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CHAPTER 23

Stem Cells
INTRODUCTION
ew issues in cell biology have garnered more public attention in recent years than stem
F cells. Research into stem cells holds the promise for novel therapeutic interventions
that could revolutionize the treatment of a range of human diseases. On the other hand,
objections to the use of human embryonic stem cells for research have been raised on
religious, moral, and ethical grounds. While keeping in mind these two important aspects
of the ongoing debate, scientists have steadily learned to exploit the potential of stem
cells for unraveling the molecular mechanisms of cell differentiation and organismal
development. This chapter presents a comprehensive collection of protocols describing
the use of stem cells to study cell differentiation.

Stem cells have the dual ability to undergo self-renewal and to generate lineages of more
differentiated or mature cells. UNIT 23.1 is an overview of the current knowledge on stem
cells and their pathways of differentiation. The overview describes the properties of stem
cells, defines the meaning of totipotent, multipotent, pluripotent, and unipotent cells, and
discusses the different cell lineages that can be derived from various stem cell types.
The discussion deals with both embryonic and adult stem cells and gives examples of
their use for tissue repair. The overview ends with two sections dealing with the genetic
manipulation and potential therapeutic applications of stem cells.

UNIT 23.2 comprises a series of protocols for the production and handling of embryonic
stem cells. The initial protocols describe procedures for the derivation, culture, and
preservation of mouse embryonic stem cells. This is followed by a description of methods
for the differentiation of mouse embryonic stem cells into “embryoid bodies,” which are
spherical masses of cells with regions of lineage-specific differentiation. This unit also
includes protocols for the propagation of human stem cells and their differentiation into
embryoid bodies, though not for their initial derivation.

UNIT 23.3 begins a series of units devoted to the differentiation of embryonic stem cells into
various lineages. This unit deals specifically with the maintenance and differentiation
of mouse embryonic stem cells to generate blood vessels. These vessels form early
during embryonic development by two processes: vasculogenesis and angiogenesis. In
vasculogenesis, mesodermally derived angioblasts differentiate to form primitive blood
vessels. This is followed by angiogenesis, in which endothelial cells derived from the
angioblasts proliferate and migrate, leading to vessel expansion and sprouting. Both of
these processes can be studied by differentiation of embryoid bodies over a period of
8 days. Blood vessels can be identified by staining for various vascular markers (e.g.,
PECAM-1) or by performing the β-galactosidase reaction on differentiated cultures
generated from mouse embryonic stem cells in which the lacZ gene is driven from a
vascular-specific promoter.

UNIT 23.4 deals with the differentiation of mouse embryonic stem cells and human adult
stem cells into adipocytes. Adipocytic differentiation of mouse embryonic stem cells
also starts with the formation of embryoid bodies, which are first committed by the
addition of retinoic acid and then terminally differentiated by the addition of adipogenic

Stem Cells

Current Protocols in Cell Biology 23.0.1-23.0.2, September 2007 23.0.1


Published online September 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471143030.cb2300s36 Supplement 36
Copyright C 2007 John Wiley & Sons, Inc.
factors (e.g., insulin, triiodothyronine, rosiglitazone). Adipocytes can also be derived
from adult human multipotent adipose-derived stem (hMADS) cells or human mes-
enchymal stem (hMS) cells. For both of these adult stem cell types, differentiation does
not require retinoic acid and is achieved in monolayer culture. hMADS are driven to
adipogenic differentiation by addition of isobutylmethylxanthine (IBMX) and dexam-
ethasone, whereas adipogenic differentiation of hMS occurs simply in the presence of
fetal bovine serum. The resulting adipocytes can be easily identified through visualiza-
tion of their lipid droplets by bright-field microscopy of unstained cells or of cells stained
with the triglyceride-specific stain, Oil Red O. Adipocytic differentiation can also be
monitored by northern analysis of the expression of adipose-specific genes.

UNIT 23.5 presents protocols for the differentiation of mouse embryonic stem cells to
chondrocytes and osteocytes. The process also starts from embryoid bodies, which spon-
taneously differentiate into various cell types, including chondrogenic and osteogenic
precursors. Mesenchymal condensations containing chondrogenic precursors and, at later
stages of differentiation, cartilage nodules with mature chondrocytes and osteocytes, are
isolated using either a microdissector or a microscalpel. Differentiated cells can be stud-
ied either in the aggregates or upon dissociation with collagenase followed by monolayer
culture. Samples can be analyzed by histochemical staining, immunofluorescent staining,
in situ hybridization, or RT-PCR, to test for the expression of various markers for carti-
lage or bone. The characteristic features of ES cell-derived mesenchymal condensations
and cartilage nodules can also be visualized by light microscopy and further examined
at the ultrastructural level by electron microscopy.

UNIT 23.6 features protocols to study the differentiation of human embryonic stem cells into
hematopoietic progenitors and endothelial cells for coculture with bone marrow stromal
cell lines. Cells belonging to both lineages can be derived simultaneously and charac-
terized by immunostaining and flow cytometry. A colony-forming assay can be used to
examine the potential of hematopoietic progenitors to differentiate into erythroid, gran-
ulocytic, macrophage, and megakaryocyte cells. The various cell lineages produced by
differentiation of human embryonic stem cells can be isolated, as well as separated from
the bone marrow stromal cells, by magnetically-activated cell sorting and preparative
flow cytofluormetry.

UNIT 23.7 describes protocols for neural differentiation of human embryonic stem cells.
Pure populations of neural precursors can be derived from human embryonic stem cells
for differentiation in chemically defined serum-free culture medium containing noggin
and fibroblast growth factor. Neural precursors derived in this way can be cultured for
long periods or further induced to differentiate into mature neurons and glial cells. Other
protocols describe methods for characterizing neural precursors and their derivatives by
immunostaining for specific marker antigens followed by fluorescence microscopy or
flow cytometric analysis of stained cells.

Juan S. Bonifacino

Introduction

23.0.2
Supplement 36 Current Protocols in Cell Biology
Stem Cells: An Overview UNIT 23.1

Stem cells are specialized cells that possess 2002). They have a wide developmental po-
a capacity to undergo self-renewal while at the tential and have been demonstrated to give rise
same time having the ability to give rise to at to every cell type of the adult organism. How-
least one or more differentiated or mature cell ever, they give rise only to a restricted subset of
type. They therefore represent a fundamental their respective extraembryonic lineages rep-
cornerstone during the life of all vertebrates, resented during development. A fourth type
playing central roles in the production of new of stem cell is the totipotent stem cell, which
and replacement cells for tissues during devel- includes the fertilized oocyte and premorula
opment and homeostasis, including repair fol- blastomeres. Totipotent stem cells replicate
lowing disease or injury. Although all tissues and generate all adult and extraembryonic tis-
during vertebrate development appear to pos- sues of their species, but undergo either no
sess cells with stem cell properties, whether or only limited self-renewal. A relatively new
this is the case in the adult remains to be clearly fifth category is the cancer stem cell. These
demonstrated. Certainly, the skin, muscle, in- cells replicate and undergo self-renewal, yet
testine, hematopoietic system, and liver can their compromised self-renewal pathways re-
be regenerated following acute trauma, but it sult in neoplasia with accompanying undiffer-
is only now becoming accepted that similar entiated, partially differentiated, and/or differ-
properties may also be attributable to tissues of entiated cell types.
the brain, heart, and pancreas (Dor and Melton, Much attention has been focused on stem
2004). cells because of their wide potential for thera-
A diverse range of stem cells have been de- peutic applications and their potential for un-
scribed according to their long-term ability to raveling molecular mechanisms of develop-
maintain stem cell–like properties in vitro and ment. They possess the potential to provide
in vivo and the number and type of derivatives replacement tissue or a means to understand
that they give rise to. The first category is the disease mechanisms, thus providing cures
unipotential stem cell, which undergoes self- for numerous diseases and injuries, such as
renewal and gives rise to only one mature cell Parkinson’s disease, spinal injury, cardiac fail-
type. One example is the keratinocyte stem cell ure, and diabetes. Before this is possible, how-
of the dermis that divides to give rise to a pop- ever, and in addition to developing methods to
ulation of closely related keratinocytes. The determine how to differentiate stem cells into
second category is the multipotent stem cell, exactly the right lineage desired at a homoge-
characterized by an ability to undergo self- nous or near-homogenous frequency, a num-
renewal and the capacity to yield at least two ber of important factors need to be addressed.
or more differentiated fetal or adult cell types. Firstly, the ethics of using ES cells need to be
Examples of multipotential stem cells include discussed widely in both the scientific and the
a range of stem cells in the developing fetus general community. Secondly, issues related
and many of the adult stem cells so far de- to the immune rejection of foreign stem cells
scribed, e.g., neural stem cells (NSCs), neural need to be overcome. Thirdly, the issue of ac-
crest stem cells (NCSCs) of the nervous sys- cessibility of stem cells to appropriate regions
tem, and hematopoietic stem cells (HSCs) and of the body must be determined.
mesenchymal stem cells (MSCs) of the bone
marrow. A third category of stem cell is the THE STEM CELL NICHE
pluripotent stem cell, which is also capable of Maintenance of the stem cell phenotype
self-renewal and gives rise to a vast array of in vivo is critically dependent upon its sur-
mature cell types, but not every cell type char- rounding microenvironment or niche. This
acteristic of that species. Examples of pluripo- microenvironment is composed of a base-
tent stem cells are the in vitro–generated ment membrane, extracellular matrix, cell-cell
embryonic germ (EG) cells of the gonads, interactions, cytokines, and other effector or
embryonal carcinoma (EC) cells, which are maintenance proteins. Together, such factors
derivatives of tumorigenic germinal tissue, regulate the stem cell’s balance between self-
and embryonic stem (ES) cells. ES cells are renewal and differentiation. Functional ge-
pluripotent in vitro derivatives of the blasto- nomic attempts to determine the genetic fac-
coel inner cell mass or epiblast (Mollard et al., tors driving the balance between self-renewal
Stem Cells

Contributed by Mark Denham, Brock Conley, Fredrik Olsson, Timothy J. Cole, and 23.1.1
Richard Mollard
Current Protocols in Cell Biology (2005) 23.1.1-23.1.18
Supplement 28
Copyright 
C 2005 by John Wiley & Sons, Inc.
and differentiation in the stem cell niche have enzymes known as telomerases (Fig. 23.1.1).
been recently undertaken (Hackney et al., In the absence of telomerase activity, cells
2002). The fetal liver, for example, provides become subject to age-dependent mortality,
the early microenvironment for expansion of with shortening of the chromosomes until a
its stem cell pool required for the liver in adult threshold senescence limit is reached. At this
life. The cellular constituents of this hepatic point, cells have a reduced replicative potential
stem cell niche were examined by cloning fe- and therefore can no longer divide, an event
tal stromal cell lines that support primitive characteristic of differentiated cell types. In
hematopoietic cells in vitro (Hackney et al., order for stem cells to maintain their stem
2002). Elucidating the genetic mechanisms cell pool throughout the life of an organism
that maintain stem cell populations in their and not reach replicative senescence, a higher
stem cell state will aid in the production of telomerase activity is utilized by many stem
large quantities of stem cells for therapeutic cell populations (Morrison et al., 1996). Germ
applications. cells, cancer cells, and some stem cells, such
as HSCs, express high levels of telomerase
Stem Cell Division (Morrison et al., 1996). However, the amount
The stem cell niche provides an environ- of telomerase expressed in adult stem cells is
ment that maintains cells in an undifferentiated not sufficient to keep the telomeres of chromo-
stem cell state. Local injury in the surrounding somes at the same length. Instead, enough is
tissue releases morphogenic signals to invoke expressed to enable adult stem cells to divide
proliferative and differentiation events within and provide sufficient cells for an organism’s
the niche, allowing tissue repair. This alters life. ES cells, on the other hand, are theoreti-
the balance within the stem cell niche, and cally immortal, and thus presumably produce
through intrinsic mechanisms within the stem enough telomerase such that their telomeres
cell, a switch occurs from self-renewal (sym- never shorten. However, depending upon the
metric division) to a proliferative differenti- specific culture conditions used, it appears that
ation event (asymmetric division), leading to karyotypic abnormalities can occur (Buzzard
the production of more mature cellular deriva- et al., 2004; Draper et al., 2004).
tives.
Asymmetrical cell division produces both THE EMBRYONIC STEM CELL AS
another stem cell and a more mature cell type A PLURIPOTENT STEM CELL
known as a progenitor cell (in hematopoiesis) ES cells can be maintained indefinitely in
or transit-amplifying cell (in other tissues). an undifferentiated pluripotent state in vitro.
Progenitor or transit-amplifying cells often Their capacity to contribute to functional
undergo a more rapid expansion, producing derivatives of all cells of the body is best ex-
fully differentiated cell types with specific emplified by tetraploid embryo complementa-
characteristics appropriate to their function in tion studies in the mouse (Nagy et al., 1990,
the particular tissue. Stem cells generally dis- 1993). Mouse ES cells (mESCs) represent the
play a slower rate of cell cycle progression. best characterized ES cell lines, and in addi-
For example, stem cells of the epidermis cy- tion to their pluripotent properties described
cle slowly, displaying a long-term retention in vivo, have also been induced to differenti-
of the nuclear label bromodeoxyuridine (Cot- ate into diverse cell lineages such as cardiac
sarelis et al., 1990). Asymmetrical cell divi- muscle, neural crest cells, neural stem cells,
sions thus maintain the local stem cell pool and hematopoietic cell lineages in vitro (Fig.
while contributing mature differentiated cells 23.1.2). Human ES cells (hESCs), although
for tissue growth, turnover, or repair. Symmet- less well characterized, are also known to pos-
rical cell division produces either two iden- sess similar properties to mESCs, but they also
tical stem cells or two progenitor or transit- display some different characteristics. Inocu-
amplifying cells. Symmetrical cell divisions lation of hESCs beneath the testicular capsule
therefore maintain, increase, or deplete the of severe combined immunodeficient (SCID)
stem cell pool. mice has produced solid teratomas containing
representative cells from all three embryonic
IMMORTALITY: TELOMERASE germ cell layers, including glandular and squa-
AND STEM CELLS mous epithelia, muscle, bone, and neuroecto-
The terminal ends, or telomeres, of eukary- derm. This represents a test for pluripotency in
otic chromosomes are protected from degra- the human comparable to blastocyst injection
Stem Cells:
An Overview dation, incomplete replication, or fusion by of mESCs in mice (Reubinoff et al., 2000). In

