INDEX

 Introduction
 Summary Points
 Haematopoietic Stem Cells
 Applications of cultured HSCs
 HSCs and Gene Therapy
 Non- Haematopoietic Stem Cells
 Conclusions
 References
 STEM CELL TECHNOLOGY
INTRODUCTION
Stem cell technology is a rapidly developing field
that combines the efforts of cell biologists,
geneticists, and clinicians and offers hope of
effective treatment for a variety of malignant and
non-malignant diseases. Stem cells are defined as
totipotent progenitor cells capable of self-renewal
and multilineage differentiation. Stem cells survive
well and show stable division in culture, making
them ideal targets for in vitro manipulation.
Although early research has focused on
haematopoietic stem cells, stem cells have also been
recognised in other sites. Research into solid tissue
stem cells has not made the same progress as that on
haematopoietic stem cells. This is due to the
difficulty of reproducing the necessary and precise
three dimensional arrangements and tight cell-cell
and cell-extracellular matrix interactions that exist in
solid organs. However, the ability of tissue stem cells
to integrate into the tissue cytoarchitecture under the
control of the host microenvironment and
developmental cues makes them ideal for cell
replacement therapy.
Summary points
 Stem cells are progenitor cells that are
capable of self-renewal and
differentiation into many different cell
lineages.
 Stem cells have potential for treatment
of many malignant and non-malignant
diseases.
 Peripheral blood stem cells are used
routinely in autologous and allogeneic
bone marrow transplantation.
 Gene transfer into haematopoietic stem
cells may allow treatment of genetic or
acquired diseases.
 Embryonic stem cells may eventually
be grown in vitro to produce complex
organs.
 Neuronal stem cells are being used for
neurone replacement in
neurovegetative disorders such as
Parkinson's and Huntington's diseases.
Haematopoietic stem cells
APPLICATIONS OF CULTURED
HAEMATOPOIETIC CELLS

Haematopoietic stem cells are a somatic cell

population with highly specific homing properties
and are capable of self-renewal and differentiation
into multiple cell lineages. Human haematopoietic
progenitor cells, like stromal cell precursors in bone
marrow, express the CD34 antigen, a transmembrane
cell surface glycoprotein identified by the My10
monoclonal antibody. However, pluripotent stem
cells constitute only a small fraction of the whole
CD34+ population, which is by itself rather
heterogeneous regarding phenotype and function.

