced experimental retinopathy. Acta Ophthalmol Scand
73:155157.
Domingos PM, Steller H (2007) Pathways regulating apoptosis
during patterning and development. Curr Opin Genet Dev
17:294299.
Donovan SL, Dyer MA (2005) Regulation of proliferation dur-
ing central nervous system development. Semin Cell Dev
Biol 16:407421.
Donovan SL, Schweers B, Martins R, Johnson D, Dyer MA
(2006) Compensation by tumor suppressor genes during reti-
nal development in mice and humans. BMC Biol 4:1415.
dos Santos AA, Medina SV, Sholl-Franco A, de Araujo EG
(2003) PMA decreases the proliferation of retinal cells
in vitro: the involvement of acetylcholine and BDNF.
Neurochem Int 42:7380.
Dunwiddie TV, Masino SA (2001) The role and regulation of
adenosine in the central nervous system. Annu Rev Neurosci
24:3155.
Dupuis F, Levasseur S, Jean-Louis F, Dulery C, Praloran V,
Denizot Y, Michel L (1997) Production, metabolism and
effect of platelet-activating factor on the growth of the human
K562 erythroid cell line. Biochim Biophys Acta 1359:
241249.
13 Tissue Biology of Proliferation and Cell Death
Dyer M, Cepko CL (2000a) p57(Kip2) regulates progenitor cell
proliferation and amacrine interneuron development in the
mouse retina. Development 127:35933605.
Dyer MA, Cepko CL (2000b) Control of Mller glial cell prolif-
eration and activation following retinal injury. Nat Neurosci
3:873880.
Dyer M, Cepko CL (2001a) p27Kip1 and p57Kip2 regulate
proliferation in distinct retinal progenitor cell populations.
J Neurosci 21:42594271.
Dyer MA, Cepko CL (2001b) Regulating proliferation during
retinal development. Nat Rev Neurosci 2:333342.
Daz B, Pimentel B, de Pablo F, de la Rosa EJ (1999) Apoptotic
cell death affecting proliferating neuroepithelial cells in the
embryonic retina is prevented by insulin. Eur J Neurosci
11:16241632.
Daz B, Serna J, De Pablo F, de la Rosa EJ (2000) In vivo regu-
lation of cell death by embryonic (pro)insulin and the insulin
receptor during early retinal neurogenesis. Development
127:16411649.
DSa C, Klocke BJ, Cecconi F, Lindsten T, Thompson CB,
Korsmeyer SJ, Flavell RA, Roth KA (2003) Caspase reg-
ulation of genotoxin-induced neural precursor cell death.
J Neurosci Res 74:435445.
Ekman R, Tornqvist K (1985) Glucagon and VIP in the retina.
Invest Ophthalmol Vis Sci 26:14051409.
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A,
Nagata S (1998) A caspase-activated DNase that degrades
DNA during apoptosis, and its inhibitor ICAD. Nature
391:4350.
Erlich RB, Werneck CC, Mouro PA, Linden R (2003) Major
glycosaminoglycan species in the developing retina: synthe-
sis, tissue distribution and effects upon cell death. Exp Eye
Res 77:157165.
Feller MB, Wellis DP, Stellwagen D, Werblin FS, Shatz CJ
(1996) Requirement for cholinergic synaptic transmission
in the propagation of spontaneous retinal waves. Science
272:11821187.
Ferriero DM, Sagar SM (1989) Development of neuropeptide
Y immunoreactive neurons in the rat retina. Dev Brain Res
48:1926.
Finlay BL (2008) The developing and evolving retina: using time
to organize form. Brain Res 1192:516.
Fischer AJ, Dierks BD, Reh TA (2002a) Exogenous growth fac-
tors induce the production of ganglion cells at the retinal
margin. Development 129:22832291.
Fischer AJ, McGuire CR, Dierks BD, Reh TA (2002b) Insulin
and fibroblast growth factor 2 activate a neurogenic pro-
gram in Mller glia of the chicken retina. J Neurosci 22:
93879398.
Fischer AJ, Omar G, Walton NA, Verrill TA, Unson CG
(2005) Glucagon-expressing neurons within the retina reg-
ulate the proliferation of neural progenitors in the circum-
ferential marginal zone of the avian eye. J Neurosci 25:
1015710166.
Fischer AJ, Reh TA (2001) Mller glia are a potential source
of neural regeneration in the postnatal chicken retina. Nat
Neurosci 4:2472052.
Fischer AJ, Reh TA (2003) Growth factors induce neurogenesis
in the ciliary body. Dev Biol 259:225240.
Fletcher EL, Kalloniatis M (1997) Localisation of amino acid
neurotransmitters during postnatal development of the rat
retina. J Comp Neurol 380:449471.
221
Florian C, Langmann T, Weber BH, Morsczeck C (2008)
Murine Mller cells are progenitor cells for neuronal cells
and fibrous tissue cells. Biochem Biophys Res Commun
374:187191.
Fontaine RH, Cases O, Lelivre V, Mespls B, Renauld JC,
Loron G, Degos V, Dournaud P, Baud O, Gressens P (2008)
IL-9/IL-9 receptor signaling selectively protects cortical neu-
rons against developmental apoptosis. Cell Death Differ
15:15421552.
Frade JM, Bovolenta P, Martnez-Morales JR, Arribas A, Barbas
JA, Rodrguez-Tbar A (1997) Control of early cell death by
BDNF in the chick retina. Development 124:33133320.
Frade JM, Bovolenta P, Rodriguez-Tebar A (1999)
Neurotrophins and other growth factors in the generation of
retinal neurons. Microsc Res Tech 45:243251.
Frade JM, Marti E, Bovolenta P, Rodriguez-Pena MA, Perez-
Garcia D, Rohrer H, Edgar D, Rodriguez-Tebar A (1996a)
Insulin-like growth factor-I stimulates neurogenesis in chick
retina by regulating expression of the alpha 6 integrin sub-
unit. Development 122:24972506.
Frade JM, Rodrguez-Tbar A, Barde YA (1996b) Induction of
cell death by endogenous nerve growth factor through its p75
receptor. Nature 383:166168.
Frana GR, Freitas RC, Ventura AL (2007) ATP-induced pro-
liferation of developing retinal cells: regulation by factors
released from postmitotic cells in culture. Int J Dev Neurosci
25:283291.
Frederick JM (1987) The emergence of GABA-accumulating
neurons during retinal histogenesis in the embryonic chick.
Exp Eye Res 45:933945.
Fredholm BB (1997) Adenosine and neuroprotection. Int Rev
Neurobiol 40:259280.
Fujita S (1962) Kinetics of cellular proliferation. Exp Cell Res
28:5260.
Fukada T, Ohtani T, Yoshida Y, Shirogane T, Nishida K,
Nakajima K, Hibi M, Hirano T (1998) STAT3 orchestrates
contradictory signals in cytokine-induced G1 to S cell-cycle
transition. EMBO J 17:66706677.
Galli-Resta L, Leone P, Bottari D, Ensini M, Rigosi E, Novelli
E (2008) The genesis of retinal architecture: an emerg-
ing role for mechanical interactions? Prog Retin Eye Res
27:260283.
Galluzzi L, Vicencio JM, Kepp O, Tasdemir E, Maiuri MC,
Kroemer G (2008) To die or not to die: that is the autophagic
question. Curr Mol Med 8:7891.
Garcia M, Forster V, Hicks D, Vecino E (2003) In vivo expres-
sion of neurotrophins and neurotrophin receptors is con-
served in adult porcine retina in vitro. Invest Ophthalmol Vis
Sci 44:45324541.
Gardino PF, dos Santos RM, Hokoc JN (1993) Histogenesis
and topographical distribution of tyrosine hydroxylase
immunoreactive amacrine cells in the developing chick
retina. Brain Res Dev Brain Res 72:226236.
Gaur VP, Liu Y, Turner JE (1992) RPE conditioned medium
stimulates photoreceptor cell survival, neurite outgrowth and
differentiation in vitro. Exp Eye Res 54:645659.
Godinho L, Williams PR, Claassen Y, Provost E, Leach
SD, Kamermans M, Wong RO (2007) Nonapical
symmetric divisions underlie horizontal cell layer formation
in the developing retina in vivo. Neuron 56:597603.
Gran X, Reddy EP (1995) Cell cycle control in mammalian
cells: role of cyclins, cyclin-dependent kinases (CDK),
222
growth supressor genes and cyclin-dependent kinases
inhibitors (CDKIs). Oncogene 11:211219.
Greka A, Lipton SA, Zhang D (2000) Expression of GABA(C)
receptor rho1 and rho2 subunits during development of the
mouse retina. Eur J Neurosci 12:35753582.
Grunder T, Kohler K, Guenther E (2000a) Distribution and
developmental regulation of AMPA receptor subunit proteins
in rat retina. Invest Ophthalmol Vis Sci 41:36003606.
Grunder T, Kohler K, Kaletta A, Guenther E (2000b) The dis-
tribution and developmental regulation of NMDA receptor
subunit proteins in the outer and inner retina of the rat. J
Neurobiol 44:333342.
Guilarducci-Ferraz CV, da Silva GM, Torres PM, Dos Santos
AA, Araujo EG (2008) The increase in retinal cells prolif-
eration induced by FGF2 is mediated by tyrosine and PI3
kinases. Neurochem Res 33:754764.
Guimaraes CA, Linden R (2004) Programmed cell deaths.
Apoptosis and alternative deathstyles. Eur J Biochem
271:16381650.
Guizzetti M, Costa P, Peters J, Costa LG (1996) Acetylcholine
as a mitogen: muscarinic receptor-mediated proliferation of
rat astrocytes and human astrocytoma cells. Eur J Pharmacol
297:265273.
Guizzetti M, Wei M, Costa LG (1998) The role of protein kinase
C alpha and epsilon isozymes in DNA synthesis induced by
muscarinic receptors in a glial cell line. Eur J Pharmacol
359:223233.
Gutierrez GJ, Ronai Z (2006) Ubiquitin and SUMO systems in
the regulation of mitotic checkpoints. Trends Biochem Sci
31:324332.
Gtz M, Huttner WB (2005) The cell biology of neurogenesis.
Nat Rev Mol Cell Biol 6:777788.
Hadjiconstantinou M, Mariani AP, Panula P, Joh TH, Neff
NH (1984) Immunohistochemical evidence for epinephrine-
containing retinal amacrine cells. Neuroscience 13:547551.
Hajra KM, Liu JR (2004) Apoptosome dysfunction in human
cancer. Apoptosis 9:691704.
Hakem R, Hakem A, Duncan GS, Henderson JT, Woo M,
Soengas MS, Elia A, de la Pompa JL, Kagi D, Khoo W,
Potter J, Yoshida R, Kaufman SA, Lowe SW, Penninger JM,
Mak TW (1998) Differential requirement for caspase 9 in
apoptotic pathways in vivo. Cell 94:339352.
Hamassaki-Britto DE, Gardino PF, Hokoc JN, Keyser KT,
Karten HJ, Lindstrom JM, Britto LR (1994) Differential
development of alpha-bungarotoxin-sensitive and alpha-
bungarotoxin-insensitive nicotinic acetylcholine receptors in
the chick retina. J Comp Neurol 347:161170.
Han SJ, Conti M (2006) New pathways from PKA to the
Cdc2/cyclin B complex in oocytes: Wee1B as a potential
PKA substrate. Cell Cycle 5:227231.
Hao Z, Duncan GS, Chang CC, Elia A, Fang M, Wakeham A,
Okada H, Calzascia T, Jang Y, You-Ten A, Yeh WC, Ohashi
P, Wang X, Mak TW (2005) Specific ablation of the apoptotic
functions of cytochrome C reveals a differential require-
ment for cytochrome C and Apaf-1 in apoptosis. Cell 121:
579591.
Hardy P, Beauchamp M, Sennlaub F, Gobeil F Jr, Tremblay
L, Mwaikambo B, Lachapelle P, Chemtob S (2005) New
insights into the retinal circulation: inflammatory lipid medi-
ators in ischemic retinopathy. Prostaglandins. Leukot Essent
Fatty Acids 72:301325.
R. Linden et al.
Harper JV, Brooks G (2005) The mammalian cell cycle: an
overview. Methods Mol Biol 296:113153.
Hartveit E, Brandstatter JH, Sassoe-Pognetto M, Laurie DJ,
Seeburg PH, Wassle H (1994) Localization and devel-
opmental expression of the NMDA receptor subunit
NR2A in the mammalian retina. J Comp Neurol 348:
570582.
Hartwell LH, Weinert TA (1989) Checkpoints: controls that
ensure the order of cell cycle events. Science 246:
629634.
Haydar TF, Wang F, Schwartz ML, Rakic P (2000) Differential
modulation of proliferation in the neocortical ventricular and
subventricular zones. J Neurosci 20:57645774.
Hayes NL, Nowakowski RS (2000) Exploiting the dynamics of
S-phase tracers in the developing brain: Interkinetic nuclear
migration for cells entering versus leavinhg the S-phase. Dev
Neurosci 22:4455.
Henriquez M, Armisen R, Stutzin A, Quest AF (2008) Cell death
by necrosis, a regulated way to go. Curr Mol Med 8:187206.
Heo JS, Han HJ (2006) ATP stimulates mouse embryonic stem
cell proliferation via PKC, PI3K/Akt, and MAPKs signaling
pathways. Stem Cells 12:26372648.
Hernandez-Sanchez C, Lopez-Carranza A, Alarcon C, de La
Rosa EJ, de Pablo F (1995) Autocrine/paracrine role of
insulin-related growth factors in neurogenesis: local expres-
sion and effects on cell proliferation and differentiation in
retina. Proc Natl Acad Sci U S A 92:98349838.
Herzog KH, Schulz A, Buerkle C, Gromoll C, Braun JS (2007)
Radiation-induced apoptosis in retinal progenitor cells is
p53-dependent with caspase-independent DNA fragmenta-
tion. Eur J Neurosci 25:13491356.
Hicks D (1998) Putative functions of fibroblast growth factors
in retinal development, maturation and survival. Semin Cell
Dev Biol 9:263269.
Hicks D, Courtois Y (1992) Fibroblast growth factor stimu-
lates photoreceptor differentiation in vitro. J Neurosci 12:
20222033.
Hill DR (1985) GABAB receptor modulation of adenylate
cyclase activity in rat brain slices. Br J Pharmacol 84:
249257.
Hokoc JN, Ventura AL, Gardino PF, de Mello FG (1990)
Developmental immunoreactivity for GABA and GAD in
the avian retina: possible alternative pathway for GABA
synthesis. Brain Res 532:197202.
Holtkamp GM, Kijlstra A, Peek R, de Vos AF (2001) Retinal
pigment epithelium-immune system interactions: cytokine
production and cytokine-induced changes. Prog Retin Eye
Res 20:2948.
Honda Z-I, Takano T, Gotoh Y, Nishida E, Ito K, Shimizu T
(1994) Transfected platelet-activating factor receptor acti-
vates mitogen-activated protein (MAP) kinase and MAP
kinase kinase in Chinese hamster ovary cells. J Biol Chem
269:23072315.
Hoyer D, Hannon JP, Martin GR (2002) Molecular, phar-
macological and functional diversity of 5-HT receptors.
Pharmacol Biochem Behav 71:533554.
Huang BO, Redburn DA (1996) GABA-induced increases in
[Ca2+ ]i in retinal neurons of postnatal rabbits. Vis Neurosci
13:441447.
Hurley PJ, Bunz F (2007) ATM and ATR: components of an
integrated circuit. Cell Cycle 6:414417.
13 Tissue Biology of Proliferation and Cell Death
Ilia M, Jeffery G (1999) Retinal mitosis is regulated by dopa, a
melanin precursor that may influence the time at which cells
exit the cell cycle: analysis of patterns of cell production in
pigmented and albino retinae. J Comp Neurol 405:394405.
Inatani M, Tanihara H (2002) Proteoglycans in retina. Prog Retin
Eye Res 21:429447.
Ingham PW, McMahon AP (2001) Hedgehog signaling in ani-
mal development: paradigms and principles. Genes Dev
15:30593087.
Isayama T, Hurst WJ, McLaughlin PJ, Zagon IS (1995)
Ontogeny of the opioid growth factor, [Met5]-enkephalin,
and its binding activity in the rat retina. Visual Neurosci
12:939950.
Isayama T, McLaughlin PJ, Zagon IS (1991) Endogenous opi-
oids regulate cell proliferation in the retina of developing rat.
Brain Res 544:7985.
Isayama T, McLaughlin PJ, Zagon IS (1996) Ontogeny of pre-
proenkephalin mRNA expression in the rat retina. Visual
Neurosci 13:695704.
Ishi I, Shimizu T (2000) Platelet-activating factor receptor and
genetically engineered PAF receptor mutant mice. Prog Lipid
Res 39:4182.
Izumi T, Shimizu T (1995) Platelet-activating factor receptor:
gene expression and signal transduction. Biochim Biophys
Acta 1259:317333.
Jacobson M (1991) Developmental Neurobiology. Plenum Press,
New York, 1776.
Jadhav AP, Cho S, Cepko CL (2006a) Notch activity permits
retinal cells to progress through multiple progenitor states
and acquire a stem cell property. Proc Natl Acad Sci U S
A 103:1899819003.
Jadhav AP, Mason HA, Cepko CL (2006b) Notch 1 inhibits pho-
toreceptor production in the developing mammalian retina.
Development 133:913923.
James J, Das AV, Rahnenfuhrer J, Ahmad I (2004) Cellular
and molecular characterization of early and late retinal stem
cells/progenitors: differential regulation of proliferation and
context dependent role of Notch signaling. J Neurobiol
61:359376.
Jaynes CD, Turner JE (2000) Isolation of a retinal pigment
epithelial cell-derived fraction which promotes Mller cell
proliferation. Dev Brain Res 120:267271.
Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J,
Jackson SP (2006) ATM- and cell cycle-dependent regula-
tion of ATR in response to DNA double-strand breaks. Nat
Cell Biol 8:3745.
Jensen AM, Wallace VA (1997) Expression of Sonic hedge-
hog and its putative role as a precursor cell mitogen in the
developing mouse retina. Development 124:363371.
Jeon CJ, Strettoi E, Masland RH (1998) The major cell popula-
tions of the mouse retina. J Neurosci 18:89368946.
Jin Z, El-Deiry WS (2005) Overview of cell death signaling
pathways. Cancer Biol Ther 4:139163.
Jotwani G, Itoh K, Wadhwa S (1994) Immunohistochemical
localization of tyrosine hydroxylase, substance P,
neuropeptide-Y and leucine-enkephalin in developing
human retinal amacrine cells. Dev Brain Res 77:285289.
Kaldis P (1999) The cdk-activating kinase (CAK): from yeast to
mammals. Cell Mol Life Sci 55:284296.
Kang MK, Kang SK (2008) Interleukin-6 induces
proliferation in adult spinal cord-derived neural
223
progenitors via the JAK2/STAT3 pathway with EGF-
induced MAPK phosphorylation. Cell Prolif 41:
377392.
Kerr JF (2002) History of the events leading to the formulation
of the apoptosis concept. Toxicology 181182:471474.
Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic bio-
logical phenomenon with wide-ranging implications in tissue
kinetics. Br J Cancer 26:23957.
Kim R (2005) Recent advances in understanding the cell
death pathways activated by anticancer therapy. Cancer 103:
15511560.
Kim IB, Lee EJ, Kim MK, Park DK, Chun MH (2000) Choline
acetyltransferase-immunoreactive neurons in the developing
rat retina. J Comp Neurol 427:604616.
Kim IB, Park DK, Oh SJ, Chun MH (1998) Horizontal cells
of the rat retina show choline acetyltransferase- and vesicu-
lar acetylcholine transporter-like immunoreactivities during
early postnatal developmental stages. Neurosci Lett 253:
8386.
Kolb H (1997) Amacrine cells of the mammalian retina: neuro-
circuitry and functional roles. Eye 11:904923.
Kolb H, Linberg KA, Fisher SK (1992) Neurons of the human
retina: a Golgi study. J Comp Neurol 318:147187.
Korenjak M, Brehm A (2005) E2F-Rb complexes regulating
transcription of genes important for differentiation and devel-
opment. Curr Opin Genet Dev 15:520527.
Koulen P (1999) Postnatal development of dopamine D1 recep-
tor immunoreactivity in the rat retina. J Neurosci Res
56:397404.
Kralj-Hans I, Tibber M, Jeffery G, Mobbs P (2006) Differential
effect of dopamine on mitosis in early postnatal albino and
pigmented rat retinae. J Neurobiol 66:4755.
Kriegstein AR, Gtz M (2003) Radial glia diversity: a matter of
cell fate. Glia 43:3743.
Kriegstein AR, Noctor S, Martinez-Cerdeno M (2006) Patterns
of neural stem and progenitor cell division may underlie evo-
lutionary cortical expansion. Nat Rev Neurosci 7:883890.
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES,
Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein
P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton
SA, Malorni W, Nuez G, Peter ME, Tschopp J, Yuan J,
Piacentini M, Zhivotovsky B, Melino G (2008) Classification
of cell death: recommendations of the Nomenclature
Committee on Cell Death 2009. Cell Death Differ 16:
311.
Krysko DV, Vanden Berghe T, Parthoens E, DHerde K,
Vandenabeele P (2008) Methods for distinguishing apoptotic
from necrotic cells and measuring their clearance. Methods
Enzymol 442:307341.
Kuan CY, Roth KA, Flavell RA, Rakic P (2000) Mechanisms
of programmed cell death in the developing brain. Trends
Neurosci 23:291297.
Kubo F, Takeichi M, Nakagawa S 2003. Wnt2b controls retinal
cell differentiation at the ciliary marginal zone. Development
130(3):587598.
Kubo F, Nakagawa S (2008) Wnt signaling in retinal stem cells
and regeneration. Dev Growth Differ 50(4):245251.
Kubota R, Hokoc JN, Moshiri A, McGuire C, Reh TA (2002)
A comparative study of neurogenesis in the retinal ciliary
marginal zone of homeothermic vertebrates. Dev Brain Res
134:3141.
224
Kubrusly RC, Guimaraes MZ, Vieira AP, Hokoc JN, Casarini
DE, de Mello MC, de Mello FG (2003) L-DOPA supply
to the neuro retina activates dopaminergic communication
at the early stages of embryonic development. J Neurochem
86:4554.
Kubrusly RC, Ventura AL, de Melo Reis RA, Serra GC,
Yamasaki EN, Gardino PF, de Mello MC, de Mello FG
(2007) Norepinephrine acts as D1-dopaminergic agonist
in the embryonic avian retina: late expression of beta1-
adrenergic receptor shifts norepinephrine specificity in the
adult tissue. Neurochem Int 50:211218.
Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Karasuyama H,
Su MS, Rakic P, Flavell RA (1998) Reduced apoptosis and
cytochrome c-mediated caspase activation in mice lacking
caspase 9. Cell 94:325337.
Kuida K, Zheng TS, Na S, Kuan C, Yang D, Karasuyama H,
Rakic P, Flavell RA (1996) Decreased apoptosis in the brain
and premature lethality in CPP32-deficient mice. Nature
384:368372.
Kumar S (2007) Caspase function in programmed cell death.
Cell Death Differ 14:3243.
Kumar S, Kahn MA, Dinh L, de Vellis J (1998) NT-3-mediated
TrkC receptor activation promotes proliferation and cell sur-
vival of rodent progenitor oligodendrocyte cells in vitro and
in vivo. J Neurosci Res 54:754765.
Kunchithapautham K, Rohrer B (2007) Apoptosis and
autophagy in photoreceptors exposed to oxidative stress.
Autophagy 3:433441.
Kuruvilla A, Pielop C, Shearer WT (1994) Platelet-activating
factor induces the tyrosine phosphorylation and activation of
phospholipase C-gamma 1, Fyn and Lyn kinases, and phos-
phatidylinositol 3-kinase in a human B cell line. J Immunol
153:54335442.
Kuwayama Y, Ishimoto I, Fukuda M, Shimiza Y, Shiosaka
S, Inagaki S, Senba E, Sakanaka M, Takagi H, Takatsuki
K, Hara Y, Kawai Y, Tohyama M (1982) Overall distri-
bution of glucagon-like immunoreactivity in the chicken
retina: an immunohistochemical study with flat-mounts.
Invest Ophthalmol Vis Sci 22:681 686.
Laasberg T (1990) Ca2(+)-mobilizing receptors of gastrulating
chick embryo. Comp Biochem Physiol C 97:912.
Langman J, Guerrant RL, Freeman BG (1966) Behavior of
neuro-epithelial cells during closure of the neural tube. J
Comp Neurol 127:399411.
Lavin MF, Kozlov S (2007) ATM activation and DNA damage
response. Cell Cycle 6:931942.
Layer PG (1991) Cholinesterases during development of the
avian nervous system. Cell Mol Neurobiol 11:733.
Lee MY, Heo JS, Han HJ (2006) Dopamine regulates cell cycle
regulatory proteins via cAMP, Ca(2+)/PKC, MAPKs, and
NF-kappaB in mouse embryonic stem cells. J Cell Physiol
208:399406.
Levine EM, Green ES (2004) Cell-intrinsic regulators of prolif-
eration in vertebrate retinal progenitors. Semin Cell Dev Biol
15:6374.
Levine EM, Roelink H, Turner J, Reh TA (1997) Sonic hedgehog
promotes rod photoreceptor differentiation in mammalian
retinal cells in vitro. J Neurosci 17:62776288.
Li, Y, Yang X, Ma C, Qiao J, Zhang C (2008) Necroptosis con-
tributes to the NMDA-induced excitotoxicity in rats cultured
cortical neurons. Neurosci Lett 447:120123.
R. Linden et al.
Liang Q, Li W, Zhou B (2008) Caspase-independent apoptosis
in yeast. Biochim Biophys Acta 1783:13111319.
Liljekvist-Soltic I, Olofsson J, Johansson K (2008) Progenitor
cell-derived factors enhance photoreceptor survival in rat
retinal explants. Brain Res 1227:226233.
Lillien L (1995) Changes in retinal cell fate induced by overex-
pression of EGF receptor. Nature 377:158162.
Lillien L (1998) Neural progenitors and stem cells: mechanisms
of progenitor heterogeneity. Curr Opin Neurobiol 8:3744.
Lillien L, Cepko C (1992) Control of proliferation in the retina:
temporal changes in responsiveness to FGF and TGF alpha.
Development 115:253266.
Lillien L, Wancio D (1998) Changes in epidermal growth factor
receptor expression and competence to generate glia regulate
timing and choice of differentiation in the retina. Mol Cell
Neurosci 10:296308.
Lin SC, Skapek SX, Papermaster DS, Hankin M, Lee EY (2001)
The proliferative and apoptotic activities of E2F1 in the
mouse retina. Oncogene 20:70737084.
Linden R (2000) The anti-death league: associative control of
apoptosis in developing retinal tissue. Brain Res Rev 32:
146158.
Linden, R. Reese BE (2006) Programmed cell death. In: Retinal
Development Evelyn Sernagor; Stephen Eglen; William
A. Harris; Rachel Wong. (Org.), p. 208241. Cambridge
University Press, Cambridge.
Linden R, Cavalcante LA, Barradas PC (1986) Mononuclear
phagocytes in the retina of developing rats. Histochemistry
85:335339.
Linden R, Martins RA, Silveira MS (2005) Control of pro-
grammed cell death by neurotransmitters and neuropeptides
in the developing mammalian retina. Prog Retin Eye Res
24:457491.
Linden R, Rehen SK, Chiarini LB (1999) Apoptosis in develop-
ing retinal tissue. Prog Retin Eye Res 18:133165.
Liu B, Nakashima S, Kanoh H, Takano T, Shimizu T, Nozawa
Y (1994) Activation of phospholipase D in Chinese ham-
ster ovary cells expressing platelet-activating factor receptor.
J Biochem 116:882891.
Livesey FJ, Cepko CL (2001) Vertebrate neural cell-fate deter-
mination: lessons from the retina. Nat Rev Neurosci 2:
109118.
Locker M, Agathocleous M, Amato MA, Parain K, Harris WA,
Perron M (2006) Hedgehog signaling and the retina: insights
into the mechanisms controlling the proliferative properties
of neural precursors. Genes Dev 20:30363048.
Lograno MD, Tricarico D, Masciopinto V, Scuderl AC (2000)
Specific binding of nicergoline on an alpha1-like adrenore-
ceptor in the rat retina. J Pharm Pharmacol 52:207211.
Lombardini JB (1991) Taurine: retinal function. Brain Res Brain
Res Rev 16:151169.
Lonze BE, Ginty D (2002) Function and regulation of CREB
family transcription factors in the nervous system. Neuron
35:605623.
Lorenzo HK, Susin SA, Penninger J, Kroemer G (1999)
Apoptosis inducing factor (AIF): a phylogenetically old,
caspase-independent effector of cell death. Cell Death Differ
6:516524.
Lossi L, Merighi A (2003) In vivo cellular and molecular mech-
anisms of neuronal apoptosis in the mammalian CNS. Prog
Neurobiol 69:287312.
13 Tissue Biology of Proliferation and Cell Death
LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR
(1995) GABA and glutamate depolarize cortical progenitor
cells and inhibit DNA synthesis. Neuron 15:12871298.
Low CP, Shui G, Liew LP, Buttner S, Madeo F, Dawes IW, Wenk
MR, Yang H (2008) Caspase-dependent and -independent
lipotoxic cell-death pathways in fission yeast. J Cell Sci
121:26712684.
Low CP, Yang H (2008) Programmed cell death in fission
yeast Schizosaccharomyces pombe. Biochim Biophys Acta
1783:13351349.
Luk KC, Kennedy TE, Sadikot AF (2003) Glutamate promotes
proliferation of striatal neuronal progenitors by an NMDA
receptor-mediated mechanism. J Neurosci 23:22392250.
Lunyak VV, Rosenfeld MG (2008) Epigenetic regulation of stem
cell fate. Hum Mol Genet 17:R28R36.
Ma C, Papermaster D, Cepko CL (1998) A unique pattern of
photoreceptor degeneration in cyclin D1 mutant mice. Proc
Natl Acad Sci 95:99389943.
Maandag EC, van der Valk M, Vlaar M, Feltkamp C, OBrien J,
van Roon M (1994) Developmental rescue of an embryonic-
lethal mutation in the retinoblastoma gene in chimeric mice.
EMBO J 13:42604268.
MacLaren RE, Pearson RA (2007) Stem cell therapy and the
retina. Eye 21:13521359.
MacPherson D, Sage J, Kim T, Ho D, McLaughlin ME, Jacks T
(2004) Cell type-specific effects of Rb deletion in the murine
retina. Genes Dev 18:16811694.
Makman MK, Dvorkin B (1997) Presence of nociceptin
(orphanin FQ) receptors in rat retina: comparison with recep-
tors in striatum. Eur J Pharmacol 338:171176.
Marigo V (2007) Programmed cell death in retinal degeneration:
targeting apoptosis in photoreceptors as potential therapy for
retinal degeneration. Cell Cycle 6:652655.
Marquardt T (2003) Transcriptional control of neuronal diversi-
fication in the retina. Prog Retin Eye Res 22:567577.
Marquardt T, Gruss P (2002) Generating neuronal diversity in
the retina: one for nearly all. Trends Neurosci 25:3238.
Martins RA, Pearson RA (2008) Control of cell proliferation by
neurotransmitters in the developing vertebrate retina. Brain
Res 1192:3760.
Martin-Martinelli E, Simon A, Vigny A, Nguyen-Legros J
(1989) Postnatal development of tyrosine-hydroxylase
hydroxylaseimmunoreactive cells in the rat retina.
Morphology and distribution. Dev Neurosci 11:1125.
Martins RA, Linden R, Dyer MA (2006) Glutamate regulates
retinal progenitors cells proliferation during development.
Eur J Neurosci 24:969980.
Martins RA, Pearson RA (2008) Control of cell proliferation by
neurotransmitters in the developing vertebrate retina. Brain
Res 1192:3760.
Martn-Partido G, Rodrguez-Gallardo L, Alvarez IS, Navascus
J (1988) Cell death in the ventral region of the neural retina
during the early development of the chick embryo eye. Anat
Rec 222:272281.
Masai I, Yamaguchi M, Tonou-Fujimori N, Komori A, Okamoto
H (2005) The hedgehog-PKA pathway regulates two
distinct steps of the differentiation of retinal ganglion cells:
the cell-cycle exit of retinoblasts and their neuronal matura-
tion. Development 132:15391553.
Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER 3rd,
Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N,
225
Lerenthal Y, Shiloh Y, Gygi SP, Elledge SJ (2007) ATM and
ATR substrate analysis reveals extensive protein networks
responsive to DNA damage. Science 316:11601166.
McCabe KL, Gunther EC, Reh TA (1999) The development
of the pattern of retinal ganglion cells in the chick retina:
mechanisms that control differentiation. Development 126:
57135724.
McFarlane S, Zuber ME, Holt CE (1998) A role for the fibroblast
growth factor receptor in cell fate decisions in the developing
vertebrate retina. Development 125:39673975.
McKinnon LA, Nathanson NM (1995) Tissue-specific regula-
tion of muscarinic acetylcholine receptor expression during
embryonic development. J Biol Chem 270:2063620642.
Mione MC, Cavanagh JF, Harris B, Parnavelas JG (1997)
Cell fate specification and symmetrical/asymmetrical divi-
sions in the developing cerebral cortex. J Neurosci 17:
20182029.
Miranda-Contreras L, Benitez-Diaz PR, Mendoza-Briceno RV,
Delgado-Saez MC, Palacios-Pru EL (1999) Levels of amino
acid neurotransmitters during mouse cerebellar neurogene-
sis and in histotypic cerebellar cultures. Dev Neurosci 21:
147158.
Miranda-Contreras L, Mendoza-Briceno RV, Palacios-Pru EL
(1998) Levels of monoamine and amino acid neurotrans-
mitters in the developing male mouse hypothalamus and
in histotypic hypothalamic cultures. Int J Dev Neurosci
16:403412.
Miranda-Contreras L, Ramirez-Martens LM, Benitez-Diaz PR,
Pena-Contreras ZC, Mendoza-Briceno RV, Palacios-Pru EL
(2000) Levels of amino acid neurotransmitters during mouse
olfactory bulb neurogenesis and in histotypic olfactory bulb
cultures. Int J Dev Neurosci 18:8391.
Mitchell CK, Huang B, Redburn-Johnson DA (1999) GABA(A)
receptor immunoreactivity is transiently expressed in the
developing outer retina. Vis Neurosci 16:10831088.
Mitchell CK, Redburn DA (1996) GABA and GABA-A recep-
tors are maximally expressed in association with cone
synaptogenesis in neonatal rabbit retina. Dev Brain Res 95:
6371.
Miyata T (2008) Development of three-dimensional architec-
ture of the neuroepithelium: role of pseudostratification and
cellular community. Dev Growth Differ 50:S105S112.
Miyata T, Kawaguchi A, Okano H, Ogawa M (2001)
Asymmetric inheritance of radial glial fibers by cortical
neurons. Neuron 31:727741.
Miyata T, Kawaguchi A, Saito K, Kuramochi H, Ogawa M
(2002) Visualization of cell cycling by an improvement in
slice culture methods. J Neurosci Res 69:861868.
Montrucchio G, Lupia E, Battaglia E, Del Sorbo L, Boccellino
M, Biancone L, Emanuelli G, Camussi G (2000) Platelet-
activating factor enhances vascular endothelial growth
factor-induced endothelial cell motility and neoangiogenesis
in a murine matrigel model. Arterioscler Thromb Vasc Biol
20:8088.
Morgan DO (1997) Cyclin-dependent kinases: engines, clocks,
and microprocessors. Annu Rev Cell Dev Biol 13:261291.
Muotri AR, Gage FH (2006) Generation of neuronal variability
and complexity. Nature 441:10871093.
Murphy SN, Miller RJ (1989) Two distinct quisqualate receptors
regulate Ca2+ homeostasis in hippocampal neurons in vitro.
Mol Pharmacol 35:671680.
226
Mller F, Albert S, Blader P, Fischer N, Hallonet M, Strahle
U (2000) Direct action of the nodal-related signal cyclops
in induction of sonic hedgehog in the ventral midline of the
CNS. Development 127:38893897.
Nagahara H, Ezhevsky SA, Vocero-Akbani AM, Kaldis P,
Solomon MJ, Dowdy SF (1999) Transforming growth fac-
tor beta targeted inactivation of cyclin E:cyclin-dependent
kinase 2 (Cdk2) complexes by inhibition of Cdk2 acti-
vating kinase activity. Proc Natl Acad Sci U S A 96:
1496114966.
Nakanishi M, Niidome T, Matsuda S, Akaike A, Kihara T,
Sugimoto H (2007) Microglia-derived interleukin-6 and
leukaemia inhibitory factor promote astrocytic differentia-
tion of neural stem/progenitor cells. Eur J Neurosci 25:
649658.
Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T,
Shishido N, Horii I, Loy D, Nakayama K (1996) Mice lack-
ing p27Kip1 display increased body side, multiple organ
hyperplasia, retinal dysplasia and pituitary tumors. Cell
85:707720.
Neves DD, Rehen SK, Linden R (2001) Differentiation-
dependent sensitivity to cell death induced in the developing
retina by inhibitors of the ubiquitin-proteasome proteolytic
pathway. Eur J Neurosci 13:19381944.
Newman EA (2001) Propagation of intercellular calcium waves
in retinal astrocytes and Mller cells. J Neurosci 21:
22152223.
Nguyen-Legros J, Vigny A, Gay M (1983) Post-natal develop-
ment of TH-like immunoreactivity in the rat retina. Exp Eye
Res 37:2332.
Nickerson PEB, Da Silva N, Myers T, Stevens K, Clarke DB
(2008) Neural progenitor potential in cultured Mller glia:
effects of passaging and exogenous growth factor exposure.
Brain Res 1230:112.
Nishi S, Minota S, Karczmar AG (1974) Primary afferent neu-
rones: the ionic mechanism of GABA-mediated depolariza-
tion. Neuropharmacology 13:215219.
Niwa H, Burdon T, Chambers I, Smith A (1998) Self-renewal of
pluripotent embryonic stem cells is mediated via activation
of STAT3. Genes Dev 12:20482060.
Njaine B, Martins RAP, Santiago MF, Linden R, Silveira MS
(2009) Pituitary adenylyl cyclase-activating polypeptide con-
trols the proliferation of retinal progenitor cells through
downregulation of cyclin D1. Submitted.
North RA, Williams JT, Surprenant A, Christie MJ, (1987) Mu
and delta receptors belong to a family of receptors that are
coupled to potassium channels. Proc Natl Acad Sci USA
84:54875491.
Nunes PH, Calaza Kda C, Albuquerque LM, Fragel-Madeira
L, Sholl-Franco A, Ventura AL (2007) Signal transduction
pathways associated with ATP-induced proliferation of cell
progenitors in the intact embryonic retina. Int J Dev Neurosci
25:499508.
Oh S, Dangelo I, Lee E, Chun M, Brecha NC (2002)
Distribution and synaptic connectivity of neuropeptide Y-
immunoreactive amacrine cells in the rat retina. J Comp
Neurol 446:219234.
Ohta K, Ito A, Tanaka H (2008) Neuronal stem/progenitor cells
in the vertebrate eye. Dev Growth Differ 50:253259.
Oku H, Ikeda T, Honma Y, Sotozono C, Nishida K, Nakamura
Y, Kida T, Kinoshita S (2002) Gene expression of
R. Linden et al.
neurotrophins and their high-affinity Trk receptors in cul-
tured human Muller cells. Ophthalmic Res 34:3842.
Olianas MC, Ingianni A, Sogos V, Onali P (1997) Expression of
pituitary adenylate cyclase-activating polypeptide (PACAP)
receptors and PACAP in human retina. J Neurochem
69:12131218.
Ooto S, Akagi T, Kageyama R, Akita J, Mandai M, Honda Y,
Takahashi M (2004) Potential for neural regeneration after
neurotoxic injury in the adult mammalian retina. Proc Natl
Acad Sci U S A 101:1365413659.
Oppenheim RW, Blomgren K, Ethell DW, Koike M,
Komatsu M, Prevette D, Roth KA, Uchiyama Y,
Vinsant S, Zhu C (2008) Developing postmitotic
mammalian neurons in vivo lacking Apaf-1 undergo
programmed cell death by a caspase-independent, non-
apoptotic pathway involving autophagy. J Neurosci 28:
14901497.
Osakada F, Ooto S, Akagi T, Mandai M, Akaike A, Takahashi M
(2007) Wnt signaling promotes regeneration in the retina of
adult mammals. J Neurosci 27:42104219.
Osborne NN, Patel S, Vigny A (1984) Dopaminergic neu-
rones in various retinas and the postnatal development of
tyrosine-hydroxylase immunoreactivity in the rabbit retina.
Histochemistry 80:389393.
Otaegi G, de Pablo F, Vicario-Abejn C, de la Rosa EJ
(2007) Retinal and olfactory bulb precursor cells show dis-
tinct responses to FGF-2 and laminin. Cell Biol Int 31:
752758.
Otteson DC, Cirenza PF, Hitchcock PF (2002) Persistent neuro-
genesis in the teleost retina: evidence for regulation by the
growth-hormone/insulin-like growth factor-I axis. Mech Dev
117:137149.
Paes-De-Carvalho R (2002) Adenosine as a signaling molecule
in the retina: biochemical and developmental aspects. An
Acad Bras Cienc 74:437451.
Page AM, Hieter P (1997) The anaphase promoting complex. In:
Checkpoint Controls and Cancer (Kastan MB, ed.), pp. 133
150. Cold Spring Harbor Lab. Press, Cold Spring Harbor,
NY.
Patel A, McFarlane S (2000) Overexpression of FGF-2 alters cell
fate specification in the developing retina of Xenopus laevis.
Dev Biol 222:170180.
Patterson SL, Pittenger C, Morozov A, Martin KC, Scanlin H,
Drake C, Kandel ER (2001) Some forms of cAMP-mediated
long-lasting potentiation are associated with release of
BDNF and nuclear translocation of phospho-MAP kinase.
Neuron 32:123140.
Pearson R, Catsicas M, Becker D, Mobbs P (2002) Purinergic
and muscarinic modulation of the cell cycle and calcium
signaling in the chick retinal ventricular zone. J Neurosci
22:75697579.
Pearson RA, Dale N, Llaudet E, Mobbs P (2005a) ATP released
via gap junction hemichannels from the pigment epithe-
lium regulates neural retinal progenitor proliferation. Neuron
46:731744.
Pearson RA, Lneborg NL, Becker DL, Mobbs P (2005b) Gap
junctions modulate interkinetic nuclear movement in retinal
progenitor cells. J Neurosci 25:1080310814.
Pellegrini-Giampietro DE, Bennett MV, Zukin RS (1992) Are
Ca(2+)-permeable kainate/AMPA receptors more abundant
in immature brain? Neurosci Lett 144:6569.
13 Tissue Biology of Proliferation and Cell Death
Pootanakit K, Brunken WJ (2000) 5-HT(1A) and 5-HT(7)
receptor expression in the mammalian retina. Brain Res
875:152156.
Pootanakit K, Brunken WJ (2001) Identification of 5-HT(3A)
and 5-HT(3B) receptor subunits in mammalian retinae:
potential pre-synaptic modulators of photoreceptors. Brain
Res 896:7785.
Pootanakit K, Prior KJ, Hunter DD, Brunken WJ (1999)
5-HT2a receptors in the rabbit retina: potential presynaptic
modulators. Visual Neurosci 16:221230.
Pourcho RG (1996) Neurotransmitters in the retina. Curr Eye
Res 15:797803.
Pow DV, Crook DK, Wong RO (1994) Early appearance
and transient expression of putative amino acid neuro-
transmitters and related molecules in the developing rab-
bit retina: an immunocytochemical study. Vis Neurosci 11:
11151134.
Prada C, Puga J, Prez-Mndez L, Lpez R, Ramrez G (1991) Russell C (2003) The roles of hedgehogs and fibroblast growth
Spatial and temporal patterns of neurogenesis in the chick
retina. Eur J Neurosci 3:559569.
Prezeau L, Carrette J, Helpap B, Curry K, Pin JP, Bockaert
J (1994) Pharmacological characterization of metabotropic
glutamate receptors in several types of brain cells in primary
cultures. Mol Pharmacol 45:570577.
Price J, Turner D, Cepko C (1987) Lineage analysis in the ver-
tebrate nervous system by retrovirus-mediated gene transfer.
Proc Natl Acad Sci U S A 84:156160.
Pruss RM, Akeson RL, Racke MM, Wilburn JL (1991) Agonist-
activated cobalt uptake identifies divalent cation-permeable
kainate receptors on neurons and glial cells. Neuron 7:
509518.
Raff MC (1992) Social controls on cell survival and cell death.
Nature 356:397400.
Rapaport DH, Robinson SR, Stone J (1984) Cell movement and
birth in the developing cat retina. In: Development of Visual
Pathways in Mammals (Stone J, ed.), pp. 2338. Alan R.
Liss, New York.
Rapaport DH, Robinson SR, Stone J (1985) Cytogenesis in the
developing retina of the cat. Aust N Z J Ophthalmol 13:
113124.
Rapaport DH, Stone J (1983) The topography of cytogenesis in
the developing retina of the cat. J Neurosci 3:18241834.
Rapaport DH, Vietri AJ (1991) Identity of cells produced by
two stages of cytogenesis in the postnatal cat retina. J Comp
Neurol 312:341352.
Reale M, Iarlori C, Thomas A, Gambi D, Perfetti B, Di
Nicola M, Onofrj M (2008) Peripheral cytokines profile in
Parkinsons disease. Brain Behav Immun 23:5563.
Reh TA, Fischer AJ (2006) Retinal stem cells. Methods Enzymol
419:5273.
Reh TA, Levine EM (1998) Multipotential stem cells and pro-
genitors in the vertebrate retina. J Neurobiol 36:206220.
Rehen SK, Cid M, Fragel-Madeira L, Linden R (2002)
Differential effects of cyclin-dependent kinase blockers upon
cell death in the developing retina. Brain Res 947:7883.
Rehen SK, Diniz DM, Fragel-Madeira L, Brito LRG, Linden
R (1999) Selective sensitivity of early postmitotic cells to
apoptosis induced by inhibition of protein synthesis. Eur J
Neurosci 11:349356.
Riedl SJ, Salvesen GS (2007) The apoptosome: signalling plat-
form of cell death. Nat Rev Mol Cell Biol 8:405413.
227
Robanus-Maandag E, Dekker M, van der Valk M, Carrozza ML,
Jeanny JC, Dannenberg JH (1998) p107 is a suppressor of
retinoblastoma development in pRb-deficient mice. Genes
Dev 12:15991609.
Rodgers EE, Theibert AB (2002) Functions of PI 3-kinase in
development of the nervous system. Int J Dev Neurosci
20:187197.
Roegiers F, Jan YN (2004) Asymmetric cell division. Curr Opin
Cell Biol 16:195205.
Rompani SB, Cepko CL (2008) Retinal progenitor cells can pro-
duce restricted subsets of horizontal cells. Proc Natl Acad Sci
U S A 105:192197.
Roos WP, Kaina B (2006) DNA damage-induced cell death by
apoptosis. Trends Mol Med 12:440450.
Rudolph U, Crestani F, Mohler H (2001) GABA(A) receptor
subtypes: dissecting their pharmacological functions. Trends
Pharmacol Sci 22:188194.
factors in eye development and retinal cell rescue. Vision Res
43:899912.
Rutishauser U (2008) Polysialic acid in the plasticity of the
developing and adult vertebrate nervous system. Nat Rev
Neurosci 9:2635.
Ryu JK, Choi HB, Hatori K, Heisel RL, Pelech SL, McLarnon
JG, Kim SU (2003) Adenosine triphosphate induces pro-
liferation of human neural stem cells: role of calcium
and p70 ribosomal protein S6 kinase. J Neurosci Res 72:
352362.
Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto
Y (1999) Acinus is a caspase-3-activated protein required for
apoptotic chromatin condensation. Nature 401:168173.
Saito K, Kawaguchi A, Kashiwagi S, Yasugi S, Ogawa M,
Miyata T (2003) Morphological asymmetry in dividing reti-
nal progenitor cells. Develop Growth Differ 45:219229.
Sakaki Y, Fukuda Y, Yamashita M (1996) Muscarinic and
purinergic Ca2+ mobilizations in the neural retina of early
embryonic chick. Int J Dev Neurosci 14:691699.
Sampath D, and Plunkett W (2001) Design of new anticancer
therapies targeting cell cycle checkpoint pathways. Curr
Opin Oncol 13(6):484490
Sanches G, de Alencar LS, Ventura AL (2002) ATP induces pro-
liferation of retinal cells in culture via activation of PKC
and extracellular signal-regulated kinase cascade. Int J Dev
Neurosci 20:2127.
Sanchez I, Dynlacht BD (2005) New insights into cyclins, CDK,
and cell cycle control. Semin Cell Dev Biol 16:311321.
Santella L, Kyozuka K, De Riso L, Carafoli E (1998) Calcium,
protease action, and the regulation of the cell cycle. Cell
Calcium 23:123130.
Santos PF, Caramelo OL, Carvalho AP, Duarte CB (1999)
Characterization of ATP release from cultures enriched in
cholinergic amacrine-like neurons. J Neurobiol 41:340348.
Sassoe-Pognetto M, Wassle H (1997) Synaptogenesis in the
rat retina: subcellular localization of glycine receptors,
GABA(A) receptors, and the anchoring protein gephyrin.
J Comp Neurol 381:158174.
Sauer FC (1935) Mitosis in the neural tube. J Comp Neurol
62:377405.
Sauer ME, Chittenden AC (1959) Deoxyribonucleic acid content
of cell nuclei in the neural tube of the chick embryo: evidence
for intermitotic migration of nuclei. Exp Cell Res 16:16.
228
Sauer ME, Walker BE (1959) Radioautographic study of interki-
netic nuclear migration in the neural tube. Proc Soc Exp Biol
Med 101:557560.
Schfers M, Sorkin L (2008) Effect of cytokines on neuronal
excitability. Neurosci Lett 437:188193.
Seki T, Shioda S, Nakai Y, Arimura A, Koide R (1998)
Distribution and ultrastructural localization of pituitary
adenylate cyclase-activating polypeptide (PACAP) and its
receptor in the rat retina. Ann N Y Acad Sci 865:408411.
Seki T, Shioda S, Ogino D, Nakai Y, Arimura A, Koide R (1997)
Distribution and ultrastructural localization of a receptor for
pituitary adenylate cyclase activating polypeptide and its
mRNA in the rat retina. Neurosci Lett 238:127130.
Shao-Min Zhang S, Liu M-G, Kano A, Zhang C, Fu X-Y,
Barnstable CJ (2005) STAT3 activation in response to growth
factors or cytokines participates in retina precursor prolifera-
tion. Exp Eye Res 81:103115.
Sharma RK, Johnson DA (2000) Molecular signals for devel-
opment of neuronal circuitry in the retina. Neurochem Res
25:12571263.
Shelke RR, Lakshmana MK, Ramamohan Y, Raju TR (1997)
Levels of dopamine and noradrenaline in the developing
of retina effect of light deprivation. Int J Dev Neurosci
15:139143.
Shen Y, Liu XL, Yang XL (2006) N-methyl-D-aspartate recep-
tors in the retina. Mol Neurobiol 34:163179.
Sherr CJ (1993) Mammalian G1 Cyclins. Cell 73:10591065.
Sherr CJ (1996) Cancer cell cycles. Science 274:16721677.
Sherr CJ, Roberts JM (1995) Inhibitors of mammalian G1
cyclin-dependent kinases. Genes Dev 9:11491163.
Shibuya EK (2003) G2 cell cycle arrest-a direct link between
PKA and Cdc25C. Cell Cycle 2:3941.
Shiloh Y (2003) ATM and related protein kinases: safeguarding
genome integrity. Nat Rev Cancer 3:155168.
Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, Gill DL
(1993) Intracellular Ca2+ pool content is linked to control of
cell growth. Proc Natl Acad Sci U S A 90:49864990.
Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner
H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA
(1995) Cyclin D1 provides a link between development and
oncogenesis in the retina and breast. Cell 82:621630.
Sidman RL (1961) Histogenesis of mouse retina studied with
thymidine-H3. In: The Structure of the Eye (Smelser GK,
ed.), pp. 487506. Academic Press, London.
Silva AG, Campello-Costa P, Linden R, Sholl-Franco A (2008)
Interleukin-4 blocks proliferation of retinal progenitor cells
and increases rod photoreceptor differentiation through dis-
tinct signaling pathways. J Neuroimmunol 196:8293.
Silveira dos Santos Bredariol A, Hamassaki-Britto DE (2001)
Ionotropic glutamate receptors during the development of the
chick retina. J Comp Neurol 441:5870.
Silveira MS, Costa MR, Bozza M, Linden R (2002) Pituitary
adenylyl cyclase-activating polypeptide prevents induced
cell death in retinal tissue through activation of cyclic AMP-
dependent protein kinase. J Biol Chem 277:1607516080.
Silver J, Robb RM (1979) Studies on the development of the
eye cup and optic nerve in normal mice and in mutants with
congenital optic nerve aplasia. Dev Biol 68:175190.
Stenkamp DL, Frey RA, Prabhudesai SN, Raymond PA (2000)
Function for hedgehog genes in zebrafish retinal develop-
ment. Dev Biol 220:238252.
R. Linden et al.
Stewart ZA, Westfall MD, Pietenpol JA (2003) Cell-cycle dys-
regulation and anticancer therapy. Trends Pharmacol Sci
24:139145.
Sturman JA (1988) Taurine in development. J Nutr 118:
11691176.
Sucher NJ, Kohler K, Tenneti L, Wong HK, Grunder T, Fauser
S, Wheeler-Schilling T, Nakanishi N, Lipton SA, Guenther E
(2003) N-methyl-D-aspartate receptor subunit NR3A in the
retina: developmental expression, cellular localization, and
functional aspects. Invest Ophthalmol Vis Sci 44:44514456.
Sugioka M, Fukuda Y, Yamashita M (1996) Ca2+ responses to
ATP via purinoceptors in the early embryonic chick retina.
J Physiol 493:855863.
Sugioka M, Zhou WL, Hofmann HD, Yamashita M (1999a)
Ca2+ mobilization and capacitative Ca2+ entry regulate DNA
synthesis in cultured chick retinal neuroepithelial cells. Int J
Dev Neurosci 17:163172.
Sugioka M, Zhou W, Hofmann H, Yamashita M (1999b)
Involvement of P1 purinoceptors in the regulation of DNA
synthesis in the neural retina of chick embryo. Int J Devl
Neurosci 17:135144.
Sun H, Chang Y, Schweers B, Dyer MA, Zhang X, Hayward SW,
Goodrich DW (2006) An E2F binding-deficient Rb1 protein
partially rescues developmental defects associated with Rb1
nullizygosity. Mol Cell Biol 26:15271537.
Takahashi T, Nowakowski RS, Caviness VS Jr (1996) The
leaving or Q fraction of the murine cerebral proliferative
epithelium: a general model of neocortical neurogenesis.
J Neurosci 16:61836196.
Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito
S, Anderson CW, Appella E, Nakanishi M, Suzuki H,
Nagashima K, Sawa H, Ikeda K, Motoyama N (2002)
Chk2-deficient mice exhibit radioresistance and defective
p53-mediated transcription. EMBO J 21:51955205.
Takeda M, Takamiya A, Jiao JW, Cho KS, Trevino SG, Matsuda
T, Chen DF (2008) alpha-Aminoadipate induces progen-
itor cell properties of Muller glia in adult mice. Invest
Ophthalmol Vis Sci 49:11421150.
Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME
(1998) Ca2+ influx regulates BDNF transcription by a CREB
family transcription factor-dependent mechanism. Neuron
20:709726.
Taomoto M, McLeod DS, Merges C, Lutty GA (2000)
Localization of adenosine A2a receptor in retinal develop-
ment and oxygen-induced retinopathy. Invest Ophthalmol
Vis Sci 41:230243.
Tappaz ML (2004) Taurine biosynthetic enzymes and tau-
rine transporter: molecular identification and regulations.
Neurochem Res 29:8396.
Tasdemir E, Galluzzi L, Maiuri MC, Criollo A, Vitale I, Hangen
E, Modjtahedi N, Kroemer G (2008) Methods for assess-
ing autophagy and autophagic cell death. Methods Mol Biol
445:2976.
Thoreson WB, Witkovsky P (1999) Glutamate receptors and
circuits in the vertebrate retina. Prog Retin Eye Res 18:
765810.
Thurston AW Jr, Rhee SG, Shukla SD (1993) Role of guanine
nucleotide-binding protein and tyrosine kinase in platelet-
activating factor activation of phospholipase C in A431
cells: proposal for dual mechanisms. J Pharmacol Exp Ther
266:11061112.
13 Tissue Biology of Proliferation and Cell Death
Tibber MS, Whitmore AV, Jeffery G (2006) Cell division and
cleavage orientation in the developing retina are regulated by
L-DOPA. J Comp Neurol 496:369381.
Tornqvist K, Ehinger B (1983) Glucagon immunoreactive neu-
rons in the retina of different species. Graefes Arch Clin Exp
Ophthalmol 220:15.
Tornqvist K, Loren I, Hakanson R, Sundler F (1981) Peptide-
containing neurons in the chicken retina. Exp Eye Res 33:
5564.
Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ,
McInnes RR, van der Kooy D (2000) Retinal stem cells in
the adult mammalian eye. Science 287:20322036.
Turner DL, Snyder EY, Cepko CL (1990) Lineage-independent
determination of cell type in the embryonic mouse retina.
Neuron 4:833845.
Ueno M, Katayama K, Yamauchi H, Nakayama H, Doi K (2006)
Cell cycle progression is required for nuclear migration of
neural progenitor cells. Brain Res 1088:5767.
Upton AL, Salichon N, Lebrand C, Ravary A, Blakely
R, Seif I,Gaspar P (1999) Excess of serotonin (5-HT)
alters the segregation of ipsilateral and contralat-
eral retinal projections in monoamine oxidase A
knock-outmice: possible role of 5-HT uptake in reti-
nal ganglion cells during development. J Neurosci 19:
70077024.
Vallires L, Campbell IL, Gage FH, Sawchenko PE (2002)
Reduced hippocampal neurogenesis in adult transgenic
mice with chronic astrocytic production of interleukin-6. J
Neurosci 22:486492.
van Koppen CJ, Kaiser B (2003) Regulation of mus-
carinic acetylcholine receptor signaling. Pharmacol Ther 98:
197220.
Vela JM, Molina-Holgado E, Arvalo-Martn A, Almazn G, Xiao Z, Chen Z, Gunasekera AH, Sowin TJ, Rosenberg SH,
Guaza C (2002) Interleukin-1 regulates proliferation and dif-
ferentiation of oligodendrocyte progenitor cells. Mol Cell
Neurosci 20:489502.
Versaux-Botteri C, Pochet R, Nguyen-Legros J (1989)
Immunohistochemical localization of GABA-containing
neurons during postnatal development of the rat retina. Invest
Ophthalmol Vis Sci 30:652659.
Vidal A, Koff A (2000) Cell-cycle inhibitors: three families
united by a common cause. Gene 247:115.
von Bartheld CS (1998) Neurotrophins in the developing and
regenerating visual system. Histol Histopathol 13:437459.
von Bartheld CS, Byers MR, Williams R, Bothwell M (1996)
Anterograde transport of neurotrophins and axodendritic
transfer in the developing visual system. Nature 379:
830833.
Vugler A, Lawrence J, Walsh J, Carr A, Gias C, Semo M,
Ahmado A, da Cruz L, Andrews P, Coffey P (2007)
Embryonic stem cells and retinal repair. Mech Dev 124:
807829.
Wallace VA (2008) Proliferative and cell fate effects of hedgehog
signaling in the vertebrate retina. Brain Res 1192:6175.
Walworth NC (2000) Cell cycle checkpoint kinases: checking in
on the cell cycle. Curr Opin Cell Biol 12:697704.
Wamsley JK, Palacios JM, Kuhar MJ (1981) Autoradiographic
localization of opioid receptors in the mammalian retina.
Neurosci Lett 27:1924.
Wang X, Fu S, Wang Y, Yu P, Hu J, Gu W, Xu XM,
Lu P (2007) Interleukin-1beta mediates proliferation and
229
differentiation of multipotent neural precursor cells through
the activation of SAPK/JNK pathway. Mol Cell Neurosci 36:
343354.
Waschek JA, Dicicco-Bloom E, Nicot A, Lelievre V (2006)
Hedgehog signaling new targets for GPCRs coupled to
cAMP and protein kinase A. Ann N Y Acad Sci 1070:
120128.
Weinberg RA (1995) The retinoblastoma protein and cell cycle
control. Cell 81:323330.
Whitaker M, Larman MG (2001) Calcium and mitosis. Semin
Cell Dev Biol 12:5358.
Willer GB, Lee VM, Gregg RG, Link BA (2005) Analysis of the
zebrafish perplexed mutation reveals tissue-specific roles for
de novo pyrimidine synthesis during development. Genetics
170:18271837.
Woldemussie E, Wijono M, Pow D (2007) Localization of alpha
2 receptors in ocular tissues. Vis Neurosci 24:745756.
Wong RO (1995a) Cholinergic regulation of [Ca2+ ]i during
cell division and differentiation in the mammalian retina.
J Neurosci 15:26962706.
Wong RO (1995b) Effects of glutamate and its analogs on intra-
cellular calcium levels in the developing retina. Vis Neurosci
12:907917.
Wu DK, Cepko CL (1993) Development of dopaminergic neu-
rons is insensitive to optic nerve section in the neonatal rat
retina. Dev Brain Res 74:253260.
Wu Y, Cutting GR (2001) Developmentally regulated expression
of GABA receptor rho1 and rho2 subunits, L7 and cone-
rod homeobox (CRX) genes in mouse retina. Brain Res 912:
18.
Wymann MP, Pirola L (1998) Structure and function of phospho-
inositide 3-kinases. Biochim Biophys Acta 1436:127150.
Fesik S, Zhang H (2003) Chk1 mediates S and G2 arrests
through CDC25A degradation in response to DNA-damaging
agents. J Biol Chem 278:2176721773.
Yamasaki EN, Barbosa VD, De Mello FG, Hokoc JN (1999)
GABAergic system in the developing mammalian retina:
dual sources of GABA at early stages of postnatal develop-
ment. Int J Dev Neurosci 17:201213.
Yamashita M, Fukuda Y (1993a) Calcium channels and GABA
receptors in the early embryonic chick retina. J Neurobiol
24:16001614.
Yamashita M, Yoshimoto Y, Fukuda Y (1994) Muscarinic
acetylcholine responses in the early embryonic chick retina.
J Neurobiol 25:11441153.
Yang XL (2004) Characterization of receptors for glutamate and
GABA in retinal neurons. Prog Neurobiol 73:127150.
Yaron O, Farhy C, Marquardt T, Applebury M, Ashery-Padan R
(2006) Notch1 functions to suppress cone-photoreceptor fate
specification in the developing mouse retina. Development
133:13671378.
Yeo GW, Coufal N, Aigner S, Winner B, Scolnick JA, Marchetto
MC, Muotri AR, Carson C, Gage FH (2008) Multiple lay-
ers of molecular controls modulate self-renewal and neuronal
lineage specification of embryonic stem cells. Hum Mol
Genet 17:R67R75.
Yew DT, Luo CB, Zheng DR, Guan YL, Tsang D, Stadlin
A (1991) Immunohistochemical localization of substance P,
enkephalin and serotonin in the developing human retina. J
Hirnforsch 32:6167.
230
Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R,
Penninger JM, Mak TW (1998) Apaf1 is required for mito-
chondrial pathways of apoptosis and brain development. Cell
94:739750.
Yoshikawa K (2000) Cell cycle regulators in neural stem cells
and postmitotic neurons. Neurosci Res 37:114.
Young RW (1985a) Cell proliferation during postnatal develop-
ment of the retina in the mouse. Brain Res 353:229239.
Young RW (1985b) Cell differentiation in the retina of the
mouse. Anat Rec 212:199205.
Young TL, Cepko CL (2004) A role for ligand-gated ion chan-
nels in rod photoreceptor development. Neuron 41:867879.
Yu J, Zhang L (2003) No PUMA, no death: implications for p53-
dependent apoptosis. Cancer Cell 4:248249.
Zarbin MA, Wamsley JK, Palacios JM, Kuhar MJ (1986)
Autoradiographic localization of high affinity GABA, benzo-
diazepine, dopaminergic, adrenergic and muscarinic cholin-
ergic receptors in the rat, monkey and human retina. Brain
Res 374:7592.
Zezula J, Freissmuth M (2008) The A2A-adenosine receptor:
a GPCR with unique features? Br J Pharmacol 153:S184
S190.
Zhang H, Ching S, Chen Q, Li Q, An Y, Quan N (2008)
Localized inflammation in peripheral tissue signals the CNS
for sickness response in the absence of interleukin-1 and
R. Linden et al.
cyclooxygenase-2 in the blood and brain. Neuroscience
157:895907.
Zhang J, Gray J, Wu L, Leone G, Rowan S, Cepko CL, Zhu
X, Craft CM, Dyer MA (2004a) Rb regulates proliferation
and rod photoreceptor development in the mouse retina. Nat
Genet 36:351360.
Zhang J, Schweers B, Dyer MA (2004b) The first knockout
mouse model of retinoblastoma. Cell Cycle 3:952959.
Zhang L, Spigelman I, Carlen PL (1991) Development of
GABA-mediated, chloride-dependent inhibition in CA1
pyramidal neurones of immature rat hippocampal slices.
J Physiol 444:2549.
Zhao S, Barnstable CJ (1996) Differential effects of bFGF on
development of the rat retina. Brain Res 723:169176.
Zhao JJ, Lemke G (1998) Selective disruption of neuregulin-1
function in vertebrate embryos using ribozyme-tRNA trans-
genes. Development 125:18991907.
Zhu CY, Shukla SD (1993) Increased tyrosine kinase activity
in pp60c-src immunoprecipitate from platelet activating fac-
tor stimulated human platelets: in vitro phosphorylation of a
synthetic peptide. Life Sci 53:175183.
Zygar CA, Colbert S, Yang D, Fernald RD (2005) IGF-1
produced by cone photoreceptors regulates rod progenitor
proliferation in the teleost retina. Brain Res Dev Brain Res
154:91100.
Chapter 14