23.1.2
Supplement 28 Current Protocols in Cell Biology
Figure 23.1.1 Eukaryotic cells express telomerase. Telomeres are located at the end of each
chromosome in eukaryotic cells. Telomeres shorten with each cell division. Stem cells express
telomerase, an enzyme that maintains telomere length during division. Maintenance of telomere
length is crucial to the prevention of cell senescence.

addition to the blastocyst, isolation of hESCs cific culture conditions, give rise to a number
has also been reported recently from the inner of distinct differentiated cell types. The isola-
cell mass of human morula (Strelchenko et al., tion of relatively pure populations of several of
2004). With the demonstrated pluripotency of these desired cell types has been subsequently
mESCs in vivo and their amenability to ma- achieved by culture with growth factors to en-
nipulation in vitro, it is believed that hESCs hance certain cell populations, by culture in
may be induced in vitro to differentiate into selection media, or by fluorescence-activated
any desired cell of the body for potential use cell sorting (FACS) for lineage-specific mark-
therapeutically. ers or lineage-specific reporter gene expres-
A common method for differentiating sion. In addition to EB formation, directed
hESCs is embryoid body (EB) formation, differentiation of ES cells into desired lin-
which can be achieved through several dif- eages has been achieved by coculture with
ferent techniques such as suspension culture selected tissues. For example, differentiation
or culture in methylcellulose-containing me- of hESCs into immature cardiomyocyte lin-
dia (Itskovitz-Eldor et al., 2000; Conley et al., eages has been reported following culture on
2004). ES cells cultured in these conditions a cell layer of a spontaneously differentiat-
form spherical structures which are called em- ing visceral endoderm-like cell line derived
bryoid bodies (EBs), which refers to the ap- from the P19 embryonal carcinoma cell line
pearance of lineage-specific regions of cell (Mummery et al., 2002). Similarly, culture of
differentiation and a gene-expression profile hESCs on a layer of γ-irradiated S17 murine
similar to that found in the early embryo. bone marrow stromal cell line or the C166
These EBs contain cells representative of all yolk sac endothelial cell line has been reported
three embryonic germ cell layers (ectoderm, to induce formation of hematopoietic colonies
mesoderm, and endoderm), and, under spe- that give rise to erythroid, myeloid, and Stem Cells

23.1.3
Current Protocols in Cell Biology Supplement 28
Figure 23.1.2 Pluripotency of embryonic stem cells. Embryonic stem cells have been reported
to differentiate into a vast array of cells types in vitro. Differentiated cell types include: germ cells,
respiratory cells, hepatocytes, cardiomyocytes, osteoblasts of the bone, neural cells, skeletal and
smooth muscle, islet cells of the pancreas, keratinocytes, and hematopoietic cells.

megakaryocyte colonies following transfer to strated to specifically innervate muscle. In an-


semisolid media in the presence of hematopoi- other recent example, mESCs were treated
etic growth factors (Kaufman et al., 2001). with the phosphoinositide-3-kinase (PI3-K)
Furthermore, prolonged cultivation of hESCs inhibitor (LY294002) to induce differentia-
to a higher cell density has been shown to in- tion of ES cells into islet-like cells, which
duce the formation of (1) contracting cells that could be transplanted into the pancreas of
stained positively for muscle-specific forms of mice, thereby rescuing several effects associ-
actin and (2) cells possessing elongated pro- ated with induced diabetes mellitus (Lumelsky
cesses that stain positively for neurofilament et al., 2001; Hori et al., 2002).
proteins and N-CAM, a neural cell adhesion
molecule (Schuldiner et al., 2001). The dif-
ferentiation of ES cells into motor neurons MULTIPOTENT STEM CELLS
has been achieved by first producing EBs, A potential source of cells for replace-
ment therapeutics, apart from ES cells, is that
then culturing them in the presence of retinoic
acid (RA) to produce caudal neural cells, and of multipotent stem cells, in particular the
finally adding an Shh signaling antagonist hematopoietic and mesenchymal stem cells
(HSCs and MSCs, respectively) of the bone
(Hh-Ag1.3) to induce the formation of ven-
tral motor neurons. This procedure mimics marrow.
the molecular signals known to promote nor-
mal development of motor neurons (Wichterle Hematopoietic Stem Cells
Stem Cells: et al., 2002). Following implantation into the In terms of molecular properties, ease
An Overview chick spinal cord, these cells were demon- of isolation, developmental potential, and
23.1.4
Supplement 28 Current Protocols in Cell Biology
Figure 23.1.3 The hematopoietic hierarchy. Long-term hematopoietic stem cells (LT-HSCs) are located at the top of
the hierarchy and give rise to short-term hematopoietic stem cells (ST-HSCs). In turn, ST-HSCs give rise to multipotent
progenitors (MPPS) that generate the two distinct common myeloid and lymphoid progenitor populations (CMPs and
CLPs, respectively). CMPs give rise to erythrocytes, platelets, granulocytes, and macrophages, whereas CLPs give rise
to natural killer (NK), B cells, and T cells.

thrapeutic applications, HSCs represent the Goodell et al., 1996, 1997; Christensen and
best characterized and understood of all stem Weissman, 2001; Scharenberg et al., 2002).
cells (Orkin, 2000). HSCs found within adult Multipotential progenitors derived from ST-
bone marrow can give rise to all lymphoid HSCs do not undergo self-renewal, but instead
and myeloid blood cell types and can be they provide both myeloid and lymphoid pre-
divided into two subtypes: long-term HSCs cursors that rapidly divide and give rise to all
(LT-HSCs), which undergo extensive self- blood cell types (Kondo et al., 1997; Akashi
renewal, and their progeny, short-term re- et al., 1999a,b; Fig. 23.1.3). Although both
populating HSCs (ST-HSCs), which undergo LT-HSC and ST-HSC equivalents have been
more limited self-renewal prior to becom- identified in both mouse and human, a num-
ing multipotential lymphoid or myeloid pro- ber of cell surface markers have been shown
genitors. At a single-cell level, LT-HSCs to differ (Okuno et al., 2002).
can be defined as being able to reconsti- With respect to therapies, both syngeneic
tute all blood types of lethally irradiated (i.e., usually from twins) and allogeneic HSC
mice (Osawa et al., 1996; Morrison et al., transplantations can be used following sys-
1997). Although numerous definitions ex- temic chemotherapy and chemoradiotherapy
ist, LT-HSCs have also been defined within to replace the depleted hematopoietic sys-
both the CD34+ /lin− population and Hoechst tems and to induce immune tolerance of
33342/rhodamine 1223 effluxing (Sca-1+ c- patients with severe aplastic anemias (80%
kit+ CD43+ CD45+lin− ) side population (SP) of cases have neutropenia and megakary-
of the bone marrow (Krause et al., 1994; ocytic anemia), fatal leukemias and other Stem Cells

23.1.5
Current Protocols in Cell Biology Supplement 28
hematological malignancies (lymphomas and from the medium. However, differentiation of
myelomas), clinically severe autoimmune dis- MSC into chondrocytes does not require all of
ease, and thalassemias. Although success these methods to be combined; chondrocyte
rates vary widely (between 15% and 20% differentiation can be achieved with the addi-
for patients with acute leukemia and ∼80% tion of TGF-β or by growth in 3-D culture,
for patients with chronic myeloid leukemia) but if TGF-β is added to the 3-D culture, in-
more than 20,000 transplants were performed creased chondrocyte differentiation will result
worldwide in 2004 alone (see American Can- (Johnstone et al., 1998; Li et al., 2005). Dif-
cer Society, Cancer Facts and Figures, 2005; ferentiation of MSCs into adipocytes has been
http://www.cancer.org). reported to be induced by monolayer culture of
MSCs in the presence of isobutylmethylxan-
thine (Suzawa et al., 2003). Differentiation of
Mesenchymal Stem Cells MSCs into skeletal muscle induced by culture
MSCs were first identified as a mixed
in the presence of amphotericin B has also
population of fibroblast-like plastic-adherent
been reported (Phinney et al., 1999). Thera-
cells that separated from the nonadherent
peutically, MSCs have been reported to con-
hematopoietic bone marrow fraction in culture
tribute to the repair of many tissues; this will
(Friedenstein et al., 1966). Reports demon-
be discussed below.
strate that residual pre-B progenitor cells and
granulocytic cells can best be removed on the
basis of CD34/CD45/CD11b immunodeple- Neural Stem Cells
tion (Ortiz et al., 2002). MSCs provide a sup- NSCs comprise the bulk of early fetal
portive role to HSCs during hematopoiesis, brain cells, and despite some conjecture, they
secreting growth factors and effecting direct have been reported to arise in the adult brain
cell-cell interactions, which play a role in in the hippocampal dentate gyrus subgranu-
HSC proliferation and differentiation. MSCs lar zone and the subventricular/subependymal
additionally possess the capacity to differen- zone of the lateral ventricles (Lois and
tiate into a range of mature connective tissue Alvarez-Buylla, 1993; Doetsch et al., 1999;
cell types including chondrocytes, osteocytes, Johansson et al., 1999; Gage, 2000; Seaberg
and adipose and smooth muscle cells. MSCs and van der Kooy, 2002). Linear differenti-
are also called bone marrow stromal cells, as ation of multipotent NSCs through more re-
they are most often isolated from the tibia, stricted progenitor populations provides all
fibula, iliac crest, or the thoracic and lum- differentiated cells of the fetal neural system
bar spine. However, MSCs that possess the during development, as well as neurons and
same multipotency have similarly been iso- glia (oligodendrocytes and astrocytes) as re-
lated from skeletal muscle, adipose tissue, de- quired for learning and memory and follow-
ciduous teeth, and the synovium (Poulsom ing functional loss of established systems in
et al., 2002; Barry and Murphy, 2004). MSCs the adult (Reynolds and Weiss, 1996; Shors
have therefore also been named mesenchymal et al., 2001; Carlen et al., 2002; Nakatomi
progenitor cells because of the ability, for ex- et al., 2002). The regulation of NSCs and
ample, of MSCs isolated from adipose tissue to their progenitors into neuronal, astrocytic, or
produce adipocytes and chondrocytes. The ca- oligodendrocytic fates occurs according to the
pacity to direct differentiation of MSCs prefer- specificities of developmentally patterned
entially into bone, cartilage, adipose tissue, or transcriptional programming and the neural-
muscle has been demonstrated to be dependent specific microenvironmental niche (Song
upon the source tissue for extracting MSCs. et al., 2002; Doetsch, 2003; Shen and Zhang,
Furthermore, different culture conditions can 2004). In addition to contributing to daily
also prejudice the percentage or preference cellular turnover, neural cells possess a lim-
of differentiation into a specific lineage. For ited replacement potential following injury,
example, osteoblastic differentiation can be but they are unable to restore function after
achieved by supplementing the medium with damage.
fetal bovine serum, dexamethasone, ascorbic Fetal neuronal transplantation has demon-
acid, and β-glycerol phosphate in combination strated some success in restoring motor func-
(Rodriguez et al., 2004). Preferential differ- tion, memory, and spatial learning in rats fol-
entiation into chondroblastic lineages can be lowing cortical grafting in focal ischemia–
achieved by the addition of TGF-β, growth in induced injury within the forebrain (Hodges
Stem Cells: a 3-D ultrastructure, and the removal of serum et al., 1996). Furthermore, although some
An Overview