The best way to define haematopoietic stem cells is

from their functional biology. They are known to
restore multilineage, long term haematopoietic cell
differentiation, and maturation in lethally cytoablated
hosts. Haematopoietic stem cells can be obtained
from bone marrow, peripheral blood, umbilical cord
blood, and foetal liver.
The use of peripheral blood stem cells in both
autologous and allogeneic transplantation has
become routine as they can be collected on an
outpatient basis and also promote a consistent
acceleration in haematopoietic reconstitution
after engraftment. Umbilical cord blood stem
cells have been used progressively in paediatric
patients, from both related and unrelated Human
leukocyte antigen (HLA)-matched donors.
In recipients with severe T cell
immunodeficiency disorders, fast engraftment is
required together with a low risk of graft versus
host disease and a low viral transmission rate.
Since umbilical cord blood stem cells can be
expanded in vitro or frozen for storage in cell
banks they have been used in clinical trials for
both autologous and allogeneic haematopoietic
stem cell transplantation.
The bone marrow is a mesenchyme derived
tissue consisting of a complex haematopoietic
cellular component supported by a
microenvironment composed of stromal cells
embedded in a complex extracellular matrix.
This extracellular matrix has an important role
in the facilitation of cell-to-cell interaction, in
addition to a more complex role in the binding
and presentation of cytokines to the
haematopoietic progenitor cells.
The cytokine milieu and extracellular matrix
interaction provides the “road map” for
maturation and differentiation of stem cells,
which should be instrumental for their in vitro
manipulation before therapeutic use. For
example, haematopoietic stem cells can be
manipulated in vitro to generate dendritic cells,
the most potent antigen presenting cells.
Dendritic cells have a pivotal role in the elicitation
and regulation of antigen specific, major
histocompatibility complex-restricted T cell
responses and are thought to be the only antigen
presenting cells able to prime naive T cells. Dendritic
cells can be derived from CD34+ precursors in
response to granulocyte macrophage colony
stimulating factor and tumour necrosis factor α and
from monocytes cultured with granulocyte
macrophage colony stimulating factor and
interleukin-4.
In vitro generated dendritic cells that have been
transduced with genes coding for tumour specific
antigens or pulsed with tumour specific antigen or
peptide could be useful for induction of cytotoxic T
cell responses. Dendritic cell tumour vaccines could
be important future therapeutic tools; phase II
clinical trials are under way and show limited
efficacy. On the other hand, the migration and
function of dendritic cells derived from liver in an
allogeneic environment may be seminal in the
development of donor specific tolerance. Genetic
engineering of dendritic cells to express
immunosuppressive or immunoregulatory molecules
may provide a novel method to promote graft
tolerance, reducing dependence on systemic
immunosuppression.
HAEMATOPOIETIC STEM CELLS AND
GENE THERAPY
Haematopoietic stem cells themselves are also a
promising target for gene therapy. Haematopoietic
stem cells have been made resistant to one or more
cytotoxic drugs with retroviral transfection of the
multidrug resistance gene (MDR1). This should help
circumvent the myelosuppressive effects of standard
regimens of chemotherapy. Haematopoietic stem
cells can also be genetically marked to allow
assessment of patterns of cell survival, localisation,
and function after bone marrow transplantation. This
strategy has already been used with retroviral
vectors.
Double genetic marking is also being used to
determine the long term effects of different protocols
of cytokines given to promote bone marrow
regeneration after cytoablative treatment. Gene
transfer into haematopoietic stem cells represents a
novel approach for treating some genetic or acquired
diseases. So far, transduction of genes into humans
using haematopoietic stem cells has shown low
efficiency, especially in the quiescent stem cell
population.
Recent advances in reproductive biology and
gene therapy have used ex vivo transduced
autologous umbilical cord blood cells or direct
targeting in utero as a potential means to correct
haematopoietic, immunological, and metabolic
single gene disorders. This technique has the
advantage of using normal haematological
development, which induces the foetus to allow
space for a new cell population and promotes
tolerance in the developing immune system.

Unfortunately, infection and graft versus host

disease are still potential risks for both the
mother and the foetus. However, advances in the
understanding of dose requirements and
manipulation of peripheral blood sources to
enrich for stem cells may provide strategies to
overcome these problems.
Figure: Characteristic dendritic cells derived from CD34+
haematopoietic stem cells and propagated in granulocyte
macrophage colony stimulating factor.