Potential Application of Very Small Embryonic Like (VSEL)

Stem Cells in Neural Regeneration

Mariusz Z. Ratajczak, Ewa Zuba-Surma, Magda Kucia, Przemyslaw Nowacki,

and Bogdan Machalinski

Contents mice, the stroke-related stress may trigger mobiliza-

14.1 Introduction . . . . . . . . . . . . . . . . .
14.2 Identification of Very Small Embryonic Like
Stem Cells (VSEL) in Adult Murine Bone
Marrow . . . . . . . . . . . . . . . . . . .
14.3 Identification of VSEL in Adult Murine Organs
Including Adult Brain . . . . . . . . . . . .
14.4 Bone-Marrow-Derived VSEL as Population of
Circulating Pluripotent Stem Cells . . . . . .
14.5 Biological Properties of VSEL . . . . . . . .
14.6 Cells that Express VSEL Markers are Mobilized
into PB in Patients After Stroke . . . . . . .
14.7 Conclusions . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .
tion of these cells into peripheral blood. We noticed
232 in stroke patients an increase in the number of circu-
lating primitive cells expressing the VSEL phenotype.
Additionally, we noticed a positive correlation between
233
stroke extensiveness, SDF-1 concentration in serum,
234 and the number of VSEL circulating in the peripheral
blood. We conclude that VSEL could have a potential
237 prognostic value in stroke patients, and more impor-
239
tant that their role in brain regeneration requires further
240 study.
241
241 Keywords CXCR4 Mobilization Pluripotent stem
cells Stroke VSEL

Abstract We found that murine bone marrow con-

tains a mobile population of Oct-4+ CXCR4+ SSEA- Abbreviations
1+ Sca-1+ lin CD45 very small embryonic like stem
cells (VSEL) that are mobilized into peripheral blood 7-AAD 7-aminoactinomycin D
(PB) e.g., after pharmacological mobilization with BM bone marrow
G-CSF or in murine model of stroke. We postu- BMNC bone-marrow nucleated cells
late that, VSEL are a mobile population of epi- CB cord blood
blast/germ line derived stem cells and play an CXCR4 CXC chemokine receptor 4
important role as organ-residing reserve population ESC embryonic stem cells
of pluripotent stem cells that gives rise to stem FACS fluorescence activated cell sorting
cells committed to particular organs and tissues FSC forward scatter
G-CSF granulocyte colony stimulating factor
including neural tissue. Moreover, since a similar
GFAP glial fibrillary acidic protein
population of small CXCR4+ CD133+ CD34+ SSEA-
HGF hepatocyte growth factor
4+ Oct-4+ lin CD45 cells resides also in human bone
HGF/SF hepatocyte growth factor/scatter factor
marrow, we asked whether in humans, similarly as in
HSC hematopoietic stem cells
ISS ImageStream
LACI lacunar infarcts
LIF leukemia inhibitory factor
M.Z. Ratajczak ()
Stem Cell Institute at James Graham Brown Cancer Center, MAPC multipotent adult progenitor cells
University of Louisville, Louisville, KY, USA MSC mesenchymal stem cells
e-mail: mzrata01@louisville.edu PACI partial anterior circulation infarcts

H. Ulrich (ed.), Perspectives of Stem Cells, 231

DOI 10.1007/978-90-481-3375-8_14, Springer Science+Business Media B.V. 2010
232
PB peripheral blood
PBNC peripheral blood nucleated cells
PGC primordial germ cells
POCI posterior circulation infarcts
PSC pluripotent stem cells(s)
RBC red blood cells
SC stem cell
SDF-1 stromal derived factor-1
SSC side scatter
SSEA-1 stage specific embryonic antigen-1
TACI total anterior ciculation infarcts
TC computer tomography
VEGF vascular endothelial growth factor
VSEL very small embryonic like stem cell(s)
VSEL-DS very small embryonic like stem
cells-derived spheres
14.1 Introduction
The occlusion of a cerebral artery leads to focal
ischemia and stroke that results in damage to neu-
rons and glial cells (Dirnagl et al., 1999; Endres
and Dirnagl, 2002). Thus the goal of regenerative
medicine is to ameliorate irreversible destruction of
brain tissue by harnessing the power of stem cells in
process of neurogenesis (Kalluri and Dempsey, 2008;
Komitova et al., 2005). Neurogenesis supplies new
neurons and glial cells (astrocytes and oligodendro-
cytes) and occurs in the mammalian brain throughout
life, and so far has been clearly demonstrated at two
locations in subventricular zone of the lateral ven-
tricles and olfactory bulb as well as in the subgranu-
lar zone of the dentate gyrus in hippocampus (Duan
et al., 2008; Encinas and Enikolopov, 2008). Data from
mouse and rats have shown that stroke and subsequent
death of neurons leads to increased proliferation of
neural precursors that are located in the subventricu-
lar zone, olfactory bulb and hippocampus (Komitova
et al., 2005; Zhang et al., 2001). Unfortunately, this
response is not effective enough to fully restore mor-
phology and function of neural tissue damaged by
stroke.
Whether neurogenesis also occurs in areas of the
mammalian brain other than hippocampus and sub-
ventricular region remains controversial. Interestingly,
mice with cyclin D2 deficiency that have severely
reduced number of stem cells in subventricular zone,
olfactory bulb and hippocampus, yet possess normally
M.Z. Ratajczak et al.
developed brains and brain-derived cells from these
animals are able to grow neurospheres (Walzlein et al.,
2008). This suggests a potential involvement in brain
development and contribution to neural tissue home-
ostasis, stem cells that reside in other locations. These
cells could reside in other brain areas or translocate
to the brain using peripheral blood (PB) from other
organs/tissues most likely bone marrow (BM) (Hess
and Borlongan, 2008; Shyu et al., 2006).
To support this last notion, it has been shown that in
response to organ damage, stem cells could be mobi-
lized into PB from BM and other tissue-specific niches
as well. These circulating stem cells potentially would
home to the damaged tissues and attempt to contribute
to regeneration (Shyu et al., 2006; Quesenberry et al.,
2007). We observed that the number of circulating
stem cells expressing pluripotent and tissue-specific
markers increases in PB after granulocyte-colony
stimulating factor (G-CSF) administration, heart
infarct in humans and mice, and in murine model of
stroke (Kucia et al., 2004a, b, 2008b; Wojakowski
et al., 2004; Zuba-Surma et al., 2008b). While these
cells may act somehow in regeneration from small tis-
sue injuries, their contribution toward repairing more
extensive tissue damages requires further study. Larger
injuries (e.g., massive stroke) by creating highly pro-
teolytic environment may perturb mechanisms that
regulate chemotaxis and homing of these cells to the
damaged organ.
Recently, we identified a population of very small
embryonic-like (VSEL) stem cells in adult murine BM
that express several pluripotent stem cells (PSC) mark-
ers and purified them at the single cell level (Kucia
et al., 2006a; Zuba-Surma et al., 2008a). We noticed
that VSEL are mobilized into PB after G-CSF admin-
istration, during toxic liver and skeletal muscle damage
in mice (Kucia et al., 2008b) and after heart infarct and
stroke both in humans and mice (Wojakowski et al.,
2004; Zuba-Surma et al., 2008b). VSEL are present in
versatile adult organs and interestingly, our data indi-
cates that the brain contains a high number of cells that
display VSEL phenotype (Ratajczak et al., 2008b, d).
Thus the role of both circulating primitive VSEL as
well as those VSEL that reside in normal brain in
regeneration of damaged neural tissue requires further
studies. In this chapter we will present the concept that
these cells could be employed as a prognostic marker
as well as could be harnessed in regenerative medicine
as a potential source of stem cells for neurogenesis.
14 Potential Application of VSEL Stem Cells in Neural Regeneration
14.2 Identification of Very Small
Embryonic Like Stem Cells (VSEL)
in Adult Murine Bone Marrow
Based on our initial work suggesting that BM may
contain unusually small cells (36 m in diame-
ter) that are (i) negative for lineage markers (lin ),
(ii) CD45 , (iii) express Sca-1 antigen (mouse) and
CXC chemokine receptor 4 (CXCR4), CD133, CD34
antigens (mouse and humans) and (iv) are pos-
itive for markers of PSC (Oct-4, SSEA) (Kucia
et al., 2006a; Zuba-Surma et al., 2008a; Kucia et al.,
2007a) we develop a sorting strategy of BM-
derived cells controlled by the beads with prede-
fined sizes (1, 2, 4, 6, 10, and 15 m in diameter)
(Zuba-Surma et al., 2008a). Generally, this strategy
allowed us to sort cells (i) very small in size (~3
5 m and 46 m mouse and human respec-
tively); (ii) Oct-4+ CXCR4+ SSEA-1+ Sca-1+ CD45 lin
(mouse) Oct-4+ CXCR4+ SSEA-4+ CD133+ CD45 lin
(human) (iii) possessing large nuclei containing
A B
SSC
SSC
Beads in size:
2-10 m
R1
FSC
R1 & R2 & R3
VSEL
- - +
CD45 /Lin /Sca-1
R1 & R2 & R4
HSC
+ -
CD45 /Lin /Sca-1
Fig. 14.1 Gating strategy for VSEL sorting by FACS. BM-
derived VSEL were isolated from immunofluorescence stained
murine BM nucleated cells by FACS. Panel A: Agranular,
small events ranging from 2 to 10 m were included into gate
R1 after comparison with six differently sized bead particles
with standard diameters of 1, 2, 4, 6, 10, and 15 m (Flow
Cytometry Size beads, Invitrogen; Molecular Probes, Carlsbad,
CA, USA). Panel B: BM nucleated cells were visualized by
dot plot showing forward scatter (FSC) vs. side scatter (SSC)
signals, that are parameters related to the size and granular-
ity/complexity of the cell, respectively. Panel C: Cells from
233
unorganized chromatin (euchromatin) (Zuba-Surma
et al., 2008a; Kucia et al., 2007b).
An example of this novel fluorescence activated cell
sorting (FACS) approach controlled by size bead mark-
ers is shown at Fig. 14.1. The first step in this strategy
was to gate in regions containing small events (2
10 m) shown as region R1 on the dot plot (Fig.
14.1 panel A and B). This region mostly contains cell
debris, erythrocytes and large blood platelets but also
as we expected includes some rare nucleated small
cells. The events enclosed in region R1 (Fig. 14.1
panel A and B), which include an average of ~50%
of total events scored by cytometer, are further ana-
lyzed for the expression of Sca-1 and lineage markers
(lin). The Sca-1+ /Lin events shown in region R2 (Fig.
14.1 panel D) consist of 0.30 0.05% of total analyzed
BM nucleated cells on average. Cells from region R2
are subsequently sorted according to the expression
of CD45 antigen as Sca-1+ /lin /CD45 (region R3)
and Sca-1+ /lin /CD45+ (region R4) subpopulations
(Fig. 14.1 panel C).
C
Lineage
R2
0.300.05%
R1 47.2 2.1%
FSC Sca-1
D
R3 R4
0.03 0.01% 0.35 0.06%
Counts
+
CD45
region R1 were further analyzed for Sca-1 and Lin expres-
sion and only Sca-1+ /Lin events were included into region R2.
Population from region R2 was subsequently sorted based on
CD45 antigen expression into CD45 and CD45+ subpopula-
tions visualized on histogram (Panel D, regions R3 and R4,
respectively). Sca-1+ /Lin /CD45 cells (VSEL) were sorted as
events enclosed in logical gate including regions R1, R2, and
R3, while Sca-1+ /Lin /CD45+ cells (HSC) from gate including
regions R1, R2, and R4. Percentages show the average content
of each cellular subpopulation ( SEM) in total BM nucleated
cells
234
The first population shown in R3 (Sca-1+ /lin /
CD45 ) contains VSEL that highly express early
embryonic markers Oct-4 and SSEA-1 as revealed by
subsequent immunofluorescence analysis, and a sec-
ond one shown in R4 (Sca-1+ /lin /CD45+ ) is highly
enriched for hematopoietic stem cells (HSC). Direct
transmission electron microscopy (TEM) analysis con-
firmed that Sca-1+ /lin /CD45 cells display several
features typical for embryonic stem cells such as small
size, a large nucleus surrounded by a narrow rim
of cytoplasm, and open-type chromatin (euchromatin)
(Kucia et al., 2006a). In contrast Sca-1+ lin CD45+
cells display heterogeneous morphology and are larger.
They measure on average 810 m in diameter;
possess scattered chromatin and prominent nucleoli
(Kucia et al., 2006a). We found that VSEL com-
prise ~0.03% while HSC are ~0.35% of total BM
nucleated cells (Fig. 14.1 panel C). We also noticed
that 95% of Sca-1+ /lin /CD45 (VSEL) are located
within the 26 m size range, while 86% of Sca-
1+ /lin /CD45+ (HSC) are found in the 610 m size
range (Zuba-Surma et al., 2008a). Thus, by employ-
ing flow cytometry and the size marker beads, we
have confirmed that the majority of Sca-1+ /lin /CD45
cells isolated from adult BM are unusually small cells
(<6 m) (Zuba-Surma et al., 2008a), and as expected
similar to those observed in our previous chemotactic
isolation experiments.
In the next step a novel imaging strategy called
ImageStream system (ISS) analysis was employed to
better enumerate and evaluate morphological features
of VSEL (Zuba-Surma et al., 2008a). This novel ISS-
based analysis involves flow cytometry combined with
microscopy and allows for statistical analysis of var-
ious cellular parameters as well as visualization of
cells in suspension during flow analysis via high res-
olution brightfield, darkfield and fluorescence images
(Basiji et al., 2007; Ortyn et al., 2006; Zuba-Surma
et al., 2007, 2008c). The high resolution of ISS imag-
ing enables the identification of objects even as small
as 1 m in diameter. Thus this imaging strategy is ideal
to analyze small cellular events in a range of 26 m
on a flow cytometry cytogram, where VSEL are to be
found (Zuba-Surma et al., 2008a).
Thus, we employed ISS to verify the results
obtained from flow classical FACS analysis to visu-
alize better events of interest. For ISS analysis, BM
nucleated cells (BMNC) were fixed and stained with
DNA-binding fluorescence dye 7-aminoactinomycin
M.Z. Ratajczak et al.
(7-AAD) cells to visualize better nucleated cells
(Fig. 14.2 panel A). By employing this staining,
the nuclear area was calculated by automatic masks
created on the 7-AAD nuclear images (morphology
mask). Cytoplasmic area was computed by subtract-
ing the nuclear area from the total area of the cell.
The N/C ratio was computed as the ratio between
nuclear and cytoplasmic areas. Moreover, based on
morphological features of acquired objects and their
direct microscopic visualization by the ISS, we were
able to exclude debris and artifacts and nucleated,
intact cells only were selected for further analysis
(Fig. 14.2).
By employing ISS analysis that allows the analysis
of gallery of pictures of acquired cells, we confirmed
with greater precision that murine BM-derived VSEL
are ~3.6 m in diameter, while Sca-1+ /lin /CD45+
HSC are larger at ~6.5 m in diameter (Zuba-Surma
et al., 2008a). We also noticed that VSEL have signif-
icantly higher nuclear/cytoplasmic ratio as compared
with HSC (1.5 0.2 and 0.8 0.03, respectively).
Furthermore, VSEL had significantly lower cytoplas-
mic area as compared with HSC (5.4 0.6 and
33.8 1.7, respectively) (Zuba-Surma et al., 2008a).
Despite their small size, VSEL when evaluated by
FACS for DNA content possess diploid DNA. They
do not express MHC-I and human leukocyte antigen-
D related (HLA-DR) antigens on the surface and
are CD90 CD105 CD29 (Kucia et al., 2008a).
Furthermore, as directly confirmed by ISS analysis
BM-derived VSEL are larger than peripheral blood
platelets and smaller than erythrocytes (Fig. 14.3).
14.3 Identification of VSEL in Adult
Murine Organs Including Adult
Brain
As described in previous paragraph, VSEL isolated
from murine BM are Oct-4 positive subpopulation
of Sca-1+ Lin CD45 cells. Thus in the next step
we evaluated a number of VSEL in versatile adult
murine organs (Ratajczak et al., 2008d, a). In order
to analyze adult organs for the presence of Sca-1+
lin CD45 cells, mice were perfused to remove resid-
ual VSEL that as we described may also circulate
at a very low level in peripheral blood. Removed
14 Potential Application of VSEL Stem Cells in Neural Regeneration
A VSEL
10m
RBC 4.94 0.46 m
10m
B RBC VSEL
Synthetic beads
10m 6m 4m
Fig. 14.2 Morphological comparison between murine
VSEL, HSC, erythrocytes, and platelets by ISS. Panel A
presents murine VSEL stained for Sca-1 (FITC, green), Lin
(PE, orange), and CD45 (PE-Cy5, magenta), and blood-derived
erythrocytes and platelets stained for Ter119 (PE, orange)
and CD41 (FITC, green), respectively. Following fixation, all
samples were stained with 7-AAD (red) to visualize nuclei.
Erythrocytes and platelets do not possess nuclei, while VSEL
organs were subsequently enzymatically homogenized
and isolated nucleated cells washed and stained with
specific antibodies for flow cytometric and ISS anal-
ysis. Flow cytometric analyses were performed on
freshly isolated or fixed cells co-stained as in case
of BM-derived cells with nuclear DNA-binding dye
7-AAD. Inclusion of 7-ADD into the staining pro-
tocol of fixed cells as mentioned above allowed us
to visualize real nucleated events and exclude arti-
facts. By employing classical flow cytometry and ISS
we found that all analyzed organs contain popula-
tion of Sca-1+ lin CD45 cells. Furthermore, inclu-
sion of nuclear dye (7-ADD) into staining protocol
significantly improved the accuracy of our analysis
by distinguishing nucleated objects from anucleated
7-ADD-negative debris. ISS allowing visualization of
acquired objects by showing their real images gave us
opportunity to not only distinguish nucleated objects
from anucleated debris, but as mentioned above also
235
3.63 0.27 m
Platelets 2.19 0.71 m
10m
Platelets
2m 1m
show cellular structure containing nuclei. Average size of each
population was computed as the length of the minor cellular axis
based on the mask covering the brightfield image of the cell
(Mean SEM). Scale bars show 10 m. Panel B shows cells
from all three populations (VSEL, red blood cells [RBC] and
platelets) when compared with size predefined beads in the same
scale
positive cells from falsely positive artifacts (Ratajczak
et al., 2008d, a). As expected we detected a presence of
small nucleated Oct-4+ Sca-1+ lin CD45 cells in all
analyzed organs and tissues including brain (Fig. 14.4).
W found that pancreas, brain, skeletal muscles and kid-
neys are the organs with the highest percent content of
these cells (0.3300.099, 0.1100.027, 0.0820.018
and 0.0560.004%, respectively), while bone mar-
row, thymus and spleen contain the lowest percentage
of Oct-4+ Sca-1+ Lin CD45 cells (0.00180.0003,
0.00180.0003 and 0.0050.001%, respectively).
Next, we calculated the absolute number of these
cells/organ (Table 14.1). We found that the high-
est number of small nucleated Oct-4+ Sca-1+ lin
CD45 cells/organ is in the brain, kidneys, skeletal
muscles, pancreas and bone marrow (43.9712.38,
19.872.03, 15.186.79, 9.414.71 and 8.392.00
103 , respectively). On the other hand, heart, thy-
mus, testes and spleen contained the lowest numbers of
236 M.Z. Ratajczak et al.