23.1.6
Supplement 28 Current Protocols in Cell Biology
patients display graft rejection and others de- ADULT STEM CELLS DISPLAYING
velop severe dyskinesis, striatal grafting of fe- UNEXPECTED
tal mesencephalic precursors or dopaminer- MULTIPOTENTIALITY
gic neurons can restore dopamine release to In addition to the ability of NSCs to un-
near-normal levels in patients with Parkin- dergo linear differentiation into the three neu-
son’s disease (Freed et al., 2001; Lindvall and ral derivatives (neuron, astrocyte, and oligo-
Bjorklund, 2004). It is unlikely, however, that dendrocyte) and the ability of HSCs to dif-
fetal neural tissue will become a routine source ferentiate into all known hematopoietic lin-
of material for treating neural disease, due to eages, as well as the capacity of MSCs to
lack of availability and difficulties in stan- give rise to a restricted subset of mesodermal-
dardizing functional outcome (Lindvall and like derivatives, evidence has appeared in the
Bjorklund, 2004). Several workers are there- literature suggesting an unexpected increased
fore investigating the use of transplantable plasticity of these cell types (Bjornson et al.,
NSCs and their derivatives as a means to re- 1999; Clarke et al., 2000; Lagasse et al., 2000;
pair damage associated with diseases such as Willenbring and Grompe, 2004). These re-
Parkinson’s disease and multiple sclerosis. ports indicate that both bone marrow (BM)–
NSCs isolated from fetal and adult brains derived stem cells and NSCs may in fact also
of mice or humans can be expanded in culture be capable of giving rise to a wider array of
from single cells in the presence of epidermal fully differentiated cell types, not previously
growth factor (EGF), fibroblast growth factor expected.
(FGF), or both EGF and FGF together, to form
neurospheres (Reynolds and Weiss, 1992;
Richards et al., 1992; Carpenter et al., 1999; Repair of Liver Tissue by Adult
Tropepe et al., 1999; Piper et al., 2001). Al- Stem Cells
though some differences exist between NSCs Following liver damage, a subpopulation
isolated from humans and mice, expanded neu- of hepatocytes with regenerative potential and
rospheres from both species maintain a small oval cells recruited from the biliary duct ep-
percentage of multipotent NSCs and can be ithelium were traditionally believed to poten-
plated to give rise to neurons, oligodendro- tiate hepatic regeneration and restore vital liver
cytes, and astrocytes, or dissociated into single function (Overturf et al., 1997; Alison et al.,
cells for serial neurosphere propagation. NSCs 1998). With reports that BM-derived cells
derived from fetal brain provide stable lines displayed greater multipotency than previ-
after extensive culture, and they maintain the ously recognized, the contribution of specific
capacity to engraft as neurons and glia follow- bone marrow populations to liver regeneration
ing transplantation to the 6-hydroxydopamine- was investigated. Informative studies have
lesioned striata of cyclosporin-treated rats taken advantage of fumarylacetoacetate hy-
(Vescovi et al., 1999). Furthermore, multi- drolase (FAH)–deficient (Fah−/− ) mice, which
potential cells isolated from the mouse optic normally display a lethal tyrosinemia mani-
nerve, hippocampus, and adult subventricular fested as progressive liver failure and chronic
zone demonstrate remyelination of damaged renal tubular damage unless maintained
axons, repopulation of the hippocampus, and on 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-
rostral migration to the olfactory bulb where cyclohexanedione (NTBC) in their drinking
they produce two different types of olfactory water (Grompe et al., 1995). Lethally irradi-
neurons, respectively (Crang et al., 1992; Lois ated Fah−/− mice reconstituted intravenously
and Alvarez-Buylla, 1994; Ray and Rodrigues, with whole bone marrow or HSCs purified
1995; Yandava et al., 1999). Perinatal intracal- for c-kithigh Thylow Lin− Sca-1+ (Lin, lineage
losal xenograft of more committed oligoden- markers CD2, CD3, CD4, CD5, CD8, NK1.1,
drocyte progenitor cells from both the late- B220, Ter119, GR-1, and Mac-1) could sur-
gestation and adult human ventricular zone vive after being withdrawn from NTBC
into myelin basic protein–deficient shiverer (Lagasse et al., 2000).
mice (shi−/− ), a model of perinatal leukodys- Subsequent studies by the same group and
trophy, generated myelinating oligodendro- others, aimed at understanding the underlying
cytes (Windrem et al., 2004). Thus, several mechanisms of the apparent transdifferentia-
strategies aimed at developing transplantation tion events, demonstrated that appearance of
therapies for correcting neurological disease hepatic phenotypes from the BM-derived cells
and genetic deficits are underway. could be best explained in terms of cell-cell
Stem Cells