Figure: Bone Marrow Stem Cells

Non-haematopoietic stem cells

The adult bone marrow also contains mesenchymal

stem cells which are involved in the regeneration of
mesenchymal tissues such as bone, cartilage, muscle,
ligament, tendon, adipose tissue, and stroma.
Although human mesenchymal stem cells have been
isolated, it remains unclear how basal nutrients, cell
density, spatial organisation, mechanical forces,
growth factors, and cytokines control their
differentiation. Transplantation of mesenchymal stem
cells into tendon defects in rabbits can significantly
improve biomechanical properties of the damaged
area.
Embryonic stem cells were first isolated in 1981
through studies focusing primarily on murine
blastocysts. Embryonic stem cells are derived from
totipotent cells of the early mammalian embryo and
are capable of unlimited, undifferentiated
proliferation in vitro.
Human embryonic stem cells can express high levels
of telomerase activity, comparable with that
expressed by cells isolated from germ lines and
embryonic tissues. They can also form several cell
types and simple tissue.
Further understanding of cell tissue interactions
and their relation with the extracellular matrix may
eventually enable in vitro production of complex
organs. In vitro manipulation of embryonic stem
cells can be enhanced by nuclear transfer and
cloning.
Advances in stem cell technology have stimulated
rapid growth in the understanding of embryonic
and postnatal neural development. A population of
neuronal stem cells capable of extended self-
renewal in addition to subsequent differentiation
into both neurones and glia has already been
identified. These common neurohaematopoietic
stem cells can be isolated from the subventricular
zone in the wall of the lateral ventricle of the
brain. They divide in response to epidermal growth
factor and fibroblast growth factor-2. Transplanted
neuronal stem cells can integrate into an intact
brain and differentiate into neuronal and glial cells.
They may also function as haematopoietic stem
cells when infused into irradiated mice.
This last finding is controversial and may
represent an artefact of the experimental
technique, since only one contaminating
haematopoietic stem cell would be needed to
repopulate a mouse haematopoietic system.
However, the reverse has also been shown, with
stem cells derived from bone marrow entering
through the brain-blood barrier, becoming fully
differentiated and displaying macrophage-like
function (microglia).
Initial clinical trials have shown that neurone
replacement for neurovegetative diseases such as
Parkinson's and Huntington's diseases is now
feasible. Reports of transplantation of foetal
striatal tissue in patients with mild to severe
Huntington's disease suggest that transplantation
of neuronal stem cells may improve some of the
cognitive symptoms associated with the condition,
as well as potentially modifying its clinical course.
Neuronal stem cells can also be manipulated
before grafting; they have been shown to respond
to stimulation with fibroblast growth factor-2, and
immortalised neural stem-like cells infected with
viral vectors have been found to express factors
that are related to neural repair.
The existence of prostate stem cells is still a matter
of debate. Nevertheless, the concept is interesting;
mainly because of the possible analogy between
effects of androgens on the prostate stem cells and
that of cytokines on haematopoietic stem cells.
Prostate stem cells could have important
implications in the development of prostatic
carcinoma.
Understanding of liver regeneration has improved
greatly since the initial description of oval cells as
progeny of facultative stem cells. Hepatic stem
cells, which are found in the interlobular bile ducts
and possibly also in the canals of Hering, could
have a large impact on the pathophysiology of
hepatocellular carcinomas and
cholangiocarcinomas.
Limbal stem cells have been described at the
junction between the cornea and sclerae. These
cells are known to be progenitors of corneal
epithelium. Keratolimbal allografts are a
promising treatment for bilateral blindness, and
limbal stem cells can now be safely obtained from
living related donors.
Figure: The localization and cell surface markers for liver
stem/progenitor cells (LSPCs).

Figure: Potential hepatic stem cells reside in EpCAM+ cells of

normal and injured mouse liver
 Conclusions
Stem cell technology is progressing as the result of
multidisciplinary effort, with clinical applications of
manipulated stem cells combining developments in
transplantation and gene therapy. There are rather complex
ethical issues related to the applications of cloning and
nuclear transfer in human stem cells. Successful ex vivo
manipulation of stem cells will depend on improved
understanding of the interactions between cytokines and the
extracellular matrix. Cytokines may decrease binding forces
between stem cells and components of the stromal
microenvironment, thus facilitating the migration of stem cells
into the peripheral blood. Improvements in mobilisation
schedules using growth factors, stem cell isolation, and
purification procedures and techniques for both positive and
negative purging (to reduce tumour cell contamination or to
deplete T lymphocytes) are emerging. The possibility of ex
vivo multiplication of stem cells to accelerate haematopoietic
recovery or to provide sufficient stem cells from one
extraction to support several cycles of high dose
chemotherapy is under investigation. The applications for
autologous stem cell transplantation should increase as it
avoids the use of non-specific immunosuppressive therapy.
Peripheral blood stem cells have advantages over bone
marrow cells for autologous transplantation in that they show
consistently faster haematopoietic reconstitution and can be
collected on an outpatient basis.
Stem cells originating from solid tissue can potentially be
applied in tissue repair. Techniques by which new genetic
material is introduced into stem cells are being developed, and
may lead to the cure of various inherited diseases by somatic
gene therapy.