A a) b)
700
B
a) Sca-1+/Lin-/CD45- cell

AR of Brightfield
1 R1 -
600
R2 CD45
R8
.8

Frequency
R1 500

.6 400

300
.4
200
.2
100

0
Single cells b) Negative cell with positive fragment
0
0 2 00 40 0 600 8 00 1e3 -100 0 0 1 000 1e4 1e 5 1e6

Area of nucleus (7-AAD) CD45 (PE-Cy5)

c) d)
250 90
c) Dead/ degrading cell
200 R3 R7 -
Lin Lin-/CD45-/Sca-1+
Frequency

Frequency
60
150 R4
100
30
50
d) Cellular fragment/ debris
0 0
-100 0 0 1 000 1e4 1 e5 1 e6 0 10 00 1e4 1e 5 1 e6

Lineage (PE) Sca-1 (FITC)

a) b) c) d)

Fig. 14.3 ImageStream system analysis of kidney- derived of CD45 (Panel A.b) and Lin (Panel A.c). Lin /CD45 objects
Sca-1+ /Lin /CD45 cells. Panel A shows analysis of whole from logical gate including regions R2 and R3 are visualized
population of cells isolated from kidney following enzymatic on the histogram presenting their Sca-1 expression (Panel A.d).
digestion of the tissue and the staining for Sca-1, CD45 and Sca-1+ /Lin /CD45 cells are included in region R4. Panel B
hematopoietic lineages markers (Lin). (Panel A.a) shows all shows normal, nucleated Sca-1+ /Lin /CD45 cells (Panel B.a)
acquired objects according to their morphological parameters when compared to the objects imitating them phenotypically.
including the area of the nucleus and the aspect ratio (AR) of Selected images of falsely positive artifacts (Panel B.b), dam-
brightfield. The aspect ratio (AR) is calculated based on bright- aged/degradating cells (Panel B.c) and cellular debris (Panel
field image of each cell as the ratio of cellular minor axis (width) B.d) are shown on the next panels. Each photograph presents
to major axis (height). Round, non-elongated cells possess an brightfield image as well as separate fluorescent images related
aspect ratio close to 1.0, while the elongated cells or clumps to nuclear image (7-AAD; red) and expression of Sca-1 (FITC,
lower than 1.0. Round, single cells containing DNA are included green), Lin (PE; orange) and CD45 (PE-Cy5; yellow). Moreover
in region R1 for further analysis. Objects from Region R1 are magnified combined images of each presented object are shown
subsequently visualized on histograms based on the expression on the lower panel. The scale bar indicates 10 m

these cells/organ (1.350.56, 2.030.37, 2.381.25 and heart (3.780.64 and 4.740.93 m, respectively)
and 3.860.43 103 , respectively) (Table 14.1). (Zuba-Surma et al., 2008a). At the same time Oct-
Thus, by employing flow cytometry- and ISS-based 4+ Sca-1+ Lin CD45 cells detected in other organs
approaches, we identified Oct-4+ cells correspond- were also small (<6 m). Furthermore, relatively high
ing phenotypically to VSEL in various adult organs nuclear to cytoplasmic ratio confirmed primitive char-
(Ratajczak et al., 2008d, a). Furthermore, ISS allowed acter of analyzed populations. BM Bone marrow and
us again to distinguish Oct-4+ Sca-1+ lin CD45 testes were identified as organs containing Oct-4+ Sca-
VSEL from cellular debris and artifacts commonly 1+ Lin CD45 cells with the highest N/C (2.120.33
present in enzymatically digested samples. and 2.110.40, respectively). Cells with the lower N/C
In the next step, we employed collected images of ratio were observed in all other organs. The presence of
these cells to calculate their morphological features small Oct-4+ Sca-1+ lin CD45 cells detected by ISS
such as average size and nuclear to cytoplasmic (N/C) was subsequently confirmed by confocal microscopy
ratio which were computed based on brightfield and on Sca-1+ lin CD45 cells sorted from various organs
nuclear images of cells. We noticed that the small- and we identified small (<5m) Oct-4+ cells with the
est Oct-4+ Sca-1+ lin CD45 cells reside in the BM VSEL phenotype in all organs tested (Ratajczak et al.,
14 Potential Application of VSEL Stem Cells in Neural Regeneration 237

A Heart (1.1%) a highest number of Oct-4 mRNA transcripts in Sca-1+

Thymus (1.7%) Lin CD45 cells isolated from BM.
100% Testes (2.0%)
Spleen (3.3%)
80% Lungs (4.5%)
Liver (5.9%)
60% 14.4 Bone-Marrow-Derived VSEL
Bone marrow (7.1%)
40% Pancreas (7.9%) as Population of Circulating
S. Muscles (12.8%) Pluripotent Stem Cells
20% Kidneys (16.7%)
Brain (37.0%)
0%
The peripheral blood could be envisioned as a high-
Lin/
way by which stem cells are trafficking in the body to
B BF Oct-4 CD45 7-AAD Sca-1 combo keep in balance a pool of stem cells located in different
niches in peripheral tissues (Fig. 14.5). In this con-
Bone marrow 10m
text, BM has been proposed to be a main reservoir that
Brain contains and releases during mobilization these circu-
lating cells (Levesque et al., 2007; Moog, 2006). To
Pancreas
support this it is well known that BM secretes several
Kidneys chemoattractants not only for HSC but also for other
Liver
non-hematopoietic stem cells including VSEL e.g.,
stromal derived factor-1 (SDF-1), hepatocyte growth
factor/scatter factor (HGF/SF), leukemia inhibitory
Fig. 14.4 Oct-4 + VSEL present in adult murine tis-
sues. Panel A shows percentage distribution of Oct-4+ /Sca-
factor (LIF), and vascular endothelial growth fac-
1+ /Lin /CD45 VSEL between analyzed organs which was tor (VEGF) (Ratajczak et al., 2006; Hill et al.,
computed based on the ImageStream system analysis and their 2004; Ponomaryov et al., 2000; Tacchini et al.,
absolute numbers in each tissue. Panel B presents representative 2003). Therefore, stem cells circulate in PB both
images of small Oct-4+ VSEL identified in selected analyzed
organs by ISS. VSEL are stained for Oct-4 expression (FITC,
during fetal development or postnatal while relo-
green). They are negative for Lin markers and CD45 (PE, cating between tissue specific niches may become
orange) and express Sca-1 (PE-Cy5, magenta). Scale bars show chemoattracted from circulating PB to settle down
10 m in BM, where they find a permissive environment.
This also explains why in addition to HSC, several
2008d, a). Furthermore, Oct-4 expression was also other types of non-hematopoietic stem cells have been
confirmed at mRNA level by employing normal RT- described in the BM including mesenchymal stem cells
PCR and real-time PCR performed on sorted by FACS (MSC) (Peister et al., 2004; Prockop, 1997), multi-
populations. Of note, real-time-PCR analysis revealed potent adult progenitor cells (MAPC) (Jiang et al.,

Table 14.1 Content of Absolute number of Oct-4+ VSEL [103 ]

Oct-4+ /Sca-1+ /Lin /CD45
Organ/tissue Per organ Per 1 g of tissue
cells in adult murine organs
and tissues analyzed by ISS. MeanSEM MeanSEM
Cells were isolated from Bone marrow 8.392.00 10.912.61
tissues of adult 48 week old Thymus 2.030.37 24.444.42
mice (C57BL/6) by enzymatic Spleen 3.860.43 47.105.21
digestion. Analysis was Pancreas 9.414.71 27.5313.78
performed three times (N=3) Brain 43.9712.38 116.0132.67
on samples prepared from
Kidneys 19.872.03 46.104.70
organs of three independent
animals. In case of geminate Lungs 5.390.33 34.112.07
organs, the absolute numbers Heart 1.350.56 9.934.13
of cells were calculated per Skeletal m. 15.186.79 4.161.86
one animal Testes 2.381.25 9.5510.04
Liver 6.981.38 4.790.95
238
Fig. 14.5 BM-derived VSEL circulate in peripheral blood.
The concept is presented based on the assumption that CXCR4+
VSEL circulate in the PB. Although the percentage of these cells
circulating in PB is much lower than that of early hematopoietic
stem/progenitor cells, these mobilized non-HSC may have an
important role in tissue repair following injury. The mobilization
of these cells occurs during tissue damage or injury (e.g., stroke,
heart infarct, toxic liver damage). It is postulated that after
mobilization into PB, CXCR4+ non-HSC may subsequently be
chemoattracted to the damaged tissues by the gradient of SDF-
1 and other chemokines. In this context, BM tissue becomes a
2002), marrow-isolated adult multilineage inducible
(MIAMI) (DIppolito et al., 2004), multipotent adult
stem cells (Beltrami et al., 2007) and identified by our
group VSEL (Kucia et al., 2006a, 2007a).
The circulating BM-derived stem/progenitor cells
could play an important role in turn-over of stem cells
in peripheral tissues. In accordance with this notion we
recently reported that in mice after an experimental
stroke or heart infarct a number of cells circulat-
ing in PB (Zuba-Surma et al., 2008b; Kucia et al.,
2006b), enriched for several tissue specific develop-
mental markers including neural ones as well as PSC
markers increases. Accordingly, we noticed that in
mice after an experimental stroke these cells not only
express neural lineage markers (-III-tubulin, nestin,
neuronal nuclear antigen [NeuN] and glial fibrillary
acidic protein [GFAP]) and PSC marker Oct-4, but
more importantly form neurospheres in vitro (Zuba-
Surma et al., 2008b; Kucia et al., 2006b). These
cells are present in significant amounts in BM har-
vested from young mice and respond to SDF-1, HGF,
M.Z. Ratajczak et al.
Circulation of Stem Cells in Blood
Network of Interactions between
Stem Cells and Niches in Peripheral
Tissues
Gradient of Stem Cells
Chemoatractants: with Receptors:
SDF 1
SDF-1 CXCR4
HGF C-met
LIF LIFR
VEGF VEGFRs
hiding place for both HSC and circulating CXCR4+ non-HSC
(e.g. EPC, MSC and VSEL). These cells are deposited early in
development and consequently reside in the BM. We hypothe-
size that they could serve an important role in tissue/organ repair
as a mobile reserve pool of PSC. Besides the SDF-1-CXCR4
axis, the trafficking of these cells involves several other moto-
morphogens such as HGF, VEGF, and leukemia inhibitory factor
(LIF) that create unique network guiding the circulation of stem
cells between various tissues/ organs through the blood stream
and LIF gradient but their abundance and responsive-
ness to gradients of SDF-1, HGF, and LIF, decreases
with age (Kucia et al., 2006a, b; Zuba-Surma et al.,
2009). Subsequent flow cytometric analysis, com-
bined with analysis of neural markers at the mRNA
and protein levels, revealed that these cells reside in
the non-hematopoietic CXCR4+ Sca-1+ lin CD45
BMNC fraction that is enriched in VSEL (Kucia et al.,
2006a).
In the murine model of stroke, we demonstrated
that the expression of SDF-1 around stroke area as
well as serum SDF-1 level increase during the first
week after stroke and that SDF-1 was one of the
important chemoattractants for recruiting stem cells to
supernatants from damaged brain tissue (Kucia et al.,
2006b). Of note, the importance of SDF-1-CXCR4
axis in development of brain has been demonstrated
in murine embryos with CXCR4 or SDF-1 knockout
which display several defects in their ability to develop
BM, brain, and cerebellum tissues (Vilz et al., 2005;
Zhu et al., 2002). Thus, SDF-1 signaling regulates
14 Potential Application of VSEL Stem Cells in Neural Regeneration
key events during early cortical development in vitro
and maintains proliferation of cortical progenitors,
possibly through a mechanism involving connexin 43-
mediated intercellular coupling (Pritchett et al., 2007).
Moreover, SDF-1 signaling upregulates the differenti-
ation of cortical gamma-aminobutyric acid outputting
(GABA-ergic) neurons, which is independent of the
sonic signaling pathway and enables the elongation
and branching of axons of cortical glutamatergic neu-
rons (Bhattacharyya et al., 2008; Kasiyanov et al.,
2008). Furthermore, we noticed that in addition to
SDF-1 also HGF/SF and LIF are important chemoat-
tractants for recruiting VSEL to supernatants from
damaged brain tissue (Ratajczak et al., 2006; Kucia
et al., 2006b).
In sum, we demonstrated that VSEL are mobi-
lized into the peripheral blood following stroke and
are subsequently chemoattracted to the damaged neu-
ral tissue in an SDF-1-CXCR4-, HGF/SF-c-Met-, and
LIF-LIF-receptor-dependent manner. This supports a
concept that chemotactic factors that are upregulated in
damaged brain orchestrate release of VSEL from BM
into PB.
14.5 Biological Properties of VSEL
VSEL residing in adult tissues and circulating in
mobilized peripheral blood could be potentially har-
nessed to regenerate damaged organs. We noticed
that these cells proliferate/differentiate relatively eas-
ily in co-cultures with other adherent cell populations
(e.g., C2C12 myoblasts, mesenchymal stem cells or
OP-9 stroma cells) (Kucia et al., 2006a; Ratajczak
et al., 2008c). For example, if plated over a C2C12
murine sarcoma cell feeder layer, ~510% of puri-
fied VSEL are able to form spheres that resemble
embryoid bodies (Kucia et al., 2006a). Cells from
these VSEL-derived spheres (VSEL-DS) are com-
posed of immature cells with large nuclei containing
euchromatin and are CXCR4+ SSEA-1+ Oct-4+ , just
like purified VSEL. Cells in these spheres however
already enlarge and show some signs of differentia-
tion. More important, cells from VSEL-DS are capable
to differentiate into all three germ layers (e.g., neu-
ral tissue, myocardium and pancreas) (Kucia et al.,
2006a). Furthermore, as reported both murine BM-
derived and human cord blood (CB)-derived VSEL are
239
able to grow neurospheres in co-cultures with bone
marrow derived mesenchymal stem cells (Kucia et al.,
2006a).
Interestingly, formation of VSEL-DS was associ-
ated with a young age in mice and no VSEL-DS
were observed in cells isolated from older mice (> 2
years) (Kucia et al., 2006a; Zuba-Surma et al., 2009).
This age-dependent content of VSEL in BM may
explain why the regeneration process is more efficient
in younger individuals. There are also differences in
the content of these cells among BMNC between long-
and short-lived mouse strains. The concentration of
these cells is much higher in BM of long-lived (e.g.,
C57Bl6) as compared to short-lived (DBA/2J) mice
(Kucia et al., 2006a). This suggests that some genes
could be responsible for developmental tissue distribu-
tion and expansion of these cells, and that these genes
could be involved in controlling the regeneration abil-
ities (e.g., for neural tissues) and thus mammalian life
span.
Since VSEL express several markers of primor-
dial germ cells (PGC) such as fetal-type alkaline
phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella,
Fragilis, Nobox and Hdac6, we envision that they
could be closely related to a population of epiblast-
derived PGC, a very first population of stem cells,
that is specified in developing embryo during early
embryogenesis (Kucia et al., 2006a; Ratajczak et al.,
2008c). VSEL are also highly mobile and respond
robustly to an SDF-1 gradient, adhere to fibronectin
and fibrinogen, and may interact with BM-derived
stromal fibroblasts. Confocal microscopy and time-
lapse studies revealed that these cells attach rapidly
to, migrate beneath, and undergo emperipolesis in
marrow-derived fibroblasts (Ratajczak et al., 2008c,
2007). Since fibroblasts secrete SDF-1 that as men-
tioned above binds to CXCR4 receptor, they may cre-
ate a homing environment for small CXCR4+ VSEL.
This robust interaction of VSEL with BM-derived
fibroblasts has an important implication, namely that
isolated BM stromal cells may be contaminated by
these tiny cells from the beginning. This observation
may somehow explain the unexpected plasticity of
marrow-derived fibroblastic cells, e.g., mesenchymal
stem cells (MSC) or MAPC. Recently, we noticed that
similar counterpart of murine VSEL is also present
in the human CB and BM. Furthermore, similar cells
increases in number and circulate in PB in patients
after acute myocardial infarction.
240
14.6 Cells that Express VSEL Markers
are Mobilized into PB in Patients
After Stroke
We become interested if VSEL could be mobilized into
PB in patients after stroke, similarly seen in patients
after acute heart infarct. To address this question,
a total of 44 patients afflicted with ischemic stroke
that were admitted within 24 h of the first symptom
onset and 22 healthy control subjects were enrolled.
In each case, the stroke had been precisely docu-
mented in clinic by computer tomography (CT) scan.
Based on clinical examination and cranial CT findings,
including volumetric analysis, patients were classi-
fied into the following four clinical subgroups: total
anterior circulation infarcts (TACI), partial anterior cir-
culation infarcts (PACI), posterior circulation infarcts
(POCI), and lacunar infarcts (LACI). Additionally, we
distinguished two subgroups of patients with differ-
ent extensiveness of stroke (e.g., group A small and
medium: patients diagnosed as LACI and PACI, and
group B large: patients diagnosed as TACI). The
patients diagnosed as POCI, were included into group
A or B on the basis of clinical examination and cranial
CT analysis.
As expected we observed an increase in mRNA for
both pluripotent (Oct-4, Nanog) and neural SC mark-
ers (GFAP, nestin, -III-tubulin, Olig1, Olig2, Sox2
and Musashi-1) in peripheral blood nucleated cells cir-
culating in stroke patients (Fig. 14.6). The increased
expression of mRNA for Oct-4 and Nanog in PBNC
of patients after stroke corresponded to those observed
by us previously in an experimental murine model
of stroke. However, maximal increased expression of
neural stem cell markers in humans was delayed by
2 days (1 day mice vs. 3 days human). Moreover,
the kinetic of changes in expression of mRNA for
early stem cells correspond to changes that we recently
observed after heart infarct in humans and mice.
Furthermore, we observed differences in SC mobiliza-
tion in cases of POCI and PACI as compared to other
subtypes of stroke. Of note patients with POCI have the
best chance of recovery and PACI is associated with the
highest risk of early recurrence of stroke (i.e., within 3
months), but it is not associated with high mortality
and significant disability.
The changes in the number of CXCR4+ VSEL cir-
culating in PB were paralleled by serum changes in
M.Z. Ratajczak et al.
12
control PBNC
10 24h
3rd day after stroke
7th day
8
mRNA (fold difference)
P<0.001 vs control
6
4
2
0
Oct4 GFAP
Fig. 14.6 Changes in the expression of markers for pluripo-
tent and neural stem cells in peripheral blood nucleated cells
(PBNC) in stroke patients and control group. PBNC were har-
vested from patients that underwent ischemic stroke within 24 h
of symptom manifestation and at day+3 and day+7 post-stroke.
The expression of mRNA for pluripotent markers (Oct-4) and
neural marker (GFAP) in the same number of cells was quanti-
fied by real-time RT-PCR and compared between groups. Data
are meanSD
level of -chemokine SDF-1. As mentioned above the
upregulation of SDF-1 expression in damaged organs
acts in chemoattracting CXCR4+ stem cells (e.g.,
VSEL) necessary for organ regeneration. We found
that the serum concentration of SDF-1 increases in
patients after stroke. This may be one of the factors
that mobilized VSEL from the BM into PB. However,
we were not able to measure the concentration of
SDF-1 in the immediate vicinity of the stroke loca-
tion in these patients. However, it is most likely that the
serum increase of SDF-1 positively correlates with an
increase in expression of SDF-1 in the damaged brain.
Interestingly, we observed a positive correlation
between the number of circulating CD34+ CD133+ and
CXCR4+ CD34+ cells in PB and the serum concen-
tration of SDF-1. In the available literature, data on
the correlation between these two variables and organ
damage are contradictory. For example, in patients
following heart infarct, some authors found a direct
14 Potential Application of VSEL Stem Cells in Neural Regeneration
correlation (De Falco et al., 2004) while others deter-
mined the inverse to be true (Abbott et al., 2004).
However, increase in circulating VSEL in peripheral
blood (~ 3 times) we observed was relatively small,
it is important to remember that stem cell mobilization
and stem cell homing are dynamic processes. It is very
likely that cells released into circulation may rapidly
home to the damaged tissues or enter stem cell niches
in other organs, or even return to stem cell niches in
BM. This means that the number of cells detected in
a frozen time laps is only one of the parameters of
VSEL trafficking, and does not reflect a total number
of these cells released into PB during time course of
acute stroke. This increase in a given moment how-
ever was statistically significant and similar to those
we observed for circulating VSEL in murine mod-
els of heart infarct as well toxic skeletal muscle and
liver injuries (Kucia et al., 2008b; Zuba-Surma et al.,
2008b). Thus, our study demonstrates for the first time
that the mobilization of CXCR4+ CD45 lin VSEL and
progenitor cells expressing early neural markers
into PB occurs in patients with ischemic stroke
(Paczkowska et al., 2008).
14.7 Conclusions
In conclusion, our data indicates that VSEL could
potentially provide a real therapeutic alternative to
the controversial use of human embryonic stem cells
(ESC) and therapeutic cloning. Augmented evidence
shows that stress related to the stroke triggers the mobi-
lization of VSEL from BM and perhaps other stem
cell niches into PB. We hypothesize that if these cells
observed in mobilized PB in humans will turn out to be
a human counterparts of VSEL identified in mice they
could be potentially purified from the PB, expanded
ex vivo, and employed for regeneration of damaged
neural tissues. Hence, while the ethical debate on the
application of ESC in therapy continues, the poten-
tial of VSEL is ripe for exploration. Researchers must
determine whether these cells could be efficiently
employed in the clinic or whether they are merely
developmental remnants found in the BM that can-
not be harnessed effectively for regeneration. The
coming years will bring important answers to these
questions.
241
References
Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ
(2004) Stromal cell-derived factor-1alpha plays a critical role
in stem cell recruitment to the heart after myocardial infarc-
tion but is not sufficient to induce homing in the absence of
injury. Circulation 110:33003305.
Basiji DA, Ortyn WE, Liang L, Venkatachalam V, Morrissey P
(2007) Cellular image analysis and imaging by flow cytome-
try. Clin Lab Med 27:653670.
Beltrami AP, Cesselli D, Bergamin N, Marcon P, Rigo S,
Burelli S, Puppato E, DAurizio F, Bottecchia M, Masolini P,
Mariuzzi L, Finato N, Beltrami CA (2007) Multipotent cells
can be generated in vitro from several adult human organs
(heart, liver and bone marrow). Blood 110:34383446.
Bhattacharyya BJ, Banisadr G, Jung H, Ren D, Cronshaw DG,
Zou Y, Miller RJ (2008) The chemokine stromal cell-derived
factor-1 regulates GABAergic inputs to neural progenitors in
the postnatal dentate gyrus. J Neurosci 28:67206730.
De Falco E, Porcelli D, Torella AR, Straino S, Iachininoto MG,
Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi
MC, Pesce M (2004) SDF-1 involvement in endothelial phe-
notype and ischemia-induced recruitment of bone marrow
progenitor cells. Blood 104:34723482.
Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology
of ischaemic stroke: An integrated view. Trends Neurosci
22:391397.
Duan X, Kang E, Liu CY, Ming GL, Song H (2008)
Development of neural stem cell in the adult brain. Curr Opin
Neurobiol 18:108115.
DIppolito G, Diabira S, Howard GA, Menei P, Roos BA,
Schiller PC (2004) Marrow-isolated adult multilineage
inducible (miami) cells, a unique population of postnatal
young and old human cells with extensive expansion and
differentiation potential. J Cell Sci 117:29712981.
Encinas JM, Enikolopov G (2008) Identifying and quantitating
neural stem and progenitor cells in the adult brain. Methods
Cell Biol 85:243272.
Endres M, Dirnagl U (2002) Ischemia and stroke. Adv Exp Med
Biol 513:455473.
Hess DC, Borlongan CV (2008) Stem cells and neurological
diseases. Cell Prolif 41(Suppl 1):94114.
Hill WD, Hess DC, Martin-Studdard A, Carothers JJ, Zheng J,
Hale D, Maeda M, Fagan SC, Carroll JE, Conway SJ (2004)
SDF-1 (CXCL12) is upregulated in the ischemic penumbra
following stroke: Association with bone marrow cell homing
to injury. J Neuropathol Exp Neurol 63:8496.
Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD,
Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad
M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA,
Verfaillie CM (2002) Pluripotency of mesenchymal stem
cells derived from adult marrow. Nature 418:4149.
Kalluri HS, Dempsey RJ (2008) Growth factors, stem cells, and
stroke. Neurosurg Focus 24:E14.
Kasiyanov A, Fujii N, Tamamura H, Xiong H (2008) Modulation
of network-driven, GABA-mediated giant depolarizing
potentials by sdf-1alpha in the developing hippocampus. Dev
Neurosci 30:285292.
Komitova M, Mattsson B, Johansson BB, Eriksson PS (2005)
Enriched environment increases neural stem/progenitor cell
242
proliferation and neurogenesis in the subventricular zone of
stroke-lesioned adult rats. Stroke 36:12781282.
Kucia M, Dawn B, Hunt G, Guo Y, Wysoczynski M, Majka
M, Ratajczak J, Rezzoug F, Ildstad ST, Bolli R, Ratajczak
MZ (2004a) Cells expressing early cardiac markers reside in
the bone marrow and are mobilized into the peripheral blood
after myocardial infarction. Circ Res 95:11911199.
Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M,
Moldenhawer S, Zuba-Surma E, Czajka R, Wojakowski W,
Machalinski B, Ratajczak MZ (2007a) Morphological and
molecular characterization of novel population of CXCR4(+)
SSEA-4(+) Oct-4(+) very small embryonic-like cells puri-
fied from human cord blood preliminary report. Leukemia
21:297303.
Kucia M, Ratajczak J, Reca R, Janowska-Wieczorek A,
Ratajczak MZ (2004b) Tissue-specific muscle, neural and
liver stem/progenitor cells reside in the bone marrow,
respond to an sdf-1 gradient and are mobilized into periph-
eral blood during stress and tissue injury. Blood Cells Mol
Dis 32:5257.
Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M,
Ratajczak J, Ratajczak MZ (2006a) A population of very
small embryonic-like (vsel) CXCR4(+) SSEA-1(+) Oct-
4+ stem cells identified in adult bone marrow. Leukemia
20:857869.
Kucia M, Wysoczynski M, Ratajczak J, Ratajczak MZ (2008a)
Identification of very small embryonic like (VSEL) stem
cells in bone marrow. Cell Tissue Res 331:125134.
Kucia MJ, Wysoczynski M, Wu W, Zuba-Surma EK, Ratajczak
J, Ratajczak MZ (2008b) Evidence that very small embryonic
like (VSEL) stem cells are mobilized into peripheral blood.
Stem Cells 26:20832092.
Kucia M, Zhang YP, Reca R, Wysoczynski M, Machalinski B,
Majka M, Ildstad ST, Ratajczak J, Shields CB, Ratajczak
MZ (2006b) Cells enriched in markers of neural tissue-
committed stem cells reside in the bone marrow and
are mobilized into the peripheral blood following stroke.
Leukemia 20:1828.
Kucia M, Zuba-Surma EK, Wysoczynski M, Wu W, Ratajczak
J, Machalinski B, Ratajczak MZ (2007b) Adult marrow-
derived very small embryonic-like stem cells and tissue
engineering. Expert Opin Biol Ther 7:14991514.
Lvesque JP, Winkler IG, Larsen SR, Rasko JE (2007)
Mobilization of bone marrow-derived progenitors. Handb
Exp Pharmacol 180:336.
Moog R (2006) Mobilization and harvesting of peripheral blood
stem cells. Curr Stem Cell Res Ther 1:189201.
Ortyn WE, Hall BE, George TC, Frost K, Basiji DA, Perry
DJ, Zimmerman CA, Coder D, Morrissey PJ (2006)
Sensitivity measurement and compensation in spectral imag-
ing. Cytometry A 69A:852862.
Paczkowska E, Kucia M, Koziarska D, Halasa M, Safranow K,
Masiuk M, Karbicka A, Nowik M, Nowacki P, Ratajczak
MZ, Machalinski B (2008) Clinical evidence that very small
embryonic-like (VSEL) stem cells are mobilized into periph-
eral blood in patients after stroke. Stroke 40:12371244.
Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop
DJ (2004) Adult stem cells from bone marrow (MSCS) iso-
lated from different strains of inbred mice vary in surface
epitopes, rates of proliferation, and differentiation potential.
Blood 103:16621668.
M.Z. Ratajczak et al.
Ponomaryov T, Peled A, Petit I, Taichman RS, Habler L,
Sandbank J, Arenzana-Seisdedos F, Magerus A, Caruz A,
Fujii N, Nagler A, Lahav M, Szyper-Kravitz M, Zipori D,
Lapidot T (2000) Induction of the chemokine stromal-
derived factor-1 following DNA damage improves human
stem cell function. J Clin Invest 106:13311339.
Pritchett J, Wright C, Zeef L, Nadarajah B (2007) Stromal
derived factor-1 exerts differential regulation on distinct
cortical cell populations in vitro. BMC Dev Biol 7:31.
Prockop DJ (1997) Marrow stromal cells as stem cells for
nonhematopoietic tissues. Science 276:7174.
Quesenberry PJ, Colvin G, Dooner G, Dooner M, Aliotta JM,
Johnson K (2007) The stem cell continuum: Cell cycle,
injury, and phenotype lability. Ann N Y Acad Sci 1106:
2029.
Ratajczak MZ, Machalinski B, Wojakowski W, Ratajczak J,
Kucia M (2007) A hypothesis for an embryonic origin of
pluripotent Oct-4+ stem cells in adult bone marrow and other
tissues. Leukemia 21:860867.
Ratajczak MZ, Zuba-Surma E, Kucia M, Reca R, Wojakowski
W, Ratajczak J (2006) The pleiotropic effects of the SDF-1-
CXCR4 axis in organogenesis, regeneration and tumorigen-
esis. Leukemia 20:19151924.
Ratajczak MZ, Zuba-Surma EK, Machalinski B, Ratajczak J,
Kucia M (2008a) Very small embryonic-like (VSEL) stem
cells: Purification from adult organs, characterization, and
biological significance. Stem Cell Rev 4:8999.
Ratajczak MZ, Zuba-Surma EK, Shin DM, Ratajczak J, Kucia
M (2008b) Very small embryonic-like (VSEL) stem cells in
adult organs and their potential role in rejuvenation of tissues
and longevity. Exp Gerontol 43:10091117.
Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak
J, Kucia M (2008c) Very small embryonic-like stem cells:
Characterization, developmental origin, and biological sig-
nificance. Exp Hematol 36:742751.
Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Wan W,
Ratajczak J, Wojakowski W, Kucia M (2008d) Hunt for
pluripotent stem cell regenerative medicine search for
almighty cell. J Autoimmun 30:151162.
Shyu WC, Lee YJ, Liu DD, Lin SZ, Li H (2006) Homing genes,
cell therapy and stroke. Front Biosci 11:899907.
Tacchini L, Matteucci E, De Ponti C, Desiderio MA (2003)
Hepatocyte growth factor signaling regulates transactivation
of genes belonging to the plasminogen activation system via
hypoxia inducible factor-1. Exp Cell Res 290:391401.
Vilz TO, Moepps B, Engele J, Molly S, Littman DR, Schilling
K (2005) The SDF-1/CXCR4 pathway and the development
of the cerebellar system. Eur J Neurosci 22:18311839.
Walzlein JH, Synowitz M, Engels B, Markovic DS,
Gabrusiewicz K, Nikolaev E, Yoshikawa K, Kaminska
B, Kempermann G, Uckert W, Kaczmarek L, Kettenmann
H, Glass R (2008) The anti-tumorigenic response of neural
precursors depends on subventricular proliferation and age.
Stem Cells 26:29452954.
Wojakowski W, Tendera M, Zebzda A, Michalowska A, Majka
M, Kucia M, Maslankiewicz K, Wyderka R, Krl M, Ochala
A, Kozakiewicz K, Ratajczak MZ (2004) Mobilization
of D34/CXCR4+ , CD34/CD117+ , c-Met+ stem cells, and
mononuclear cells expressing early cardiac, muscle, and
endothelial markers into peripheral blood in patients with
acute myocardial infarction. Circulation 110:32133220.
14 Potential Application of VSEL Stem Cells in Neural Regeneration
Zhang RL, Zhang ZG, Zhang L, Chopp M (2001) Proliferation
and differentiation of progenitor cells in the cortex and
the subventricular zone in the adult rat after focal cerebral
ischemia. Neuroscience 105:3341.
Zhu Y, Yu T, Zhang XC, Nagasawa T, Wu JY, Rao Y (2002)
Role of the chemokine SDF-1 as the meningeal attrac-
tant for embryonic cerebellar neurons. Nat Neurosci 5:
719720.
Zuba-Surma EK, Kucia M, Abdel-Latif A, Dawn B, Hall
B, Singh R, Lillard JW Jr, Ratajczak MZ (2008a)
Morphological characterization of very small embryonic-like
stem cells (VSELs) by Imagestream System analysis. J Cell
Mol Med 12:292303.
Zuba-Surma EK, Kucia M, Abdel-Latif A, Lillard JW Jr,
Ratajczak MZ (2007) The Imagestream System: A key step
243
to a new era in imaging. Folia Histochem Cytobiol 45:
279290.
Zuba-Surma EK, Kucia M, Dawn B, Guo Y, Ratajczak MZ,
Bolli R (2008b) Bone marrow-derived pluripotent very
small embryonic-like stem cells (VSELs) are mobilized
after acute myocardial infarction. J Mol Cell Cardiol 44:
865873.
Zuba-Surma EK, Kucia M, Ratajczak MZ (2008c) Decoding of
dot: The Imagestream System (ISS) as a supportive tool for
flow cytometric analysis. Cent Eur J Biol 3:110.
Zuba-Surma EK, Wu W, Ratajczak J, Kucia M, Ratajczak MZ
(2009) Very small embryonic-like stem cells in adult tissues-
potential implications for aging. Mech Ageing Dev 130:
5866.
Chapter 15

Embryonic Stem Cell Transplantation for the Treatment

of Parkinsons Disease

Asuka Morizane and Jun Takahashi

Contents cells and induced pluripotent stem (iPS) cells. Both

human ES and human iPS cells can differentiate into
15.1 Introduction . . . . . . . . . . . . . . . . 246 dopamine (DA) neurons in vitro. These cells have been
15.2 Rationale for Using Transplantation as a transplanted into animal models of Parkinsons disease
Treatment for Parkinsons Disease . . . . . . 246 and behavioral recovery has been observed in some
15.3 In Vitro Differentiation of Embryonic Stem
cases. Prior to clinical application, however, several
Cells . . . . . . . . . . . . . . . . . . . . 247
15.4 Transplantation in a Parkinsons Disease Model 247 problems remain: instability of DA neurons in vivo,
15.5 Safety Issues for Clinical Application . . . . 248 risk of tumor formation by grafted cells, and the possi-
15.6 Another Donor Candidate: Induced Pluripotent ble transfer of animal-derived pathogens. When these
Stem Cell (iPS cell) . . . . . . . . . . . . . 251
problems are solved, cell transplantation will likely
References . . . . . . . . . . . . . . . . . . . . 251
become one of the standard treatments for Parkinsons
disease.
Abstract Parkinsons disease is one of the most Keywords Parkinsons disease Dopamine neuron
common movement disorders. Medical treatment and Embryonic stem cells Transplantation Clinical
deep brain stimulation (DBS) are useful in some application
patients. However, these two therapies do not recon-
stitute patients lost neuronal circuits and the disease Abbreviations
itself progresses. Thus, it is often difficult to control
the disease in late stages. Hundreds of patients have AA ascorbic Acid
been involved in clinical trials where they received cell BDNF brain-derived neurotrophic factor
transplants from aborted fetal/embryonic brain tissue. ALS amyotrophic lateral sclerosis
Although some patients exhibit enormous improve- DA dopamine
ment, this treatment is not standard due to potential dbc AMP dibutyryl cyclic AMP
problems associated with the use of aborted tissue, DBS deep brain stimulation
including a limited supply. In contrast, stem cell EB embryoid bodies
research is advancing rapidly and many people regard ES cell embryonic stem cell
stem cells as the donor source of the next genera- FACS fluorescent activated cell sorting
tion. Many laboratories have tried to generate donor FN fibronectin-coated dish
cells from stem cells, especially embryonic stem (ES) GABA -aminobutyric acid
GDNF glial cell line-derived neurotrophic factor
HLA human leukocyte antigen
iPS cells induced pluripotent stem cells
ITSF insulin, transferrin, selenite and fibronectin
J. Takahashi ()
Department of Biological Repair, Institute for Frontier Medical L laminin coated dish
Sciences, Kyoto University, Kyoto, Japan MACS magnetic activated cell sorting
e-mail: jbtaka@frontier.kyoto-u.ac.jp MEF mouse embryonic fibroblast