23.1.7
Current Protocols in Cell Biology Supplement 28
fusion events. Serial transplantation of HSCs and hematopoietic reconstitution with donor
along with karyotypic analysis and expres- whole BM (Bittner et al., 1999). BM en-
sion of donor versus host alleles in regener- graftment of infarcted cardiac tissue was
ating liver nodules confirmed that the appar- supported by several left coronary artery
ent change in the HSC derivative phenotype (LCA) ligation studies, including the intra-
and reinstated liver function was achieved by venous injection of SP BM–derived cells
a mechanism associated with cell-cell fusion (CD31–/low /c-kit+ ,Sca-1+ ) following lethal ir-
(Vassilopoulos et al., 2003; Wang et al., 2003). radiation and hematopoietic reconstitution,
Dissection of the HSC derivatives partici- and the direct peri-infarct injection of lin− c-
pating in the cell-cell fusion demonstrated that kit+ BM–derived cells (Jackson et al., 2001;
fusogenic macrophages are sufficient for the Orlic et al., 2001). In both studies, transd-
appearance of Fah+/+ hepatocytes in the re- ifferentiation of BM-derived cells into car-
cipient mice. It was further demonstrated that diomyocytes and vascular endothelium was
mature hepatocytes did not fuse (Willenbring reported. In the latter, engraftment was re-
and Grompe, 2004). Conjecture, however, re- ported to improve cardiac hemodynamics. In
mains over the precise cell type and the extent addition to providing a means of ameliorat-
of the role that a specific injury may play in ing the symptoms of heart disease, the de-
the induction of fusion between donor HSC- livery of BM cells to cardiac tissue was
derived cells and host liver. Two-dimensional suggested as a viable means of delivering
homed Fr25lin− marrow cell lines, se- dystrophin to patients with Duchenne’s mus-
lected as both functionally and phenotypically cular dystrophy.
representing HSCs, were reported to con- Subsequent and more detailed studies of
vert to a hepatocyte lineage in the absence the mechanisms underlying engraftment us-
of fusion following engraftment in carbon ing conditional transgenic mice revealed that
tetrachloride–induced hepatocyte injury in BM–derived cells and cardiomyocytes can un-
vivo (Jang et al., 2004). Similarly an inde- dergo cell-cell fusion (Alvarez-Dolado et al.,
pendent study using Z/EG Cre-reporter BM 2003). Furthermore, c-kit+ , c-kit+ lin− and
donors to reconstitute lethally irradiated β- c− kit+ lin− Thy-1lo Sca-1+ BM–derived cells
actin-Cre recipients reported transdifferenti- injected directly into ischemic hearts fol-
ation of BM-derived cells to hepatocytes in lowing LCA ligation, into c-kit+ Lin− BM-
the absence of any apparent cell-cell fusion derived cells following freeze injury, or into
(Harris et al., 2004). α-myosin-GFP c-kit+ lin− BM–derived cells
following LCA ligation or cauterization, al-
Repair of Cardiac Tissue by Adult though transiently present in the graft, did
Stem Cells not express markers characteristic of car-
Myocardial infarction (MI) is an ischemic diomyocyte differentiation (Murry et al., 2004;
insult resulting in cardiomyocyte apoptosis Nygren et al., 2004). Some benefit in lim-
and fibrous replacement. MI and its associ- iting ventricular dilation and dysfunction
ated process of scarring is a leading cause of in the long-term was observed; however,
congestive heart failure (CHF) and morbidity 30-day survival and infarct size did not dif-
in Western societies (Tang and Francis, 2003). fer significantly between control and treated
Although cardiomyocytes or resident lin− c- groups, suggesting that recruitment of resi-
kit+ CD45− cells may regenerate the heart and dent cardiomyocyte progenitors was minimal
improve function to some minor extent fol- (Murry et al., 2004). Similarly, lethal irradia-
lowing injury, this process is largely insuf- tion followed by reconstitution with β-actin-
ficient. Therefore, heart transplantation, left- GFP or α-actin-GFP c-kit+ lin− Sca-1+ CD45+
ventricular implantation devices, and pharma- BM–derived cells, then LCA ligation and mo-
ceutical intervention remain as current clinical bilization of stem cells with cytokines or re-
treatments. Novel strategies aimed at reducing constitution with α-myosin-GFP c-kit+ lin−
heart failure after MI are warranted and greatly BM–derived cells, followed by LCA lig-
needed. ation, did not show evidence for engraft-
Investigations into the possibility that cir- ment of donor cells displaying cardiac phe-
culating myogenic precursors may contribute notype within the infarct (Murry et al., 2004;
to cardiomyocyte regeneration demonstrated Nygren et al., 2004). Following lethal irra-
that cardiac and skeletal muscle of mdx diation and reconstitution, however, a small
mutant mice (a mouse model approximat- number of donor cells were observed to take
Stem Cells: ing Duchenne’s muscular dystrophy) could on characteristics of cardiomyocytes through
An Overview
be engrafted following total-body irradiation a process of cell fusion with endogenous
23.1.8
Supplement 28 Current Protocols in Cell Biology
cardiomyocytes within the peri-infarct zone. Wnt signaling (Polesskaya et al., 2003). Al-
What effect these fused cells have on car- though the precise identity of any single BM-
diac function is not known; however, it was derived cell type harboring myogenic activity
suggested that the previously reported benefit is unknown, Camargo et al. (2003) showed that
in function is most likely attributable to left- 0.03% to 0.08% of all skeletal muscle cells in
ventricular remodeling and/or the potentiation the CTX-injured tibialis anterior muscle were
of endogenous endothelial vascularization. derived from a single SP Sca1+ CD45+ HSC
Despite this conjecture in the literature with cell previously transplanted following irradia-
animal models, several clinical trials have al- tion and long-term hematopoietic chimerism.
ready been conducted, and mixed success in Furthermore, CD45+ BM cells derived from
terms of functional benefit has been reported. SP Sca1+ CD45+– reconstituted mice, injected
Injection of BM-derived cells or cytokines has into lethally irradiated mdx mice, demon-
been reported to be safe and beneficial, yet it strated a 0.13% TA myofibril contribution. No
has also been associated with occlusion of mi- donor-derived cells were found in uninjured
crocoronary circulation, induction of myocar- muscle or desmin-null muscle, and transplan-
dial infarction, increases in cardiac enzymes, tation of CD45− BM-derived cells did not re-
and increased rates of restenosis (Lee et al., sult in TA engraftment following injury. The
2004). presence of β-gal+ cells in CTX-injured TA
of bitransgenic Lysozyme M-Cre recombinase
Repair of Skeletal Muscle by Adult (LysM-Cre) × ROSAflox/STOP mice indicated
Stem Cells that engraftment by BM derivatives was at-
Self-renewal, growth, and regeneration of tributable to circulating myelomonocytic pre-
skeletal muscle is effected by slowly dividing, cursors, macrophages, and/or granulocytes.
self-renewing CD45− Sca1− /CD34+ Sca1− Furthermore, engraftment was attributable to
mononuclear satellite cells that lie between fusion with myoblasts produced from resident
the muscle fiber plasmalemma and associated satellite cells during muscle fiber regeneration
basement membrane (Campion et al., 1984; and not transdifferentiation. Thus, it still re-
Zammit and Beauchamp, 2001; Asakura mains unclear whether BM-derived stem cells
et al., 2002; Sherwood et al., 2004). Following may be exploited for myogenic therapeutics.
injury, or in patients suffering degenerative However, it appears that fractionated cell types
myopathies, satellite cells divide and fuse to with myogenic potential (with or without fu-
form multinucleated replacement myotubes. sion) can be isolated from both bone mar-
Satellite cells display a reduced self-renewal row and adult skeletal muscle, and can, to
capacity with age, resulting in their gradual varying degrees, contribute to repair in an
loss. In combination with chronic or severe injury-specific fashion. Therefore, it remains
injury, satellite cells are eventually depleted uncertain whether the incidence of BM con-
such that absent muscle tissue can be replaced tribution will be high enough to offer therapy
only with scar tissue. for muscle dystrophies and whether both sub-
In addition to satellite cells, unfractionated, sets may eventually play equal roles in future
nonadherent and adherent BM-derived cells therapeutics.
were reported to engraft cardiotoxin (CTX)–
injured skeletal muscle fibers and SP cells Neural Stem Cell Plasticity
were reported to engraft mdx null skeletal mus- In a similar fashion to BM-derived stem
cle fibers (Ferrari et al., 1998; Bittner et al., cells, mouse NSC differentiation pathways
1999; Gussoni et al., 1999; Ferrari et al., 2001). have been reported to be permissive to re-
The production of mononuclear myoblasts and programming, transdifferentiation, or trans-
subsequent skeletal muscle engraftment by un- determination in response to ectopic stimuli
fractionated whole BM following irradiation (Frisen, 2002). Adult and fetal forebrain NSC
and/or exercise-induced injury were reported derivatives were reported to reconstitute the
to occur after passage through a muscle satel- hematopoietic system following intraportal in-
lite cell intermediary phenotype (LaBarge and jection of sublethally irradiated mice. Adult
Blau, 2002). Both Sca-1+ CD45+ and Sca- NSCs were reported to give rise to repre-
1+ CD45− resident skeletal muscle cells were sentatives of all three embryonic germ cell
reported to harbor myogenic activity (Asakura layers following injection into the amniotic
et al., 2002; Polesskaya et al., 2003). With cavity of chick embryos, and intrablastocoel
respect to Sca-1+ CD45+ resident cells, myo- injection and transfer to pseudopregnant mice
genic recruitment into CTX-induced skeletal (Bjornson et al., 1999; Clarke et al., 2000).
Stem Cells
muscle injury was coupled to activation of Neurospheres derived from a human fetal brain
23.1.9
Current Protocols in Cell Biology Supplement 28
were injected into irradiated scid-hu mice and duce hyperglycemia in streptozotocin-induced
similarly displayed hematopoietic reconstitut- damage of NOD-SCID mouse pancreas by
ing activity (Shih et al., 2001). Coculture of contributing to neovasculogenesis, engrafting
adult mouse paraventricular NSCs and human the pancreas, and stimulating endogenous islet
embryonic NSCs with myoblasts and direct in- cell proliferation and insulin secretion (Hess
jection into CTX-injured tibialis anterior mus- et al., 2003). Intraventricular or systemic in-
cle were reported to result in transdifferentia- jection of adult periventricular neural progen-
tion of NSCs into skeletal muscle cells (Galli itors into myelin oligodendrocyte glycopro-
et al., 2000). NSCs were thus suggested as tein induced autoimmune encephalomyelitis,
an alternative source of cells for therapeutic which resulted in decreases in astroglyosis, de-
strategies where caveats associated with ther- myelination, axonal loss, and associated func-
apeutic cloning or immune rejection of MHC- tional impairment (Pluchino et al., 2003). Neu-
disparate donor cells limit the application of ral progenitors were reported to home to the
ES cells. site of injury, remyelinate neurons, stimulate
Theories concerning stem cell plastic- endogenous oligodendroglia, and attenuate re-
ity have not been unequivocally accepted active astrogliosis. Roughly a quarter of all
(Anderson, 2001; Morshead et al., 2002). The mice in each group were reported to recover
ability of the blastocyst environment to alter completely from the disease, whereas mice
NSC fate was considered an abnormal situa- that were either sham-transplanted or trans-
tion and repeat experiments using NSC/morula planted with other cell types showed no re-
aggregation failed to demonstrate embry- covery. Similarly, systemically administered
onic integration of NSC derivatives (Tropepe plastic adherent and CD11bCD34CD45 im-
et al., 2001). Experiments aimed at repeat- munodepleted whole BM MSCs were reported
ing hematopoietic reconstitution of irradiated to home to bleomycin-induced respiratory in-
mice using NSCs similarly failed. It was sug- jury. Notwithstanding issues of cell-cell fu-
gested that this reported property was more sion, MSCs adopted an epithelial-like mor-
likely due to a rare in vitro genetic or epi- phology and were reported to graft as type II
genetic alteration in long-term cultured NSCs epithelial cells. Furthermore, MSC admin-
or the presence of contaminating HSCs in the istration immediately following bleomycin-
original NSC culture (Morshead et al., 2002; induced respiratory injury reduced inflamma-
Magrassi et al., 2003). More recently, culture tion and fibrosis with an associated decrease
of mouse NSCs with human endothelium in- in collagen deposition and increase in metallo-
duced a type of transdifferentiation into en- proteinase gene expression (Ortiz et al., 2003).
dothelial lineages (Wurmser et al., 2004). No
fusion was observed in vitro and injection of
NSCs into the telencephalon of embryonic day GENETIC MANIPULATION OF
14 (E14) mice resulted in expression of en- EMBRYONIC STEM CELLS
dothelial markers by a fraction of the cells by The ability to manipulate mESCs has
E16. Although it is not known whether NSC proven to be a valuable tool in developing
conversion to endothelial cell phenotype rep- understanding of processes of differentiation,
resents a normal physiological process, the ap- development, and disease. The generation of
pearance of a small percentage of cells (1.6%) genetically altered cell lines is standard in
from all input cells opens up this possibility. many laboratories. The capacity to similarly
genetically manipulate hESCs is fast becom-
ing a reality. Several strategies have been de-
STEM CELLS STIMULATE veloped that allow the successful transfec-
ENDOGENOUS REPAIR tion of hESCs. These have resulted in the
Despite debates over transdifferentiation generation of ES cell lines with ubiquitous
and the capacity of transplanted adult stem or specific promoter-driven reporter genes
cells to replace damaged tissue in a func- for lineage tracing and gene-targeting events
tionally relevant context, some studies sug- such as knock-in/up/out. The first major suc-
gest adult stem cell transplantation may stim- cess came from liposome-based ExGen500
ulate endogenous repair mechanisms. These (Fermentas) technology, which produces a
studies suggest direct consequences for treat- high transfection efficiency when compared to
ing diseases such as type I diabetes, multiple both standard electroporation conditions and
sclerosis, and idiopathic pulmonary fibrosis. other liposome-mediated systems (Eiges et al.,
Stem Cells: For example, transplantation of c-kit+ adult 2001). This technique was used for the gener-
An Overview
BM-derived stem cells was reported to re- ation of stable hESCs with an EGFP reporter
23.1.10
Supplement 28 Current Protocols in Cell Biology
gene driven by the Rex-1 promoter (Eiges sion, resulting in trophectodermal differentia-
et al., 2001). An alternative approach has been tion (Velkey and O’Shea, 2003). In the human
the use of both adenoviral and lentiviral vec- context, Vallier et al. (2004) have shown that
tors. Adenoviral vectors Ad5 containing the hairpin RNAi constructs can efficiently induce
β-galactosidase reporter gene have proven the gene-specific knockdown of reporter gene ex-
most successful, generating transient transfec- pression in hESCs.
tion efficiencies of 11.2% with no apparent Advances in methods for high-efficiency
effect on differentiation status (Smith-Arica transfection of hESCs, production of novel ex-
et al., 2003). Self-inactivating lentiviral vec- pression systems, and advances in the abil-
tors have also been particularly successful in ity to direct specific activation and inacti-
generating stable transgenic hESCs (Gropp vation of gene expression provide powerful
et al., 2003; Ma et al., 2003). These viral technologies for the future of human em-
vectors have been reported to generate sta- bryonic stem cell research. These, coupled
ble transduction efficiencies of between 20% with growing databases of genes implicated in
and 80% (Gropp et al., 2003; Ma et al., pluripotency and lineage-specific differentia-
2003). More importantly, electroporation of tion, greatly enhance the ability to specifically
hESCs have resulted in both the HPRT1 and regulate ES cell differentiation and thus fully
POU5F1 loci to be successfully targeted using harness the potential of ES cells.
homologous recombination (Zwaka and
Thomson, 2003). Most recently, a novel trans-
fection method has been reported that com- THERAPEUTIC APPLICATIONS
bines electroporation with a chemical-based OF STEM CELLS
technology. This method is termed nucleo- Mouse ES cells were first described some
fection, as it enhances nuclear targeting of 17 years before hESCs, which were first iso-
exogenous DNA to the nucleus, thus en- lated in 1998 (Martin, 1981; Thomson et al.,
hancing expression and genomic integration 1998). Research using mESCs is currently
(Lakshmipathy et al., 2004). more advanced, with several in vivo exam-
The generation of defined lineage-restricted ples reporting appropriate physiological func-
populations has also been explored through tion of the transplanted cell types. Reports
the use of genetic manipulation. The directed of mESC derivatives improving heart func-
differentiation of mESCs has been success- tion (Min et al., 2002; Hodgson et al., 2004),
fully achieved through the manipulation of curing blood disease (Rideout et al., 2002),
lineage-specific transcription factors. Ectopic normalizing weight, longevity, and insulin
expression of Nurr1 in transgenic mESCs di- levels in diabetic mice (Hori et al., 2002),
rects their differentiation into a midbrain-type reducing symptoms of Parkinson’s disease
dopaminergic neuronal phenotype (Chung (Bjorklund et al., 2002; Kim et al., 2002),
et al., 2002). Similarly, forced constitu- and partially repairing damaged spinal cords
tive expression of GATA-6 and GATA-4 (McDonald et al., 1999; Liu et al., 2000)
induces mESC differentiation into extraem- have already appeared in the literature. More
bryonic endoderm (Fujikura et al., 2002). The sophisticated studies demonstrating similar
production of a more tightly regulated in- physiological function of hESC derivatives
duction has recently been reported, whereby in vivo, until now, have been lacking. Kehat
insulin-producing cells have been generated et al. (2004) recently described the produc-
from mESCs via tetracycline-off regulated ex- tion of cardiac-like cells from hESCs, and, for
ogenous Pdx-1 integrated into the ROSA26 the first time, meaningful in vivo physiolog-
locus (Miyazaki et al., 2004). Alternatively, ical function of hESC derivatives. They suc-
episomal constructs that are active as extra- cessfully treated pigs with an atrioventricular
chromosomal vectors have also been shown to block and restored a heart rhythm compati-
be effective in directing lineage-specific dif- ble with the survival of approximately half
ferentiation. In this study, the Wnt antagonist the animals tested. As acknowledged by the
Sfrp2 was used to stimulate the production of authors, this work was limited in part by the
neural progenitors (Aubert et al., 2002). failure to test for cell fusion (between the trans-
The development of new technologies such planted cells and the cells already existing in
as RNA interference (RNAi)–based protocols the pig heart) and the inability to discriminate
may also efficiently result in lineage restric- between direct electrical effects of the cells
tion of ES cells by modifying gene expression versus indirect instructive effects upon neigh-
within the cell. RNAi technology has been boring cells. This research provides important
Stem Cells
applied to mESCs to suppress Oct4 expres- evidence to support the suggestion that hESCs
23.1.11
Current Protocols in Cell Biology Supplement 28
may represent suitable candidates for reliev- being genetically identical to the patient, will
ing heart conditions and disorders, and that circumvent an immunological response. An
hESCs may one day play an important role in alternative to therapeutic cloning may be in
cell-transplantation therapeutics. the form of ES cell banks. It is envisaged
The therapeutic application of ES cells is that, by generating large numbers of ES cells
not only challenged by the ability to generate from a range of genetic backgrounds, a his-
specific cell types capable of repairing vari- tocompatible hESC line will be available for
ous tissues, but it is also confronted with the any patient (Baharvand et al., 2004; Heins
additional hurdle of immune rejection. Cur- et al., 2004). Alternatively, reprogramming
rently, histocompatible donors and immune- adult cells into the phenotype of an ES cell
suppression drugs are used to reduce these without the use of oocytes is also currently be-
effects in organ-transplantation patients. How- ing investigated. ES cell extracts may be capa-
ever, alternatives in overcoming immune rejec- ble of de-differentiating/reprogramming adult
tion will broaden the availability and success patient cells to take on an ES-like phenotype,
of organ transplantation and ES cell therapy. whereupon subsequent differentiation strate-
One approach is that of therapeutic cloning, gies can then be utilized (Tada et al., 2001).
whereby patient tissue is cloned into an oocyte A fourth approach has been that of manip-
for the generation of ES cells, which can then ulating immunological tolerance. Such meth-
be differentiated into the desired cell type for ods include second-signal blockade (Rifle and
tissue regeneration (Fig. 23.1.4). These cells, Mousson, 2002), manipulation of recipient

Figure 23.1.4 Therapeutic cloning. Therapeutic cloning involves the production of custom-
designed embryonic stem (ES) cells by the transfer of a person’s own DNA (nuclear transfer)
into a donor oocyte. Following nuclear transfer, the oocyte is incubated to develop into a blasto-
cyst, from which ES cells are derived. ES cells are then coaxed to become a desired cell type (e.g.,
Stem Cells: neural, cardiac, respiratory, pancreatic, or renal) and made available for transplantation. Because
An Overview the resulting tissue for transplant contains the same DNA as the donor, it should not be rejected.