H. Ulrich (ed.), Perspectives of Stem Cells, 245

DOI 10.1007/978-90-481-3375-8_15, Springer Science+Business Media B.V. 2010
246
MPTP 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine
Neu5Gc N-glycolyl-neuraminic acid residues
NPC Neural stem/progenitor cell(s)
O/L poly-ornithine and laminin-coated dish
6-OHDA 6-hydroxy dopamine
PD Parkinsons disease
SDIA stromal cell-derived inducing activity
SHH sonic hedgehog
TH tyrosine hydroxylase
15.1 Introduction
Parkinsons disease (PD) is the most common move-
ment disorder. About 1% of the population over 60
years is affected. Typical symptoms of Parkinsons dis-
ease include resting tremor, bradykinesia, and limb
rigidity (Samii et al., 2004). The main neuropathology
in PD is loss of dopamine (DA) neurons in the substan-
tia nigra pars compacta, which leads to decreased DA
level in the striatum. Clinical signs of PD are evident
when about 80% of striatal DA and 50% of nigral neu-
rons are lost (Fearnley and Lees, 1991). Patients are
treated with L-3, 4-dihydroxyphenylalanine (L-dopa)
and DA agonists to compensate for the loss of DA
neurotransmission in the nigrostriatal system. Most
patients that begin with DA agonist therapy will need
the addition of L-dopa within 5 years. Patients treated
with L-dopa long-term gradually develop side effects,
such as motor fluctuations and L-dopa-induced dysk-
inesia. Although medical therapy is standard, many
patients have difficulty controlling their symptoms in
the later phase of the disease.
Surgical therapy is another option. Deep brain stim-
ulation is the most common surgical procedure for PD.
Some patients exhibit beneficial effects, but selection
of suitable patients for deep brain stimulation is the
key to successful results. A good response to L-dopa
treatment is a good predictor of a positive outcome.
Although deep brain stimulation is a powerful option
for controlling PD symptoms, the procedure does not
slow down disease progression, nor is it reparative in
nature. In contrast, cell transplantation has the poten-
tial to reconstruct neuronal circuits. This procedure
was first applied clinically to PD patients in the late
1980s using fetal/embryonic brain tissues. However,
thus far, this treatment is not a standard therapy due
A. Morizane and J. Takahashi
to the potential problem associated with using aborted
tissue, including a limited supply. Many researchers
have been trying to use stem cells as another donor
cell source. In this chapter we will review cell trans-
plantation for PD, focusing on the use of ES and iPS
cells.
15.2 Rationale for Using Transplantation
as a Treatment for Parkinsons
Disease
The first clinical trial of cell transplantation for PD
was performed in 1987 using human ventral mesen-
cephalon cells from aborted embryos (Brundin et al.,
1987). To date, more than 400 PD patients around
the world have received the same type of donor tis-
sue. In small, open-label trials, some of these patients
have shown dramatic improvement after transplan-
tation, attracting interest and expectations (Brundin
et al., 2000; Hagell and Brundin, 2001; Hauser et al.,
1999; Mendez et al., 2000). Recently, three groups
reported that transplanted DA neurons can survive
over ten years after surgical postmortem examina-
tion of patients who had undergone the procedure (Li
et al., 2008; Mendez et al., 2008; Kordower et al.,
2008). However, Lewy body structures, pathological
changes often observed in the brain of PD patients,
have been detected in grafted cells in these long-
term cases (Li et al., 2008; Kordower et al., 2008).
Furthermore, two double-blind placebo-controlled tri-
als in the USA failed to observe an improvement of
symptoms at primary endpoints (Freed et al., 2001;
Olanow et al., 2003), highlighting several problems
that require consideration. The first is patient selection.
Disease stage and preoperative response to L-dopa are
important issues for selecting good candidates for the
treatment. Younger patients with less severe pathol-
ogy have increased chances for successful outcomes
after transplantation. Not all the patients suffering from
PD are suitable for this cell therapy. Other considera-
tions are problems associated with the procedure itself,
including tissue preparation, number of the donor cells,
graft implantation techniques, and immunosuppres-
sion, among others. Freshly dissected fetal ventral
mesencephalic tissue has been used in most of the
open-label trials. However, in one of the double-blind
15 ES Cell Transplantation for the Treatment of Parkinsons Disease
trials, the tissue was dissociated and expanded for up to
4 weeks before transplantation. The number of donor
embryos used for one side of the brain has ranged
from 1 to 8. Although it would be better biologically
to use fresh tissue, it is difficult in practice to prepare
enough donor tissues at the same time. Collectively,
previous clinical data indicates that cell transplantation
is effective if the PD patients and procedures are cor-
rectly selected. Due to their pluripotency and ability to
self-renew, ES or iPS cells might be better donor cell
candidates.
15.3 In Vitro Differentiation
of Embryonic Stem Cells
Embryonic stem cells can generate neurons, as well as
other type of cells, including those with mesodermal
and endodermal phenotypes. Therefore, to generate
DA neurons from ES cells, we have to begin by induc-
ing a neural cell fate, for which two methods are
reported. One is a co-culture of ES cells with feeder
cells, such as mesenchymal stromal cells (Kawasaki
et al., 2000). As for feeders, PA6 cells (Kawasaki
et al., 2000), MS5 cells (Barberi et al., 2003), Sertoli
cells (Yue et al., 2006), amniotic membrane (Ueno
et al., 2006), human telomerase-overexpressing astro-
cytes from embryonic midbrain (Roy et al., 2006)
and meninges (Hayashi et al., 2008) all have the
activity needed to induce the generation of DA neu-
rons from ES cells. The second approach for neu-
ral induction is to form aggregates called embryoid
bodies (EB) and then add soluble factors to differ-
entiate the EB into proper neuronal cells (Lee et al.,
2000). Many cytokines and growth factors that are
found in the developing midbrain of the embryo/fetus
have been tried as additional factors in the media,
including sonic hedgehog (Shh), FGF8, ascorbic acid,
interleukin-1, glial cell line-derived neurotrophic fac-
tor (GDNF), neurturin, TGF-3, dibutyryl cyclic AMP
(dbcAMP), vitamin B12, brain-derived neurotrophic
factor (BDNF), neurotrophin 3, and fibroblast growth
factor (FGF) 20, among others (Rolletschek et al.,
2001; Park et al., 2005; Yamazoe et al., 2006, Takagi
et al., 2005; Park et al., 2004).
Overexpression of transcription factors that partic-
ipate in the development of DA neurons can also
induce DA neurons. These factors include Nurr1,
247
Pitx3, Mash1, Lmx1a, and Msx1 (Kim et al., 2002;
Chung et al., 2005; Kanda et al., 2004; Andersson
et al., 2006). The most efficient protocol for induc-
ing DA neurons from murine ES cells thus far is to
use a combination of Nurr1 overexpression, PA6 cells
co-culture, and soluble factors including Shh, FGF8,
and ascorbic acid (Kim et al., 2006). Ninety percent of
neurons are dopaminergic under these conditions (see
Table 15.1). For human ES cells, a similar combina-
tion of co-culture with MS5 and soluble factors (Shh
and FGF8) generates TuJ1-immunopositive neurons,
among which 6479% of the cells express tyrosine
hydroxylase (TH), a marker for DA neurons (Perrier
et al., 2004).
Studies of ventral mesencephalon transplants in ani-
mals (Brown and Dunnett, 1989; Thompson et al.,
2005) and PD patients (Mendez et al., 2005) indi-
cate that only A9-type DA neurons can improve
Parkinsonian symptoms. A9 DA neurons express a
specific set of markers, including G-protein-coupled
inwardly rectifying potassium channel (Girk) 2, Nurr1,
Pitx3, Engrailed 1/2 (En1/2), Lmx1a, Lmx1b, and
DA transporter (Anderson et al., 2006). TH-positive
cells are also found in the olfactory bulb (A16 neu-
rons), but these cells co-express gamma-aminobutyric
acid (GABA) and neurons in the substantia nigra pars
compacta (A9 neurons) do not.
15.4 Transplantation in a Parkinsons
Disease Model
There are several animal models for PD. Treating
rats with 6-hydroxy dopamine (6-OHDA) is one of
the most common and established models. Usually
the toxin is injected into one side of the nigrostri-
atal pathway, where it causes specific degradation of
DA neurons in the substantia nigra. This hemi-lesioned
model is advantageous for functional analysis. Because
the DA system is imbalanced between the right and left
sides of the brain, the model animal rotates to one side
when tested with drugs such as amphetamine or apo-
morphine (Marshall and Ungerstedt, 1977). If grafted
cells secrete DA and compensate for decreased DA
levels, the frequency of rotation decreases. Another
PD model is based on 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP). Monkeys and mice are
sensitive to MPTP. The monkey MPTP model is
248
very similar to human PD patients biochemically and
pathologically, as well as symptomatically, displaying
rigidity, tremor and bradykinesia.
There are numbers of reports describing transplants
of ES cell-derived DA neurons, or their progenitors,
into the brains of animal PD models. Murine ES
cell-derived progenitors are able to induce behavioral
recovery in recipients (Kim et al., 2002; Nishimura
et al., 2003; Barberi et al., 2003; Baier et al., 2004;
Rodriguez-Gomez et al., 2007). Monkey ES cells
can also differentiate into DA neurons when co-
cultured with PA6 feeder cells (Kawasaki et al.,
2002). Monkeys treated with MPTP recover from
motor and posture impairments when monkey ES
cell-derived DA neurons are grafted. In positron
emission tomography scans of these monkeys, 18F-
fluorodopa uptake in the striatum is increased after
transplantation (Takagi et al., 2005). Human ES
cells are also able to differentiate into DA neurons
and survive in the striatum of rat PD models (see
Table 15.1). However, some groups have reported
no functional effect (Brederlau et al., 2006; Park
et al., 2005), or only small changes (Ben-Hur et al.,
2004), after transplantation, indicating that the num-
ber of surviving DA neurons was insufficient. Other
groups have reported that motor function improves
after cell transplantation (Sonntag et al., 2007; Yang
et al., 2007; Ko et al., 2007; Cho et al., 2008). It
appears that DA neurons derived from human ES cells
have a lower survival rate and are less functional in
rat striatum than those from murine ES cells. Although
grafts contain numerous neurons that express a marker
for neuronal nuclei (NeuN; Schulz et al., 2004; Park
et al., 2005; Brederlau et al., 2006), the number of TH-
positive neurons is several-fold lower than expected
based on grafts from murine ES cells or fetal tissue.
The mechanisms underlying the selective vulnerabil-
ity or instability of human ES cell-derived DA neurons
are not understood, but they may be related to species
differences or improper differentiation prior to trans-
plantation. Further studies are needed to solve this
important problem.
15.5 Safety Issues for Clinical
Application
Prior to the clinical application of ES cell transplanta-
tion, a number of safety issues need to be addressed.
A. Morizane and J. Takahashi
One major problem is tumor formation by the grafted
cells. This phenomenon has been reported by the use
of the term tumor (Yang et al., 2007), teratoma
(Brederlau et al., 2006; Sonntag et al., 2007), over-
growth, or neoplastic tumor mass (Roy et al., 2006;
Goldman et al., 2007). Due to pluripotency and the
ability to self-renew, the population of cells in a cul-
ture dish is heterogeneous. Some cells have advanced
in maturation, while other cells might still be imma-
ture and proliferating. Although most cells are proper
midbrain DA neurons, the population may be contami-
nated with undifferentiated or non-neural cells. If cells
with a non-neural cell fate or high proliferation activ-
ity exist in the graft, they keep growing and destroy
the surrounding host brain structures. To avoid this
phenomenon, donor cells must be prepared under con-
ditions that precisely control the direction and extent of
differentiation. Furthermore, a strategy for purification
or selection of ideal donor cells from the differentiated
culture is essential. Cell sorting using fluorescent acti-
vated cell sorting (FACS) or magnetic activated cell
sorting (MACS) has been used for positive (Fukuda
et al., 2006; Chung et al., 2006; Pruszak et al., 2007) or
negative selection (Shibata et al., 2006). Using chem-
icals or drugs to eliminate undesired cells is another
approach (Bieberich et al., 2004). If we can purify
suitable donor cells and prepare a completely homoge-
neous population before grafting, DA neuron survival
after transplantation would increase and the risk of
tumor formation would decrease.
For clinical use of ES cells, we also need to consider
the issue of non-human materials. From the derivation
of human ES cells to their maintenance and differenti-
ation into DA neurons, we use materials derived from
non-human species, such as mouse embryonic fibrob-
lasts and fetal bovine serum. Exposing human ES
cells to animal derivatives allows them to incorporate
N-glycolyl-neuramninic acid residues (Neu5Gc) that
are present in most mammals, with the exception of
humans (Martin et al., 2005). Because contamination
of the xenogenic material might cause transfer of ani-
mal pathogens or an immunological reaction, we need
to establish a protocol that uses only human-derived or
chemically defined materials.
Genetic instability of human ES cells is another
issue that requires consideration. Karyotypic changes
in several human ES cell lines have been reported
(Cowan et al., 2004; Maitra et al., 2005; Herszfeld
et al., 2006; Draper et al., 2004). Changes commonly
Table 15.1 Induction and transplantation of human ES cell-derived DA neurons
Study Cell lines Methods Culture TH/Tub
days III (%) Transplantation
NPC, DA No. of cells Follow- Function Tumor
neuron transplanted Stage up Histology (rotation) formation
Zeng et al. BG01 SDIA (PA6) 12, 18 n.d. n.d. 8- or 5 No Oct4+ n.d. n.d.
(2004) days 22-day- weeks cells, but
old cells SMA+
cells and
TH+
cells
Schulz BG01, EB (HepG2 10, 28 74 1,000 3- or 8 A few n.d. 1 out of 23
et al. BG03 CM/ FGF2 or days 20,000 4-week- weeks TH+
(2004) DMEM/N2) old cells cells
O/L (serum,
BDNF, GDNF)
Ben-Hur HES-1 MEF (serum, 180, 187 19 400,000 6-weeek- 12 389 TH+ Partial PCNA 0.2%
et al. Noggin) days old weeks cells recovery
(2004) sphere cells
(FGF2)
15 ES Cell Transplantation for the Treatment of Parkinsons Disease

L (FGF
withdrawn)
Park et al. HSF-6, SDIA (PA6) 14, 30 41 500,000 Stage 2 A few No PCNA 53%
(2005) SNU- N2, AA, days 2 or 3 weeks TH+ recovery
hES-3, FGF2 cells cells
Miz- FN (BDNF,
hES-1 GDNF, NT-3,
Shh, FGF8)
Brederlau SA002.5 SDIA (PA6) 14, 28 65 100,000 23-day- 13 1050 No +
et al. O/L days old cells weeks TH+ recovery
(2006) cells
Roy et al. H1, H9 EB ITSF 14, 36 67 500,000 Stage 5 10 58,273 Recovery Overgrowth
(2006) medium O/L days cells weeks TH+
(FGF2, SHH, cells
FGF8, human
midbrain
astrocytes)
249
 250

Table 15.1 (continued)

Study Cell lines Methods Culture TH/ Tub
days III (%) Transplantation
NPC, DA No. of cells Follow- Function Tumor
neuron transplanted Stage up Histology (rotation) formation
Sonntag et H7, H9 SDIA (MS5- 21, 49 23 100,000 42-day- 12 160 TH+ Recovery +
al. (2007) Wnt1 days (TH/ old cells weeks cells
Noggin) total
SHH, FGF8, cells)
AA, BDNF
O/L (BDNF,
GDNF,
TGF, AA)
Iacovitti et H9, EB ITSF 14, 21 n.d. 2500,000 Stage 4 23 Many n.d. Ki67+ but no
al. (2007) BG01, medium days cells weeks TH+ cells Oct4 or
HUES7, (Noggin) SSEA4
HUES8 FGF2
Ornithine
(dcAMP)
Yang et al. H9 EB (FGF2) 38, 52 43 200,000 5 1273 Recovery Overgrowth
(2007) adherent days months TH+ cells
(FGF8, SHH)
O/L (Wnt3a,
AA, cAMP,
TGF, BDNF,
GDNF)
Ko et al. HSF-6, SDIA (PA6 or >2 45 1,500,000 Progenitors 8 200 Recovery
(2007) H9 MS5, ITS months weeks TH+ cells
medium,SHH,
AA) O/L
(ITS+FGF2)
ITS (AA,BDNF,
dbcAMP)
Cho et al. SNU- EB Matrigel 30, 44 86 500,000 37-day-old 12 10,732 Recovery
(2008) hES- (FGF2) days cells weeks TH+ cells
1,3,16 suspension
(FGF2)
Matrigel (SHH,
FGF8, AA)
In these studies, human ES cell-derived DA neurons were transplanted into 6-OHDA-lesioned striatum of rats. Brain sections were stained immunohistochemically
with TH antibody, and apomorphine or amphetamine was used to test rotational behavior.
A. Morizane and J. Takahashi
15 ES Cell Transplantation for the Treatment of Parkinsons Disease
occur in chromosomes 12 and 17 (Baker et al., 2007).
We do not fully understand the influence of these
chromosomal abnormalities, but they may affect sur-
vivability and risk of tumor formation.
15.6 Another Donor Candidate: Induced
Pluripotent Stem Cell (iPS cell)
In 2006, Yamanakas group succeeded in generating
iPS cells from mouse fibroblasts by transducing four
transcriptional factors, Oct3/4, Sox2, c-Myc and Klf4
(Takahashi and Yamanaka, 2006). It took only one year
to establish human iPS cells (Takahashi et al., 2007;
Yu et al., 2007). The cells are pluripotent and pos-
sess the potency to differentiate into DA neurons in
vitro (Takahashi et al., 2007). Although iPS cells retro-
virally integrate extra genes, cells from patients with
genetic disorders might be helpful for understanding
disease mechanisms, screening drugs, and measuring
toxicity. Several groups have started to establish iPS
cell lines from patients with genetic diseases such
as Gaucher disease type , Parkinsons disease, Type
1 diabetes, Lesch-Nyhan syndrome, and amyotrophic
lateral sclerosis (ALS), among others (Park et al.,
2008; Dimos et al., 2008). It may also be possible
to use iPS cells for transplantation, rather than ES
cells. A rat PD model recovers following transplant
of DA neurons derived from mouse iPS cells (Wernig
et al., 2008). Theoretically, it is possible to gener-
ate iPS cells from the host patient. This autologous
transplantation only causes a minimal immunological
response, and reduces the risk of infection from other
people. Therapeutic cloning of ES cells for the treat-
ment of PD is also a possibility. Nuclear transferred ES
cells have been generated from 24 PD model mice. The
cells were successfully differentiated into DA neurons
and transplanted back into each host mouse individu-
ally (Tabar et al., 2008). However, due to time and cost
considerations, it is not practical at this point to estab-
lish, and verify the safety of, an individual iPS cell line
for each patient. Due their genetic make-up, iPS cells
from a patient may be more sensitive to the disease
environment or may even cause the disease. Thus, it
might be better to use iPS cells derived from healthy
individuals and establish a bank of iPS cell lines to
251
cover all types of human leukocyte antigen (HLA)
populations.
The risk of tumor formation is another problem
associated with using iPS cells as the donor for
transplantation. Originally, iPS cells were established
using retrovirus and transducing four genes including
c-myc oncogene. Because of this genetic manipu-
lation, iPS cells may have a higher risk of tumor
or cancer generation after grafting than hES cells.
Mouse and human iPS cells can now be estab-
lished without the Myc gene, thereby reducing this
risk (Nakagawa et al., 2008). Furthermore, genomic
integration of these genes by retrovirus is not
always needed for generating iPS cells. iPS cells
have been established by tentative expression of
these four genes using adenovirus (Stadtfeld et al.,
2008) or a plasmid expression system (Okita et al.,
2008).
Currently, iPS cells are most useful for drug screen-
ing and/or researching genetic disorders. However,
because the technology in this field is advancing
rapidly, we can expect safer methods for generating
human iPS cells and can apply them for cell therapy
in the near future.
References
Andersson E, Tryggvason U, Deng Q, Friling S, Alekseenko
Z, Robert B, Perlmann T, Ericson J (2006) Identification of
intrinsic determinants of midbrain dopamine neurons. Cell
124:393405.
Baier PC, Schindehutte J, Thinyane K, Flugge G, Fuchs E,
Mansouri A, Paulus W, Gruss P, Trenkwalder C (2004)
Behavioral changes in unilaterally 6-hydroxy-dopamine
lesioned rats after transplantation of differentiated mouse
embryonic stem cells without morphological integration.
Stem Cells 22:396404.
Baker DE, Harrison NJ, Maltby E, Smith K, Moore HD, Shaw
PJ, Heath PR, Holden H, Andrews PW (2007) Adaptation to
culture of human embryonic stem cells and oncogenesis in
vivo. Nat Biotechnol 25:207215.
Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H,
Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N, Tabar V,
Harrison NL, Beal MF, Moore MA, Studer L (2003) Neural
subtype specification of fertilization and nuclear transfer
embryonic stem cells and application in parkinsonian mice.
Nat Biotechnol 21:12001207.
Ben-Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, Itzik A,
Reubinoff BE (2004) Transplantation of human embryonic
stem cell-derived neural progenitors improves behavioral
deficit in Parkinsonian rats. Stem Cells 22:12461255.
252
Bieberich E, Silva J, Wang G, Krishnamurthy K, Condie BG
(2004) Selective apoptosis of pluripotent mouse and human
stem cells by novel ceramide analogues prevents teratoma
formation and enriches for neural precursors in ES cell-
derived neural transplants. J Cell Biol 167:723734.
Brederlau A, Correia AS, Anisimov SV, Elmi M, Roybon
L, Paul G, Morizane A, Bergquist F, Riebe I, Nannmark
U, Carta M, Hanse E, Takahashi J, Sasai Y, Funa K,
Brundin P, Eriksson PS, Li JY (2006) Transplantation of
human embryonic stem cell-derived cells to a rat model
of Parkinsons disease: effect of in vitro differentiation
on graft survival and teratoma formation. Stem Cells 24:
14331440.
Brown VJ, Dunnett SB (1989) Comparison of adrenal and foetal
nigral grafts on drug-induced rotation in rats with 6-OHDA
lesions. Exp Brain Res 78:214218.
Brundin P, Pogarell O, Hagell P, Piccini P, Widner H, Schrag
A, Kupsch A, Crabb L, Odin P, Gustavii B, Bjorklund A,
Brooks DJ, Marsden CD, Oertel WH, Quinn NP, Rehncrona
S, Lindvall O (2000) Bilateral caudate and putamen grafts
of embryonic mesencephalic tissue treated with lazaroids in
Parkinsons disease. Brain 123: 13801390.
Brundin P, Strecker RE, Lindvall O, Isacson O, Nilsson
OG, Barbin G, Prochiantz A, Forni C, Nieoullon
A, Widner H, et al. (1987) Intracerebral grafting of
dopamine neurons. Experimental basis for clinical trials
in patients with Parkinsons disease. Ann N Y Acad Sci
495:473496.
Cho MS, Lee YE, Kim JY, Chung S, Cho YH, Kim DS, Kang
SM, Lee H, Kim MH, Kim JH, Leem JW, Oh SK, Choi YM,
Hwang DY, Chang JW, Kim DW (2008) Highly efficient
and large-scale generation of functional dopamine neurons
from human embryonic stem cells. Proc Natl Acad Sci USA
105:33923397.
Chung S, Hedlund E, Hwang M, Kim DW, Shin BS, Hwang DY,
Jung Kang U, Isacson O, Kim KS (2005) The homeodomain
transcription factor Pitx3 facilitates differentiation of mouse
embryonic stem cells into AHD2-expressing dopaminergic
neurons. Mol Cell Neurosci 28:241252.
Chung S, Shin BS, Hedlund E, Pruszak J, Ferree A, Kang UJ,
Isacson O, Kim KS (2006) Genetic selection of sox1GFP-
expressing neural precursors removes residual tumorigenic
pluripotent stem cells and attenuates tumor formation after
transplantation. J Neurochem 97:14671480.
Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J,
Zucker JP, Wang S, Morton CC, McMahon AP, Powers D,
Melton DA (2004) Derivation of embryonic stem-cell lines
from human blastocysts. N Engl J Med 350:13531356.
Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto
H, Chung W, Croft GF, Saphier G, Leibel R, Goland
R, Wichterle H, Henderson CE, Eggan K (2008) Induced
pluripotent stem cells generated from patients with ALS
can be differentiated into motor neurons. Science 321:1218
1221.
Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson
J, Meisner L, Zwaka TP, Thomson JA, Andrews PW (2004)
Recurrent gain of chromosomes 17q and 12 in cultured
human embryonic stem cells. Nat Biotechnol 22:5354.
Fearnley JM, Lees AJ. Ageing and Parkinsons disease: sub-
stantia nigra regional selectivity (1991) Brain 114 (Pt 5):
22832301.
A. Morizane and J. Takahashi
Freed CR, Greene PE, Breeze RE, Tsai WY, DuMouchel W,
Kao R, Dillon S, Winfield H, Culver S, Trojanowski JQ,
Eidelberg D, Fahn S (2001) Transplantation of embryonic
dopamine neurons for severe Parkinsons disease. N Engl
J Med 344:710719.
Fukuda H, Takahashi J, Watanabe K, Hayashi H, Morizane
A, Koyanagi M, Sasai Y, Hashimoto N (2006)
Fluorescence-activated cell sorting-based purification
of embryonic stem cell-derived neural precursors averts
tumor formation after transplantation. Stem Cells 24:
763771.
Goldman SA, Roy NS, Beal MF, Cleren C (2007) Large stem
cell grafts could lead to erroneous interpretations of behav-
ioral results? Nat Med 13:118119.
Hagell P, Brundin P. Cell survival and clinical outcome follow-
ing intrastriatal transplantation in Parkinson disease (2001)
J Neuropathol Exp Neurol 60:741752.
Hauser RA, Freeman TB, Snow BJ, Nauert M, Gauger L,
Kordower JH, Olanow CW (1999) Long-term evaluation of
bilateral fetal nigral transplantation in Parkinson disease.
Arch Neurol 56:179187.
Hayashi H, Morizane A, Koyanagi M, Ono Y, Sasai Y,
Hashimoto N, Takahashi J (2008) Meningeal cells induce
dopaminergic neurons from embryonic stem cells. Eur J
Neurosci 27: 261268.
Herszfeld D, Wolvetang E, Langton-Bunker E, Chung TL,
Filipczyk AA, Houssami S, Jamshidi P, Koh K, Laslett AL,
Michalska A, Nguyen L, Reubinoff BE, Tellis I, Auerbach
JM, Ording CJ, Looijenga LH, Pera MF (2006) CD30 is
a survival factor and a biomarker for transformed human
pluripotent stem cells. Nat Biotechnol 24:351357.
Iacovitti L, Donaldson AE, Marshall CE, Suon S, Yang MA
(2007) Protocol for the differentiation of human embryonic
stem cells into dopaminergic neurons using only chemically
defined human additives: studies in vitro and in vivo. Brain
Res 1127:1925.
Kanda S, Tamada Y, Yoshidome A, Hayashi I, Nishiyama T
(2004) Over-expression of bHLH genes facilitate neural for-
mation of mouse embryonic stem (ES) cells in vitro. Int
J Dev Neurosci 22:149156.
Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana
Y, Nakanishi S, Nishikawa SI, Sasai Y (2000) Induction of
midbrain dopaminergic neurons from ES cells by stromal
cell-derived inducing activity. Neuron 28:3140.
Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano
F, Ichinose H, Haruta M, Takahashi M, Yoshikawa K,
Nishikawa S, Nakatsuji N, Sasai Y (2002) Generation of
dopaminergic neurons and pigmented epithelia from primate
ES cells by stromal cell-derived inducing activity. Proc Natl
Acad Sci USA 99:15801585.
Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin
D, Lumelsky N, Lee SH, Nguyen J, Sanchez-Pernaute R,
Bankiewicz K, McKay R (2002) Dopamine neurons derived
from embryonic stem cells function in an animal model of
Parkinsons disease. Nature 418:5056.
Kim DW, Chung S, Hwang M, Ferree A, Tsai HC, Park
JJ, Chung S, Nam TS, Kang UJ, Isacson O, Kim KS
(2006) Stromal cell-derived inducing activity, Nurr1, and
signaling molecules synergistically induce dopaminergic
neurons from mouse embryonic stem cells. Stem Cells 24:
557567.
15 ES Cell Transplantation for the Treatment of Parkinsons Disease
Ko JY, Park CH, Koh HC, Cho YH, Kyhm JH, Kim YS,
Lee I, Lee YS, Lee SH (2007) Human embryonic stem
cell-derived neural precursors as a continuous, stable, and on-
demand source for human dopamine neurons. J Neurochem
103:14171429.
Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW
(2008) Lewy body-like pathology in long-term embryonic
nigral transplants in Parkinsons disease. Nat Med 14:
504506.
Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD
(2000) Efficient generation of midbrain and hindbrain neu-
rons from mouse embryonic stem cells. Nat Biotechnol
18:675679.
Li JY, Englund E, Holton JL, Soulet D, Hagell P, Lees AJ,
Lashley T, Quinn NP, Rehncrona S, Bjorklund A, Widner
H, Revesz T, Lindvall O, Brundin P (2008) Lewy bodies in
grafted neurons in subjects with Parkinsons disease suggest
host-to-graft disease propagation. Nat Med 14:501503.
Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V,
Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN, Noaksson
K, Hyllner J, Schulz TC, Zeng X, Freed WJ, Crook J,
Abraham S, Colman A, Sartipy P, Matsui S, Carpenter M,
Gazdar AF, Rao M, Chakravarti A (2005) Genomic alter-
ations in cultured human embryonic stem cells. Nat Genet
37:10991103.
Marshall JF, Ungerstedt U (1977) Striatal efferent fibers play
a role in maintaining rotational behavior in the rat. Science
198:6264.
Martin MJ, Muotri A, Gage F, Varki A (2005) Human embryonic
stem cells express an immunogenic nonhuman sialic acid.
Nat Med 11:228232.
Mendez I, Dagher A, Hong M, Hebb A, Gaudet P, Law A,
Weerasinghe S, King D, Desrosiers J, Darvesh S, Acorn
T, Robertson H (2000) Enhancement of survival of stored
dopaminergic cells and promotion of graft survival by expo-
sure of human fetal nigral tissue to glial cell line-derived neu-
rotrophic factor in patients with Parkinsons disease. Report
of two cases and technical considerations. J Neurosurg
92:863869.
Mendez I, Sanchez-Pernaute R, Cooper O, Vinuela A, Ferrari D,
Bjorklund L, Dagher A, Isacson O (2005) Cell type analysis
of functional fetal dopamine cell suspension transplants in
the striatum and substantia nigra of patients with Parkinsons
disease. Brain 128:14981510.
Mendez I, Vinuela A, Astradsson A, Mukhida K, Hallett P,
Robertson H, Tierney T, Holness R, Dagher A, Trojanowski
JQ, Isacson O (2008) Dopamine neurons implanted into peo-
ple with Parkinsons disease survive without pathology for
14 years. Nat Med 14:507509.
Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka
T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka
S (2008) Generation of induced pluripotent stem cells with-
out Myc from mouse and human fibroblasts. Nat Biotechnol
26:101106.
Nishimura F, Yoshikawa M, Kanda S, Nonaka M, Yokota H,
Shiroi A, Nakase H, Hirabayashi H, Ouji Y, Birumachi J,
Ishizaka S, Sakaki T (2003) Potential use of embryonic
stem cells for the treatment of mouse parkinsonian mod-
els: improved behavior by transplantation of in vitro
differentiated dopaminergic neurons from embryonic stem
cells. Stem Cells 21:171180.
253
Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S
(2008) Generation of mouse induced pluripotent stem cells
without viral vectors. Science 322:949953.
Olanow CW, Goetz CG, Kordower JH, Stoessl AJ, Sossi V,
Brin MF, Shannon KM, Nauert GM, Perl DP, Godbold J,
Freeman TB (2003) A double-blind controlled trial of bilat-
eral fetal nigral transplantation in Parkinsons disease. Ann
Neurol 54:403414.
Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura
A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ
(2008) Disease-specific induced pluripotent stem cells. Cell
134:877886.
Park S, Lee KS, Lee YJ, Shin HA, Cho HY, Wang KC, Kim
YS, Lee HT, Chung KS, Kim EY, Lim J (2004) Generation
of dopaminergic neurons in vitro from human embryonic
stem cells treated with neurotrophic factors. Neurosci Lett
359:99103.
Park CH, Minn YK, Lee JY, Choi DH, Chang MY, Shim JW,
Ko JY, Koh HC, Kang MJ, Kang JS, Rhie DJ, Lee YS, Son
H, Moon SY, Kim KS, Lee SH (2005) In vitro and in vivo
analyses of human embryonic stem cell-derived dopamine
neurons. J Neurochem 92:12651276.
Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf
N, Harrison NL, Studer L (2004) Derivation of midbrain
dopamine neurons from human embryonic stem cells. Proc
Natl Acad Sci USA 101:1254312548.
Pruszak J, Sonntag KC, Aung MH, Sanchez-Pernaute R, Isacson
O (2007) Markers and methods for cell sorting of human
embryonic stem cell-derived neural cell populations. Stem
Cells 25:22572268.
Rodriguez-Gomez JA, Lu JQ, Velasco I, Rivera S, Zoghbi SS,
Liow JS, Musachio JL, Chin FT, Toyama H, Seidel J, Green
MV, Thanos PK, Ichise M, Pike VW, Innis RB, McKay
RD (2007) Persistent dopamine functions of neurons derived
from embryonic stem cells in a rodent model of Parkinsons
disease. Stem Cells 25:918928.
Rolletschek A, Chang H, Guan K, Czyz J, Meyer M, Wobus
AM (2001) Differentiation of embryonic stem cell-derived
dopaminergic neurons is enhanced by survival-promoting
factors. Mech Dev 105:93104.
Roy NS, Cleren C, Singh SK, Yang L, Beal MF, Goldman
SA (2006) Functional engraftment of human ES cell-
derived dopaminergic neurons enriched by coculture with
telomerase-immortalized midbrain astrocytes. Nat Med
12:12591268.
Samii A, Nutt JG, Ransom BR (2004) Parkinsons disease.
Lancet 363:17831793.
Schulz TC, Noggle SA, Palmarini GM, Weiler DA,
Lyons IG, Pensa KA, Meedeniya AC, Davidson BP,
Lambert NA, Condie BG (2004) Differentiation of
human embryonic stem cells to dopaminergic neu-
rons in serum-free suspension culture. Stem Cells 22:
12181238.
Shibata H, Ageyama N, Tanaka Y, Kishi Y, Sasaki K, Nakamura
S, Muramatsu S, Hayashi S, Kitano Y, Terao K, Hanazono
Y (2006) Improved safety of hematopoietic transplantation
with monkey embryonic stem cells in the allogeneic setting.
Stem Cells 24:14501457.
Sonntag KC, Pruszak J, Yoshizaki T, van Arensbergen J,
Sanchez-Pernaute R, Isacson O (2007) Enhanced yield of
neuroepithelial precursors and midbrain-like dopaminergic
254
neurons from human embryonic stem cells using the BMP
antagonist noggin. Stem Cells 25:411418.
Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K
(2008) Induced pluripotent stem cells generated without viral
integration. Science 322:945949.
Tabar V, Tomishima M, Panagiotakos G, Wakayama S, Menon
J, Chan B, Mizutani E, Al-Shamy G, Ohta H, Wakayama T,
Studer L (2008) Therapeutic cloning in individual parkinso-
nian mice. Nat Med 14:379381.
Takagi Y, Takahashi J, Saiki H, Morizane A, Hayashi T, Kishi Y,
Fukuda H, Okamoto Y, Koyanagi M, Ideguchi M, Hayashi
H, Imazato T, Kawasaki H, Suemori H, Omachi S, Iida
H, Itoh N, Nakatsuji N, Sasai Y, Hashimoto N (2005)
Dopaminergic neurons generated from monkey embryonic
stem cells function in a Parkinson primate model. J Clin
Invest 115:102109.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T,
Tomoda K, Yamanaka S (2007) Induction of pluripotent stem
cells from adult human fibroblasts by defined factors. Cell
131:861872.
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by
defined factors. Cell 126:663676.
Thompson L, Barraud P, Andersson E, Kirik D, Bjorklund
A (2005) Identification of dopaminergic neurons of nigral
and ventral tegmental area subtypes in grafts of fetal
ventral mesencephalon based on cell morphology, pro-
tein expression, and efferent projections. J Neurosci 25:
64676477.
Ueno M, Matsumura M, Watanabe K, Nakamura T, Osakada
F, Takahashi M, Kawasaki H, Kinoshita S, Sasai Y (2006)
A. Morizane and J. Takahashi
Neural conversion of ES cells by an inductive activity on
human amniotic membrane matrix. Proc Natl Acad Sci USA
103:95549559.
Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner
F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch
R (2008) Neurons derived from reprogrammed fibroblasts
functionally integrate into the fetal brain and improve symp-
toms of rats with Parkinsons disease. Proc Natl Acad Sci
USA 105:58565861.
Yamazoe H, Kobori M, Murakami Y, Yano K, Satoh M,
Mizuseki K, Sasai Y, Iwata H (2006) One-step induction
of neurons from mouse embryonic stem cells in serum-free
media containing vitamin B12 and heparin. Cell Transplant
15:135145.
Yang D, Zhang ZJ, Oldenburg M, Ayala M, Zhang SC (2007)
Human embryonic stem cell-derived dopaminergic neurons
reverse functional deficit in parkinsonian rats. Stem Cells
26:5563.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J,
Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart
R, Slukvin II, Thomson JA (2007) Induced pluripotent stem
cell lines derived from human somatic cells. Science 318:
19171920.
Yue F, Cui L, Johkura K, Ogiwara N, Sasaki K (2006) Induction
of midbrain dopaminergic neurons from primate embry-
onic stem cells by coculture with sertoli cells. Stem Cells
24:16951706.
Zeng X, Cai J, Chen J, Luo Y, You ZB, Fotter E, Wang Y, Harvey
B, Miura T, Backman C, Chen GJ, Rao MS, Freed WJ
(2004) Dopaminergic differentiation of human embryonic
stem cells. Stem Cells 22:925940.
Chapter 16

Functional Multipotency of Neural Stem Cells

and Its Therapeutic Implications

Yang D. Teng, Serdar Kabatas, Jianxue Li, Dustin R. Wakeman, Evan Y. Snyder,
and Richard L. Sidman

Contents macro-environment towards the formation and self-

maintenance of a physiologically functioning adult
16.1 Background . . . . . . . . . . . . . . . . 256 nervous system. We believe that embracing this view
16.2 The Neural Stem Cell . . . . . . . . . . . . 257 of the NSCs multipotency is pivotal for correctly,
16.2.1 Biological Definition . . . . . . . . 257 efficiently, and optimally exploiting stem cell biology
16.2.2 Issues of Cell Identification:
for therapeutic applications, including reconstituting
Cross-Differentiation and Cell Fusion . 257
16.3 Analysis of Neurogenesis and Neural Stem the dysfunctional CNS.
Cell Fate . . . . . . . . . . . . . . . . . . 258
16.3.1 In Vivo . . . . . . . . . . . . . . . 258 Keywords Homeostasis Multipotency Neural stem
16.3.2 In Vitro . . . . . . . . . . . . . . 259 cells Spinal cord injury Transplantation
16.4 Clinically Oriented Investigations . . . . . . 261
16.4.1 Spinal Cord Injury . . . . . . . . . . 261
16.4.2 Neurodegenerative Diseases . . . . . 264 Abbreviations
16.4.3 Stroke . . . . . . . . . . . . . . . 265
16.5 Conclusion . . . . . . . . . . . . . . . . . 266
References . . . . . . . . . . . . . . . . . . . . 266
AD Alzheimers disease
ALS amyotrophic lateral sclerosis
bFGF basic fibroblast growth factor
Abstract In this review, we propose an updated con- BrdU bromodeoxyuridine
cept of the neural stem cell (NSC). New data of our CNTF ciliary neurotrophic factor
own and others suggest that the fields conventional CS-PG chondroitin sulphate proteoglycans
view which has touched principally on the essential DAT dopamine transporter
multipotency of lineage phenotypes (i.e., the ability DDC dopa-decarboxylase
of NSC to differentiate into all neural cells), should EAE experimental allergic encephalomyelitis
be broadened to include the emerging recognition of EdU 5-ethynyl-2-deoxyuridine
the biofunctional multipotency of the NSC to medi- EGF epidermal growth factor
ate systemic homeostasis. Under this new conceptual GAP growth-associated protein
context, one may begin to appreciate and seek the GRP glial restricted precursors
logic and teleology behind the wide range of molec- HD Huntingtons disease
ular tactics the NSC appear to serve at each devel- hNSC human neural stem cells
opmental stage as they integrate into and prepare, HSC hematopoietic stem cells
modify, and guide the surrounding CNS micro- and LIF leukaemia inhibitory factor
LSD lysosomal storage diseases
MP methylprednisolone
MS multiple sclerosis
Y.D. Teng () MPTP 1-methyl-4-phenyl-1,2,3,6-tetra-
Department of Neurosurgery, HMS/CHB/BWH, Boston, MA, hydropyridine
USA
e-mail: yang_teng@hms.harvard.edu NRP neuronal restricted precursors

H. Ulrich (ed.), Perspectives of Stem Cells, 255

DOI 10.1007/978-90-481-3375-8_16, Springer Science+Business Media B.V. 2010
256
NSC Neural stem cell(s)
PD Parkinsons disease
SN substantia nigra
16.1 Background
In 1959, a new method of marking dividing cells in
the mammalian brain was developed (Sidman et al.,
1959). The method was based on the already known
fact (Hughes et al., 1958) that [3 H]-thymidine injected
systemically in mammals would be incorporated selec-
tively into DNA replicating during S-phase of the cell
cycle (Howard and Pelc, 1953), and their positions and
numbers ascertained by autoradiography.
The availability of this technology set an impor-
tant process in motion, through which the conven-
tional doctrine of neurogenesis was reexamined (Ming
and Song, 2005). The idea that no new neurons are
formed after birth became dogma in neuroscience
since Santiago Ramon y Cajal, in 1913, hypothe-
sized that neurons are generated exclusively during the
prenatal phase of development. Although some investi-
gators held different opinions, it was impossible, with
the resources and methods obtainable then, to prove
that postnatally born cells were actually neurons and
not glia.
Using the [3 H]-thymidine labeling technique, pat-
terns of neuron genesis and migration were demon-
strated in several developing areas of the mouse brain
(Angevine and Sidman, 1961; Miale and Sidman,
1961; Sidman, 1961), and in the adult mouse brain
(Smart, 1961). Soon after, it was suggested that not
only astrocytes and microglial cells, but also oligo-
dendrocytes (Altman, 1962a) and neurons proliferate
(Altman, 1962b) in adult mammals. Subsequently,
Angevine (1965) reported that granule cell neurons
were still forming in the mouse at least until post-
natal day 20, the oldest age he examined, in and
near the dentate gyrus of the hippocampal formation,
and Altman and colleagues reported evidence of new
neurons forming in various regions of the adult rat
brain including the dentate gyrus of the hippocampus
(Altman and Das, 1965), the neocortex (Altman, 1966)
and the olfactory bulb, the latter after cell proliferation
in the wall of the anterior horn of the lateral ventri-
cle and migration via a pathway he named the rostral
migratory stream (Altman, 1969).
Y.D. Teng et al.
A decade later, Nottebohm and collaborators
demonstrated telencephalic neuronal replacement in
the adult avian brain related to seasonal song learn-
ing (Graziadei and Graziadei, 1979; Goldman and
Nottlebohm, 1983; Nottlebohm, 2004). Around the
same time, it was observed that newborn neurons in the
adult rodent hippocampus appeared to receive synap-
tic inputs (Kaplan and Bell, 1983), and later it was
established that they also extended axon projections to
their target area (Stanfield and Trice, 1988). Parallel to
these studies, a new cell tracer was being explored that
would accelerate research in the stem/progenitor cell
field (Gratzner, 1982). The marker, bromodeoxyuri-
dine (BrdU), is a synthetic thymidine analogue that
also becomes incorporated into a cells DNA during the
S-phase of the cell cycle, much like [3 H]-thymidine,
but could be easily and inexpensively detected by
immunocytochemistry, in contrast to [3 H]-thymidine.
A new technique based on incorporation of the thymi-
dine analogue 5-ethynyl-2 -deoxyuridine (EdU), with
subsequent detection by click chemistry, is a very
sensitive, rapid method, independent of fixation or
harsh denaturing treatment of the tissue, and appli-
cable to thick sections or whole mounts (Salic and
Mitchison, 2008; Buck et al., 2008). By staining a
given cell for dual expression of BrdU or EdU and
a cell type-specific marker, the phenotypic fate of a
newly divided cell can be readily analyzed and quan-
tified by unbiased stereological methods. This tech-
nique has become a mainstay in stem cell research.
Only recently has there been concern that BrdU and
other thymidine analogs might be passed from labeled
grafted stem cells to dividing host cells, allowing for an
element of misidentification unless adequate controls
are invoked (Burns et al., 2006). Also, labeling might
represent DNA repair in a non-mitotic cell (Bauer and
Patterson, 2005), and cell cycle activity with labeling
by thymidine analogs can be a prelude not only to
cell division, but also to cell death of neurons (Herrup
et al., 2004).
In 1992, adult and fetal neural stem cells from the
CNS of rodents were isolated (Reynolds and Weiss,
1992; Snyder et al., 1992) and, in 1998, human neu-
ral stem cells (Flax et al., 1998; Kukekov et al., 1999).
Also in 1998, taking advantage of the fortuitous use of
a BrdU-like chemotherapeutic agent in the treatment
of a head and neck cancer, the presence of neurogene-
sis and rostral migratory stream in adult humans was
confirmed, further suggesting that concepts regarding
16 Functional Multipotency of Neural Stem Cells
stem cell biology being formulated in rodents may be
evolutionarily conserved (Eriksson et al., 1998; Curtis
et al., 2007).
Also in the late 1980s and early 1990s, comple-
mentary methods for identifying and tracking newly-
mitotic cells were being developed and employed for
tracing the lineage of cells. The most powerful of
these was the use of replication incompetent retro-
viral vectors that could transduce a reporter trans-
gene to the genome of cells passing through S-phase
such that this marker would become permanently inte-
grated in a unique chromosomal site and passed to
all progeny and generations (Price et al., 1987; Sanes
et al., 1986). Unlike BrdU, which becomes diluted
50% with each cell division, retroviral-transduced
genes are not diluted. Combining such marked cells
with electrophysiological analysis could provide con-
vincing evidence that newborn neurons in the adult
mammalian CNS are functional and synaptically inte-
grated (Belluzzi et al., 2003; Carleton et al., 2003, van
Praag et al., 2002). Yet, though it is now known that
neurogenesis persists in some regions of the human
adult CNS and that these new neurons are functional
and connected, it remains not entirely certain what
biological roles are served by the newborn cells in
these few neurogenic hot-spots (Zhao et al., 2008),
and especially in regions such as the adult spinal cord
where stem cell proliferation only occurs transiently
following trauma or other non-physiological influences
(Teng et al., 2006).
16.2 The Neural Stem Cell
16.2.1 Biological Definition
Neural stem cells (NSC) are the most primordial and
uncommitted cells of the nervous system, and are
believed to give rise to the vast array of more special-
ized cells of the CNS and peripheral nervous system
(PNS). To be considered a neural stem cell in
contrast to a progenitor cell (i.e., cells that have
already become lineage-committed to give rise to only
one category of neural component, e.g., glial cells
versus neurons) that cell must be capable of (i)
generating all neural lineages (neurons, astrocytes and
oligodendrocytes) throughout the nervous system, (ii)
having some capacity for self-renewal and (iii) giving
257
rise to cell types in addition to themselves through
asymmetric cell division (Gage, 2000).
Stem cells are defined according to their repertoires.
A totipotent stem cell, if implanted in the uterus
of a living animal can give rise to a full organism
and all its organ systems, including CNS and PNS.
A pluripotent stem cell, in the simplest definition,
is similar to the totipotent cell, except that it can-
not give rise to trophoblasts of the placenta. Such
cells have been convincingly affirmed to exist only
in the inner cell mass of the blastocyst, although a
series of recent controversial papers have suggested
that such pluripotent cells may be harbored in the
amniotic fluid, placenta, umbilical cord, and bone mar-
row mesenchyme (Ortiz-Gonzalez et al., 2004). The
next developmental stage in the progressive restric-
tion of potency is the multipotent somatic stem cell,
which is capable of differentiating into all cell types
of a given organ or tissue and into only cells of that
organ or tissue (Martinez-Serrano et al., 2001). Even
though that is the traditional and most widely accepted
definition of the multipotent stem cell, recent find-
ings have forced a reexamination of this biological
concept, based on experiments showing possible cross-
differentiation, although some such instances have
been found to be artifacts caused by cell fusion, as
discussed in the next section. Hence, while an active
area of inquiry at the time of this writing, the distinc-
tions above remain the lexicon of the field. Importantly,
a common belief among stem cell biologists is that
the distinction between totipotence, pluripotence, and
multipotence is not discrete, but rather a continuum
in development. Some investigators also define adult
brain-derived stem cells as neural progenitor cells
(Shihabuddin et al., 2004). In addition, it is currently
not clear if any specific nomenclature will be desig-
nated for neural stem/progenitor-like cells that are
derived from induced pluripotent stem (iPS) cells (e.g.,
naming them as neural precursor cells; Wernig et al.,
2008).
16.2.2 Issues of Cell Identification:
Cross-Differentiation
and Cell Fusion
Somatic stem cells sometime erroneously called
adult stem cells in the lay press have been viewed
258
as exceptionally plastic but nevertheless restricted to
the production of cells from the tissue of origin but not
cells of non-related tissues. For example, NSC give rise
to the three main types of nervous system cells (neu-
ron, oligodendrocyte and astrocyte), while hematopoi-
etic stem cells (HSC) produce only blood derived cells,
etc. However, various reports in the last few years
have challenged this central dogma by demonstrating
that adult stem cells, under certain microenvironmen-
tal (often very non-physiological) conditions, generate
cell types besides those in the tissue of origin, pos-
sibly indicating that they can switch cell fate. HSC,
for instance, in addition to forming blood cells, have
been reported to develop into liver cells (Petersen et al.,
1999) and NSC to give rise not only to nerve cells
but also to early hematopoietic precursors (Bjornson
et al., 1999). These cell behaviors have been termed
cross-differentiation or stem cell plasticity. Such
reports have generated excitement as well as skepti-
cism in the field of stem cell biology, as the concept
of plasticity defies the developmental biology principle
that lineage restriction is imparted during morpho-
genesis. However, if correct, the ability of somatic
stem cells to change fate also holds immense ther-
apeutic potential as well in circumventing the con-
cerns by some of having to obtain pluripotent cells
from human embryos (Lakshmipathy and Verfaillie,
2005).
The controversy comes when more recent stud-
ies suggest that such stem cell plasticity might
not actually exist. Rather, the illusion of cross-
differentiation/transdifferentiation might actually
result from the fusion of donor cells with host cells
conferring on these host cells the transplanted cells
reporter gene and creating the misperception that
these host cells are donor-derived. In other words, the
markers used to visualize the grafted cells in vivo or
in vitro have simply been taken up by the host cells
but no differentiation of donor cells into differentiated
organ-specific cells had actually occurred. It is also
possible that cell fusion may cause a change in the
phenotype and/or the function of cells (through, e.g.,
nuclear reprogramming) and may also explain the
apparent inherent plasticity of committed cells. While
cell fusion may have created, in many instances, the
misleading appearance of cross-differentiation, the
prevalence of this process itself has caused stem cell
biologists to speculate whether fusion may actually
play a natural role in some normal biological as well
Y.D. Teng et al.
as in pathophysiological functions. This process, as
suggested by some investigators, could provide a
strategy for de-differentiating committed cells that
might then be reprogrammed for tissue reconstitution
and reversal or repair of injury (Ogle et al., 2005).
Resolution of the cross-differentiation controversy
will obviously have a major impact on our current
definitions of stem cells.
16.3 Analysis of Neurogenesis
and Neural Stem Cell Fate
16.3.1 In Vivo
As briefly described, the analysis of neurogenesis
in vivo was largely established by the use of [3 H]-
thymidine in 1959 and bromodeoxyuridine (BrdU)
after 1982 as markers of cell division. Those label-
ing techniques made it possible to track endogenous as
well as donor stem/progenitor cells and do quantitative
analysis of proliferation, differentiation, and survival
of all cells born after the injection or prelabeling of
such a marker (Kempermann et al., 1997; Miller and
Nowakowski, 1988; Redmond et al., 2007; Sidman,
1970).
Because all marking techniques carry caveats and
require rigorous controls, a practical principle is to use
at least two independent markers and marking tech-
niques for identifying cells. For example, a strategy
to label newly born cells might include BrdU or EdU
incorporation and/or the tagging of a cell with a genetic
marker carried in a replication-incompetent, helper-
virus-free retroviral vector that lacks nuclear import
mechanisms. With the latter technique, viral integra-
tion occurs only when the nuclear membrane breaks
down during mitosis. Also the transgene becomes
transduced only when the vector adheres to a spe-
cific cell surface receptor. Therefore, they are good,
non-diffusible, non-dilutable markers of dividing cells
(Lewis and Emerman, 1994; Ming and Song, 2005).
Other methods for distinguishing donor cells from
host cells in transplantation studies might include mis-
matching the species or sex between the two; for
example, transplanting human cells into a rodent organ
or implanting a male cell (recognized by its Y chromo-
some) into a female host (e.g., Li et al., 2006b).
16 Functional Multipotency of Neural Stem Cells
Some research might employ cell type- or develop-
mental stage-specific markers to track the development
and differentiation of a stem cell. For example, a
developmental neuron is defined as immature when
it expresses immature markers but lack mature ones.
Therefore, antibodies directed against those imma-
ture markers would stain only newborn, still imma-
ture cells that are undergoing neuronal differentiation.
Oligodendrocyte (Olg) development can be used to
illustrate the use of this technique. Even though Olgs
express proteolipid protein (PLP) and DM20 (myelin
proteins) during most of their development, the ratio
between these two proteins varies during differenti-
ation (Yu et al., 1994; Timsit et al., 1995). While
DM20s expression precedes that of PLP and is kept
high during the first stages of differentiation, PLP is
expressed in higher levels only at the latter stages of
differentiation and, therefore, defines a mature myelin-
forming oligodendrocyte (Meng et al., 2005). Multiple
steps at multiple CNS sites have been defined for this
cell type (Miller, 2002, 2005). Similarly, other markers
for other cell types can be used to track the devel-
opment of a particular cell type. It is important to
reiterate that this method is best used in combination
with some or all of the others described above, because
antibodies to developmental markers are not always
ideally specific (Brandt et al., 2003; Kempermann
et al., 2004).
16.3.2 In Vitro
The isolation and culture of NSC in vitro is of great
value, providing an opportunity to scrutinize individ-
ual cells and their differentiation more closely, as well
as to prepare them for research and therapeutic appli-
cations. It also allows one to exclude confounding
variables (e.g., intervention by the immune system)
and to manipulate the microenvironment in system-
atic and prescribed ways. Stem cells can be propagated
as clonal populations in vitro by many effective and
safe means that include both epigenetic and genetic
strategies.
16.3.2.1 Epigenetic
The identification of cytokines and growth factors that
affect survival and proliferation of neural stem cells
259
has been very important for cell isolation and culture.
In the late 1980s and early 1990s it became evident
that most regions of the CNS contain a small popula-
tion of individual cells that could be isolated and could
give rise to clonally-related populations consisting of
multiple neural cell types that heretofore had not been
regarded as deriving from a common progenitor. These
regions included olfactory bulb and cerebellum (Ryder
et al., 1990; Snyder et al., 1992); postnatal (Altman,
1966) as well as embryonic neocortex (where 18% of
the cells could generate both neurons and oligodendro-
cytes in vitro) (Williams et al., 1991), hippocampus
(Renfranz et al., 1991; Gage et al., 1995); and sep-
tum (Temple, 1989). From these studies, however, it
became clear that neural progenitors or stem cells,
when isolated from the CNS, had a predisposition to
exit the cell cycle and differentiate unless an inter-
vention was imposed upon them to remain cycling
and hence hold commitment in abeyance. For exam-
ple, cells cultured from the embryonic rat septum were
incapable of more than one cycle of cell division unless
they were co-cultured with fetal striatal tissue (Temple,
1989). When they were prodded to continue prolifer-
ating, some of the cells appeared to be multipotent.
Subsequent studies were launched to identify exoge-
nous growth factors and/or internal genes that could
be used to maintain multipotent cells in a proliferative
state that might allow them to be expanded and used
for developmental or therapeutic studies. The princi-
pal mitogens studied and most commonly used to date
are basic fibroblast growth factor (bFGF) (Richards
et al., 1992), epidermal growth factor (EGF) (Reynolds
and Weiss, 1992) and leukemia inhibitory factor (LIF)
(Smith et al., 1988).
As described above, one of the first difficulties
encountered in attempting to culture NSC was that
isolated single cells tended to cease dividing and differ-
entiate. Only cells surrounded by other cells remained
mitotic, suggesting that cell-to-cell contact was impor-
tant for cell proliferation (Temple and Qian, 1995).
Further investigation suggested that a likely candi-
date for mediating this inter-cellular interaction was
basic fibroblast growth factor (bFGF). Besides being
known for its association with extracellular matrix and
cell membranes, bFGF was surprisingly found to be
present throughout CNS development (Baird, 1994;
Kilpatrick et al., 1995). Previous studies had already
shown that the addition of exogenous bFGF to cultures
of cortical neuroectoderm cells appeared to stimulate
260
their proliferation and lead to increased numbers of
neurons (Gensburger et al., 1987), but, it wasnt until
the 1990s that the issue was addressed of which type
of cortical cell was the target of bFGF. It was found
that bFGF would stimulate multipotent stem cells from
many regions of the CNS (Temple and Qian, 1995;
Gritti et al., 1996).
In addition to bFGF, studies in the 1980s had
already shown that epidermal growth factor (EGF)
bound to CNS cells and stimulated their prolifera-
tion (Simpson et al., 1982; Anchan et al., 1991). EGF
induced proliferation of progenitor cells that began to
divide and would come to form a cluster of undifferen-
tiated cells that expressed nestin, an intermediate fila-
ment present in neuroepithelial stem-like cells. These
cells, when they exited the cell cycle, would differenti-
ate into neurons and astrocytes (Reynolds et al., 1992).
These EGF-expanded cells, when then stimulated by
bFGF, would yield two progenitor cell sub-types: one
giving rise to neurons and astrocytes, the other gener-
ating only neurons (Vescovi et al., 1993). Addition of
both bFGF and EGF simultaneously in the same cell
culture yielded better cell survival than either agent
alone, and would also better maintain their ability to
differentiate into neurons, oligodendrocytes and astro-
cytes (Vescovi et al., 1999). The notion emerged that
the most immature and hence most multipotent
neural stem/progenitor cells bore receptors for both
mitogens. Selecting for the presence of both receptors
(and hence responsiveness to both mitogens) became
the basis of a quick and easy screening technique for
selecting neural stem cell populations from a hetero-
geneous CNS culture (including from human material;
Flax et al., 1998).
The addition of leukaemia inhibitory factor (LIF)
to cell cultures yielded better long term growth than
bFGF and EGF alone. No difference was noted in early
stage cultures, but, after 5060 days in vitro, the cul-
tures without LIF consistently showed slower expan-
sion while those with all three mitogens continued to
expand (Carpenter et al., 1999).
Interestingly, it has been demonstrated that under
the presence of bFGF, multipotent NSC (i.e., tripotent
cells) normally produce specified neural progenitors
through a bipotent intermediate cell type. In fact, the
tripotent state may be reset at each passage. The cil-
iary neurotrophic factor (CNTF) is believed to instruct
multipotent cells to an astrocytic fate. More recently, it
was reported that CNTF could direct astrogliogenesis
Y.D. Teng et al.
from tripotent cells, bypassing two of the three normal
bipotent intermediates. It also promotes the expansion
of specified astrocytic progenitors (Ravin et al., 2008).
Therefore, the method most used now for expand-
ing and maintaining NSC cultures by epigenetic means
involves addition of bFGF, EGF and LIF to a serum-
free basal medium. Many studies (as well as our own
experience) suggest that, (at least for short periods),
bFGF alone (at 20 ng/ml) is sufficient. As noted above,
in addition to stimulating proliferation of NSC, respon-
siveness to these factors may be one technique for
selecting NSC from a mixed population of cells at
various degrees of maturation, potency, and lineage
commitment.
Culturing cells with mitogens alone is a double-
edged sword. Because one does not by intent genet-
ically manipulate the cells, approval by regulatory
agencies for translation to the clinic may be eas-
ier to obtain. On the downside, however, obtaining a
sufficient number of cells while avoiding senescence
(particularly of human cells) can be quite challeng-
ing. Furthermore, one may be selecting for cells with
particular avidity for these factors and hence may
be more prone to neoplastic transformation. Genetic
modifications that might be imposed by tonic expo-
sure to high concentrations of a mitogen are mainly
unknown and less controllable than the some of the
well-studied self-regulated or regulatable genetic aug-
mentations described in the next section (Snyder et al.,
1992; Roy et al., 2004).
16.3.2.2 Genetic
The genetic strategy for culturing NSC in vitro seeks
to augment and prolong the expression of stemness
genes. Myc represents such a gene; it is becoming rec-
ognized as important to the proliferative state of stem
cells. When its expression is prolonged, cells remain
in the cell cycle and hence their differentiation is held
in abeyance. When they exit the cell cycle, they may
still respond to regional epigenetic cues and differ-
entiate appropriately spontaneously and inherently
downregulating myc translation. In other words, the
exogenous extra copy of myc may be regulated by the
cell as it does its cellular myc. Interestingly, embry-
onic stem cells and iPS cells are colloquially said to
be naturally immortalized, with myc being one the
16 Functional Multipotency of Neural Stem Cells
four genes first used to induce pluripotency in postmi-
totic cells (Takahashi and Yamanaka, 2006). However,
the goal, in most studies, for augmenting certain genes
in somatic stem cells is to temporarily and control-
lably co-opt that quality safely and effectively. The
NSC are usually transfected using a retroviral vector
encoding the stemness gene. These clonal NSC have
the advantage of being easily expanded to large num-
bers that typically remain stable and homogenous over
long periods of time from experiment to experiment
and recipient to recipient without senescing. Such cells
have not only proved to be an ideal model for under-
standing fundamental aspects of stem cell biology, but,
because they have been, in many published cases, used
safely and effectively as transplant material for a wide
variety of disease models over the past two decades,
they have established a good reference of what ther-
apeutic benefit should be safely achievable by a cell
with stem-like qualities (Parker et al., 2005). In fact,
whatever expansion technique is selected for use by
an investigator should be judged in part by its ability
to attain a commonly acceptable degree of safety and
efficacy in models of disease and injury.
Of course, when contemplating clinical strategies,
a complete knowledge of the fate of these cells as
for any implanted cell must be vouchsafed. As noted
above, while such cells have proven exceptionally safe,
regulatory agencies generally feel most comfortable
approving the use of non-genetically modified cells.
Ultimately, however, a well-characterized, uniform,
stable, readily available, inexhaustible supply of clon-
ally related NSC may prove to be most efficacious
and practical (Vescovi and Snyder, 1999). Other genes
(e.g., telomerase) have been explored and proven valu-
able (Roy et al., 2004). To maximize comfort with
stem cells, their stemness genes may be placed under
regulatable control or put in tandem with suicide genes.
16.4 Clinically Oriented Investigations
Although the field of NSC biology is very young,
strategies by which these cells and their emerging
properties might be harnessed to ameliorate a range of
neurological disorders have already captured the imag-
ination of scientists, clinicians and the public, which
has helped fuel the new field of regenerative medicine.
261
Broadly, there are two fundamental interlocking strate-
gies for using NSC therapeutically. The first seeks
to use the stem cell to provide replacement cells for
those that have become dysfunctional or lost (Muotri
and Gage, 2006). The second exploits the observation
that stem cells (particularly in their non-neuronal state)
constitutively produce neuroprotective, immunoregu-
latory and homeostatic molecules that serve to reduce
host cell loss and inhibit the formation of barriers to
self-repair (Teng et al., 2002, 2006). We will review
both lines of research as they apply to the most fre-
quently studied diseases and where NSC seem to be
most promising.
16.4.1 Spinal Cord Injury
Experimental treatment strategies aimed at the acute
stage of the injury process currently include neu-
ronal protection and preservation of residual axons
and white matter. Several approaches have been
proposed and applied to this end, most notably anti-
secondary injury therapy using high dose methyl-
prednisolone (MP) (Hall and Springer, 2004). Ideally,
with effective neuroprotection in place, treatments
to promote axonal regeneration can be started
simultaneously or immediately thereafter. In pur-
suing this possibility, experimental use of neu-
rotrophins or neurotrophic factors to increase neuro-
protection and axonal growth, and neutralizing anti-
bodies to Nogo and other oligodendrocyte-related
inhibitory molecules have been investigated (Blesch
and Tuszynski, 2002; Schwab, 2004). While some
produced encouraging outcomes, evidence to the con-
trary has also been reported. The conflicting findings
do not negate the potential for this therapy but sug-
gest that this strategy, when used in isolation, may
not be sufficient to promote functionally meaning-
ful axonal regrowth in the injured spinal cord. For
example, enzymatic or genetic antagonism to chon-
droitin sulphate proteoglycans (CS-PG) has offered
some encouraging results. This approach was found,
however, to exert neuroprotection (Carter et al., 2008),
and could be strengthened when it was jointly applied
with maneuvers to prompt adult CNS neurons to
reenter growth mode (Silver and Miller, 2004).
Hence, combined therapies are likely to be most
effective.
262
Despite the fact that various treatment strategies
have shown benefit in experimental animal models,
there is still no effective therapy for clinical spinal
cord injury (SCI). This difficult situation, in our opin-
ion, is attributable to the following realities. First,
there has been no conclusive evidence favoring one
process as the predominant pathophysiological mech-
anism which can account for all the spinal dysfunction
seen following SCI. Most of the pathophysiological
processes (e.g., secondary molecular events: glutamate
toxicity, sodium and calcium influx, free radical insult,
cytochrome c release; secondary pathophysiological
events: ischemia, anoxia, apoptosis, etc.) apparently
exist either simultaneously or sequentially in an inter-
locked manner throughout the evolution of the injury
and represent different facets of this complicated dis-
ease entity (Tator and Fehlings, 1991; Teng et al.,
2004). Most interventions reported to date target solely
one facet of the injury process which, in isolation, is
doomed to have limited benefit. To further complicate
the situation, a given approach that may be useful when
used alone, may become ineffective or even detrimen-
tal when used in combination with other interventions,
perhaps working at cross purposes. Hence, it is crit-
ical to understand the intricate interactions between
these options and identify the underlying mechanisms
of their actions so that they may be orchestrated in a
safe, synergetic, and clinically feasible fashion (Reier,
2004).
Apropos to this point, we have come to view NSC
as the glue that can bind and integrate multiple
approaches. Several studies have suggested that NSC,
when transplanted into the injured brain or spinal cord
of rodents, migrate preferentially to and become inte-
grated within the damaged areas. A subpopulation
of the transplanted NSC is re-directed to differenti-
ate into cell types that might replace the diseased or
degenerated host cells (Snyder et al., 1997; Rosario
et al., 1997; Yandava et al., 1999; Park et al., 1999,
2002b). More intriguingly and, ultimately, of proba-
bly greater importance and utility is the observation
that undifferentiated NSC or NSC that have pursued
a glial lineage seem to recondition the host CNS
microenvironment and promote functional recovery by
protecting pre-existent but threatened host neurons and
circuitry (Teng et al., 2002). The impact of this action
is probably greater than neuronal replacement. The
precise mechanism by which NSC exert this homeo-
static pressure is unclear, though it is likely attributable
Y.D. Teng et al.
to a large degree by intrinsic ability of NSC to secrete
neurotrophic factors, and/or immunomodulators, and
form gap junctions with host cells (as demonstrated by
Teng et al., 2002 and subsequently reported by many
others, including Ourednik et al., 2002; Lu et al., 2003;
Pluchino et al., 2005; Llado et al., 2004; Yan et al.,
2004; Li et al., 2006, Bjugstad et al., 2005; Teng et al.,
2006; Redmond et al., 2007; Jderstad et al., 2010).
Thus, preserving the multipotency of these cells as
opposed to attempting to direct them invariantly down
the differentiation pathway of a single cell type might
offer the greatest chance for cell-based therapies of
the different inter-locked stages of SCI, but in a par-
simonious fashion. Harnessing the potentially broad
therapeutic capacity of the NSC for use in an intelligent
and rationale manner requires learning the principles
that can govern interaction between the pathologic tar-
get and host environmental components, the NSC, and
other therapeutic reagents.
We believe that the innate biology of NSC (i.e.,
their default production and secretion of various neu-
rotrophic factors and other molecules in a differentia-
tion stage-dependent fashion) enables them to interact
with the surrounding environment, including releasing
trophic factors in an appropriate, regulated, stimulus-
appropriate manner. These factors, in our view, are
components of the stem cells inherent developmen-
tal program which calls upon it to exert homeostatic
forces upon a dynamically growing nervous system
which, otherwise, could become dysequilibrated. The
result of this inherent program a dividend, so
to speak, from developmental biology is to pro-
mote, enable, induce, or catalyze the host to attempt
to reconstitute its own tissue, to minimize barriers to
this process, and to protect endangered cells from cell
death or other toxic influences. Methods to optimize
this process i.e., to work in concert with normal
developmental tendencies, is undoubtedly desirable for
optimizing repair.
An example of harnessing and exploiting such
inherent stem cell programs will be presented here.
To direct neural repair more effectively following SCI,
we cultured NSC ex vivo upon a biosynthetic scaffold
that mimicked the general structure of a healthy spinal
cord. It had an inner section, engineered to emulate the
gray matter with an isotropic pore structure of 250
500 m in diameter to facilitate seeding of the NSC.
The outer section of the scaffold, modeled to emu-
late the white matter, had long, axially oriented pores
16 Functional Multipotency of Neural Stem Cells
for axonal guidance and radial porosity to allow fluid
transport while inhibiting the ingrowth of scar tissue
(Teng et al., 2002). Implantation of the scaffold-NSC
unit into an adult rat hemi-section model of SCI pro-
moted long-term improvement in function (persistent
for 1 year in a group of animals specifically designated
for long-term studies) relative to control groups. At 70
days post injury, animals implanted with scaffold-plus-
NSC exhibited coordinated, weight-bearing hindlimb
stepping. Histological and immunocytochemical anal-
ysis suggested that this recovery was not caused by
neural cell replacement; rather, it is attributable pre-
dominantly to a reduction in host tissue loss from
secondary injury processes as well as diminished glial
scarring. This work is the first to demonstrate explic-
itly the chaperone neuroprotective effects of the
NSC. Tract tracing demonstrated spared corticospinal
tract fibers coursing by the injury epicenter and their
caudal ends expressing classic growth cone-type vari-
cosities, a finding not observed in untreated groups.
Together with evidence of enhanced local growth-
associated protein (GAP)-43 expression by axons, a
marker associated with regenerative processes (also
not seen in controls), these findings suggest donor-
derived neuroprotection and promotion of local neural
regenerative and plastic processes that might have
also contributed to functional recovery. These results,
besides suggesting a novel approach to SCI, may more
broadly serve as a prototype for the use of NSC
to anchor multidisciplinary strategies in regenerative
medicine, including gene therapy, material science,
growth factor delivery, anti-inflammation and anti-
scarring strategies, and pharmacological interventions
against secondary injury.
The use of scaffolds and cellular bridges are well-
suited for lesions in which there is large parenchymal
loss (as was created experimentally with the hemisec-
tion model described above) or where a syrinx might
otherwise form because of extensive cell death follow-
ing contusion. For injuries where the amount of tissue
loss is less dramatic, the implantation of cells alone
(without a template) may be useful. Murine embryonic
stem cells (ESC) were observed to survive and promote
recovery in the contused spinal cord (McDonald et al.,
1999). Although this recovery was at first attributed
to the few neurons that appeared to emerge, a more
detailed study showed that functional impact may, in
fact, have been more plausibly due to oligodendrocytes
that myelinated some traumatized host axons (Liu
263
et al., 2000). Indeed, it has been suggested that the
injured cord offers a microenvironment that is not
favorable to the differentiation of multipotent NSC into
neurons (Cao et al., 2002). Rather, it has been proposed
that transplanting neuronal and glial restricted precur-
sors (NRP/GRP) is more tractable, i.e., pre-committing
the cells to a particular lineage ex vivo rather than
letting the in vivo environment direct their differenti-
ation. The transplantation of NRP/GRP into the post-
contusion spinal cord did improve motor and sensory
function. Histological analysis showed that a subset of
the NRP/GRP survived, filled the lesion site, differen-
tiated into neurons and glia, and migrated selectively
(Mitsui et al., 2005; Cao et al., 2005). Interestingly,
the volume of spinal cord spared was increased in
NRP/GRP recipients, suggesting that their action may
nevertheless have been attributable in a large part
to local neuroprotection. The actual role that donor-
derived neurons played in recreating neurocircuitry is
presently not determined.
We are coming to learn that replacing lost neural
tissue with its complex connections is more challeng-
ing than once assumed, even using a cell that can yield
immunocytochemically- and electrophysiologically-
proven neurons in vivo. We do believe that, as we better
understand the molecular milieu of the injured cord,
some degree of reconstruction will be achievable per-
haps not of long projection neurons, but perhaps of
interneurons with shorter axons. However, it is fortu-
itous that the very same exogenous cells that we hope
will participate in circuit reconstitution may also con-
currently be providing trophic and immunoregulatory
factors that can preserve host circuits. Future research
must clearly pay attention not only to how the donor
cells are changed by the host but also how the host
is changed by the donor cells i.e., placing a greater
research emphasis on dynamic donor-host interactions.
Such an understanding may allow this dynamic to be
optimized such that a greater number of endogenous
stem cells may be effectively mobilized (Konya et al.,
2008).
Endogenous NSC have been established to reside
in a few well-characterized secondary germinal zones
of the adult nervous system, most notably the sub-
granular zone (SGZ) of the dentate gyrus of the hip-
pocampus and the subventricular zone (SVZ) along the
anterior part of the lateral ventricle in the forebrain.
Endogenous NSC may also reside in the ependymal
region of the spinal cord. In response to injuries like
264
stroke, adult hippocampal NSC may proliferate and
differentiate into new, functioning neurons (Schaffer
and Gage, 2004). However, NSC in the adult spinal
ependymal region do not seem to differentiate into
neurons when they reside in their normal spinal cord
niche. Nevertheless, when these same NSC are trans-
planted into the SGZ, they do yield neurons (Doetsch
et al., 1999; Johansson et al., 1999; Shihabuddin
et al., 2000). Hence, limitations to neurogenesis must
emanate in large part from the microenvironment of
the adult spinal cord rather than from the cells them-
selves. Therefore, it seems feasible that either altering
the milieu or changing the cells to respond differently
to that milieu may permit these endogenous spinal
NSC to play a more prominent role in neuronal recon-
stitution in the adult cord (Danilov et al., 2006). To
overcome the normal impediments to adult neurogene-
sis will require a better understanding of the biological
roles of spinal NSC, especially their proliferation in
response to injury, inflammation, and neuroactivity
all significantly unexplored (Teng et al., 2006).
16.4.2 Neurodegenerative Diseases
Neurodegenerative diseases are a prominent target for
stem cell therapy in general and NSC in particu-
lar. Although adult-onset Alzheimers disease (AD),
Parkinsons disease (PD), amyotrophic lateral scle-
rosis (ALS), and Huntingtons disease (HD) tend to
receive the most attention, there is a growing recog-
nition that some of the lysosomal storage diseases
(LSD) affecting the nervous system in childhood are
attractive targets because each disease requires the
replacement of a single well-characterized, usually dif-
fusible, enzyme without a significant need for cell
replacement if treated early. In the case of the LSD,
the enzymes to be replaced are typically produced
constitutively by the NSC as part of its normal house-
keeping (e.g., Shihabuddin et al., 2004; Sidman et al.,
2007). In special cases, as when exogenous NSC gain
access to a normal neuronal precursor pool, they may
differentiate abundantly into neurons (Rosario et al.,
1997; Flax et al., 1998).
The notion that using NSC for molecular ther-
apies is likely more tractable even for neurode-
generative diseases that do ostensibly require cell
replacement has begun to spill over into the adult
Y.D. Teng et al.
literature, as well. Although traditionally, for dis-
eases like PD, this has entailed arming cells to pro-
vide substrates for dopamine production, such as
tyrosine hydroxylase (TH) and dopa-decarboxylase
(DDC) (e.g., Kim et al., 2000), we have come to view
molecular therapies more broadly as described
above for SCI, using the NSCs intrinsic propen-
sity to supply homeostasis-promoting, neuroprotec-
tive, and neurotrophic agents that directly benefit
host neural functioning. In preliminary studies, as
proof-of-concept of this notion using human NSC
(hNSC), we have approached a model of PD that
most faithfully mirrors the actual human entity: non-
human primates (African green monkeys, a macaque)
exposed to the complex I mitochondrial toxin 1-
methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP).
hNSC were implanted into the left and right cau-
date nucleus and the right substantia nigra (SN) of
eight MPTP-treated, severely Parkinsonian monkeys.
By 4 months (and clearly by 7 months) post transplan-
tation, the majority of hNSC were found bilaterally
along the nigrostriatal pathway and in the SN. In the
presence of NSC, the number and size of host TH-
and dopamine transporter (DAT)-expressing neurons
in SN, typically poisoned by MPTP, returned to
nearly non-MPTP-exposure control levels. Host TH+
neurons in the caudate, whose size-to-number ratio
had become dysequilibrated, also returned to their nor-
mal ratio, as if forced towards equipoise by stem cell
transplant (Bjugstad et al., 2005). The host-nigral stri-
atal pathway, normally attenuated and dysfunctional
in the MPTP-lesioned monkey (as in PD) was largely
preserved (or restored; Bjugstad et al., 2008). Taken
together, these actions resulted in significant and long-
term diminution in Parkinsonian symptoms. Therefore,
a broader view of the role of stem cells even for
diseases characterized by degeneration of a particu-
lar neuronal subtype, as in PD may make these
diseases a more tractable therapeutic target than we
initially presumed (Redmond et al., 2007). Exogenous
NSC may similarly rescue neurons at genetic risk of
degenaration (Li et al., 2006b)
Diseases characterized by glial dysfunction might
be viewed in a similar manner. Although we
had established long ago that NSC could yield
effectively remyelinating oligodendrocytes through-
out the brains of poorly myelinated mouse mutants
(Yandava et al., 1999), some of the most preva-
lent white matter diseases of adulthood such
16 Functional Multipotency of Neural Stem Cells
as multiple sclerosis (MS) are characterized by
an environment that is likely inhospitable to both
host and donor-derived oligodendrocytes. For exam-
ple in the experimental allergic encephalomyelitis
(EAE) model of MS (characterized by chronic CNS
inflammation, multifocal demyelination, and axonal
loss), syngeneic undifferentiated neural precursor cells
injected intravascularly, transited from the vascular
space into the inflamed intracerebral microenviron-
ment (via constitutively activated integrins and func-
tional chemokine receptors), accumulated and survived
within perivascular areas (in the company of reactive
astrocytes, inflamed endothelial cells and encephalito-
genic T cells that produced neurogenic and gliogenic
regulators) and helped to preserve host oligodendro-
cytes (more so than replacing oligodendrocytes) by
exerting an anti-inflammatory effect (Pluchino et al.,
2003, 2005), thus protecting against chronic neural
tissue loss. We had previously observed this anti-
inflammatory action of NSC in a cerebral-ischemia
model (Park et al., 2002a); now it was being reca-
pitulated in a bona fide inflammatory disease. In the
EAE model, the NSC appeared to counteract the
inflammation by inducing apoptosis of blood-borne
CNS-infiltrating encephalitogenic T cells. In addition,
there was a significant reduction in astrogliosis as we
had noted also in traumatic diseases and described
above (Teng et al., 2002; Park et al., 2002a). Taken
together, these actions resulted in a marked decrease
in the extent of demyelination and axonal loss, and,
in turn, disease-related disability, both clinically and
neurophysiologically (Pluchino et al., 2003).
16.4.3 Stroke
The presumptive goal in using cell-based therapies in
ischemic (stroke) or hypoxic-ischemic injury would
be to replace infarcted CNS tissue in an appropriate
organotypic manner. Given the data presented above,
however, it seems plausible to expect that cell-based
approaches might also provide trophic and neuro-
protective support to tissue at risk in the penumbra
surrounding the infarct, inhibit inflammation and scar-
ring, promote angiogenesis, and help promote the
mobilization, migration, survival, and differentiation
of endogenous precursor cells (Bramlett and Dietrich,
2004; Hass et al., 2005).
265
Stem cell therapy for stroke may be divided into two
approaches: the first focuses on mobilizing endoge-
nous NSC and the second depends on providing exo-
geneous NSC. Obviously, as suggested above, not only
will both approaches likely be required for optimal
restitution of function, but the two strategies likely act
synergistically.
Typically, the strategy for exploiting the popu-
lation of endogenous NSC has been to attempt to
stimulate their proliferation and neuronal differentia-
tion by administering exogenous growth factors and
other pharmacological agents (e.g., bFGF, transform-
ing growth factor (TGF)-, erythropoietin). Although,
some human studies suggest safety and efficacy of this
approach (Ehrenreich et al., 2002), the misadventures
of the neurotrophic factor field in the 1980s and 1990s,
where the unanticipated pleiotrophic actions of sys-
temically administered growth factors produced unto-
ward effects, suggests extreme caution before large
scale clinical application.
With regard to using exogenous NSC for ther-
apy against ischemic injury, a number of interesting
insights have emerged that draw on the growing field
of tissue engineering. It was observed that, in con-
ditions where the likelihood of parenchymal loss is
greatest, use of a biodegradable synthetic scaffold to
support exogenous NSC transiently within the injured
terrain served to prolong the reciprocal interaction
between the donor and host, fix instructive molecules
emanating from both, abet the inhibition of astroglial
and inflammatory host reactions, and provide a tem-
plate for donor-derived and host-fiber regrowth. Using
hypoxic-ischemic injury as a prototype for insults char-
acterized by extensive tissue loss, Park et al. (2002a)
seeded NSC onto a polymer scaffold that was subse-
quently implanted into the infarction cavities. Indeed,
not only did this approach serve a dramatic therapeutic
function, but it allowed the investigators to document
for the first time the multiple reciprocal interactions
that spontaneously ensue between NSC and the exten-
sively damaged brain: parenchymal loss was dramati-
cally reduced, an intricate meshwork of many highly
arborized neurites of both host- and donor-derived
neurons emerged, and some anatomical connections
appeared to be reconstituted. The NSC-scaffold com-
plex altered the trajectory and complexity of host
cortical neurites. Reciprocally, donor-derived neurons
appeared to be capable of directed, target-appropriate
neurite outgrowth. These biobridges appeared to
266
unveil or augment a constitutive reparative response by
facilitating a series of reciprocal interactions between
NSC and host, including promoting neuronal differen-
tiation, enhancing the elaboration of neural processes,
fostering the re-formation of cortical tissue, promoting
connectivity and prompting revascularization of new
parenchyma by the host. Inflammation and scarring
were also reduced.
Another interesting observation is that NSC admin-
istered intravenously in the systemic circulation may
migrate into lesioned brain sites, differentiate into neu-
rons and glia and improve functional deficits in rat
models of focal ischemia or cerebral hemorrhage (Chu
et al., 2003; Jeong et al., 2003; Kim, 2004). This
approach to ischemic injury using an intravascular
route for the delivery of NSC intracranially extends
the observations first made using animal models of
intracranial brain tumors (Aboody et al., 2000) and the
above-described EAE (Pluchino et al., 2003, 2005).
Although results in animal models of stroke
seem promising, challenges remain before attempting
human therapies. For example, obtaining the requisite
number of the proper cells that circumvent immunore-
jection yet interact effectively with host neurocircuitry
and limit their impact and distribution solely to the
CNS are important considerations. Some answers may
be found through a better understanding of the molec-
ular events that underlie each of the key responses of
the injured adult brain to donor NSC and vice versa.
16.5 Conclusion
Even though studies regarding neurogenesis date from
1913, only recently has the cellular and molecular
basis of this inborn plasticity begun to be under-
stood. The stem cell appears to be the repository of
much of this plasticity. The ability to identify, select,
isolate, clone, culture, differentiate, genetically manip-
ulate, and transplant NSC has clearly advanced our
understanding of this biology. Indeed, given that the
NSC is the first stem cell isolated from a solid organ,
insights derived from studying these cells have and
will continue to help advance our understanding of
development and repair of other solid organs.
Although research into the use of NSC, dating back
to the late 1980s, initially focused solely on their
potential for replacing injured or dysfunctional neu-
Y.D. Teng et al.
rons, we have come to recognize that the original view
was overly narrow and simplistic. The inherent biology
of the NSC the richness and complexity of which we
are only now beginning to appreciate offers many
other therapeutic tools, including effecting neuropro-
tection and immunoregulation, as well as direct cell to
cell communication (i.e., via gap junctions), induction
of and inhibition of obstacles to inborn regenerative
processes, axonal guidance, promotion of angiogene-
sis, exertion of homeostatic pressure, and presumably
other actions yet to be unveiled. These multifaceted
actions of NSC make them ideally suited for anchor-
ing the multi-modal interventions that we are coming
to recognize will be needed to restore function in most
neurological disorders. Complex diseases appear to
require complex solutions. NSC, as we have described
in this review, have interfaced and worked syner-
gistically with gene and growth factor therapy, anti-
apoptotic and neuroprotective strategies, stimulation
of neurogenesis, anti-inflammatory and anti-scarring
approaches, material science and tissue engineering.
We, therefore, propose an updated concept of the
NSC. The fields conventional view which has touched
principally on the essential multipotency of lineage
phenotypes (i.e., the ability of NSC to differentiate
into all neural cells), should be broadened to include
the emerging recognition on the biofunctional multi-
potency of the NSC to mediate systemic homeostasis.
Under this new conceptual context, one may begin to
appreciate and seek the logic and teleology behind
the wide range of molecular tactics the NSC appears
to serve at each developmental stage as it integrates
into and prepares, modifies, and guides the surround-
ing CNS micro- and macro-environment towards the
formation and self-maintenance of a physiologically
functioning adult nervous system. We believe that
embracing this view of the NSCs multipotency is
pivotal for correctly, efficiently, and optimally exploit-
ing stem cell biology for therapeutic applications
including reconstituting the dysfunctional CNS.
References
Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W,
Small JE, Herrlinger U, Ourednik V, Black PM, Breakefield
XO, Snyder EY (2000) Neural stem cells display extensive
tropism for pathology in adult brain: evidence from intracra-
nial tumors. Proc Natl Acad Sci USA 97:1284612851.
16 Functional Multipotency of Neural Stem Cells
Altman J (1962a) Autoradiographic study of degenerative and
regenerative proliferation of neuroglia cells with tritiated
thymidine. Exp Neurol 5:302318.
Altman J (1962b) Are neurons formed in the brains of adult
mammals? Science 135:11271128.
Altman J (1966) Autoradiographic and histological studies of
postnatal neurogenesis. II. A longitudinal investigation of the
kinetics, migration and transformation of cells incorporating
tritiated thymidine in infant rats, with special reference to
postnatal neurogenesis in some brain regions. J Comp Neurol
126:431474.
Altman J (1969) Autoradiographic and histological studies of
postnatal neurogenesis. IV. Cell proliferation and migration
in the anterior forebrain, with special reference to persist-
ing neurogenesis in the olfactory bulb. J Comp Neurol 137:
433457.
Altman J, Das GD (1965) Autoradiographic and histological evi-
dence of postnatal hippocampal neurogenesis in rats. J Comp
Neurol 124:319335.
Anchan RM, Reh TA, Angello J, Balliet A, Walker M (1991)
EGF and TGF-alpha stimulate retinal neuroepithelial cell
proliferation in vitro. Neuron 6:923936.
Angevine JB Jr (1965) Time of neuron origin in the hippocampal
region. An autoradiographic study in the mouse. Exp Neurol
Suppl 2:170.
Angevine JB Jr, Sidman RL (1961) Autoradiographic study of
cell migration during histogenesis of cerebral cortex in the
mouse. Nature 192:766768.
Baird A (1994) Fibroblast growth factors: activities and signifi-
cance of non-neurotrophin neurotrophic growth factors. Curr
Opin Neurobiol 4:7886.
Bauer S, Patterson PH (2005) The cell cycle-apoptosis connec-
tion revisited in the adult brain. J Cell Biol 71:641650.
Belluzzi O, Benedusi M, Ackman J, LoTurco JJ (2003)
Electrophysiological differentiation of new neurons in the
olfactory bulb. J Neurosci 23:1041110418.
Bjornson CRR, Rietze RL, Reynolds BA, Magli MC, Vescovi
AL (1999) Turning brain into blood: a hematopoietic fate
adopted by adult neural stem cells in vivo. Science 283:
534537.
Bjugstad KB, Redmond DE Jr, Teng YD, Elsworth JD, Roth RH,
Blanchard BC, Snyder EY, Sladek JR Jr (2005) Neural stem
cells implanted into MPTP-treated monkeys increase the size
of endogenous tyrosine hydroxylase-positive cells found in
the striatum: a return to control measures. Cell Transplant
14:183192.
Bjugstad KB, Teng YD, Redmond DE Jr, Elsworth JD, Roth
RH, Cornelius SK, Snyder EY, Sladek JR Jr (2008) Human
neural stem cells migrate along the nigrostriatal pathway
in a primate model of Parkinsons disease. Exp Neurol
211:362369.
Blesch A, Tuszynski MH (2002) Spontaneous and neurotrophin-
induced axonal plasticity after spinal cord injury. Prog Brain
Res 137:415423.
Bramlett HM, Dietrich WD (2004) Pathophysiology of cere-
bral ischemia and brain trauma: similarities and differences.
J Cereb Blood Flow Metab 24:133150.
Brandt MD, Jessberger S, Steiner B, Kronenberg G, Reuter
K, Bick-Sander A, von der Behrens W, Kempermann
G (2003) Transient calretinin expression defines early
postmitotic step of neuronal differentiation in adult hip-
267
pocampal neurogenesis of mice. Mol Cell Neurosci 24:
603613.
Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A
(2008) Detection of S-phase cell cycle progression using 5-
ethynyl-2-deoxyuridine incorporation with click chemistry,
an alternative to using 5-bromo-2-deoxyuridine antibodies.
Biotechniques 44:927929.
Burns TC, Ortiz-Gonzalez XR, Gutierrez-Perez M, Keene CD,
Sharda R, Demorest ZL, Jiang Y, Nelson-Holte M, Soriano
M, Nakagawa Y, Luquin MR, Garcia-Verdugo JM, Prsper
F, Low WC, Verfaillie CM (2006) Thymidine analogs are
transferred from pre-labeled donor to host cells in the cen-
tral nervous system after transplantation: a word of caution.
Stem Cells 24:11211127.
Cao QL, Howard RM, Dennison JB, Whittemore SR (2002)
Differentiation of engrafted neuronal-restricted precursor
cells is inhibited in the traumatically injured spinal cord. Exp
Neurol 177:349359.
Cao Q, Xu XM, Devries WH, Enzmann GU, Ping P, Tsoulfas P,
Wood PM, Bunge MB, Whittemore SR (2005) Functional
recovery in traumatic spinal cord injury after transplanta-
tion of multineurotrophin-expressing glial-restricted precur-
sor cells. J Neurosci 25:69476957.
Carleton A, Petreanu LT, Lansford R, Alvarez-Buylla A, Lledo
PM (2003) Becoming a new neuron in the adult olfactory
bulb. Nat Neurosci 6:507518.
Carpenter MK, Cui X, Hu ZH, Jackson J, Sherman S, Seiger
A, Wahlberg LA (1999) In vitro expansion of a multipo-
tent population of human neural progenitor cells. Exp Neurol
158:265278.
Carter LM, Starkey ML, Akrimi SF, Davies M, McMahon SB,
Bradbury EJ (2008) The yellow fluorescent protein (YFP-H)
mouse reveals neuroprotection as a novel mechanism under-
lying chondroitinase ABC-mediated repair after spinal cord
injury. J Neurosci 28:1410714120.
Chu K, Kim M, Jeong SW, Kim SU, Yoon BW (2003) Human
neural stem cells can migrate, differentiate, and integrate
after intravenous transplantation in adult rats with transient
forebrain ischemia. Neurosci Lett 343:129133.
Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ,
Wikkelso C, Holts S, van Roon-Mom WM, Bjrk-Eriksson
T, Nordborg C, Frisn J, Dragunow M, Faull RL, Eriksson
PS (2007) Human neuroblasts migrate to the olfactory bulb
via a lateral ventricular extension. Science 315:12431249.
Danilov AI, Covacu R, Moe MC, Langmoen IA, Johansson CB,
Olsson T, Brundin L (2006) Neurogenesis in the adult spinal
cord in an experimental model of multiple sclerosis. Eur J
Neurosci 23:394400.
Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-
Buylla A (1999) Subventricular zone astrocytes are neural
stem cells in the adult mammalian brain. Cell 97:703716.
Ehrenreich H, Hasselblatt M, Dembowski C, Cepek L, Lewczuk
P, Stiefel M, Rustenbeck HH, Breiter N, Jacob S, Knerlich
F, Bohn M, Poser W, Rther E, Kochen M, Gefeller O,
Gleiter C, Wessel TC, De Ryck M, Itri L, Prange H,
Cerami A, Brines M, Sirn AL (2002) Erythropoietin ther-
apy for acute stroke is both safe and beneficial. Mol Med 8:
495505.
Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM,
Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in
the adult human hippocampus. Nat Med 4:13131317.
268
Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst
LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY
(1998) Engraftable human neural stem cells respond to devel-
opmental cues, replace reurons and express foreign genes.
Nat Biotechnol 16:10331039.
Gage FH (2000) Mammalian neural stem cells. Science
287:14331438.
Gage FH, Ray J, Fisher LJ (1995) Isolation, characterization,
and use of stem cells from the CNS. Ann Rev Neurosci 18:
159192.
Gensburger C, Labourdette G, Sensenbrenner M (1987) Brain
basic fibroblast growth factor stimulates the proliferation of
rat neuronal precursor cells in vitro. FEBS Lett 217:15.
Goldman SA, Nottebohm F (1983) Neuronal production, migra-
tion, and differentiation in a vocal control nucleus of the adult
female canary brain. Proc Natl Acad Sci USA 80:23902394.
Gratzner HG (1982) Monoclonal antibody to 5-bromo- and
5-iododeoxyuridine: a new reagent for detection of DNA
replication. Science 218:474475.
Graziadei GA, Graziadei PP (1979) Neurogenesis and neu-
ron regeneration in the olfactory system of mammals. II.
Degeneration and reconstitution of the olfactory sensory
neurons after axotomy. J Neurocytol 8:197213.
Gritti A, Parati EA, Cova L, Frolichsthal P, Galli R, Wanke E,
Faravelli L, Morassutti DJ, Roisen F, Nickel DD, Vescovi AL
(1996) Multipotential stem cells from the adult mouse brain
proliferate and self-renew in response to basic fibroblast
growth factor. J Neurosci 16:10911100.
Hall ED, Springer JE (2004) Neuroprotection and acute spinal
cord injury: a reappraisal. NeuroRx 1:80100.
Hass S, Weidner N, Winkler J (2005) Adult stem cell therapy in
stroke. Curr Opin Neurol 18:5964.
Herrup K, Neve R, Ackerman SL, Copani A (2004) Divide
and die: cell cycle events as triggers of nerve cell death. J
Neurosci 24:92329239.
Howard A, Pelc SR (1953) Synthesis of desoxyribonucleic acid
in normal and irradiated cells and its relation to chromosome
breakage. Heredity 6:261273.
Hughes WL, Bond VP, Brecher G, Cronkite ER, Painter RB,
Quastler H, Sherman FG (1958) Cellular proliferation in
the mouse as revealed by autoradiography with tritiated
thymidine. Proc Natl Acad Sci USA 44:476483.
Jderstad J, Jderstad LM, Li J, Chintawar S, Salto C, Pandolfo
M, Ourednik V, Teng YD, Sidman RL, Arenas E, Snyder
EY, Herlenius E (2010) Communication via gap junctions
underlies early functional & beneficial interactions between
grafted neural stem cells & the host. Proc Natl Acad Sci USA
107 (in press).
Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK (2003)
Human neural stem cell transplantation promotes functional
recovery in rats with experimental intracerebral hemorrhage.
Stroke 34:22582263.
Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U,
Frisen J (1999) Identification of a neural stem cell in the adult
mammalian central nervous system. Cell 96:2534.
Kaplan MS, Bell DH (1983) Neuronal proliferation in the 9-
month-old rodent-radioautographic study of granule cells in
the hippocampus. Exp Brain Res 52:15.
Kempermann G, Jessberger S, Steiner B, Kronenberg G (2004)
Milestones of neuronal development in the adult hippocam-
pus. Trends Neurosci 27:447452.
Y.D. Teng et al.
Kempermann G, Kuhn HG, Gage FH (1997) More hippocam-
pal neurons in adult mice living in an enriched environment.
Nature 386:493495.
Kilpatrick TJ, Richards LJ, Bartlett PF (1995) The regulation of
neural precursor cells within the mammalian brain. Mol Cell
Neurosci 6:215.
Kim SU (2004) Human neural stem cells genetically modified
for brain repair in neurological disorders. Neuropathology
24:159171.
Kim SU, Nakagawa E, Nagai A, Snyder EY, Ryu JK, Kim J, Lee
MA, Mook IH, Jin BK (2000) Generation of human neural
stem cell lines and brain transplantation in animal models
of Parkinson disease and multiple sclerosis. Arch Neurol
57:1238.
Konya D, Liao WL, Choi H, Yu D, Woodard MC, Newton KM,
King AM, Pamir NM, Black PM, Frontera WR, Sabharwal S,
Teng YD (2008) Functional recovery in T13-L1 hemisected
rats resulting from peripheral nerve rerouting: role of central
neuroplasticity. Regen Med 3:309327.
Kukekov VG, Laywell ED, Suslov O, Davies K, Scheffler B,
Thomas LB, OBrien TF, Kusakabe M, Steindler DA (1999)
Multipotent stem/progenitor cells with similar properties
arise from two neurogenic regions of adult human brain. Exp
Neurol 156:333344.
Lakshmipathy U, Verfaillie C (2005) Stem cell plasticity. Blood
Rev 19:2938.
Lewis PF, Emerman M (1994) Passage through mitosis is
required for oncoretroviruses but not for the human immun-
odeficiency virus. J Virol 68:510516.
Li J, Imitola J, Snyder EY, Sidman RL (2006b) Neural stem
cells rescue nervous Purkinje neurons by restoring molecular
homeostasis of tissue plasminogen activator and downstream
targets. J Neurosci 26:78397848.
Li J, Ma Y, Teng YD, Zheng K, Vartanian TK, Snyder EY,
Sidman RL (2006a) Purkinje neuron degeneration in nervous
(nr) mutant mice is mediated by a metabolic pathway involv-
ing excess tissue plasminogen activator. Proc Natl Acad Sci
USA 103:78477852.
Liu S, Qu Y, Stewart TJ, Howard MJ, Chakrabortty S, Holekamp
TF, McDonald JW (2000) Embryonic stem cells differen-
tiate into oligodendrocytes and myelinate in culture and
after spinal cord transplantation. Proc Natl Acad Sci USA
97:61266131.
Llado J, Haenggeli C, Maragakis NJ, Snyder EY, Rothstein JD
(2004) Neural stem cells protect against glutamate-induced
excitotoxicity and promote survival of injured motor neurons
through secretion of neurotrophic factors. Mol Cell Neurosci
27:322331.
Lu P, Jones LL, Snyder EY, Tuszynski MH (2003) Neural stem
cells constitutively secrete neurotrohic factors and promote
extensive host axonal growth after spinal cord injury. Exp
Neurol 181:115129.
Martinez-Serrano A, Rubio FJ, Navarro B, Bueno C, Villa
A (2001) Human neural stem and progenitor cells: in
vitro and in vivo properties, and potential gene therapy
and cell replacement in the CNS. Curr Gene Ther 1:
279299.
McDonald JW, Liu XZ, Qu Y, Liu S, Mickey SK, Turetsky D,
Gottlieb DI, Choi DW (1999) Transplanted embryonic stem
cells survive, differentiate and promote recovery in injured
rat spinal cord. Nat Med 5:1410.
16 Functional Multipotency of Neural Stem Cells
Meng F, Zolova O, Kokorina NA, Dobretsova A, Wight PA
(2005) Characterization of an intronic enhancer that reg-
ulates myelin proteolipid protein (Plp) gene expression in
oligodendrocytes. J Neurosci Res 82:346356.
Miale IL, Sidman RL (1961) An autoradiographic analy-
sis of histogenesis in the mouse cerebellum. Exp Neurol
4:277296.
Miller RH (2002) Regulation of oligodendrocyte development in
the vertebrate CNS. Progress Neurobiol 67:451467.
Miller RH (2005) Dorsally derived oligodendrocytes come of
age. Neuron 45:13.
Miller MW, Nowakowski RS (1988) Use of bromodeoxyuridine-
immunohistochemistry to examine the proliferation, migra-
tion and time of origin of cells in the central nervous system.
Brain Res 457:4452.
Ming GL, Song H (2005) Adult neurogenesis in the mammalian
central nervous system. Annu Rev Neurosci 28:223250.
Mitsui T, Shumsky JS, Lepore AC, Murray M, Fischer I (2005)
Transplantation of neuronal and glial restricted precursors
into contused spinal cord improves bladder and motor
functions, decreases thermal hypersensitivity, and modifies
intraspinal circuitry. J Neurosci 25:96249636.
Muotri AR, Gage FH (2006) Generation of neuronal variability
and complexity. Nature 441:10871093.
Nottebohm F (2004) The road we travelled: discovery, choreog-
raphy, and significance of brain replaceable neurons. Ann N
Y Acad Sci 1016:628658.
Ogle BM, Cascalho M, Platt JL (2005) Biological implications
of cell fusion. Nat Rev Mol Cell Biol 6:567575.
Ortiz-Gonzalez XR, Keene CD, Verfaillie CM, Low WC (2004)
Neural induction of adult bone marrow and umbilical cord
stem cells. Curr Neurovasc Res 3:207213.
Ourednik J, Ourednik V, Lynch WP, Schachner M, Snyder
EY (2002) Neural stem cells display an inherent mecha-
nism for rescuing dysfunctional neurons. Nat Biotechnol
20:11031110.
Park KI, Liu S, Flax JD, Nissim S, Stieg PE, Snyder EY (1999)
Transplantation of neural progenitor and stem cells: devel-
opmental insights may suggest new therapies for spinal cord
and other CNS dysfunction. J Neurotrauma 16: 675687.
Park KI, Ourednik J, Ourednik V, Taylor RM, Aboody KA,
Auguste KI, Lachyankar M, Redmond DE, Snyder EY
(2002b) Global gene and cell replacement strategies via stem
cells. Gene Ther 9:613624.
Park KI, Teng YD, Snyder EY (2002a) The injured brain
interacts reciprocally with neural stem cells supported by
scaffolds to reconstitute lost tissue. Nat Biotechnol 20:
11111117.
Parker MA, Anderson JK, Corliss DA, Abraria VE, Sidman
RL, Park KI, Teng YD, Cotanche DA, Snyder EY (2005)
Expression profile of an operationally-defined neural stem
cell clone. Exp Neurol 194:320332.
Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK,
Murase N, Boggs SS, Greenberger JS, Goff JP (1999) Bone
marrow as a potential source of hepatic oval cells. Science
284:11681170.
Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G,
Galli R, Del Carro U, Amadio S, Bergami A, Furlan R, Comi
G, Vescovi AL, Martino G (2003) Injection of adult neu-
rospheres induces recovery in a chronic model of multiple
sclerosis. Nature 422:688694.
269
Pluchino S, Zanotti L, Rossi B, Brambilla E, Ottoboni L,
Salani G, Martinello M, Cattalini A, Bergami A, Furlan
R, Comi G, Constantin G, Martino G (2005) Neurosphere-
derived multipotent precursors promote neuroprotection by
an immunomodulatory mechanism. Nature 436: 266271.
Price J, Turner D, Cepko C (1987) Lineage analysis in the ver-
tebrate nervous system by retrovirus-mediated gene transfer.
Proc Natl Acad Sci USA 84:156160.
Ravin R, Hoeppner DJ, Munno DM, Carmel L, Sullivan J,
Levitt DL, Miller JL, Athaide C, Panchision DM, McKay
RD (2008) Potency and fate specification in CNS stem cell
populations in vitro. Cell Stem Cell 3:670680.
Redmond DE Jr, Bjugstad KB, Teng YD, Ourednik V, Ourednik
J, Wakeman DR, Parsons XH, Gonzalez R, Blanchard BC,
Kim SU, Gu Z, Lipton SA, Markakis EA, Roth RH, Elsworth
JD, Sladek JR Jr, Sidman RL, Snyder EY (2007) Behavioral
improvement in a primate Parkinsons model is associated
with multiple homeostatic effects of human neural stem cells.
Proc Natl Acad Sci USA 104:1217512180.
Reier PJ (2004) Cellular transplantation strategies for spinal cord
injury and translational neurobiology. NeuroRx 1:424451.
Renfranz PJ, Cunningham MG, McKay RD (1991) Region-
specific differentiation of the hippocampal stem cell line
HiB5 upon implantation into the developing mammalian
brain. Cell 66:713729.
Reynolds BA, Tetzlaff W, Weiss S (1992) A multipotent EGF-
responsive striatal embryonic progenitor cell produces neu-
rons and astrocytes. J Neurosci 12:45654574.
Reynolds BA, Weiss S (1992) Generation of neurons and astro-
cytes from isolated cells of the adult mammalian central
nervous system. Science 255:17071710.
Richards LJ, Kilpatrick TJ, Bartlett PF (1992) De novo genera-
tion of neuronal cells from the adult mouse brain. Proc Natl
Acad Sci USA 89:85918595.
Rosario CM, Yandava BD, Kosaras B, Zurakowski D, Sidman
RL, Snyder EY (1997) Differentiation of engrafted mul-
tipotent neural progenitors towards replacement of miss-
ing granule neurons in meander tail cerebellum may help
determine the locus of mutant gene action. Development
124:42134224.
Roy NS, Nakano T, Keyoung HM, Windrem M, Rashbaum
WK, Alonso ML, Kang J, Peng W, Carpenter MK, Lin J,
Nedergaard M, Goldman SA (2004) Telomerase immortal-
ization of neuronally restricted progenitor cells derived from
the human fetal spinal cord. Nat Biotechnol 22: 297305.
Ryder EF, Snyder EY, Cepko CL (1990) Establishment and char-
acterization of multipotent neural cell lines using retrovirus
vector-mediated oncogene transfer. J Neurobiol 21: 356375.
Salic A, Mitchison TJ (2008) A chemical method for fast and
sensitive detection of DNA synthesis in vivo. Proc Natl Acad
Sci USA 105:24152420.
Sanes JR, Rubenstein JL, Nicolas JF (1986) Use of a recom-
binant retrovirus to study postimplantation cell lineage in
mouse embryos. EMBO J 5:31333142.
Schaffer DV, Gage FH (2004) Neurogenesis and neuroadapta-
tion. Neuromolecular Med 5:19.
Schwab ME (2004) Nogo and axon regeneration. Curr Opin
Neurobiol 14:118124.
Shihabuddin LS, Horner PJ, Ray J, Gage FH (2000) Adult spinal
cord stem cells generate neurons after transplantation in the
adult dentate gyrus. J Neurosci 20:87278735.
270
Shihabuddin LS, Numan S, Huff MR, Dodge JC, Clarke J,
Macauley SL, Yang W, Taksir TV, Parsons G, Passini MA,
Gage FH, Stewart GR (2004) Intracerebral transplantation of
adult mouse neural progenitor cells into the Niemann-Pick-
A mouse leads to a marked decrease in lysosomal storage
pathology. J Neurosci 24:1064210651.
Sidman RL (1961) Histogenesis of mouse retina studied with
thymidine-H3 . In: The Structure of the Eye (Smelser GK,
ed.), pp. 487506. Academic Press, New York.
Sidman RL (1970) Autoradiographic methods and principles
for study of the nervous system with thymidine-H3 . In:
Contemporary Research Methods in Neuroanatomy (Nauta
WJ, Ebbesson SOE, eds.), pp. 252274. Springer-Verlag,
New York.
Sidman RL, Li J, Stewart GR, Clarke J, Yang W, Snyder EY,
Shihabuddin LS (2007) Injection of mouse and human neural
stem cells into neonatal Niemann-Pick A model mice. Brain
Res 1140:195204.
Sidman RL, Miale IL, Feder N (1959) Cell proliferation in
the primitive ependymal zone; an autoradiographic study
of histogenesis in the nervous system. Exp Neurol 1:
322333.
Silver J, Miller JH (2004) Regeneration beyond the glial scar.
Nat Rev Neurosci 5:146156.
Simpson DL, Morrison R, de Vellis J, Herschman HR (1982)
Epidermal growth factor binding and mitogenic activity on
purified populations of cells from the central nervous system.
J Neurosci Res 8:453462.
Smart I (1961) The subependymal layer of the mouse brain
and its cell production as shown by autography after [H3]-
thymidine injection. J Comp Neurol 116:325327.
Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl
M, Rogers D (1988) Inhibition of pluripotential embryonic
stem cell differentiation by purified polypeptides. Nature
336:688690.
Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartweig
EA, Cepko CL (1992) Multipotent neural cell lines can
engraft and participate in development of mouse cerebellum.
Cell 68:3351.
Snyder EY, Yoon CH, Flax JD, Macklis JD (1997) Multipotent
neural progenitors can differentiate toward replacement
of neurons undergoing targeted apoptotic degeneration in
adult mouse neocortex. Proc Natl Acad Sci USA 94:
1166311668.
Stanfield BB, Trice JE (1988) Evidence that granule cells gen-
erated in the dentate gyrus of adult rats extend axonal
projections. Exp Brain Res 72:399406.
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by
defined factors. Cell 126:663676.
Tator CH, Fehlings MG (1991) Review of the secondary injury
theory of acute spinal cord trauma with emphasis on vascular
mechanisms. J Neurosurg 75:1526.
Temple S (1989) Division and differentiation of isolated CNS
blast cells in microculture. Nature 340:471473.
Temple S, Qian X (1995) bFGF, neurotrophins, and the control
of cortical neurogenesis. Neuron 15:249252.
Teng YD, Choi H, Onario RC, Zhu S, Desilets FC, Lan
S, Woodard EJ, Snyder EY, Eichler ME, Friedlander RM
(2004) Minocycline inhibits contusion-triggered mitochon-
drial cytochrome c release and mitigates functional deficits
Y.D. Teng et al.
after spinal cord injury. Proc Natl Acad Sci USA 101:
30713076.
Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D,
Langer R, Snyder EY (2002) Functional recovery following
traumatic spinal cord injury mediated by a unique polymer
scaffold seeded with neural stem cells. Proc Natl Acad Sci
USA 99:30243029.
Teng YD, Liao W-L, Choi C, Konya D, Sabharwal D,
Langer L, Sidman RL, Snyder EY, Frontera WR (2006)
Physical activity-mediated functional recovery after spinal
cord injury: potential roles of neural stem cells. Reg Med
1:763776.
Teng YD, Yu D, Song G, Poon C-S (2006) NSC-mediated
multimechanistic repair of respiratory function following
spinal cord injury: neurotrophic factors and gap junction.
Symposium on hot topics in stem cell research. The 36th
annual meeting of the Society for Neuroscience.
Timsit SG, Martinez S, Alliquant B, Peyron F, Puelles L, Zalc
B (1995) Oligodendrocytes originate in a restricted zone of
the embryonic ventral neural tube defined by DM-20 mRNA
expression. J Neurosci 15:10121024.
van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD,
Gage FH (2002) Functional neurogenesis in the adult hip-
pocampus. Nature 415:10301034.
Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke
E, Frlichsthal-Schoeller P, Cova L, Arcellana-Panlilio M,
Colombo A, Galli R (1999) Isolation and cloning of mul-
tipotential stem cells from the embryonic human CNS and
establishment of transplantable human neural stem cell lines
by epigenetic stimulation. Exp Neurol 156:7183.
Vescovi AL, Reynolds BA, Fraser DD, Weiss S (1993) bFGF
regulates the proliferative fate of unipotent (neuronal) and
bipotent (neuronal/astroglial) EGF-generated CNS progeni-
tor cells. Neuron 11:951966.
Vescovi AL, Snyder EY (1999) Establishment and properties of
neural stem cell clones: plasticity in vitro and in vivo. Brain
Pathol 9:569598.
Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner
F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch
R (2008) Neurons derived from reprogrammed fibroblasts
functionally integrate into the fetal brain and improve symp-
toms of rats with Parkinsons disease. Proc Natl Acad Sci
USA 105:58565861.
Williams BP, Read J, Price J (1991) The generation of neurons
and oligodendrocytes from a common precursor cell. Neuron
7:685693.
Yan J, Welsh AM, Bora SH, Snyder EY, Koliatsos VE (2004)
Differentiation and tropic/trophic effects of exogenous neural
precursors in the adult spinal cord. J Comp Neurol 480:101
114.
Yandava BD, Billinghurst L, Snyder EY (1999) Global cell
replacement is feasible via neural stem cell transplantation:
evidence from the dysmyelinated shiverer mouse brain. Proc
Natl Acad Sci USA 96:70297034.
Yu W-P, Collarini EJ, Pringle NP, Richardson WD (1994)
Embryonic expression of myelin genes: evidence for a focal
source of oligodendrocyte precursors in the ventricular zone
of the neural tube. Neuron 23:13531362.
Zhao C, Deng W, Gage FH (2008) Mechanisms and
functional implications of adult neurogenesis. Cell 132:
645660.
Chapter 17
Dual Roles of Mesenchymal Stem Cells in Spinal Cord Injury:
Cell Replacement Therapy and as a Model System
to Understand Axonal Repair
Cecile King, Shyam Patel, Treena Livingston Arinzeh, and Pranela Rameshwar
Contents
17.1 Mesenchymal Stem Cells (MSC) . . . . . . .
17.2 Biology of Spinal Cord Injury . . . . . . . .
17.3 Current Interventions for Spinal Cord Injury
17.4 Cytokines and Soluble Factors . . . . . . .
17.4.1 Tumor Necrosis Factor Alpha (TNF-) .
17.4.2 Leukemia Inhibitory Factor (LIF) . . .
17.4.3 Interlekin-6 (IL-6) . . . . . . . . . .
17.4.4 Interleukin-1 (IL-1 ) . . . . . . . .
17.4.5 Transforming Growth Factor 1 (TGF-1)
17.5 Prospects for Axonal Regeneration in the CNS
17.6 Stem Cell Therapy for Spinal Cord Injury . .
17.7 Transdifferentiation of Mesenchymal Stem Cells
to Neurons . . . . . . . . . . . . . . . . .
17.8 Other Neurodegenerative Disorders . . . . .
17.9 Limitations to Stem Cell Therapeutics . . . .
17.10 An Interdisciplinary Approach . . . . . . .
17.11 Experimental Models for SCI . . . . . . . .
17.12 On the Frontier of Stem Cell Therapy for
Neural Dysfunction . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .
Abstract Mesenchymal stem cells (MSC) are
pluripotent cells that differentiate into cells of meso-
dermal origin and transdifferentiate into ectodermal
and endodermal cell types. MSC can transdifferentiate
with high efficiency to functional neurons, microglia
and oligodendrocytes. MSC and neurons can respond
to environmental cues such as cytokines, which
also affect the development to neural cells. Since
cytokines and other inflammatory mediators are also
P. Rameshwar ()
UMDNJ-New Jersey Medical School, Department of Medicine,
Newark, NJ, USA
e-mail: rameshwa@umdnj.edu
H. Ulrich (ed.), Perspectives of Stem Cells,
DOI 10.1007/978-90-481-3375-8_17, Springer Science+Business Media B.V. 2010
expected at sites of spinal cord injury (SCI), simple
implantation of MSC and/or their differentiated cells
272 might be premature unless the basic science precedes
273 translational science. This is particularly true since
273 an individuals immune response might be unique.
274
Similar argument could be made for embryonic stem
274
275 cells (ESC). The unstable behavior of ESC to form
275 tumors, as well as rapid generation of mixed cell types
275 makes MSC desirable stem cells for translational
275
science. This review discusses varied interdisciplinary
275
276 approaches by which MSC can be applied to SCI
repair, including bioengineering approach. The mech-
277 anism by which microenvironmental factors such as
277
inflammatory mediators can affect stem cell therapies
278
279 is also discussed. In addition to the potential of direct
280 application of stem cells or their differentiated cells,
MSC can be used as models to understand axonal
281
repair, which could lead to the development of new
281
drugs for SCI. The model could test novel factors
to repair neurons. Any identified factor could be
delivered directly or by gene therapy. The co-culture
method is significant to long-term investigation for
rapid screening of compounds, with simultaneous
understanding of repair mechanisms of axotomized
neurons. The model could be translated in parallel
with other stem cell therapies for SCI repair. We also
discuss the potential of stem cell therapies for other
neurodegenerative diseases.
Keywords Cytokines Mesenchymal stem cells
Neurons Spinal cord injury Tissue repair
Abbreviations
BDNF brain-derived neurotrophic factor
ESC embryonic stem cells
271
272 C. King et al.