23.1.12
Supplement 28 Current Protocols in Cell Biology
antigen-presenting cells, and chimerism of the Fusion of bone-marrow-derived cells with Purk-
immune system. Chimerism involves the in- inje neurons, cardiomyocytes and hepatocytes.
Nature 425:968-973.
jection of donor bone marrow into the recipi-
ent in order to improve organ allograft survival Anderson, D.J. 2001. Stem cells and pattern forma-
tion in the nervous system: The possible versus
(Monaco and Wood, 1970; Deng et al., 2004).
the actual. Neuron 30:19-35.
It has been suggested that a combination
Asakura, A., Seale, P., Girgis-Gabardo, A., and
of donor myeloid (immature) dendritic cells
Rudnicki, M.A. 2002. Myogenic specification
(Rifle and Mousson, 2002) and donor T cells of side population cells in skeletal muscle. J.
aids in the induction of transplantation toler- Cell Biol. 159:123-34.
ance (Tian et al., 2004). This technique, com- Aubert, J., Dunstan, H., Chambers, I., and Smith,
bined with ES cell therapy, could aid in long- A. 2002. Functional gene screening in embry-
term survival for donor cells in patients and in onic stem cells implicates Wnt antagonism in
overcoming the requirements for ES cell banks neural differentiation. Nat. Biotechnol. 20:1240-
1245.
and therapeutic cloning.
Baharvand, H., Ashtiani, S.K., Valojerdi, M.R.,
Shahverdi, A., Taee, A., and Sabour, D. 2004.
CONCLUSION Establishment and in vitro differentiation of a
Current research on the culture and dif- new embryonic stem cell line from human blas-
ferentiation of both adult and embryonic tocyst. Differentiation 72:224-229.
stem cells has allowed advances in unravel- Barry, F.P. and Murphy, J.M. 2004. Mesenchymal
ing the molecular mechanisms behind specific stem cells: Clinical applications and biologi-
cellular differentiation events. In vitro expan- cal characterization. Int. J. Biochem. Cell Biol.
36:568-584.
sion of stem cell populations, combined with
differentiation protocols and genetic manip- Bittner, R.E., Schofer, C., Weipoltshammer, K.,
Ivanova, S., Streubel, B., Hauser, E., Freilinger,
ulation of ES cells, has enabled the tracing
M., Hoger, H., Elbe-Burger, A., and Wachtler,
of various cell lineage differentiation events F. 1999. Recruitment of bone-marrow-derived
and the development of an understanding of cells by skeletal and cardiac muscle in adult
several of the molecular pathways involved. dystrophic mdx mice. Anat. Embryol. (Berl.)
Ethical issues surrounding the use of ES cells 199:391-396.
may be resolved by current research into adult Bjorklund, L.M., Sanchez-Pernaute, R., Chung, S.,
stem cell plasticity, which offers a potential Andersson, T., Chen, I.Y., McNaught, K.S.,
Brownell, A.L., Jenkins, B.G., Wahlestedt, C.,
source of cells for replacement therapies in
Kim, K.S., and Isacson, O. 2002. Embryonic
certain diseases and the ability to overcome stem cells develop into functional dopaminergic
immune-rejection issues. Overall, it is hoped neurons after transplantation in a Parkinson rat
that homogenous differentiation of stem cells model. Proc. Natl. Acad. Sci. U.S.A. 99:2344-
into a desired precursor, or specific differen- 2349.
tiated cell lineage, will facilitate the develop- Bjornson, C.R., Rietze, R.L., Reynolds, B.A.,
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brain into blood: A hematopoietic fate adopted
a wide range of diseases, and also increase
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Buzzard, J.J., Gough, N.M., Crook, J.M., and
Colman, A. 2004. Karyotype of human ES
cells during extended culture. Nat. Biotechnol.
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Stem Cells:
An Overview

23.1.18
Supplement 28 Current Protocols in Cell Biology
Mouse Embryonic Stem Cell Derivation, UNIT 23.2
and Mouse and Human Embryonic Stem
Cell Culture and Differentiation as
Embryoid Bodies
Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blas-
tocysts in vitro that maintain long-term self renewal and the capacity to give rise to all
cell types in the adult body (including some extraembryonic cell types) when subjected
to the appropriate conditions. It is envisaged that the development of methods enabling
controlled differentiation of mouse ES cell counterparts from human blastocysts would
enable the provision of an unlimited supply of tissue for cell and tissue transplanta-
tion therapies for the repair and replacement of diseased, injured, and senescent tissue.
Furthermore, derivation of mouse ES cells has allowed for the generation of thousands
of gene-targeted mouse mutants. Culture of mouse ES cells as embryoid bodies (EBs)
has provided a convenient system for studying early mouse developmental processes,
including several aspects of extraembryonic lineage and axis formation associated with
the pre- and peri-gastrulating mouse embryo. Relatively little is known regarding the
corresponding development of the early human embryo due to limitations associated
with the acquisition of relevant tissue material for study. The transfer of methods such
as EB formation to human systems should, by association, facilitate a more advanced
understanding of similar processes associated with early human development. This unit
describes protocols for isolating mouse embryonic stem cells and methods for propagat-
ing, freezing, and producing EBs from both mouse and human embryonic stem cells.

The protocols in this unit are designed to provide a basis for the derivation of mouse
ES cells (see Basic Protocol 1) and the successful propagation of both mouse (see Basic
Protocol 1 and Alternate Protocol 1) and human ES cells (see Basic Protocol 3 and
Alternate Protocol 2) in culture. In addition, protocols for the production of embryoid
bodies (EBs) from both mouse (see Basic Protocol 2) and human (see Basic Protocol
4 and Alternate Protocol 4) ES cells are described. Descriptions of mouse embryonic
fibroblast (MEF) feeder layer preparation (see Support Protocol 1), inactivation by mit-
omycin C (see Support Protocol 2) or γ-irradiation (see Support Protocol 3), mouse
and human ES cell media preparation, methods for propagating ES cells on MEFs (see
Basic Protocols 1 and 3), gelatin, and Matrigel (see Alternate Protocol 3), methods for
passaging human ES cells by mechanical dissection (see Basic Protocol 4) and enzymatic
digestion (see Alternate Protocol 3), conditioned medium preparation and storage (see
Alternate Protocol 2), mouse blastocyst derivation, handling and cultivation techniques,
vitrification, freezing and thawing protocols for human ES cells (see Basic Protocol 5),
mouse ES cells and MEFs (see Support Protocol 4), and mouse (see Basic Protocol 2) and
human EB (see Basic Protocol 3) formation using hanging drop and suspension culture
are presented.

NOTE: For all procedures described in this unit, tissue culture, reagent preparation,
and wash-up and sterilizing facilities are required. Experiments should be performed
under sterile conditions in either Class II Biological Hazard Flow Hoods or laminar
flow horizontal draft hoods. When working with human embryonic stem cells, Class II
Biological Hazard Flow Hoods are recommended.

NOTE: Ethics approval for the described protocols is usually required from the appro-
priate institutional research office.
Stem Cells

Contributed by Brock J. Conley, Mark Denham, Lerna Gulluyan, Fredrik Olsson, Timothy J. Cole, 23.2.1
and Richard Mollard
Current Protocols in Cell Biology (2005) 23.2.1-23.2.22
Supplement 28
Copyright 
C 2005 by John Wiley & Sons, Inc.
NOTE: All incubations should be performed in a humidified 37◦ C, 5% CO2 incubator
unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2
to maintain pH 7.4.

BASIC DERIVING, CULTURING, AND FREEZING MOUSE EMBRYONIC


PROTOCOL 1 STEM CELLS
Mouse embryonic stem cells (mES) are currently the best defined ES or ES-like cells.
Due to the relative ease of working with mouse ES cells when compared to their human
counterparts, it may be convenient for researchers working for the first time in this
discipline to set up both systems in the laboratory. Basic culture techniques for working
with mouse ES cells are outlined below. For further information, see Key References.

NOTE: All protocols using live animals must first be reviewed and approved by an Insti-
tutional Animal Care and Use Committee (IACUC) and must conform to governmental
regulations for the care and use of laboratory animals.

Materials
3.5 days post coitum (dpc) pregnant strain 129sv mouse
M2 medium (Sigma), sterile
M16 medium (Sigma), sterile
Cell culture–grade distilled water (JRH Biosciences), sterile
Organ culture dishes treated with 0.1% (w/v) gelatin and coated with mitotically
inactivated MEFs (see Support Protocol 2 or 3)
mES cell medium (see recipe), sterile
PBS, calcium- and magnesium-free (CMF-PBS; Invitrogen), sterile
0.025% trypsin/0.04% EDTA (see recipe), sterile
Cell culture–grade distilled water (JRH Biosciences), sterile
1-ml syringe and 26-G needle
Petri dishes
Organ culture dish (Becton Dickinson)
Finely drawn glass capillary pipets, 1 mm i.d.
Microscope
25-cm2 tissue culture flask (Becton Dickinson), optional

Collect embryos
1. Sacrifice a 3.5-days dpc pregnant mouse by cervical dislocation. Fill a 1-ml syringe
with M2 medium, attach a 26-G needle, then insert the needle into the oviduct of the
mouse and gently flush out the embryos into a petri dish containing M2 medium.
2. Transfer embryos to 1 ml M16 medium in the central well of an organ culture dish
and incubate at a 37◦ C until the embryos become expanded blastocysts. Add 5 ml
cell culture–grade sterile distilled water to the outer compartment of organ culture
dish to help maintain humidity during culture.
Some freshly isolated embryos may already be expanded blastocysts, ∼1 day in culture
should be sufficient for all blastocysts to expand.

Isolate ICMs
3. Place four embryos in an organ culture dish treated with 0.1% (w/v) gelatin and
mitotically inactivated MEFs in 1 ml mES cell medium (see Support Protocol 2 or 3).
Derivation, Embryos should attach within 2 days. The trophectoderm cells will be seen to migrate out
Culture, and onto the feeder cells and the inner cell mass (ICM) will be seen to expand.
Differentiation
of Embryonic
Stem Cells

23.2.2
Supplement 28 Current Protocols in Cell Biology
4. Pick off each ICM with a finely drawn glass capillary and place in a microdrop (10
to 20 µl) of 0.025% trypsin/EDTA at room temperature.
5. Disaggregate the ICM with a finely drawn glass capillary into single cells, then
replate onto a new organ culture dish coated with MEFs in 1 ml mES cell medium.
Culture one dissociated ICM per MEF-coated organ culture dish.
The ICM cells should proliferate in clumps as ES cells. Some non-ES cells may persist
for a few days.

Grow ES cells
6. Change mES medium daily, removing and replacing 1 ml per central well.
7. After an additional 2 to 3 days, remove the medium from the plate, wash by
adding 1 ml CMF-PBS and then removing the buffer, then add 200 µl of 0.025%
trypsin/EDTA. Incubate 5 to 10 min at 37◦ C.
8. Observe cells detaching under a microscope and, prior to their disaggregation to
single cells, add 600 µl mES cell medium while gently pipetting up and down with
a finely drawn glass pipet.
9. Transfer cells to a fresh MEF-coated organ culture dish, incubate at 37◦ C, and change
mES cell medium the following day.
10. Two days later, treat cells with 0.025% trypsin/EDTA as per previous steps 7 and 8.
11. After transferring the collected cells to a new MEF-coated organ culture dish, add
500 µl of 0.025% trypsin/EDTA to the old plate and incubate for an additional 5 to
10 min at 37◦ C.
12. Add 1 ml mES cell medium to stop reaction, transfer remaining cells to a second
new MEF-coated organ culture dish and incubate overnight at 37◦ C.
13. Change medium the following day on both dishes and analyze under a microscope.
The first plate should contain ES cell colonies, the second plate should not. If the second
plate contains ES cell colonies, repeat previous steps 7 to 9.

Passage ES cells
14. When colonies have expanded (2 to 3 days), treat with 0.025% trypsin/EDTA as
described in steps 7 and 8, and transfer to a fresh MEF-coated organ culture dish or
a 25-cm2 tissue culture flask coated with MEFs.
15. Trypsinize cells and transfer to a new dish or flask no longer than every 3 days for the
first few passages. Observe cells each day and transfer when 80% to 90% confluent.
For purposes of tracking passage number, begin counting passages the first time single
cells are plated, i.e., step 5.
To scale up the ES culture, cells can be split into two or more flasks or into larger flasks.

16. Freeze cells at regular intervals to prevent loss of cell line (see Support Protocol 4).
It is recommended to freeze ES cells following the second or third passage at step 10.

17. Monitor ES cells at regular intervals via karyotypic analysis (G-banding; UNIT 22.3)
and undifferentiated marker analysis.
See Conley (2004a) for discussion of markers.
The morphology of a typical mouse ES cell colony grown on MEFs can be seen in Figure
23.2.1C.

Stem Cells

23.2.3
Current Protocols in Cell Biology Supplement 28
Figure 23.2.1 Photomicrographs of human ES cell and mouse ES cell propagation and growth
as EBs. (A) Human ES cells (HES-2) propagated on MEFs (40× magnification). (B) An EB formed
from human ES cells following mechanical dissection and culture in suspension for 7 days (2.5×
magnification). (C) Zin40 mouse ES cells (see Munsie et al., 1998) propagated on gelatin in the
presence of LIF (40× magnification). (D) An EB formed from Zin40 ES cells following 7 days of
culture in a hanging drop (2.5× magnification).

ALTERNATE MOUSE ES CELL CULTURE IN THE ABSENCE OF FEEDER CELLS


PROTOCOL 1
Subsequent to their derivation, mES cells can be maintained in the absence of an MEF
feeder layer in mES cell medium supplemented with 2000 U/ml leukemia inhibitory
factor (LIF). Cells are grown in flasks and passaged when 75% to 85% confluent.
Medium should be changed every 1 to 2 days.

SUPPORT PREPARATION OF MOUSE EMBRYONIC FIBROBLASTS


PROTOCOL 1
Mouse embryonic fibroblasts (MEFs) are used as a feeder support layer for the derivation
of mouse ES cells and the propagation of both mouse and human ES cells. This protocol
requires the investigator to possess basic animal handling, dissection, and tissue culture
skills.
Derivation,
Culture, and NOTE: All protocols using live animals must first be reviewed and approved by an Insti-
Differentiation tutional Animal Care and Use Committee (IACUC) and must conform to governmental
of Embryonic
Stem Cells regulations for the care and use of laboratory animals.

23.2.4
Supplement 28 Current Protocols in Cell Biology
Materials
12.5 to 13.5 days post coitum (dpc) pregnant strain 129sv females
PBS, calcium- and magnesium-free (CMF-PBS; Invitrogen), sterile
0.025% (w/v) trypsin/EDTA (see recipe), sterile
MEF medium (see recipe), sterile
90-mm bacteriological petri dish
Scalpel, sterile
20-G needle (Becton Dickinson)
75-cm2 tissue culture flasks (Becton Dickinson/Falcon)
Platform shaker
15-ml tubes

Dissect and process embryos


1. Dissect the pregnant females at 12.5 to 13.5 days post coitus and remove the fetuses
from the uterine horns into 90-mm bacteriological petri dishes containing sterile
CMF-PBS.
2. Decapitate and eviscerate the fetuses to isolate the carcasses. Slice the carcasses into
small, ∼0.1-cm2 fragments with a sterile scalpel.
3. Digest the carcass slices in 0.025% trypsin/EDTA for ∼5 min at 37◦ C.
In this and the following step, enough trypsin solution should be used to immerse the
tissue.

4. Dissociate the tissue further by passing through a 20-G needle and then incubate the
tissue in 0.025% trypsin/EDTA for an additional 10 min at 37◦ C.
5. Plate all of the cells from one carcass in a 75-cm2 tissue culture flask containing
20 ml of MEF medium. Incubate at 37◦ C.
Passage MEFs
6. When the cells reach 80% to 90% confluency (∼1 to 2 days), aspirate and dis-
card the MEF medium, rinse with 2 ml CMF-PBS, replace with 1.5 ml of 0.025%
trypsin/EDTA, and incubate 1 to 2 min at room temperature with agitation.
7. Inactivate trypsin by adding 1 ml MEF culture medium down the growing surface to
dislodge all cells. Transfer cells to a 15-ml tube.
8. Centrifuge cells 2 min at ∼700 × g, room temperature, to pellet. Remove and discard
supernatant and resuspend the cells in 8 ml MEF medium.
9. Dispense cell suspension into 2-ml aliquots and add each aliquot to 18 ml of pre-
warmed MEF medium in 75-cm2 tissue culture flasks. Incubate at 37◦ C (1:4 split).
10. Allow cells to reach 80% to 90% confluency (1 to 3 days) and passage again according
to steps 6 to 9.
11. Passage cells four to five times to ensure minimal non-fibroblast contamination and
to create a stockpile of genetically equivalent MEFs.
For the purpose of counting passage number, begin counting following step 9.
MEFs can be utilized following three or four passages, but for maximum efficiency, utilize
them at passages five and six. After about seven passages, MEFs begin to lose their ability
to support ES cells.
MEFs can be frozen for storage at each passage number according to the freezing protocol
(see Support Protocol 4).
Stem Cells

23.2.5
Current Protocols in Cell Biology Supplement 28
Other strains of mice can be used to derive MEFs; however, these must be batch tested
with ES cells to assess compatibility. Mouse ES cell derivation efficiency is severely
reduced when not using the inbred 129sv mice. Mouse ES cells and MEFs need not be
strain-matched. MEFs used for human ES cell culture provides a potential means for
the xenobiotic transfer of pathogens from the mouse to human tissue, thus making these
cells unsuitable for human therapeutic use. Human fetal fibroblasts have been utilized for
propagation of human ES cells; however, currently, the use of MEFs in human ES cell
culture is the most accessible and effective means for their propagation (Richards et al.,
2002).

SUPPORT PASSAGING AND MITOTIC INACTIVATION OF MEFs BY MITOMYCIN C


PROTOCOL 2
Before MEFs can be used as a feeder layer for ES cells, they must be mitotically
inactivated to prevent overgrowth or contamination of ES cells. They can be mitotically
inactivated by treatment with mitomycin C or γ-irradiation (see Support Protocol 3).
Mitomycin C treatment is an efficient and effective way to inactivate MEFs. Following
treatment, ∼90% to 95% of cells are seen to be inactivated.

Materials
MEF cultures in 75-cm2 culture flasks (see Support Protocol 1)
MEF medium (see recipe)
Mitomycin C (see recipe)
0.1% (w/v) gelatin (see recipe)
PBS, calcium- and magnesium-free (CMF-PBS; Invitrogen)
0.025% (w/v) trypsin/EDTA (see recipe)
Cell culture–grade water (JRH Biosciences), sterile
Organ culture dishes
15-ml centrifuge tubes
Additional reagents and equipment for counting cells (UNIT 1.1)

Inactivate MEFs
1. Incubate all MEFs from one 75-cm2 culture flask at passage 4 or 5 with 20 ml of
MEF medium containing 10 µg/ml mitomycin C for a minimum of 2.5 hr (maximum
3 hr) at 37◦ C.
2. During incubation, pretreat the central compartment of organ culture dishes with 1
ml of 0.1% gelatin for 30 min at room temperature. After 30 min, aspirate the gelatin
and allow the plates to dry at room temperature.
3. Aspirate MEF medium containing mitomycin C from MEFs and wash cells one time
with 20 ml of prewarmed MEF medium at 37◦ C.
Collect cells
4. Wash three times each time by adding and then removing 20 ml of CMF-PBS at
room temperature.
5. Detach cells from the flasks by incubating with 1.5 ml of 0.025% trypsin/EDTA for
1 to 2 min at room temperature with agitation.
6. Inactivate the trypsin by adding 6 ml of pre-warmed MEF medium at 37◦ C. Pipet up
and down the growing surface of the flask to ensure all cells are dislodged.
7. Transfer cells to a 15-ml centrifuge tube and centrifuge 2 min at ∼700 × g, room
Derivation,
Culture, and temperature, to pellet cells.
Differentiation
of Embryonic
Stem Cells

23.2.6
Supplement 28 Current Protocols in Cell Biology
Set up organ culture dishes
8. Resuspend the cells in 8 ml of MEF or human ES culture medium. Pipet a 10-µl
aliquot and count total cell number using a hemacytometer (UNIT 1.1).
9. Plate MEFs onto the pretreated, gelatin-coated central portions of organ culture
dishes (from step 2) at a concentration of 1.75–1.8 × 105 cells/ml per dish.
10. Pipet 5 ml of tissue culture–grade distilled water into the outer section of the organ
culture dish to maintain humidity. Place dishes at 37◦ C.
ES cells may be added 24 hr after the MEFs are plated (i.e., after the MEFs have attached).

PASSAGING AND MITOTIC INACTIVATION OF MEFs BY γ-IRRADIATION SUPPORT


PROTOCOL 3
γ-Irradiation can be used to mitotically inactivate MEFs. This method is as effective as
mitomycin C inactivation provided that an appropriate level of radiation is utilized. A
disadvantage to this method is that it requires additional equipment to irradiate MEFs,
whereas mitomycin C is a reagent that can be directly applied in almost any laboratory.

Materials
MEF cultures (for fresh MEF, see Support Protocol 1 or for frozen MEF, see
Support Protocol 4)
0.025% (w/v) trypsin/EDTA (see recipe)
MEF medium (see recipe)
Human ES (hES) cell culture medium (see recipe)
75-cm2 culture flasks
50-ml centrifuge tube
γ irradiator (Gammacell 1000, Nordion)
150- and 100-mm gelatin-coated tissue culture dishes (optional)
Organ culture dishes
1. Grow MEFs at passage 4 to 5 or a thawed frozen vial of primary MEFs (from an
earlier preparation, see Support Protocol 4) to confluence in 75-cm2 culture flasks
or on 150-mm gelatin-coated tissue culture dishes.
2. Detach cells using 0.025% trypsin/EDTA as described in Support Protocol 2, steps
4 to 6, and collect in a 50-ml centrifuge tube.
3. Centrifuge cells 2 min at ∼700 × g, room temperature, to pellet and resuspend pellet
in 25 ml MEF medium.
4. Irradiate each tube of cells with 3000 to 10,000 rads of γ-irradiation.
This varies with the cell line used, therefore, a titration should be performed to determine
the effective dose.

5. Plate irradiated cells onto gelatin-coated plates at 3–4 × 106 cells per 100-mm
gelatin-coated dish, or 1.8 × 105 cells per organ culture dish, in MEF or hES culture
medium.

FREEZING MEFS SUPPORT


PROTOCOL 4
Early passaged MEFs can be frozen to provide cells for use at a later point.

Materials
Freezing solution (see recipe)
MEF cultures in 75-cm2 tissue culture flasks (see Support Protocol 1)
Stem Cells

23.2.7
Current Protocols in Cell Biology Supplement 28
PBS, calcium- and magnesium-free (CMF-PBS; Invitrogen)
0.05% (w/v) trypsin/EDTA (Invitrogen)
MEF medium (see recipe)
Liquid nitrogen
1-ml cryovial
1. Prior to freezing cells, store the freshly prepared freezing solution on ice. Label
1.0-ml cryovials with passage number and cell line and chill on ice (use two vials
per 75-cm2 tissue culture flask).
2. Aspirate medium from 80% to 90% confluent MEFs cultured in 75-cm2 tissue culture
flasks and wash gently with 2 ml CMF-PBS to remove all traces of medium.
Freeze aliquots of MEFs from passage 1 to 5 for subsequent thawing and inactivation.

3. Add 1.5 ml of 0.05% trypsin/EDTA to cells and incubate for 1 to 2 min at room
temperature. Tap flask/dish to gently dislodge the cells.
4. Add 6 ml MEF medium to inactivate the trypsin. Pellet cells by centrifuging 2 min
at ∼700 × g, room temperature.
5. Aspirate supernatant from the cell pellet and resuspend cells in 2 ml freezing solution.
6. Transfer 1 ml freezing solution and cells to each 1.0-ml cryovial and place at −80◦ C.
After 24 hr, transfer the cryovials to liquid nitrogen.
MEFs can be stored indefinitely in liquid nitrogen. Upon thawing, cells may take 1 to 3
days to recover to 80% to 90% confluency for subsequent use.

BASIC MOUSE EMBRYOID BODY FORMATION (HANGING DROP CULTURE)


PROTOCOL 2
Mouse embryoid body (EB) formation is routinely utilized as an initial differentiation
method as it initiates differentiation into a variety of cell types that can be subsequently
purified and analyzed. This method is for use with mES cells propagated in the absence
of a feeder layer. It is important that the mES cell medium used in EB formation lacks
LIF. A typical mouse EB can be seen in Figure 23.2.1D.