HPC hematopoietic progenitor cell amounts of bone marrow aspirates. More specifi-
IFN- interferon- cally, the total numbers cells that can be attained
IL-6 interlekin-6 and expanded from one donor are immense. Another
IL-1 interleukin-1 attractive property of MSC is the versatility in their
LIF leukemia inhibitory factor immune properties. MSC are both immune suppressive
MEM microelectricomechanical as well as immune-enhancing (Castillo et al., 2007;
systems Chan et al., 2006; Potian et al., 2003; Stagg et al.,
MSC mesenchymal stem cells 2006; Ong et al., 2006). This factor is important as the
NEM nanoelectromechanical microenvironment of sites of tissue injury could affect
systems the immune response of MSC through the complex
NGF nerve growth factor
array of factors present. Thus, implantation of MSC,
NSC neural stem cell(s)
either as undifferentiated or differentiated cells might
NT-3 neurotrophin-3
be premature unless basic science research precedes
PNS peripheral nervous system
the translational research. In this chapter, the potential
PEG poly-ethylene glycol
PGA polyglycolic acid use of MSC in spinal cord injury (SCI) cases will be
PLA polylactic acid discussed.
SCI spinal cord injury In general MSC represent a promising source for
STAT3 signal transducer and activator of tran- various CNS impairments. However, the potential use
scription of MSC for SCI is especially exciting due to the mostly
TGF-1 transforming growth factor 1 irreversible and severe effects of such injuries (Hardy
TNF- tumor necrosis factor- et al., 2008). Previous studies have shown, MSC medi-
ated functional recovery in animal models of SCI
however the specific mechanism of improvement is
not understood (Hardy et al., 2008). Most scientists
believe that release of various cytokines and soluble
17.1 Mesenchymal Stem Cells (MSC) factors from MSC catalyze and support the repair of
the areas of injury (Hardy et al., 2008). Another less
MSC are pluripotent cells that can differentiate into supported hypothesis implies the transdifferentiation
various cell types including adipocytes, chondrocytes of MSC into neurons in addition to cell fusion events
and stroma (Fig. 17.1) (Castillo et al., 2007; Bianco may be the cause of the functional recovery (Hardy
et al., 2001). MSC also demonstrate the ability to trans- et al., 2008).
differentiate into cells of endodermal and ectodermal The microenvironment of any damaged tissue gen-
lineages such as hepatocytes and neurons (Ong et al., erates a complex array of factors. Thus, implantation of
2006; Greco and Rameshwar, 2007). MSC are found MSC, either as undifferentiated or differentiated cells
in adult and fetal tissues. In adults, the bone marrow could begin a crosstalk with endogenous cells. In this
is the major location of MSC (Lee et al., 2004) how- chapter, various representative factors and their effects
ever, MSC are also found in umbilical cord blood, on endogenous cells will be discussed. A proposed
although at lower frequencies (Bieback et al., 2004). interdisciplinary approach will bring an understand-
Morphologically, MSC are symmetrical cells display- ing on the interactions between tissue microenviron-
ing a fibroblastoid appearance (Blanc and Pittenger, ment and MSC. Studies using such an approach will
2005). Phenotypically, MSC express CD44, CD29, lead to safe and effective delivery of MSC to SCI
CD105, CD73, and CD166 and lack markers that are patients and will determine whether MSC should be
consistent with hematopoietic cells, in particular CD45 transplanted as undifferentiated or transdifferentiated
(Pittenger et al., 1999; Martinez et al., 2007). MSC also (partly vs. fully), and allow for personalized treat-
express neural-associated markers, such as neural gan- ment by incorporating mathematics and engineering.
glioside, GD2 (Deng et al., 2006; Padovan et al., 2003; Future delivery of MSC or other non-embryonic stem
Caplan, 1994). cell will also require the aid of biomaterials that
The advantage in pursuing research on MSC is could assists in dictating the behavior of stem cells
due to their ease in expansion from relatively small in vivo.
17 Dual Roles of MSC in Spinal Cord Injury 273

Fig. 17.1 Human

mesenchymal stem cells
(hMSC) are pluripotent and
can differentiate into cells of
mesodermal origin and
transdifferentiate into cells
of ectodermal and
enodermal lineages. MSC
show high efficiency in the
generation of functional
neurons, microglia and
oligodendrocytes. These
characteristics make MSC a
promising therapy for spinal
cord injury and other central
nervous system disorders

17.2 Biology of Spinal Cord Injury
Spinal Cord Injury (SCI) is characterized by com-
pression or tension applied to the spinal cord which
results in damage to the nerves, vessels or spinal fluid
(Fig. 17.2) (Divani et al., 2007). The applied force
causes a loss of function or mobility whether it is tran-
sient or permanent. Minor spinal cord injuries result in
compression of the spinal cord and give rise to mostly
temporary neurological deficits (Kwon et al., 2004). In
contrast, severe trauma directed to the spinal cord has
a resultant damage or axotomy of neurons and often,
if not always leads to permanent paralysis (Divani
et al., 2007). Cases of severe SCI injury are character-
ized by two stages a primary stage and a secondary
stage (Divani et al., 2007). The primary phase of
injury is denoted by the initial infarct, which cause a
series of events resulting ultimately in the initiation of
the secondary phase (Kwon et al., 2004; Sekhon and
Fehlings, 2001). Secondary stage injuries include: vas-
cular irregularities, abnormal ion fluctuations, edema
formation, glutamate release and accumulation, free
radical formation, necrosis and apoptosis of cells
and inflammation (Kwon et al., 2004; Sekhon and
Fehlings, 2001). The prevention or treatment of sec-
ondary complications is essential if irreversible paral-
ysis is to be avoided.
17.3 Current Interventions for Spinal
Cord Injury
For the past three decades, physicians have employed
the use of corticosteroids, specifically intravenous
methylprednisolone, to reduce the inflammation asso-
ciated with SCIs (Ducker and Hamit, 1969; Young,
2000; Ducker and Zeidman, 1994). The justification
is that the anti-inflammatory properties of such agents
would lead not only to the decreased inflammation and
edema, but also to a reduction in neurotoxic agents,
modulation of immune cells, enhanced vascular per-
fusion and inhibition of calcium influx (Divani et al.,
274 C. King et al.