Materials
mES cell culture in the absence of a feeder layer (see Alternate Protocol 1) in a
25-cm2 tissue culture flask
PBS, calcium- and magnesium-free (CMF-PBS; Invitrogen)
0.025% (w/v) trypsin/EDTA (see recipe)
mES medium (see recipe) without LIF
Cell culture–grade distilled water (JRH Biosciences), sterile
15-ml centrifuge tubes
90-mm bacterial culture plate
Additional reagents and equipment for counting cells (UNIT 1.1)

Collect cells
1. Aspirate the medium from a 25-cm2 tissue culture flask of undifferentiated mES
cells.
2. Rinse the growing surface with 2 ml CMF-PBS at room temperature.

Derivation, 3. Apply 1 ml of 0.025% trypsin/EDTA and incubate 3 to 7 min at room temperature


Culture, and or until cells have detached.
Differentiation
of Embryonic 4. Inactivate trypsin by adding 6 ml mES medium without LIF down the growing
Stem Cells
surface to collect all cells.
23.2.8
Supplement 28 Current Protocols in Cell Biology
5. Transfer cells and medium to a 15-ml centrifuge tube and centrifuge 2 min at
∼700 × g, room temperature.
6. Aspirate supernatant and resuspend pellet in 3 ml mES medium without LIF. Count
cells using a hemacytometer (UNIT 1.1).
7. Dilute cells in mES medium without LIF to obtain a concentration of 1 × 104 cells/ml.
Set up cultures for EB formation
8a. For hanging drop cultures: Apply 30- to 50-µl droplets (300 to 500 cells/droplet)
onto the lid of a 90-mm bacterial culture plate (ensuring sufficient space is left
between the droplets to avoid fusion of neighboring drops on lid—between 30 to 35
individual drops per lid) and flip lid in a smooth, steady manner to invert. Place the
lid onto the bacterial dish, which has been half filled with cell culture–grade distilled
water to maintain humidity.
8b. For solution culture: Alternatively, produce EBs by transfering dissociated ES cells
to a bacteriological culture dish at a concentration of 2 × 106 cells in 10 ml mES
cell medium without LIF.
EB formation is slower with this method and smaller spheres are observed after 2 to 3
days in culture.

9. Incubate at 37◦ C for desired time.


After 1 to 2 days of incubation, spheres can be observed; these increase in size.
Cell types observed in EBs may vary between cell lines used and between methods used.

HUMAN EMBRYONIC STEM CELL PROPAGATION BASIC


PROTOCOL 3
Human ES cell handling techniques are a little more challenging than mouse ES cell
handling techniques. The following protocols are modifications of those described in
Reubinoff et al. (2000) and those found at http://www.geron.com/PDF/scprotocols.pdf.
The morphology of a typical human ES cell colony grown on MEFs can be seen in Figure
23.2.1A. The mechanical human ES cell propagation procedure is illustrated in Figure
23.2.2.

Materials
Mitomycin C–treated MEFs in organ culture dish (see Support Protocol 2)
Human ES cell medium (see recipe)
Cultures of human ES cells
Finely drawn glass capillaries, 1.0-mm o.d. (Clark Electromagnetic Industries)
1. On the day prior to human ES cell transfer, equilibrate mitomycin C–treated MEFs
by incubating in human ES cell medium at 37◦ C.
2. At ∼7 days of culture, mechanically dissect the human ES cell colonies into ∼0.1-
cm2 morphologically designated undifferentiated fragments using a glass capillary
finely drawn over a blue flame of a Bunsen burner. Gently lift the fragment from the
plate with the end of a 20-µl micropipettor tip.
Morphologically undifferentiated fragments, visualized through a dissection microscope,
appear as a uniform and consistent white color with distinct edges, whereas differentiated
regions are more heterogenous and irregular in appearance often with cystic (fluid-
filled) regions. Differentiated regions have indistinct edges and often display processes
extending away from the colony. Often colonies of hES cells will contain a densely packed
region in the center, from which the colony has initially arisen. This region also contains
differentiated cells and thus is not transferred to a new dish.
Stem Cells

23.2.9
Current Protocols in Cell Biology Supplement 28
Figure 23.2.2 Schematic representation of the human ES cell mechanical dissociation culture procedure. (A, B, and C)
Human ES cell colonies are dissected with a drawn glass micropipet according to the dotted lines. Isolated undifferentiated
regions are gently lifted with a micropipettor to separate them from any differentiated cells. The undifferentiated regions
are then transferred to wash plates. (D) Approximately six to nine regions of undifferentiated cells from the original colony
are transferred to a new MEF layer for growth.

3. Transfer the undifferentiated fragments to a fresh organ culture dish containing


1 ml room temperature hES medium, then transfer to a second organ culture dish
containing 1 ml medium for a second wash.
4. Transfer six to nine fragments to an equilibrated MEF-coated organ culture dish.
5. Change the medium daily (1 ml per dish) until the following transfer at ∼7 days.
Depending on the number of fragments seeded, one organ culture dish after 7 days should
yield 25 to 35 colonies.

ALTERNATE PREPARATION OF CONDITIONED MEDIUM FEEDER-LAYER-FREE


PROTOCOL 2 CULTURE OF HUMAN ES CELLS
Human ES (hES) cells can be cultured in the absence of a feeder layer using MEF cell
conditioned medium supplemented with FGF-2 on an extracellular matrix (see Alternate
Protocol 3). This method is efficient for larger cultures; however, prolonged culture in
this manner (>23 passages) has been shown to produce chromosomal abnormalities
(Mitalipova et al., 2005).

Materials
Derivation, Mitotically inactivated MEFs (see Support Protocol 2 or 3)
Culture, and MEF medium (see recipe)
Differentiation
of Embryonic Serum-free medium (see recipe)
Stem Cells

23.2.10
Supplement 28 Current Protocols in Cell Biology
0.1 mg/ml human recombinant basic fibroblast growth factor stock (hbFGF;
Invitrogen): reconstitute 10 µg in 100 µl of 10 mM Tris·Cl, pH 7.6 (APPENDIX 2A),
store at −20◦ C
hES cells
25-cm2 tissue culture flasks
0.22-µm filter
1. Plate mitomycin C–treated or γ-irradiated MEFs at 5.6 × 104 cells/cm2 in 5 ml MEF
medium in 25-cm2 tissue culture flasks. Incubate overnight at 37◦ C.
2. Replace the MEF medium the following day with 5 ml of serum-free medium.
Incubate overnight at 37◦ C.
3. Collect medium from the flask, filter through a 0.22-µm filter, and add 8 ng/ml
hbFGF. Transfer the new conditioned medium (CM) either to human ES cells (see
Basic Protocol 3) or freeze for storage.
CM can be stored for 1 month at −20◦ C.

4. Add 5 ml of fresh serum-free medium to the flask and incubate cells overnight at
37◦ C.
5. Repeat steps 3 and 4 for up to 7 days.

ENZYMATIC PASSAGE OF HUMAN ES CELLS FOR CULTURE ON ALTERNATE


FEEDER LAYERS AND ON MATRIGEL PROTOCOL 3
Human ES cells have also been propagated more recently using enzymatic methods as
well as growth in the absence of feeders on the extracellular matrix substrate Matrigel.
Some controversy remains with respect to the reliability of this culture method, however,
especially in regard to human ES cell karyotype integrity following prolonged culture
(see Mitalipova et al., 2005). Nevertheless, these protocols provide a more time-efficient
method for regular maintenance of human ES cells in short-term cultures.

Materials
hES cell cultures in Matrigel-coated 4-well plates (see recipe for plates)
1 mg/ml collagenase IV (see recipe)
PBS, calcium- and magnesium-free (CMF-PBS; Invitrogen)
Serum-free medium (see recipe)
Conditioned medium (CM, see Alternate Protocol 2)
Matrigel-coated 4-well plates (see recipe)
15-ml centrifuge tubes
1. Replace the medium on human ES cells with 0.5 ml of 200 U/ml collagenase IV per
well of a Matrigel-coated 4-well plate.
2. Incubate for 5 to 10 min at 37◦ C or until colonies just begin to detach from plate.
3. Gently remove collagenase IV and wash gently with 1 ml CMF-PBS.
4a. For passage onto MEFs: Replace CMF-PBS with 1 ml serum-free medium equili-
brated at 37◦ C and proceed to Basic Protocol 3, step 4.
4b. For passage onto Matrigel: Remove CMF-PBS and add 1 ml CM to each well.
5. Gently pipet cells up and down, scraping with the pipet to dislodge cells, and transfer
to a 15-ml centrifuge tube.
Stem Cells

23.2.11
Current Protocols in Cell Biology Supplement 28
6. Dissociate cells with gentle pipetting to small clusters (50 to 400 cells), then transfer
between 1/3 and 1/6 of the suspension to a Matrigel-coated well containing 1 ml of
CM medium.
Colonies should appear within days and should continue to expand.

7. Passage when human ES cells comprise 80% of the surface area of the well.

BASIC PRODUCING HUMAN ES CELL EMBRYOID BODIES


PROTOCOL 4 BY MECHANICAL DISSOCIATION
Mechanical dissection is more laborious but more selective with the ability to select
for morphologically undifferentiated regions whereby enzymatic dissociation obtains
both differentiated and undifferentiated cells together. This results in a difference in cell
potential and thus a difference in EBs produced.

Materials
Cultures of human ES cell colonies in organ culture dishes on MEF feeder layer, 6
to 9 colonies/dish
Human ES cell medium (see recipe)
Finely drawn glass capillary (1.0-mm o.d.)
90-mm bacteriological petri dish
1. Dissociate human ES cells colonies with a finely drawn glass capillary to obtain
small undifferentiated regions of between 100 and 300 cells.
2. Transfer as many of these aggregates as required to 90-mm bacteriological petri
dishes containing 10 ml human ES cell medium and culture as a suspension (see
Conley et al., 2004b).
3. Incubate at 37◦ C.
A typical 7-day-old EB formed from human ES cells can be seen in Figure 23.2.1B.

ALTERNATE PRODUCING HUMAN ES CELL EMBRYOID BODIES


PROTOCOL 4 BY ENZYMATIC DIGESTION
Human EBs can also be produced following enzymatic digestion. EBs are produced this
way from smaller digested fragments of ES cells and thus are generally smaller. Digestion
additionally leads to more variability in ES cell fragment size as well as resultant EB
size.

Materials
Human ES cell colonies
Serum-free medium (see recipe)
1 mg/ml collagenase/dispase stock (see recipe)
Certified fetal bovine serum (Invitrogen)
Human ES cell medium (see recipe)
Finely drawn glass capillary (1.0-mm o.d.)
15-ml centrifuge tube
90-mm bacteriological petri dish
1. Mechanically dissect each colony with a finely drawn glass capillary to obtain
Derivation,
Culture, and undifferentiated areas (see Basic Protocol 3).
Differentiation
of Embryonic 2. Transfer fragments to a 15-ml centrifuge tube and centrifuge 2 min at ∼700 × g,
Stem Cells room temperature.
23.2.12
Supplement 28 Current Protocols in Cell Biology
3. Aspirate as much medium as possible and resuspend pellet in 500 µl serum-free
medium.
4. Add 500 µl of 1 mg/ml collagenase IV/dispase stock (final concentration 0.5 mg/ml
for each enzyme) and mix gently with a pipet.
5. Incubate 3 min at 37◦ C and then agitate with a pipet ten times.
6. Add 5 ml certified fetal bovine serum and invert gently three to five times to mix.
FBS should be batch tested for compatibility with ES cells.

7. Add 7 ml human ES cell medium, mix, and centrifuge 3 min at ∼700 × g, room
temperature.
8. Resuspend the pellet in 5 ml human ES cell medium and transfer to a 90-mm
bacteriological petri dish containing 3 to 5 ml medium to allow aggregation as EBs
in suspension.
EBs can be seen to form after ∼2 days.

VITRIFICATION/THAWING OF HUMAN ES CELLS BASIC


PROTOCOL 5
The vitrification protocol described is the one that can be found in Reubinoff et al. (2001).
It is used to maintain frozen stocks of human ES cell lines.