Fig. 17.2 Spinal cord

injury occurs when
significant amounts of force
are applied to the spinal
cord. This force can lead to
two initially indistinguishable
outcomes: axotomy or
compression of neurons or
spinal fluid. Severe SCI injury
is characterized by two
phases the primary injury
phase and the secondary
injury phase

2007; Chi et al., 2008). In addition to corticosteroid
administration, decompressive surgery may be per-
formed to reduce edema and ultimately decrease the
risk of further compression of the spinal cord. Other
than these two interventional procedures, physicians
may perform anatomic realignments to reduce dislo-
cation, suggest bed rest to decrease opportunities for
further damage and ultimately suggest physical ther-
apy to assist patients with coping with the disability. In
general, none of these practices restore full mechanis-
tic functionalities to SCI patients but only act to reduce
the advancement of secondary injuries.
17.4 Cytokines and Soluble Factors
Cytokines, soluble factors and other inflammatory
mediators are expected at sites of spinal cord injury.
A representative list of some of these factors with
corresponding descriptions is presented in the follow-
ing section.
17.4.1 Tumor Necrosis Factor Alpha
(TNF-)
TNF- is an inflammatory cytokine has been reported
to play a dual role in SCI. It is produced by various
endogenous cells in the site of injury including neu-
rons, glial and vascular endothelial cells (Chi et al.,
2008). During the secondary injury phase of SCI TNF-
acts by directing apoptosis events (Chi et al., 2008).
More specifically, studies utilizing neutralizing anti-
bodies to TNF- caused significant reduction in apop-
totic cells (Yune et al., 2003). Another role of TNF- in
secondary stage SCI is the induction of the expression
of endothelial leukocyte adhesion molecules. These
molecules release inflammatory mediators such as
reactive oxygen species (ROS) and neutrophil elas-
tase which activate neutrophils and ultimately lead to
increased edema (Carlos and Harlan, 1994; Hernandez
et al., 1987). A recent article detailed the dual role of
TNF- in SCI injury. These researchers characterize
the cytokine as having an initial deleterious role in the
17 Dual Roles of MSC in Spinal Cord Injury
acute phase and a beneficial role in the chronic phase.
Early inhibition of this cytokine decreases apoptotic
events while later secretion of the cytokine is linked
to neuroprotective effects (Chi et al., 2008).
17.4.2 Leukemia Inhibitory Factor (LIF)
LIF is a cytokine originally discovered to play a role
in the inhibition of myeloid leukemic cells (Gearing
et al., 1987; Pan et al., 2006). Knockout mice stud-
ies show that LIF is imperative to the development
and survival of motor neurons (Li et al., 1995; Pan
et al., 2006). More recently it has been proven by var-
ious studies that this cytokine plays an important role
in SCI. Studies have also shown that after one day of
SCI, both transcription of LIF and number of binding
sites for exogenous LIF are increased (Kurek et al.,
1998a; Pan et al., 2006). During SCI, LIF is secreted
by endogenous cells, specifically fibroblasts, leading
to a marked increase in axonal sprouting and release of
neurotrophin-3 (Blesch et al., 1999; Pan et al., 2006).
Also, oliodendrocyte survival is enhanced by exoge-
nous LIF and has been linked to aiding in functional
recovery (Butzkueven et al., 2002; Kurek et al., 1998b;
Pan et al., 2006).
17.4.3 Interlekin-6 (IL-6)
IL-6 belongs to the family of interleukin cytokines and
it has been implicated in spontaneous recovery after
SCI (Kang and Kang, 2008; Zai et al., 2005). After
spinal cord injury IL-6 levels rapidly increase (Kang
and Kang, 2008; Hayashi et al., 2000) however the spe-
cific function of IL-6 is not fully understood. Studies
have shown that the cytokine is involved both in neu-
ronal cell death and differentiation (Bonni et al., 1997;
Gadient and Otten, 1997) and also in induction of the
functional recovery of neurons (Yamauchi et al., 2006).
17.4.4 Interleukin-1 (IL-1 )
IL-1 belongs to the family of IL-1 cytokines (Allan
et al., 2005). The IL-1 family of cytokines is central
to inflammation and host defense. IL-1 and IL-1
appear to exhibit similar effects. Each binds to the
type I IL-1 receptor. The type II receptor subtype lacks
275
the intracellular signaling domain and therefore cannot
initiate intracellular signaling. The IL-1 cytokines are
induced by other inflammatory mediators and by tis-
sue insults such as infectious agents, hypoxia, and at
sites of tissue injuries (Allan et al., 2005). The effects
of IL-1 could be direct or indirectly mediated by other
cytokines (Moore, 2002; Laver and Moore, 1989).
Neurons can be affected directly by IL-1 through the
type I receptor (Allan et al., 2005). In addition, IL-1
could cause indirect effects on neurons by inducing
other cytokines in the surrounding tissues, which in
turn can bind to neurons (Dinarello, 2005; Laver and
Moore, 1989; Dinarello, 1996; Bagby, 1989).
17.4.5 Transforming Growth Factor 1
(TGF-1)
TGF-1 belongs to a superfamily of proteins including
the activins, inhibins and bone morphogenic proteins
(Roberts and Sporn, 1993). TGF- receptors are ubiq-
uitously expressed on healthy and malignant cells
(Massague et al., 1992; Massague and Weis-Garcia,
1996). TGF-1 interacts with two receptor subtypes:
I and II (Shi and Massague, 2003). Type I signals
through intracellular pathways involving four mem-
bers of the Smad transcription factors (Massague and
Wotton, 2000; Sato et al., 2003; Mehra and Wrana,
2002). The only reported function for type II recep-
tor is their ability to activate the type I TGF- receptor
(Wrana, 2000). The family of TGF- proteins has been
linked to developmental processes such as embryoge-
nesis and development of neural cells (Roberts and
Sporn, 1993; Golestaneh and Mishra, 2005). TGF-
1 functions as immune modulators, inhibitors of cell
proliferation, differentiation and apoptosis (Sokol and
Schiemann, 2004; Downing, 2004). TGF-1 has been
determined to exhibit both tumor suppressor and onco-
genic properties and can therefore inhibit cell prolif-
eration and promotes malignancy (Kim and Letterio,
2003).
17.5 Prospects for Axonal Regeneration
in the CNS
The process of axotomy has been shown to alter
gene expression in neurons (Tonge et al., 2008).
276
Specifically, the regeneration-associated genes play
a pivotal role in axonal regeneration (Tonge et al.,
2008). The process is speculated to involve reverse
axonal transport of particular signaling molecules,
such as signal transducer and activator of tran-
scription (STAT3) (Tonge et al., 2008). Prior in
vitro conditioning of axotomized nerves with neu-
rotrophins causes enhanced axonal growth (Tonge
et al., 2008). Such neurotrophins include brain-derived
neurotrophic factor, which increases axonal length by
over 90%. In addition, environmental factors serve to
guide axons upon regeneration. For example, the Slit
family functions in repulsing axonal growth (Fujiwara
et al., 2008) while HIP/PAP1 function in the formation
and regeneration of motor neurons (Tebar et al., 2008).
Animal models have also assisted in understanding
axonal regeneration. The role of the immune response
in CNS regeneration has been recently characterized in
a medicinal leech model. The production of antibacte-
rial peptides by neurons demonstrates that a controlled
microbial response facilitates regeneration of nervous
system function.
Axotomy has been shown to serve as one compo-
nent, along with motor neuron dysfunction, to induce
neural precursor cells (Azari et al., 2008). Neural pre-
cursor cells are positive for nestin, an intermediate
filament protein expressed in stem cells of neurolog-
ical origin. The process of axotomy in the presence
of motor neuron disease mobilizes neural precursors
to migrate to the spinal cord (Azari et al., 2008).
Thus, injury to CNS structures can induce a response
from cells with stem-like characteristics, suggesting a
valuable role for stem cell therapy in the treatment
of axonal injury and nerve severance (Azari et al.,
2008).
17.6 Stem Cell Therapy for Spinal
Cord Injury
Estimates indicate that over 120 million Americans
benefit from regenerative medicine (Harris, 2008; Cho
et al., 2008; Cui et al., 2008). With respect to neuro-
logical disease, the use of stem cells may encompass
disease states such as spinal cord injury, stroke, demen-
tias, neurodegeneration, and cerebral palsy (Harris,
C. King et al.
2008). The current upsurge of interest in stem cell
applications in spinal cord injury has been attributed to
the high likelihood of permanent loss of neurological
function upon trauma (Barnab-Heider and Frisn,
2008). In this section, we explore the notion of the
clinical use of various undifferentiated cell types
with self-renewal properties in scenarios of CNS
dysfunction.
As of date, approximately 2.5 million people expe-
rience disability due to spinal cord injury (SCI) (Lo
et al., 2008). The phenomenon of SCI has been
suggested to have two stages: physical trauma and
intrinsic cellular destruction. During physical trauma
stage, various gross insults to the spinal cord such
as penetration or contusion can result in loss of
normal tract function. During the cellular destruction
stage, events including ischemia, glutamate-induced
excitotoxicity, and apoptosis lead to further damage
of the nervous system (Lo et al., 2008). Trauma in
non-nervous tissue does not pose as great a threat
as damage to nervous tissue because many tissues
can regenerate themselves and allow for survival.
The limited capacity of adult neurons to regenerate,
however, makes the prospect of mesenchymal stem
cell therapy promising for treatment of patients with
injury. Multiple reasons have been proposed for the
failure of ectodermal tissue to successfully repair
itself. Firstly, the innate capacity for neurons to
grow is less than other cells of the body (Planchamp
et al., 2008). Secondly, the microenvironment in
which neurons reside may be non-permissive for
development.
Neural stem cells (NSC) have been investigated to
determine their regeneration abilities in spinal cord
transaction. Retinoic acid, the same factor used in
the transdifferentiation of MSC into neurons, can
be used to differentiate NSC into neurons. NSC
and neurotrophin-3-expressing Schwann cells can be
grafted into a site of spinal cord transaction, result-
ing in improvement of neural function and myeli-
nation (Zhang et al., 2007). Fetal NSC have been
proposed to repair motor neuron dysfunction since
they have been shown to contact the neuromuscular
junction and develop into a functional cholinergic phe-
notype (Gao et al., 2007). Synaptic transmission is
intact upon fetal NSC transplantation, and improve-
ment in motor function can be observed (Gao et al.,
2007).
17 Dual Roles of MSC in Spinal Cord Injury
17.7 Transdifferentiation
of Mesenchymal Stem Cells
to Neurons
Since neurons do not regenerate naturally with ease
after injury, studies have investigated the role of stem
cell therapy in CNS regeneration. Of particular inter-
est are mesenchymal stem cells (MSC), since they
entail far fewer ethical concerns than embryonic stem
cells. MSC have been shown to have transdifferenti-
ation potential. This suggests that cells that are nor-
mally committed to one fate can be induced to give
rise to cells of another lineage by in vitro manipu-
lation (Greco et al., 2007). Mesenchymal stem cells,
which are derived from the mesoderm of the mam-
malian gastrula stage, normally develop into tissue
of mesodermal origin, including adipocytes, chon-
drocytes, osteoblasts, and bone marrow stromal cells
(Fig. 17.1). Recently, studies have demonstrated a role
for MSC in the development of non-mesodermal tis-
sue, including hepatocytes-like cells of the endoderm
(Kazemnejad et al., 2008).
MSC can also develop into neurons, which nor-
mally derive from the ectodermal lineage of the gas-
trula (Greco et al., 2007). The value of this con-
cept is seen in neurodegenerative conditions such as
Parkinsons disease, amyotrophic lateral sclerosis, and
Alzheimers disease, in which dopaminergic, cholin-
ergic, and motor neurons, respectively, are not func-
tioning at an optimal level (Lang et al., 2006; Perrier
and Studer, 2003; Shigetomi and Fukuchi, 1994).
Parkinsons disease has been investigated intensively
in the past few years, as stem cell therapy extends
to regeneration of dopaminergic neurons of the mid-
brain (Chiba et al., 2008). We discuss these prospects
in Section 17.8.
MSC have been shown to function in wound
repair by transdifferentiating into keratinocytes, a phe-
nomenon which functions to replace skin cells after
trauma or injury (Sasaki et al., 2008). They have the
potential for developing into cells of the endothelium
and pericytes (Sasaki et al., 2008).
Mesenchymal stem cell therapy may be approved in
the near future based on the large therapeutic poten-
tial of these cells. MSC can be obtained from adult
adipose tissue (Sasaki et al., 2008). This route of
access suggests few ethical concerns, unlike the obtain-
ment of embryonic stem cells. The in vitro use of
277
natural microenvironmental cues may allow for the use
of MSC in the clinic. Upon transdifferentiation into
neural stem cells via retinoic acid, MSC may harbor
the capacity to secrete neural-specific products. The
advantage of this process is evident in the beneficial
effects of various molecules including glial-derived
neurotrophic factor (GDNF). GDNF has been shown
to facilitate survival on neurons of various phenotypes,
including noradrenergic and dopaminergic (Lo et al.,
2008). GDNF also hinders cell death of retinal gan-
glion cells and cortical neurons, suggesting a role in
the treatment of vision loss and various dementias (Lo
et al., 2008).
Thus far, the use of MSC in SCI has not been com-
pletely successful (Saski et al., 2008). In a study that
assessed the safety of autologous MSC transplant in
patients with the motor neuron disease amyotrophic
lateral sclerosis, also known as Lou Gehrigs disease,
MSC expansion and delivery to the spinal cord of
patients showed minimal systemic toxicity at vari-
ous time points and was well tolerated. The reported
complications included sensory deficits in the lower
extremities and pain in the thoracic cavity (Mazzini
et al., 2006). In another clinical study, neural stem cells
were produced from bone marrow-derived MSC. Upon
direct administration of these MSC to the target sites
in patients with chronic SCI, no adverse effects were
reported (Moviglia et al., 2006). The effects of bone
marrow-derived hematopoietic progenitor cell (HPC)
transplant have also been evaluated in patients with
chronic SCI (Deda et al., 2008). In this study, autolo-
gous HPC administration resulted in no major adverse
effects, and patients experienced improvement in sen-
sory and motor functions (Deda et al., 2008). The
majority of investigators shed a positive perspective on
the use of MSC in SCI and neurological trauma.
17.8 Other Neurodegenerative
Disorders
Regarding the employment of stem cells for neurore-
generative purposes, stem cell therapy is not lim-
ited to spinal cord injury. Perhaps the most well-
known application of stem cell therapy to the com-
munity is regeneration of neurons for the treatment of
Parkinsons disease. Parkinsons disease affects more
than 1 out of 100 people over the age of 65. Treatment
278
of patients with this neurodegenerative condition is
arguably the most fruitful application of stem cells
(Trzaska and Rameshwar, 2007; Trazaska et al., 2007;
Sandberg, 2007). In Parkinsons disease, dopamine
neurons of the substantia nigra of the midbrain lose
function. As a result, motor and cognitive functions
are compromised, and patients display bradykinesia,
resting tremor, rigidity, shuffling gait, and mask-like
facial expression (Osawa et al., 1997). The cur-
rent standards of treatment for Parkinsons disease
include the dopamine precursor L-dopa, dopamine
receptor agonists, and cholinergic muscarinic antag-
onists (Reiss et al., 2007). Such treatments func-
tion to restore the proper balance between dopamine
and acetylcholine, but their effects tend to wear off
after some time, leading to uncomfortable symptoms
such as tardive dyskinesia and psychosis (Olanow
et al., 2006). Given these drawbacks of conventional
Parkinsons disease therapy, stem cells appear to be
critical to the improvement of the quality of patient life.
Fortunately, numerous advances have been made
in this area. Human ESC can develop into neurons
with a dopaminergic phenotype, facilitating the recov-
ery of patients with midbrain dysfunction. Dopamine-
secreting neurons can be generated from human and
murine ESC via treatment with the bone morphogenic
protein antagonist Noggin (Chiba et al., 2008). These
discoveries, along with others, underscore the potential
for stem cell treatment in Parkinsons disease.
The boundaries of the therapeutic potential for
stem cells have been extended to include treatment
of lesions of the brain stem. Pair box 7 (Pax7), a
transcription factor involved in fetal development and
cancer cell proliferation, has been shown to enhance
development of mesencephalic structures of the brain
(Thompson et al., 2008). Pax3 expression, on the other
hand, exerts antagonistic effects, maintaining a stem-
like phenotype of midbrain structures. Stem cell ther-
apy in the future can make use of the opposing roles
of Pax3 and Pax7 in differentiation of the midbrain
(Thompson et al., 2008).
Stem cell therapy extends to the treatment of cere-
brovascular accidents, commonly known as strokes.
Cerebrovascular accident is due to CNS dysfunction
resulting from ischemic or hemorrhagic events in the
brain. Adult stem cells from the bone marrow, skele-
tal muscle, peripheral blood and other sites have been
proposed to enhance recovery after a cerebrovascu-
lar accident (Roh et al., 2008). Scientists believe that
C. King et al.
stem cells may repair nonfunctional neuronal connec-
tions in chronic ischemia of the brain (Yamashita et al.,
2008; Tansey et al., 2007). Activation or transplanta-
tion of stem cells in the neural environment may assist
in increasing cerebral blood flow (Yamashita et al.,
2008). The activation strategy involves stimulating the
endogenous neural stem cells of the subventricular
zone of the brain, and stroke has been shown to induce
proliferation of these cells (Yoo et al., 2008). On the
other end of the spectrum, the transplantation strategy
employs an exogenous source of neural stem cells to
help patients recover from ischemic states (Yamashita
et al., 2008).
17.9 Limitations to Stem Cell
Therapeutics
Although numerous advances have been made in
stem cell research for neurological conditions such as
Parkinsons disease, mesencephalic dysfunction, and
cerebrovascular accidents, the field is not without its
limitations. A brief summary of prospective problems
with cell therapy for neurological disease is warranted.
Some experts in the field of stem cell research cast
doubt on the ability of MSC to develop into true neu-
ral derivatives (Hardy et al., 2008). Such qualms merit
investment of time and resources into investigations on
the therapeutic prospects of MSC therapy and stem cell
therapy in general for SCI and the other neurological
insults. In any case, the immunosuppressive properties
of MSC make their clinical use convincing (Ramasamy
et al., 2008) however, adult stem cells may be limited in
their therapeutic potential, as regenerative ability tends
to decay with age (Roh et al., 2008).
Immunological interactions in the context of stem
cell therapy may pose a potential dilemma dur-
ing treatment. Investigations into immunological phe-
nomenon have been conducted, and it has been shown
that inflammatory cytokines, such as tumor necro-
sis factor- (TNF-), interferon- (IFN-), and the
acute phase reactant interleukin-6 (IL-6) can upregu-
late MHC genes in neural stem cells and progenitor
cells. These discoveries encompass numerous models,
including monkeys, rats, and humans (Johansson et al.,
2008). The implications of immunostimulation of such
cytokines may cause complications to surface, as these
endogenous cytokines increase the probability of graft
17 Dual Roles of MSC in Spinal Cord Injury
rejection (Johansson et al., 2008). The environment
of the CNS includes the aforementioned inflammatory
cytokines and can cause immune-mediated rejection of
transplanted cells (Johansson et al., 2008).
Clever methods must be designed to counteract
this phenomenon while keeping these cytokines func-
tions intact, as they may be vital to fetal neurogen-
esis (Johansson et al., 2008; Thompson and Powrie,
2004). One prospect on preventing an unfavorable
immunological reaction involves transplantation or co-
transplantation of MSC, which are immunosuppressive
in nature (Ramasamy et al., 2008).
17.10 An Interdisciplinary Approach
Bioengineering strategies have focused on providing
a permissive environment for the regeneration of the
spinal cord and achieving integration between the CNS
and PNS (Schmidt and Leach, 2003; Nomura et al.,
2006; Willerth and Sakiyama-Elbert, 2007). A com-
mon approach is the use of biomaterials that act as
guidance channels, and/or as delivery vehicles for
cells, neuroregenerative factors and anti-inflammatory
drugs. This section will review the current work in this
area. Bioengineering approaches also focus on under-
standing injury to the spinal cord from a biomechanics
perspective, using various mathematical and experi-
mental models for analyzing the anatomical loading
distribution and resulting cellular responses (Maikos
et al., 2008; Laplaca et al., 2007). The functional
response of nerves to deleterious biomechanical load-
ing has lead to discoveries in determining the range
of loads that can, in turn, promote nerve growth,
i.e. stretch-induced growth (Bueno and Shah, 2008;
Smith et al., 2001), and may hold promise in develop-
ing transplantable nerve constructs to bridge extensive
damage.
Bioengineering research efforts have been primar-
ily concentrated on developing biomaterials that act
as scaffolds to physically guide the regeneration of
nerves across lesions, i.e. nerve guide or nerve of growth factors/drugs alone or in combination with
guidance channel (Schmidt and Leach, 2003). They
direct the axon growth as well as reduce the infiltration
of scar tissue. Natural materials, such as collagen, fib-
rin, hyaluronic acid, agarose, chitosan and alginates,
as well as degradable and nondegradable synthetic
materials, such as polylactic (PLA) and polyglycolic
279
acids (PGA), polyethylene glycol (PEG), and silicone,
have been sought and fabricated into tubes, fibers,
cross-linked hydrogels and gels containing fibers to
guide axonal growth. Although these materials show
promise, directing the axons from the nerve guide
back to the spinal cord and achieving integration
with the host synaptic pathways remains a chal-
lenge. Therefore, recent efforts have been exploring
combination strategies using guidance channels with
cells and/or neurotrophic or neuroprotective factors
(Nomura et al., 2006).
Various cell types have been explored in combina-
tion with scaffolding materials to regenerate the spinal
cord. Schwann cells are of interest since they secrete
numerous neurotrophic factors, produce an extracel-
lular matrix and express cell adhesion molecules
(Nomura et al., 2006). They have been used in com-
bination with degradable and nondegradable tubular
scaffolds; however have been found to be minimally
effective in treating the injured spinal cord without the
use of additional growth factors and other cell types
(Bunge, 2008). Other supportive cell types that provide
ensheathment for developing axons, such as olfactory-
ensheathing glial cells (OEFs), have also been explored
and show promise on scaffolds in which the biomate-
rial acts as a structure to support their cell function.
Stem cells, such as neural stem cells (NSC), MSC
and embryonic stem cells (ESC), are currently being
investigated in combination with scaffolds because
the biomaterial can aid in localizing and promoting
the differentiation and orientation of these cells at
the defect site based on the physical and chemical
characteristics of the scaffold (Engler et al., 2006;
Arinzeh et al., 2005; Xie et al., 2008). By combin-
ing the scaffold with stem/neural progenitor cells, the
cells may provide the therapeutic benefit of neuropro-
tection (Himes et al., 2006) and/or functionally inte-
grate into the spared spinal cord circuitry (e.g. form-
ing new oligodendrocytes and/or neurons) (Cummings
et al., 2005; Cizkova et al., 2006) to improve the
outcome.
Scaffolds can also be used for controlled-release
cells. By incorporating growth factors/drugs in the
scaffold during fabrication, controlled release of that
drug occurs via diffusion and is dependent upon
the physical properties of the material, such as pore
size, degree of cross-linking, and rate of degradation.
Natural materials, such as collagen, agarose and fibrin,
280
and synthetic polymers like polyethylene glycol-poly
lactic acid (PEG-PLA) have been fabricated into guid-
ance channels for the release of neurotrophin-3 (NT-3),
nerve growth factor (NGF) and brain-dervied neu-
rotrophic factor (BDNF). Results demonstrated axonal
sprouting and an increased functional recovery in SCI
models (Willerth and Sakiyama-Elbert, 2007). Other
therapeutic drugs, such as chondroitinase ABC, anti-
bodies against Nogo and cyclic AMP, have shown
promise in reducing the inhibitory environment created
in the CNS after injury (Willerth and Sakiyama-Elbert,
2007) and are currently being investigated in controlled
release strategies. Other approaches being explored
in drug delivery methods are affinity-based systems
and immobilized drug delivery systems, where the
desired drug is non-covalently bound or covalently
attached to the scaffold, respectively. Electrically con-
trolled delivery systems, where the release of the
target drug is controlled by electrical stimulation,
have been investigated for use as coatings on neu-
ral electrodes. Anti-inflammatory drugs were released
to reduce the number of reactive astrocytes and had
no negative effect on the viability of neurons (Wadha
et al., 2006). Release of NT-3 via this method has
been shown to stimulate neurite extension (Thompson
et al., 2006). Other systems under development use
micro- and nano-electromechanical systems (MEMs
and NEMs) for on-demand pulsatile or adjustable con-
tinuous administration of the drug for extended periods
using a wireless integrated system that can regulate
drug release, receive sensor feedback, and transmit
updates (Staples et al., 2006). Combining technologies
of microfabrication, information science and systems
biology will lead to more sophisticated drug delivery
systems in the future. Approaches above are still in
their early stages of investigation and will require a bet-
ter understanding of the cell source used, drug release
rates and biomaterial properties to obtain functional
recovery.
17.11 Experimental Models for SCI
SCI requires attention to major areas: repair the region
of injury; prevent rapid atrophy and cell death of dis-
tal neuronal degeneration; and regrowth of the axons.
To this end, experimental models should be established
with the SCI-associated confounds. Several research
C. King et al.
groups have been able to generate functional neu-
rons from MSC, including those from adult human
bone marrow. The neurons can be used to estab-
lish a neuro-muscular system. Skeletal muscle cells
can be generated from the same MSC to establish
an autologous system. The neurons would be able
to respond to tissue factors similar to those found
at sites of injuries, such as those discussed above.
Except for extrapolation on the roles of cytokines and
other inflammatory mediators, it is unclear how the
factors at regions of SCI interact with each other to
affect the damaged tissues. If stem cells, such as MSC
will be used in any protocol for SCI treatment, it
would be important to determine if the receptors are
clustered following responses to injuries and deter-
mine if there are synergism or antagonism among
cytokines, and how this would affect the skeletal
muscles.
An experimental model can be established in which
neurons are placed in contact with skeletal muscle
from the same source of MSC. The responses of mus-
cle, namely end plate potential, could be used as an
indication to test axonal repair if the neurons are sub-
jected to axotomy by microdissection. This type of
model would recapitulate injured neurons, similar to
SCI. By adding cytokines to the system, before and
after injury, the model would be able to mimic the
injured nerve within a milleu of tissue factors. The
model would be able to use to screen compound to
identify methods to repair axons.
The discussed model could lead to the development
of a genetic engineering tool in which human MSC
could be used to deliver the identified factors for SCI
treatment. Similar methods could be used for oligo-
dendrocytes. This in vitro system could be quickly
verified by in vivo methods. In addition, the system
could allow for experimental questions to understand
nerve regrowth, and synapse formation with skeletal
muscle.
The method discussed above would need to com-
partmentalize the neurons from skeletal muscle. We
describe a co-culture method that has been modified
from a chamber model reported by (Campenot, 1977)
(Fig. 17.3). The system allows for the neurons and
skeletal muscle cells to be cultured in their respective
media. The insert is sealed after placement on sterile
grease, as shown. Despite the compartmentalization,
the cells can form contact through tracks placed within
the gelatin that coats the surface of the Petri dish.
17 Dual Roles of MSC in Spinal Cord Injury
Fig. 17.3 Petri dishes were coated with 1 endotoxin-free
gelatin. After this, tracks were made along the dried gelatin
then the Teflon inserts were added. The insert was stabilized
with sterile Vaseline. Shown are human MSC-derived neurons.
The neurons can contact skeletal muscle in the inner chamber.
Axotomy in the neurons can affect end plate potential. This
simple co-culture model could be a tool for rapid screen of
compound for SCI repair
17.12 On the Frontier of Stem Cell
Therapy for Neural Dysfunction
At the beginning of this twenty-first century, sci-
entists can benefit from vast scientific discoveries.
Researchers must constantly ask, How can todays
advances in science lead to benefits for tomorrows
patients? The past prospects of stem cells on treat-
ment of pathological conditions of the nervous sys-
tem have shed light onto future discoveries in the
field. In any case, scientists must exert some cau-
tion concerning the therapeutic potential of stem cells,
considering the aforementioned qualms. With a con-
tinued commitment to the advancement of science
and the translation of basic science discoveries to
the clinic, stem cell therapy may one day alleviate
much suffering while improving the quality of patient
care.
Acknowledgement This work was supported by the FM Kirby
Foundation.
281
References
Allan SM, Tyrrell PJ, Rothwell NJ (2005) Interleukin-1 and
neuronal injury. Nat Rev Immunol 5:629640.
Arinzeh TL, Tran T, Mcalary J, Daculsi G (2005) A com-
parative study of biphasic calcium phosphate ceramics for
human mesenchymal stem cell-induced bone formation.
Biomaterials 26:36313638.
Azari MF, Profyris C, Zang DW, Petratos S, Cheema SS (2005)
Induction of endogenous neural precursors in mouse mod-
els of spinal cord injury and disease. Eur J Neurol 12:
638648.
Bagby GC (1989) Interleukin-1 and hematopoiesis. Blood Rev
3:152161.
Barnab-Heider F, Frisn J (2008) Stem cells for spinal cord
repair. Cell Stem Cell 3:1624.
Bianco P, Riminucci M, Gronthos S, Robey PG (2001) Bone
marrow stromal stem cells: nature, biology, and potential
applications. Stem Cells 19(3):180192.
Bieback K, Kern S, Klter H, Eichler H (2004) Critical param-
eters for the isolation of mesenchymal stem cells from
umbilical cord blood. Stem Cells 22:625634.
Blanc KL, Pittenger MF (2005) Mesenchymal stem cells:
progress toward promise. Cytotherapy 7:3645.
Blesch A, Uy HS, Grill RJ, Cheng JG, Patterson PH, Tuszynski
MH (1999) Leukemia inhibitory factor augments neu-
rotrophin expression and corticospinal axon growth after
adult CNS injury. J Neurosci 19:35563566.
Bonni A, Sun Y, Nadal-Vicens M, Bhatt A, Frank DA,
Rozovsky I, Stahl N, Yancopoulos GD, Greenberg ME
(1997) Regulation of gliogenesis in the central nervous sys-
tem by the JAK/STAT signaling pathway. Science 278:477
483.
Bueno FR, Shah SB (2008) Implications of tensile loading for
the tissue engineering of nerves. Tissue Eng Part B Rev
14:219233.
Bunge MB (2008) Novel combination strategies to repair the
injured mammalian spinal cord. J Spinal Cord Med 31:262
269.
Butzkueven H, Zhang JG, Soilu-Hanninen M, Hochrein
H, Chionh F, Shipham KA, Emery B, Turnley AM,
Petratos S, Ernst M, Bartlett PF, Kilpatrick TJ (2002)
LIF receptor signaling limits immune-mediated demyelina-
tion by enhancing oligodendrocyte survival. Nat Med 8:
613619.
Campenot RB (1977) Local control of neurite development
of nerve growth factor. Proc Natl Acad Sci U S A 74:
45164519.
Caplan AI (1994) The mesengenic process. Clin Plast Surg
21:429435.
Carlos TM, Harlan JM (1994) Leukocyte-endothelial adhesion
molecules. Blood 84:20682101.
Castillo M, Liu K, Bonilla LM, Rameshwar P (2007) The
immune properties of mesenchymal stem cells. Intl J Biomed
Sci 3:100.
Chan JL, Tang KC, Patel AP, Bonilla LM, Pierobon N,
Ponzio NM, Rameshwar P (2006) Antigen-presenting prop-
erty of mesenchymal stem cells occurs during a narrow
window at low levels of interferon-gamma. Blood 107:
48174824.
282
Chi LY, Yu J, Zhu H, Li XG, Zhu SG, Kindy MS (2008) The dual
role of tumor necrosis factor-alpha in the pathophysiology of
spinal cord injury. Neurosci Lett 438:174179.
Chiba S, Lee YM, Zhou W, Freed CR (2008) Noggin enhances
dopamine neuron production from human embryonic stem
cells and improves behavioral outcome after transplantation
into Parkinsonian rats. Stem Cells 26:28102820.
Cho SR, Yang MS, Yim SH, Park JH, Lee JE, Eom YW, Jang IK,
Kim HE, Park JS, Kim HO, Lee BH, Park CI, Kim YJ (2008)
Neurally induced umbilical cord blood cells modestly repair
injured spinal cords. Neuroreport 19:12591263.
Cizkova D, Rosocha J, Vanicky I, Jergova S, Cizek M (2006)
Transplants of human mesenchymal stem cells improve func-
tional recovery after spinal cord injury in the rat. Cell Mol
Neurobiol 26:11671180.
Cui L, Jiang J, Wei L, Zhou X, Fraser JL, Snider BJ, Yu SP
(2008) Transplantation of embryonic stem cells improves
nerve repair and functional recovery after severe sciatic nerve
axotomy in rats. Stem Cells 26:13561365.
Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand
M, Summers R (2005) Human neural stem cells differentiate
and promote locomoter recovery in spinal cord-injured mice.
Proc Natl Acad Sci U S A 102:1406914074.
Deda H, Inci MC, Kurekci AE, Kayihan K, Ozgun E,
Ustunsoy GE, Kocabay S (2008) Treatment of chronic spinal
cord injured patients with autologous bone marrow-derived
hematopoietic stem cell transplantation: 1-year follow-up.
Cytotherapy 10:565574.
Deng J, Petersen BE, Steindler DA, Jorgensen ML, Laywell
ED (2006) Mesenchymal stem cells spontaneously express
neural proteins in culture and are neurogenic after transplan-
tation. Stem Cells 24:10541064.
Dinarello CA (1996) Biologic basis for interleukin-1 in disease.
Blood 87:20952147.
Dinarello CA (2005) Blocking IL-1 in systemic inflammation. J
Exp Med 201:13551359.
Divani AA, Hussain MS, Magal E, Heary RF, Qureshi AI
(2007) The use of stem cells hematopoietic stimulating fac-
tors therapy following spinal cord injury. Ann Biomed Eng
35:16471656.
Downing JR (2004) TGF- signaling, tumor suppression, and
acute lymphoblastic leukemia. N Engl J Med 351(6):528.
Ducker TB, Hamit HF (1969) Experimental treatments of acute
spinal cord injury. J Neurosurg 30:693697.
Ducker TB, Zeidman SM (1994) Spinal cord injury. Role of
steroid therapy. Spine 19:22812287.
Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elas-
ticity directs stem cell lineage specification. Cell 126:677
689.
Fujiwara T, Kubo T, Koyama Y, Tomita K, Yano K, Tohyama
M, Hosokawa K (2008) mRNA expression changes of slit
proteins following peripheral nerve injury in the rat model. J
Chem Neuroanat 36:170176.
Gadient RA, Otten UH (1997) Interleukin-6 (IL-6) a
molecule with both beneficial and destructive potentials.
Prog Neurobiol 52:379390.
Gao J, Coggeshall RE, Chung JM, Wang J, Wu P (2007)
Functional motoneurons develop from human neural stem
cell transplants in adult rats. Neuroreport 18:565569.
Gearing DP, Gough NM, King JA, Hilton DJ, Nicola NA,
Simpson RJ, Nice EC, Kelso A, Metcalf D (1987) Molecular
C. King et al.
cloning and expression of cDNA encoding a murine
myeloid leukaemia inhibitory factor (LIF). EMBO J 6:
39954002.
Golestaneh N, Mishra B (2005) TGF-beta, neuronal stem cells
and glioblastoma. Oncogene 24:57225730.
Greco SJ, Rameshwar P (2007) Enhancing effect of IL-1alpha
on neurogenesis from adult human mesenchymal stem
cells: implication for inflammatory mediators in regenerative
medicine. J Immunol 179:33423350.
Greco SJ, Zhou C, Ye JH, Rameshwar P (2007) An interdis-
ciplinary approach and characterization of neuronal cells
transdifferentiated from human mesenchymal stem cells.
Stem Cells Dev 16:811826.
Hardy SA, Maltman DJ, Przyborski SA (2008) Mesenchymal
stem cells as mediators of neural differentiation. Curr Stem
Cell Res Ther 3:4352.
Harris DT (2008) Cord blood stem cells: a review of potential
neurological applications. Stem Cell Rev 4:269274.
Hayashi M, Ueyama T, Nemoto K, Tamaki T, Senba E
(2000) Sequential mRNA expression for immediate early
genes, cytokines, and neurotrophins in spinal cord injury. J
Neurotrauma 17:203218.
Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan
JM, Granger DN (1987) Role of neutrophils in ischemia-
reperfusion-induced microvascular injury. Am J Physiol
253:H699H703.
Himes BT, Neuhuber B, Coleman C, Kushner R, Swanger
SA, Kopen GC (2006) Recovery of function following
grafting of human bone marrow-derived stromal cells into
the injured spinal cord. Neurorehabil Neural Repair 20:
278296.
Johansson S, Price J, Modo M (2008) Effect of inflammatory
cytokines on MHC expression and differentiation of human
neural stem/progenitor cells. Stem Cells 26:24442454.
Kang MK, Kang SK (2008) Interleukin-6 induces prolifera-
tion in adult spinal cord-derived neural progenitors via the
JAK2/STAT3 pathway with EGF-induced MAPK phospho-
rylation. Cell Prolif 41:377392.
Kazemnejad S, Allameh A, Soleimani M, Gharehbaghian
A, Mohammadi Y, Amirizadeh N, Jazayery M (2008)
Biochemical and molecular characterization of hepatocyte-
like cells derived from human bone marrow mesenchy-
mal stem cells on a novel three-dimensional biocompatible
nanofibrous scaffold. J Gastroenterol Hepatol 24:278287.
Kim SJ, Letterio J (2003) Transforming growth factor-beta sig-
naling in normal and malignant hematopoiesis. Leukemia
17:17311737.
Kurek JB, Bennett TM, Bower JJ, Muldoon CM, Austin L
(1998a) Leukaemia inhibitor factor (LIF) production in a
mouse model of spinal trauma. Neurosci Lett 249:14.
Kurek JB, Radford AJ, Crump DE, Bower JJ, Feeney SJ, Austin
L, Byrne E (1998b) LIF (AM424), a promising growth factor
for the treatment of ALS. J Neurol Sci 160:S106S113.
Kwon BK, Tetzlaff W, Grauer JN, Beiner J, Vaccaro AR
(2004) Pathophysiology and pharmacologic treatment of
acute spinal cord injury. Spine J 4:451464.
LaPlaca MC, Simon CM, Prado GR, Cullen DK (2007) CNS
injury biomechanics and experimental models. Prog Brain
Res 161:1326.
Lange KW, Mecklinger L, Walitza S, Becker G, Gerlach M,
Naumann M, Tucha O (2006) Brain dopamine and kine-
17 Dual Roles of MSC in Spinal Cord Injury
matics of graphomotor functions. Human Movement Sci
25:492509.
Laver J, Moore MAS (1989) Clinical use of recombinant
human hematopoietic growth factors. J Natl Cancer Inst 81:
13701382.
Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH
(2004) Isolation of multipotent mesenchymal stem cells from
umbilical cord blood. Blood 103:16691675.
Li M, Sendtner M, Smith A (1995) Essential function of LIF
receptor in motor neurons. Nature 378:724727.
Lo WC, Hsu CH, Wu AT, Yang LY, Chen WH, Chiu WT, Lai
WF, Wu CH, Gelovani JG, Deng WP (2008) A novel cell-
based therapy for contusion spinal cord injury using GDNF-
delivering NIH3T3 cells with dual reporter genes monitored
by molecular imaging. J Nucl Med 49:15121519.
Maikos JT, Qian Z, Metaxas D, Shreiber DI (2008) Finite ele-
ment analysis of spinal cord injury in the rat. J Neurotrauma
25:795816.
Martinez C, Hofmann TJ, Marino R, Dominici M, Horwitz
EM (2007) Human bone marrow mesenchymal stromal cells
express the neural ganglioside GD2: a novel surface marker
for the identification of MSCs. Blood 109:42454248.
Massague J, Weis-Garcia F (1996) Serine/threonine kinase
receptors: mediators of transforming growth factor beta fam-
ily signals. Cancer Surv 27:4164.
Massague J, Wotton D (2000) Transcriptional control by the
TGF-beta/Smad signaling system. EMBO J 19:17451754.
Massagu J, Andres J, Attisano L, Cheifetz S, Lpez-Casillas
F, Ohtsuki M, Wrana JL (1992) TGF-beta receptors. Mol
Reprod Dev 32(2):99104.
Mazzini L, Mareschi K, Ferrero I, Vassallo E, Oliveri G,
Boccaletti R, Testa L, Livigni S, Fagioli F (2006) Autologous
mesenchymal stem cells: clinical applications in amy-
otrophic lateral sclerosis. Neurol Res 28:523526.
Mehra A, Wrana JL (2002) TGF-beta and the Smad signal
transduction pathway. Biochem Cell Biol 80:605622.
Moore MA (2002) Cytokine and chemokine networks influ-
encing stem cell proliferation, differentiation, and marrow
homing. J Cell Biochem 38:2938.
Moviglia GA, Fernandez Via R, Brizuela JA, Saslavsky J,
Vrsalovic F, Varela G, Bastos F, Farina P, Etchegaray G,
Barbieri M, Martinez G, Picasso F, Schmidt Y, Brizuela P,
Gaeta CA, Costanzo H, Moviglia Brandolino MT, Merino S,
Pes ME, Veloso MJ, Rugilo C, Tamer I, Shuster GS (2006)
Combined protocol of cell therapy for chronic spinal cord
injury. Report on the electrical and functional recovery of two
patients. Cytotherapy 8:202209.
Nomura H, Tator CH, Shoichet MS (2006) Bioengineered strate-
gies for spinal cord repair. J Neurotrauma 23:496507.
Olanow CW, Obeso JA, Stocchi F (2006) Drug insight: continu-
ous dopaminergic stimulation in the treatment of Parkinsons
disease. Nat Clin Pract Neurol 2:382392.
Ong SY, Dai H, Leong KW (2006) Hepatic differentiation poten-
tial of commercially available human mesenchymal stem
cells. Tissue Eng 12:34773485.
Osawa H, Yamabe H, Kaizuka M, Tamura N, Tsunoda S, Shirato
KI, Okumura K (1997) Systemic lupus erythematosus asso-
ciated with transverse myelitis and parkinsonian symptoms.
Lupus 6:613615.
Padovan CS, Jahn K, Birnbaum T, Reich P, Sostak P, Strupp M,
Straube A (2003) Expression of neuronal markers in differ-
283
entiated marrow stromal cells and CD133+ stem-like cells.
Cell Transplant 12:839848.
Pan W, Cain C, Yu Y, Kastin AJ (2006) Receptor-mediated trans-
port of LIF across blood-spinal cord barrier is upregulated
after spinal cord injury. J Neuroimmunol 174:119125.
Perrier AL, Studer L (2003) Making and repairing the mam-
malian brainin vitro production of dopaminergic neurons.
Semin Cell Dev Biol 14:181189.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,
Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak
DR (1999) Multilineage potential of adult human mesenchy-
mal stem cells. Science 284:143147.
Planchamp V, Bermel C, Tnges L, Ostendorf T, Kgler S, Reed
JC, Kermer P, Bhr M, Lingor P (2008) BAG1 promotes
axonal outgrowth and regeneration in vivo via Raf-1 and
reduction of ROCK activity. Brain 131:26062619.
Potian JA, Aviv H, Ponzio NM, Harrison JS, Rameshwar
P (2003) Veto-like activity of mesenchymal stem cells:
Functional discrimination between cellular responses
to alloantigens and recall antigens. J Immunol 171:
34263434.
Ramasamy R, Tong CK, Seow HF, Vidyadaran S, Dazzi
F (2008) The immunosuppressive effects of human bone
marrow-derived mesenchymal stem cells target T cell pro-
liferation but not its effector function. Cell Immunol 251:
131136.
Reis HJ, Rosa DV, Guimares MM, Souza BR, Barros AG,
Pimenta FJ, Souza RP, Torres KC, Romano-Silva MA (2007)
Is DARPP-32 a potential therapeutic target? Expert Opin
Ther Targets 11:16491661.
Roberts AB, Sporn MB (1993) Physiological actions and clinical
applications of transforming growth factor-beta (TGF-beta).
Growth Factors 8:119.
Roh JK, Jung KH, Chu K (2008) Adult stem cell transplantation
in stroke: its limitations and prospects. Curr Stem Cell Res
Ther 3:185196.
Sanberg PR (2007) Neural stem cells for Parkinsons disease: to
protect and repair. Proc Natl Acad Sci U S A 104:11869
11870.
Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H
(2008) Mesenchymal stem cells are recruited into wounded
skin and contribute to wound repair by transdifferentiation
into multiple skin cell type. J Immunol 180:25812587.
Sato M, Muragaki Y, Saika S, Roberts AB, Ooshima A (2003)
Targeted disruption of TGF-beta1/Smad3 signaling protects
against renal tubulointerstitial fibrosis induced by unilateral
ureteral obstruction. J Clin Invest 112:14861494.
Schmidt CE, Leach JB (2003) Neural tissue engineering: strate-
gies for repair and regeneration. Annu Rev Biomed Eng
5:293347.
Sekhon LH, Fehlings MG (2001) Epidemiology, demograph-
ics, and pathophysiology of acute spinal cord injury. Spine
26:S2S12.
Shi Y, Massagu J (2003) Mechanisms of TGF-beta signaling
from cell membrane to the nucleus. Cell 113:685700.
Shigetomi S, Fukuchi S (1994) Recent aspect of the role of
peripheral dopamine and its receptors in the pathogenesis of
hypertension. Fukushima J Med Sci 40:6983.
Smith DH, Wolf JA, Meaney DF (2001) A new strategy to
produce sustained growth of central nervous system axons:
continuous mechanical tension. Tissue Eng 7:131139.
284
Sokol JP, Schiemann WP (2004) Cystatin C antagonizes trans-
forming growth factor beta signaling in normal and cancer
cells. Mol Cancer Res 2:183195.
Stagg J, Pommey S, Eliopoulos N, Galipeau J (2006) Interferon-
gama-stimulated marrow stromal cells: a new type of
nonhematopoietic antigen-presenting cell. Blood 107:2570
2577.
Staples M, Daniel K, Cima MJ, Langer R (2006) Application of
micro- and nano-electromechanical devices to drug delivery.
Pharm Res 23:847863.
Tansey MG, McCoy MK, Frank-Cannon TC (2007)
Neuroinflammatory mechanisms in Parkinsons disease:
Potential environmental triggers, pathways, and targets for
early therapeutic intervention. Expl Neurol 208:125.
Tebar LA, Granton SM, Parsons-Perez C, Fisher AS, Bayne
R, Smith AJ, Turmaine M, Perez-Luz S, Sheasby A, De
Felipe C, Ruff C, Raivich G, Hunt SP (2008) Deletion
of the mouse RegIIIbeta (Reg2) gene disrupts ciliary neu-
rotrophic factor signaling and delays myelination of mouse
cranial motor neurons. Proc Natl Acad Sci U S A 105:
1140011405.
Thompson BC, Moulton SE, Ding J, Richardson R, Cameron
A, OLeary S, Wallace GG, Clark GM (2006) Optimising
the incorporation and release of a neurotrophic factor using
conducting polypyrrole. J Control Release 116:285294.
Thompson C, Powrie F (2004) Regulatory T cells. Curr Opin
Pharmacol 4:408414.
Thompson JA, Zembrzycki A, Mansouri A, Ziman M (2008)
Pax7 is requisite for maintenance of a subpopulation of supe-
rior collicular neurons and shows a diverging expression
pattern to Pax3 during superior collicular development. BMC
Dev Biol 8:62.
Tonge D, Chan K, Zhu N, Panjwani A, Arno M, Lynham S,
Ward M, Snape A, Pizzey J (2008) Enhancement of axonal
regeneration by in vitro conditioning and its inhibition by
cyclopentenone prostaglandins. J Cell Sci 121:25652577.
Trzaska KA, Kuzhikandathil EV, Rameshwar P (2007)
Specification of a dopaminergic phenotype from adult human
mesenchymal stem cells. Stem Cells 25:27972808.
Trzaska KA, Rameshwar P (2007) Current advances in the treat-
ment of Parkinsons disease with stem cells. Curr Neurovas
Res 4:99109.
C. King et al.
Wadhwa R, Lagenaur CF, Cui XT (2006) Electrochemically
controlled release of dexamethasone from conducting poly-
mer polypyrrole coated electrode. J Control Release 110:
531541.
Willerth SM, Sakiyama-Elbert SE (2007) Approaches to neu-
ral tissue engineering using scaffolds for drug delivery. Adv
Drug Dev Rev 59:325338.
Wrana JL (2000) Regulation of Smad activity. Cell 100:
189192.
Xie J, Willerth SM, Li X, Macewan MR, Rader A, Sakiyama-
Elbert SE, Younan X (2008) The differentiation of embryonic
stem cells seeded on electrospun nanofibers into neural
lineages. Biomaterials 30:354362.
Yamashita T, Deguchi K, Sehara Y, Lukic-Panin V, Zhang H,
Kamiya T, Abe K (2008) Therapeutic strategy for ischemic
stroke. Neurochem Res 34:707710.
Yamashita T, Deguchi K, Sehara Y, Lukic-Panin V, Zhang H,
Kamiya T, Abe K, Yamauchi K, Osuka K, Takayasu M,
Usuda N, Nakazawa A, Nakahara N, Yoshida M, Aoshima C,
Hara M, Yoshida J (2006) Activation of JAK/STAT signaling
in neurons following spinal cord injury in mice. J Neurochem
96:10601070.
Yoo SW, Kim SS, Lee SY, Lee HS, Kim HS, Lee YD, Suh-Kim
H (2008) Mesenchymal stem cells promote proliferation of
endogenous neural stem cells and survival of newborn cells
in a rat stroke model. Exp Mol Med 40:387397.
Young W (2000) Molecular and cellular mechanisms of spinal
cord injury therapies. In: Neurobiology of Spinal Cord Injury
(Kalb RG, Strittmatter SM, eds.), p. 241. Humana Press,
Totowa.
Yune TY, Chang MJ, Kim SJ, Lee YB, Shin SW, Rhim H,
Kim YC, Shin ML, Oh YJ, Han CT, Markelonis GJ, Oh TH
(2003) Increased production of tumor necrosis factor-alpha
induces apoptosis after traumatic spinal cord injury in rats. J
Neurotrauma 20:207219.
Zai LJ, Yoo S, Wrathall JR (2005) Increased growth factor
expression and cell proliferation after contusive spinal cord
injury. Brain Res 1052:147155.
Zhang X, Zeng Y, Zhang W, Wang J, Wu J, Li J (2007) Co-
transplantation of neural stem cells and NT-3-overexpressing
Schwann cells in transected spinal cord. J Neurotrauma
24:18631877.
Index

Acetylcholine (ACh), 53, 107, 109, 116, 121122, 124, 156, Axonal repair, 271281
162, 203204, 208, 278 Axotomy, 273 276, 280281
Acetylcholine (ACh) receptor, 121, 162
Activated microglia, 21, 176, 182, 183
B
Adenosine, 124, 162, 209
Basic fibroblast growth factor (bFGF), 20, 181, 199201, 211,
Adenosine receptor, 124, 209
259260, 265
Adenylyl cyclase-activating polypeptide (PACAP), 209210
Basic helix-loop-helix (bHLH), 38, 136139
Adipose-derived stem cells (ADSC), 119
Bergmann glia, 148149, 161
Adrenergic neurotransmitters, 207
Bipolar disorder, 104, 108
Adult neural stem cells, 21, 100
Bipotent progenitors, 50
Adult neurogenesis, 1221, 88, 93, 100, 116, 149151, 264
glial-melanocytic (GM), 50, 5355
Adult stem cell, 1415, 116, 120, 151, 238, 257258, 278
glial-myofibroblastic (GF), 50
Alkaline phosphatase, 82, 123, 239
Blastocyst, 61, 79, 119, 257
Altman, 1213, 17, 116, 149, 152, 256, 259
B-myb, 76
Alzheimers disease (AD), 14, 2425, 79, 98, 109110, 264,
Bone marrow cells, 15, 2324
277
Bone marrowderived stem/progenitor cells, 119, 238
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
Bone marrow stromal cells (BMSC), 118, 239, 277
(AMPA) receptor, 122123, 157, 204205
Bone morphogenetic protein (BMP), 38, 38, 49, 54, 99101,
-aminobutyric acid (GABA)ergic, 25, 9899, 102, 104106,
105106, 150151, 160, 183, 202
108109, 123124, 126, 137, 156, 207, 239
transforming Growth Factor beta (TGF-) superfamily, 3,
-aminobutyric-acid (GABA), 25, 9899, 101, 104106, 116,
68, 22, 99, 102103, 151, 154155, 157158, 160, 162,
120122, 124, 126, 128, 156, 206208, 239, 247
175177, 180, 182183, 200, 247, 250, 275
-aminobutyric-acid (GABA) receptor, 104, 120121, 206207
Brain derived neurotrophic factor (BDNF), 2021, 42, 99, 103,
Amniotic membrane, 247
106108, 179, 181182, 200201, 247, 249250, 276,
Amyloid- peptide, 25, 109, 157
280
Amyotrophic lateral sclerosis (ALS), 98, 105, 107, 163, 251,
Brain lipid-binding protein (BLBP), 148149, 151, 153154
264, 277
Brn3a, 105
Aneuploidy, 7383
Bromodeoxyuridine (BrdU), 1314, 1617, 19, 23, 150, 193,
Anophthalmia-microphthalmia (AM), 202
196, 203, 208, 210212, 256258
Apoptosis, 1718, 38, 78, 80, 125, 177, 179, 180, 212217,
Bub1, 75
265, 273276
Bub3, 75
Astrocyte differentiation, 120, 151154, 156158, 181, 183
BubR1, 75, 79
Astrocytes, 1517, 2122, 24, 4142, 60, 63, 65, 6768, 100,
103, 106, 117119, 124, 136, 138, 146163, 172,
176177, 180183, 232, 247, 249, 256257, 260, 265, C
280 Cajal-Retzius cells, 153
Astrocytic stem cells, 16, 150 Calcium, 104, 118, 124, 128129, 262, 273
Astroglial cells, 146152 free intracellular calcium concentration ([Ca2+]i), 120123,
Astroglia-neuron interactions, 147 125127, 203204, 207, 210, 213
Astrogliogenesis, 183, 260 Calcium binding proteins, 104, 148
ATM kinase (DNA-damage response kinase ATM), 78 calbindin (CB), 99, 104
ATP, 124125, 156, 176, 208209, 211, 215 calretinin (CR), 104
ATR kinase, 199 Calcium-induced calcium release (CICR), 122, 125, 128
Autism, 94, 128 Calcium signaling, 123, 125128
Autophagic cell death, 214

H. Ulrich (ed.), Perspectives of Stem Cells, 285

DOI 10.1007/978-90-481-3375-8,  c Springer Science+Business Media B.V. 2010
286 Index

Calcium waves, 120, 125126 Dedifferentiation, 5455, 151, 153, 183

frequency of calcium waves, 120 Deep brain stimulation (DBS), 246
Callcium oscillations in astrocytes, 159 Dendritic cell markers, 180
Camillo Golgi, 146 Dentate gyrus, 1314, 1621, 100, 104, 116, 123, 149, 153,
Cancer stem cells (CSCs), 80 182, 232, 256, 263
Caspase, 179180, 213215, 217 Developmental neural diseases, 128
Catecholaminergic, 53 Dibutyryl cyclic AMP (dbcAMP), 247, 250
Caudal ganglionic eminence (CGE), 105 Dickkopf, 49
CD34, 233, 240 Dlx1/2, 105
CD36 scavenger receptor, 180 DNA damage signaling, 7779, 214
CD45, 233238, 241, 272 Dogma of neuroscience; dogma in neuroscience, 12, 116,
Cdc2, 38, 76, 201 256
Cdc25A phosphatase, 78, 211 Dopamine (DA), 102103, 124, 203, 207208, 211, 246247,
Cdk4, 75, 197 251, 264, 278
Cdk6, 75, 77, 197 Dopaminergic, 18, 25, 52, 9899, 102104, 207208, 247,
Cell-based therapy, 74, 8283, 98, 102, 262, 265 277278
Cell cycle, 14, 18, 7476, 92, 122, 139140, 148, 150, Downs syndrome (trisomy 21), 79
193212, 215217, 256, 259260
Cell cycle checkpoints, 7476, 199
E
Cell-cycle machinery, 197, 199
E2F-DP, 7577
Cell division, 12, 14, 3839, 61, 7678, 8081, 93, 117, 121,
Ectoderm explants (animal caps), 23, 8
148, 193197, 208, 217, 256259
Embryogenesis, 55, 6167, 74, 100102, 106, 124, 175, 193,
asymmetric division, 14, 80, 117118, 136, 148, 195
239, 275
symmetric division, 14, 117, 135136
Embryoid bodies (EB), 100102, 106, 107
Cellular replacement, 18, 24, 53, 55, 103104, 107, 261,
Embryonal carcinoma (EC) cells, 79, 118120, 125
263264, 271281
Embryonic development, 7, 12, 17, 59, 61, 105, 116, 149, 204,
CENP-E, 75, 80
206
Chemokines, 2022, 180181, 183, 200, 233, 238, 240, 265
Embryonic stem (ES) cells, 15, 24, 53, 5969, 7383, 97110,
Chick embryo, 2, 4, 78
119120, 161, 211, 234, 245251, 260, 263, 277, 279
Chk1/2, 78, 199
Endonuclease (EN), 88, 91, 94
Cholecystokinin (CCK), 104
Engrailed-1, engrailed 1/2, 103, 247
Cholinergic, 53, 9899, 106, 109110, 121122, 125126, 203,
Enkephalin, 209210
276278
Epidermal growth factor (EGF), 20, 39, 101, 103, 119, 125,
Chordin, 2, 48, 49
150151, 153, 159, 200201, 211, 259260
Chromatin remodeling, 93
Epigenetic, 88, 93, 95, 155, 163, 182, 192, 259260
Chromosomal aberrations, 7475
Epigenetic gene silencing, 155
Ciliary epithelium (CE), 194
Epilepsy, 1820, 22, 25, 98, 104, 106, 163
Ciliary neurotrophic factor (CNTF), 120, 150, 154155, 211,
5-ethynyl-2-deoxyuridine (EdU), 256, 258
260
Evolution, 89, 92, 94, 163, 262
Colony stimulating factors (CSF), 175177, 179, 232
Evx1/2, 105
Compensatory neurogenesis, 19
Excitatory neurotransmisson, 122
Controlling elements, 9293
Excitotoxicity, 104105, 276
Cross-differentiation/transdifferentiation, 15, 55, 194, 257258,
Experimental allergic encephalomyelitis (EAE), 265266
272, 276277
Extracellular matrix components (ECM) in neurite outgrowth,
CXCR4, 2122, 233, 238241
42, 64, 157, 159, 160, 162, 164, 176177, 180, 259,
Cyclin
279
cyclin A, 7577
Extrinsic factors, 116118, 121, 128, 194, 205, 214
cyclin D, 75, 77, 197198
cyclin E, 7578, 197198
Cyclin-Cdk complexes, 75, 77, 199 F
Cyclin-dependent kinase inhibitors (CKI), 75, 79, 139, 198 Fetal derived striatal neurons, 106
Cyclin-dependent kinases (CDK), 7577, 139, 197199, 201, Fetal neural stem cells, 25, 256
211, 216217 Fibroblast growth factor (FGF), 4, 20, 38, 42, 100, 119, 150,
Cytokeratins, 37, 3940, 6062 181, 199, 247, 259
Cytokines, 2024, 75, 78, 154, 176, 179181, 194, 199203, FGF20, 247
210, 247, 259, 272, 274275, 278280 FGF4, 99, 108
FGF8, 38, 99, 102103, 105106, 108, 247, 249250
D Follistatin, 2, 4, 7, 49
Dbx1, 105 FOM-1, 82
Dbx2, 105 Fractalkine, 180181
Index 287

G Hormones, 20, 93, 116, 120, 150, 159, 177, 179

GABA, 25, 9899, 101, 104106, 116, 120122, 124, 126, 128,
156, 206208, 239, 247
GAD67, 99, 106, 109
GAP-43 (growth associated protein of 43-kDa molecular mass),
37
Gata2, 99, 108
Genetic mosaicism, 89
Genome integrity, 7476
Genomic shocks hypothesis, 93
GFAP (glial fibrillary acid protein), 16, 60, 6364, 66, 6869,
137152, 154158, 183, 238, 240
Glaucomatous neurodegeneration, 192
Glia-induced synaptogenesis, 161162
Glial (cell line)-derived neurotrophic factor (GDNF), 25, 99,
103, 107, 175, 181182, 247, 249250, 277
Glial tumors; gliomas, 6768, 174, 183
Gliogenic differentiation potential, 50
Gliotransmitters, 159, 163
Globose basal cells (GBC), 3637, 3940
Glutamate, 21, 102, 104, 116, 120, 122124, 126, 128, 137,
148, 156158, 162, 181, 204206, 273, 276
Glutamate-aspartate transporter (GLAST), 148150, 154156
Glutamate receptors, 122124, 204205
Glutamate transporter-1 (GLT-1), 156158
Glutamic acid decarboxylase (GAD), 104, 126, 206
Glutamine synthase, 148149
Glycine, 206207
GM-CSF, 179
GNMFCO progenitors, 51, 5354
G-protein-coupled inwardly rectifying potassium channel
(Girk), 2, 247
Group II introns, 94
Growth factors, 156158, 199203
See also Basic fibroblast growth factor (bFGF), Brain
derived neurotrophic factor (BDNF), Epidermal growth
factor (EGF), Fibroblast growth factor (FGF),
Hepatocyte growth factor (HGF), Insulin-like growth
factor (IGF), Nerve growth factor (NGF), Transforming
growth factor beta (TGF-)
Gsh2, 148
H
Hanging drop cell suspension, 119
Hayflick limit, 81
HB9, 99, 107
Hedgehog (Hh), 100101, 201202
Hematopoietic stem cells (HSC), 15, 174, 234, 238, 258
Hensens Node, 56
Hepatocyte growth factor (HGF), 183, 237239
Hes1, 137141
Hes3, 137138
Hes5, 137138
Hippocampal development, hippocampal formation, 17, 121,
123, 256
Hippocampal subgranular zone, 116
Hippocampus, 1314, 1721, 23, 25, 6667, 100, 104, 106,
109110, 123, 149151, 153, 178, 182, 232
HNK1, 52
Horizontal basal cells, 37
[3 H]-thymidine; tritiated thymidine, 1213, 16, 175176, 196,
204, 209210, 212, 256, 258
Human neural stem cells (hNSC), 25, 256, 264
Huntingtin, 104
Huntingtons disease (HD), 14, 104, 106, 163, 264
6-hydroxy dopamine (6-OHDA), 247, 250
I
ImageStream system (ISS) analysis, 234237
Immune response, 20, 23, 272, 276
Immune system, 20, 24, 88, 163, 182, 259
innate immune response, 23
Induced pluripotent stem (iPS) cells, 99, 107, 246247, 251,
257, 260
Inflammation, 2023, 103, 107, 125, 180181, 202, 215,
263266, 273, 275
inflammatory cytokines, 21, 180182, 278279
Inflammatory mediators, 274275, 280
Inner cell mass (ICM); inner cell mass of the blastocyst, 61,
119, 257
Inositol 1, 4, 5-trisphosphate (IP3), 125, 128, 203204, 209210
Insulin-like growth factor (IGF), 20, 22, 99, 199200, 210
Interferon gamma (IFN), 21, 22, 179180
Interkinetic nuclear migration (INM), 147, 193, 196197, 203,
211212
Interleukin-1 (IL-1), 21, 180, 182, 275
Intermediate filament (IF), 5969, 148, 158, 260, 276
Interneurons, 16, 25, 98, 101, 104106, 109, 137, 206, 263
-internexin, 60, 62
Intracellular calcium transients, 118, 127
Invasion routes (microglial precursors), 175
Irx3, 105
Isl1/2, 105
J
Jak/STAT pathway, 154155
Joseph Altman, 12, 116
Junk DNA, 88
K
Kainate receptors, 122123, 204205
Karyotypes, 76, 8182, 248
Karyotypic instability, 76
Keratin
keratin 5, 37
keratin 14, 37
Kinetochore-associated mitotic checkpoint machinery, 75
L
L1- enhanced green fluorescent protein (EGFP) transgenic
animal, 89
L-aromatic amino acid decarboxylase (AADC), 103
Lbx1, 105
L-DOPA, 102103, 207208, 246, 264, 278
Leukemia inhibitory factor (LIF), 154, 183, 211, 237239,
259260, 275
Lewy Body, Lewy Bodies, 108109, 246
LeX, 151152
288 Index

LFA-1/ICAM-1 (lymphocyte function-associated Mller glial cells; Mller cells, 146, 149, 161, 176, 193194,
antigen-1/lymphocytes/ intercellular adhesion 200, 202203, 205
molecule-1) adhesion system, 175 Multiple sclerosis (MS), 265
Lhx1, 105 Multipotency, 14, 55, 63, 149, 255266
Lhx2, 38 Multipotent neurospheres, 152, 182
Lhx5, 105 Multipotent somatic stem cell, 257
Lhx6, 105, 109 Muscarinic acetylcholine receptor (mAChR), 121122,
Lhx8, 99, 109 203204
Lim1/2, 105 Myc, 49, 201, 251, 260
LINE-1 (Long Interspersed Nucleotide Elements-1, or L1)
retroelements, 8895 N
Lipid mediators of inflammation, 181, 203 Nanog, 7980, 123, 240
Lmx1a, 99, 103, 108, 247 Necrosis, 2021, 67, 180, 213215, 273275, 278
Lmx1b, 99, 105, 108, 247 Necturus, 48
Lysophosphatidic acid (LPA), 160 Nerve growth factor (NGF), 42, 109, 124, 150, 160, 181182,
Lysosomal storage diseases (LSD), 264 261, 280
Nestin, 60, 6269, 102, 119, 124, 148, 150, 152, 154, 155, 183,
M 210, 238, 240, 260, 276
Macroglia, 146, 172 Neural crest cells (NCC), 4855, 6465, 68
Mad1, 75 Neural crest (NC), 4755, 6465, 68
Mad2, 75, 77 Neural crest (NC) stem cells, 5055
Mad3, 75 Neural fate, 24, 6, 8, 163
Mash1 (mammalian achaete-scute homolog 1), 3739, 105, neural default fate, 3
119, 136139, 247 Neural fold (NF), 4849, 60, 62, 182
Math1, 105, 119 Neural inducer, 2, 4, 68
Math3, 137139 Neural induction, 18, 100, 124, 182, 247
Medial ganglionic eminence (MGE), 105, 109, 148 Neural lineages, 15, 101, 238, 257
Meninges, 172175, 183, 247 Neural plate border, 49
Mesectoderm (ectomesenchyme), 48 Neural primordium, 49, 51, 55
Mesenchymal stem cells (MSC), 4851, 5355, 6162, 64, 80, Neural progenitor cells (NPC), 15, 2122, 24, 54, 74, 76,
108, 118119, 121, 128, 173, 237, 239, 247, 271281 98103, 108, 116122, 128, 135141, 147, 151,
Mesoderm, 2, 5, 4849, 6162, 6466, 100101, 118119, 182183, 194, 200, 212213, 257, 259260, 279
174175, 183, 247, 273, 277 Neural stem cells (NSC), 12, 1416, 21, 2426, 52, 55, 63, 67,
Metabotropic glutamate receptors (mGluR), 122124, 128, 157, 88, 91, 98, 100, 102, 117118, 147152, 163, 182, 211,
204 240, 255266, 276279
Metachromatic leukodystrophy, 174, 183 Neural tube, 48, 5051, 53, 101102, 105, 107, 116117,
1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), 103, 135136, 141, 147148, 153, 155, 193, 196
247248, 264 Neurite outgrowth, 21, 122125, 159, 265
Meteorin, 153 Neuroblastic layer (NBL), 193, 195196, 198, 202204,
Michael Kaplan, 13, 116 206207, 209, 212, 215, 217
Microenvironment, 25, 121, 149, 151, 179, 182, 184, 194195, Neuro D, 119
258259, 262265, 272, 276277 Neurodegenerative diseases, 2425, 4041, 98, 147, 154,
Microglial differentiation, 176177 163164, 264265
Microglial neurotoxic factors, 179181 Neurofilaments (NF), 52, 60, 62, 66, 68, 122, 183, 200
Microglial precursors, 173176 Neurogenesis, 1126, 3542, 52, 54, 64, 76, 80, 88, 93, 100,
Microglia; microglial cells, 172183, 256 102, 105, 116125, 128, 147154, 182183, 193, 195,
development, 174175, 177178, 183 202204, 232, 256261, 264, 279
origin, 172174 Neurogenic astrocytes, 149152
Mitogens, 39, 5455, 64, 75, 106, 157, 175, 178, 183, 201202, Neurogenic regions, 1317, 19, 147, 152
208, 210211, 259260 subgranular zone of hippocampal dentate gyrus, 13, 16, 17,
MNR2, 107 100, 116, 149, 232, 263
Mobile introns, 94 subventricular zone, 1314, 16, 52, 100, 116, 122123, 149,
Morphogens, 3, 38, 54, 79, 100, 116, 120121, 150, 183, 202, 176, 183, 232, 263, 278
208, 238, 258, 275, 278 Neurogenin (Ngn), 3738, 136
Motor neurons, 98, 101, 105108, 275277 Ngn1, 3739, 105, 137
Mps1, 75 Ngn2, 137, 139141
MS5 cells, 99, 247, 250 Neurological disorders, 19, 102, 119, 128, 147, 158, 163164,
Msx1, 4999, 103, 247 261, 266
Msx2, 49 Neuromodulators, 159, 162, 194, 203210
Neuron-restrictive silencer element (NRSE), 92
Index 289

Neuropeptides, 209210 PA6 cells, 247

Neuropeptide Y (NPY), 20, 104, 209, 210 Paracrine, 24, 120, 127128, 202
Neuroregulin (NRG)-ErbB2 signaling, 151, 153 Parkinsons disease (PD), 18, 98, 128, 245251, 264, 277278
Neurosphere, 14, 25, 41, 101, 119, 121, 123, 125, 151152, Parvalbumin (PV), 25, 104105
182, 200, 202, 232, 238239 Pax2, 105
Neurotensin, 178 Pax3, 49, 278
Neurotransmitter, 13, 20, 25, 53, 101, 104, 107, 115128, 152, Pax6, 39, 99, 101, 105, 107, 109, 119, 148, 151
156158, 163, 194, 203211 Pax7, 49, 105, 278
Neurotransmitter choice, 53, 126 Peripherin, 60
Neurotransmitter receptors, 116, 118, 120, 128, 156, 163 Pet1, 99, 108
Neurotrophic factors, 20, 25, 42, 103, 120, 150, 161, 175, Phenotypic fate, 116, 256
178179, 181182, 200, 211, 247, 260262, 265, Phospholipase C-, 128
276277, 279280 Photoreceptor precursors, 212
Neurotrophin (NT), 42, 106, 161, 175, 181182, 200201, 247, Phox2b, 108
261, 275276, 280 Po Del Ro Hortega, 146
Neurturin, 247 Pitx3, 99, 103, 108, 247
Neutralizer, 154, 177, 261, 274 Platelet-Activating Factor (PAF), 181, 202203, 211
Nicotine, 121122 Pluripotent, 4748, 5053, 55, 77, 80, 98, 100, 106107, 119,
Nicotinic acetylcholine receptor (nAChR), 121122, 203 232, 237239, 240, 251, 257258, 272273
Nkx2. 1, 99, 105, 109 Polydactyly, 202
Nkx2. 2, 107 Primordial germ cells (PGC), 239
Nkx6. 1, 99, 107 Programmed cell death, 19, 193, 195, 212213, 215217
N-methyl-D-aspartic acid (NMDA) receptor, 122123, 125, Progressive lineage restriction, 194
128, 157, 204205 Protein kinase A (PKA), 200201, 211
Noggin, 2, 4, 68, 49, 150151, 249250, 278 Psd-93, 88, 92
Notch, 38, 54, 100, 135141, 150, 153, 155, 183, 197, 201202 Ptf1, 105
Nottebohm, 1213, 19, 116, 149, 256 Purinergic neurotransmission, 124
NSCL2, 119 Purinergic receptor, 116, 124125, 157, 209, 211
NSC-scaffold (neural-stem cell scaffold), 265 Purines, 116, 124126, 128, 157, 209, 211
Nurr1, 99, 103, 247
Q
O Quail brain, 176
Oct-4, 82, 123, 233240 Quail embryo, 49, 51
Odorant receptors, 36, 3839
Olfactory bulb, 13, 16, 18, 3638, 4142, 150, 178, 232, 247, R
256, 259 Radial glial cell; radial glia (RG), 17, 65, 67, 101, 117118,
Olfactory ensheating cells, 37, 4142 125, 135136, 138, 146149, 152155, 164, 195
Olfactory epithelium, 16, 3542 Radial glia (RG)-astrocyte transition, 152
Olfactory sensory neurons, 3537 Radial migration, 176
Olig2, 99, 107, 148, 240 Ramified microglial cells, 172, 178179
Oligodendrocyte (Olg) development; oligodendrocyte Ramon y Cajal, 12, 87, 116, 146, 256
differentiation, 182183, 259 Raphe nuclei, 108
OMP (olfactory marker protein), 3740 RCA-1 (Ricinus communed agglutinin-1), 174
Opioid peptides, 209 Regeneration of damaged neural tissue, 159, 232
Organizer, 2, 47, 100, 103 Regenerative medicine, 49, 232, 261, 263, 276
Oscillatory expression, 139141 Regulation of gliogenesis, 182
OST (olfactory sulfotransferase), 37, 40 Resting microglia (ramified microglia), 178179
Oxidative injury, 179 Retina, 14, 63, 6566, 137, 146, 148149, 161, 163, 175176,
178179, 181, 192213, 215217
P Retinal development, 193194, 197198, 201, 204, 206207,
p16, 75, 77, 198 209, 210
p18, 75, 198 Retinal dystrophies, 192
p19 mouse embryonic carcinoma cell line, 119 Retinal progenitor cells (RPC), 137, 191218
p21, 7578, 198, 211 proliferation, 194, 199203
p27 glial fibrillary acidic protein, GFAP, 7577 survival, 212
P2X receptors, 209 Retinoblastoma (tumor suppression) protein (Rb), 75, 197, 201,
P2Y receptors, 124, 209, 210 214
P2Y1 receptors, 125 Retinoic acid (RA), 38, 101, 119, 150, 176, 211, 276277
P2Y2 receptors, 125 Retinopathy, 203
p53, 7881, 214, 216217 Retrotransposition, 8794
290 Index

Retrotransposon, 89, 9394 Telomerase-deficient mice, 81

Intracisternal A-particle (IAP) retrotransposon, 89 Telomeres, 8081
L1 retrotransposon, 8892, 94 Temporal lobe epilepsy (TLE), 20, 104
Reverse transcriptase (RT), 88, 94 Teratocarcinoma, 98, 119
reverse transcriptase (RT) phylogeny, 94 Teratoma, 248
Rostral migratory stream, 16, 151, 256 Thrombospondin (TSP), 162, 176, 182
RRMP2, 76 Thyroid hormones (TH), 177178
Ryanodine, 121122, 125 TN-C, 148
Totipotent, 257
S Transdifferentiation/cross-differentiation, 15, 55, 194, 258259,
S100, 148, 150152, 154, 156, 161162 272, 276277
Sca-1, 233238 Transforming growth factor beta (TGF-), 3, 151, 154
Schizophrenia, 89, 9394, 102, 104, 108, 163 Tripartite synapse, 161
Secondary germinal zones, 263 Trisomic zygotic rescue, 79
Selfish DNA, 89, 94 Trisomy 21 (Downs syndrome), 7980
Serotonergic, 9899, 108109 Trophic factors in CNS, 150
Serotonin (5-hydroxytryptamine, 5-HT), 208 3-tubulin, 123
Smad1, 78, 137 Tumor, 13, 2021, 24, 6769, 7475, 8081, 98100, 119, 180,
Somatostatin (SST), 104, 209 197, 200202, 214, 248251
Sonic hedgehog (Shh), 38, 54, 102, 119, 201, 247 Tyrosine hydroxylase (TH), 102, 126, 207, 247, 264
Sox2, 67, 88, 91, 149150, 152, 240, 251
Sox3, 67 U
Sox9, 4950, 54, 183 Unorganized chromatin (euchromatin), 233
Spinal cord injury (SCI), 42, 98, 261264, 271281 Untranslated region (UTR), 9092
Spinal cord lesions, 98
Spinal muscular atrophy (SMA), 105 V
Spindle assembly checkpoint (SAC), 75 Vasoactive intestinal peptide (VIP), 104, 209
SSEA-1, 82, 233234, 239 Very small embryonic like stem cells (VSELs), 233241
SSEA-4, 233 Vesicular monoamine transporter 2 (VMAT2), 103
Stat3, 141, 183, 202, 211, 276 Vimentin, 6063, 6668, 148150, 158
Stem cell therapy, 24, 107, 264265, 276, 281 Vitamin B12, 247
Stemness genes, 260261 Vitronectin receptor, 180
Stroke, 14, 174, 183, 232, 238241, 265266, 276, 278 VSEL-derived spheres, 239
Stromal derived factor-1 (SDF-1), 22, 237240
Subgranular zone (SGZ), 13, 1617, 100, 116, 149, 232, 263
W
Substantia nigra (SNi), 18, 102, 246247, 264, 278
Wnt, 8, 49, 54, 99100, 102103, 106, 119, 151, 153, 201202,
Subventricular zone (SVZ), 1314, 16, 52, 100, 116, 122123,
250
149, 176, 183, 232, 263, 278
Wnt/catenin signaling, 54, 153
Syncoilin, 60
Synemin, 60, 6268
X
Xenopus laevis, 2, 49
T
Tangential migration, 176
Target Primed Reverse Transcription (TPRT), 91 Z
Telomerase, 8081, 247, 261 Zic1, 49