Materials
Bench medium (BM; see recipe)
Vitrification solution 1 (VS1; see recipe)
Vitrification solution 2 (VS2; see recipe)
Liquid nitrogen
hES cell culture
Warming solution 1 (WS1; see recipe)
Warming solution 2 (WS2; see recipe)
Organ culture dish seeded with mitotically inactivated MEFs (see Support Protocol
2 or 3)
Human ES cell medium (see recipe)
4-well tissue culture plates (NUNC)
5-ml cryovials (NUNC)
18-G needle
Canes for liquid nitrogen tank
20-µl micropipettor
Vitrification straws (LEC Instruments)
Forceps

Prepare equipment for vitrification


1. Prior to freezing human ES cell fragments, prepare a vitrification plate. Pipet 1 ml
of each of the solutions BM, VS1, and VS2, into separate wells of a 4-well tissue
culture plate and pre-warm at 37◦ C.
2. Label a 5-ml cryovial with all details pertaining to cells being frozen. Using a flame-
heated 18-G needle, punch holes in the upper section, base, and lid of the cryovial
and attach the cryovial to a labeled cane. Submerge in a bucket of liquid nitrogen.
Prepare cells for freezing
3. Prepare human ES cell pieces for freezing by cutting fragments slightly larger than
that required for usual transfer (0.1 cm2 ). Transfer about eight fragments to the first
well of the 4-well tissue culture plate containing BM using a 20-µl micropipettor. Stem Cells

23.2.13
Current Protocols in Cell Biology Supplement 28
4. With a fresh tip, transfer all fragments to the second well containing VS1 for 1 min,
ensuring all pieces settle to the bottom of the viscous solution. During this minute,
pipet a 20-µl drop of VS2 onto the inside of the 4-well tissue culture plate lid. Ensure
a fresh drop is prepared for each straw to be frozen.
5. Transfer the human ES cell fragments to the last well containing VS2 for 25 sec and
again ensure that all pieces settle to the bottom.
6. Transfer the fragments in the least possible volume to the 20-µl VS2 drop on the
4-well tissue culture plate lid.
7. Using a second pipet set at 3 µl, pick up the fragments and make a small, high droplet
on the lid.
Collect cells in vitrification straws and freeze
8. Immediately after making the droplet, touch the narrow end of a vitrification straw
to the side of the droplet at an angle of 30◦ to 45◦ to the plane of the lid and draw up
the droplet and fragments into the straw by capillary action.
9. Plunge the straw into the bucket of liquid nitrogen at an angle of 45◦ . Without
removing the straw from the liquid nitrogen, carefully transfer into the prepared
cryovial. Repeat until all fragments have been collected (three to five straws per
cryovial). Store the cryovial in liquid nitrogen.
Thaw and process ES cells
10. Prior to thawing the cryovial containing the human ES cell straws, prepare a 4-well
tissue culture plate containing 1 ml of each of solutions WS2, WS1, and 2 wells of
BM in separate wells and prewarm at 37◦ C.
11. Collect the cryovial containing the straws to be thawed in a bucket of liquid nitrogen.
Remove a straw using forceps and hold it between the thumb and middle finger with
the index finger blocking the larger end of the straw which is devoid of cells.
12. Immediately submerge the narrow end of the straw containing the fragments into the
first well containing WS2 where, as the gas expands, the human ES cell fragments
will expel from the straw and into the well.
13. After 1 min transfer the fragments to the next well containing WS1 for 5 min.
14. Transfer the fragments into the third well containing BM for an additional 5 min.
15. Transfer fragments to the last well containing BM for an additional 5 min.
16. Transfer the fragments onto a previously prepared organ culture dish containing
mitotically inactivated MEFs (see Support Protocol 2 or 3) and 1 ml of human ES
cell medium, and culture at 37◦ C.

REAGENTS AND SOLUTIONS


Use deionized or distilled tissue culture grade water in all recipes and protocol steps. For common
stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Bench medium (BM)
Add 800 µl DMEM/F12 (Invitrogen)
200 µl FBS (20% v/v; JRH Biosciences)
Store up to 2 weeks at 4◦ C
Derivation, FBS purchased from JRH Biosciences has been traditionally used in the authors’ labora-
Culture, and
Differentiation tory for mouse ES cell and MEF cultures, while it has been observed that certain batches
of Embryonic of FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
Stem Cells

23.2.14
Supplement 28 Current Protocols in Cell Biology
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.
Collagenase IV, 1 mg/ml
Dilute 100 mg collagenase type IV (Invitrogen) in 100 ml knockout DMEM
(Invitrogen). Dispense into aliquots and store up to 6 months at −20◦ C.
Collagenase IV/dispase stock, 1 mg/ml
Add 100 mg collagenase type IV (Invitrogen) and 100 mg dispase (Invitrogen)
to 100 ml of filter-sterilized serum-free medium (see recipe). Store in aliquots at
−20◦ C.
Gelatin, 0.1% (w/v)
Dilute 5 ml of 1% (w/v) gelatin stock (see recipe) in 50 ml distilled water. Store up
to 4 weeks at 4◦ C.
Gelatin, 1% (w/v)
Dissolve 0.4 g of gelatin powder (Sigma) in 40 ml distilled water, autoclave, store
in 30- to 50-ml aliquots up to 2 months at −20◦ C.
Freezing solution
Prepare a mixture of 90% (v/v) FBS and 10% (v/v) DMSO. Prepare fresh and store
on ice.
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.

hbFGF stock, 0.1 mg/ml


Reconstitute 10 µg human basic FGF (Invitrogen) in 100 µl of 10 mM Tris·Cl, pH
7.6. Store up to 6 months in 20- to 50-µl aliquots at −20◦ C.

Human ES cell medium


High-glucose DMEM (Invitrogen) supplemented with:
20% (v/v) certified fetal bovine serum (FBS; Invitrogen)
1% (v/v) nonessential amino acids (Invitrogen)
2 mM L-glutamine (Invitrogen)
50 U/ml penicillin/50 mg/ml streptomycin (Invitrogen)
1× insulin-transferrin-selenium supplement (Invitrogen)
0.1 mM β-mercaptoethanol (Sigma)
Store up to 2 weeks at 4◦ C
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.

Matrigel-coated plates
Thaw an aliquot of Matrigel stock (see recipe) at 4◦ C. Dilute stock 1:10 in cold
knockout DMEM (Invitrogen). Apply 500 µl of diluted Matrigel to each well of a
4-well tissue culture plate. Incubate 1 hr at 37◦ C and aspirate liquid immediately
Stem Cells
prior to use.
23.2.15
Current Protocols in Cell Biology Supplement 28
Matrigel stock
Thaw 10-ml bottle of Matrigel (Becton Dickinson) overnight at 4◦ C, add 10 ml
cold knockout DMEM (Invitrogen), and mix well. Store in 10-ml aliquots up to
6 months at −20◦ C.

mES cell medium


High-glucose DMEM (Invitrogen) supplemented with:
10% (v/v) fetal bovine serum (FBS; JRH Biosciences)
2 mM L-glutamine (Invitrogen)
50 U/ml penicillin/50 mg/ml streptomycin (Invitrogen)
1% (v/v) nonessential amino acids (Invitrogen)
0.1 mM β-mercaptoethanol (Sigma)
1000 U/ml recombinant mouse leukemia inhibitory factor (LIF; Chemicon)
Store up to 2 weeks at 4◦ C
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.

MEF medium
High-glucose DMEM; (Invitrogen) supplemented with:
10% (v/v) fetal bovine serum (FBS; JRH Biosciences)
2 mM L-glutamine (Invitrogen)
50 U/ml penicillin/50 mg/ml streptomycin (Invitrogen)
Store up to 2 weeks at 4◦ C
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.

Mitomycin C
Reconstitute a 2-mg ampule of mitomycin C powder (Sigma) with 1 ml sterile
PBS using a 21-G needle, with a 26-G needle in place to vent the ampule. Remove
solution and filter through a 0.22-µm syringe filter. Store solution up to 2 weeks at
4◦ C protected from light.
CAUTION: Mitomycin C is a toxic substance, refer to the product data sheet from the
manufacturer for handling instructions.

Serum-free medium (SFM)


High-glucose DMEM (Invitrogen) supplemented with:
1% (w/v) nonessential amino acids (Invitrogen)
2 mM L-glutamine (Invitrogen)
50 U/ml penicillin/50 mg/ml streptomycin (Invitrogen)
1× insulin-transferrin-selenium supplement (Invitrogen)
mM β-mercaptoethanol (Sigma)
Store up to 2 weeks at 4◦ C
Derivation,
Culture, and
Differentiation
of Embryonic
Stem Cells

23.2.16
Supplement 28 Current Protocols in Cell Biology
Serum replacement medium
80% (v/v) knockout DMEM (Invitrogen)
20% (v/v) knockout serum replacement (Invitrogen)
1% (v/v) nonessential amino acids (Invitrogen)
2 mM L-glutamine (Invitrogen)
50 U/ml penicillin/50 mg/ml streptomycin (Invitrogen)
1× insulin-transferrin-selenium supplement (Invitrogen)
0.1 mM β-mercaptoethanol (Sigma)
Store up to 2 weeks at 4◦ C
Sucrose solution, 2 M
Dissolve 6.846 g sucrose (Sigma) in ∼5 ml DMEM/F12 (Invitrogen). Add
DMEM/F12 to a final volume of 10 ml. Prepare fresh.
Trypsin/EDTA, 0.025% (w/v)
Dilute 20 ml of 0.05% (w/v) trypsin/EDTA (Invitrogen) in 20 ml calcium- and
magnesium-free PBS (CMF-PBS; e.g., Invitrogen) for a final concentration of
0.025%. Store in aliquots up to 2 weeks at 4◦ C or up to 6 months at −20◦ C.
Vitrification solution 1 (VS1)
600 µl DMEM/F12 (Invitrogen)
200 µl FBS (Invitrogen; 20% v/v final)
100 µl DMSO (10% v/v final)
100 µl ethylene glycol (Sigma; 10% v/v final)
Prepare fresh
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.
Vitrification solution 2 (VS2)
250 µl sucrose solution (0.5 M final; see recipe for 2 M sucrose)
150 µl DMEM/F12 (Invitrogen)
200 µl FBS (Invitrogen; 20% v/v final)
200 µl DMSO (20% v/v final)
200 µl ethylene glycol (20% v/v final)
Prepare fresh
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.
Warming solution 1 (WS1)
100 µl sucrose solution (0.2 M final; see recipe for 2 M sucrose)
700 µl DMEM/F12 (Invitrogen)
200 µl FBS (Invitrogen; 20% v/v final)
Prepare fresh
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
Stem Cells
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative. 23.2.17
Current Protocols in Cell Biology Supplement 28
Warming solution 2 (WS2)
50 µl sucrose solution (0.1 M final; see recipe for 2 M sucrose)
750 µl DMEM/F12 (Invitrogen)
200 µl FBS (Invitrogen; 20% v/v final)
Prepare fresh
FBS purchased from JRH Biosciences has been traditionally used in the authors’ laboratory
for mouse ES cell and MEF cultures, while it has been observed that certain batches of
FBS purchased from Invitrogen maintained human ES cells more effectively. It has been
hypothesized that may be due to a low endotoxin level in some of the batches from Invitrogen
(however, this has not been proven definitively). Therefore, batch testing of all serum is
imperative.

COMMENTARY
Background Information Li et al., 1998; McDonald et al., 1999; Kim
Mouse embryonic stem (ES) cells are an in et al., 2002; Rideout et al., 2002; Wichterle
vitro–derived cell type isolated from the epi- et al., 2002). Failing such technological ad-
blast/inner cell mass (ICM) of mouse blas- vances and due to the limitations that result
tocysts (Evans and Kaufman, 1981; Mar- from currently applied ethical regulations, the
tin, 1981). mES cells are said to possess true pluripotential identity of human ES cell
properties consistent with an immortal phe- lines derived to date remains obscure and many
notype: demonstrating continual self-renewal researchers still refer to human ES cells as
and maintaining a normal chromosomal con- human ES-like cells (see Buehr and Smith,
tent over extended periods in culture. mES 2003).
cells further retain several properties of their The ability to manipulate mouse ES cell
in vivo predecessors, including a primitive fate in vitro and in vivo, however, has driven
pluripotent phenotype enabling differentiation the belief that ES-like cells derived from hu-
into all functional cell types of the adult and man blastocysts (human ES cells) may one
some extraembryonic tissues given the appro- day be similarly manipulated and thus used
priate stimuli (see Nagy et al., 1990, 1993). to produce sufficient tissue for replacement
The isolation of ES cells, or ES-like cells, therapeutics aimed at treating a variety of
has more recently been reported from other human diseases and injuries. Technological
species including human and nonhuman pri- advances supporting this notion include the
mates (see Thomson et al., 1995, 1998). Func- development of strategies describing human
tional studies, however, remain more advanced ES cell differentiation into several desired
for mouse ES cell systems because of the rel- lineages (for a review, see Pera and Troun-
atively long time since their first description son, 2004), clonal (albeit inefficient) deriva-
some 24 years ago and, when compared to hu- tion (Amit et al., 2000), cryopreservation
man ES cell work, the relatively relaxed ethical (Reubinoff et al., 2001), knock-ins and knock-
regulations associated with their research (for outs using homologous recombination (Zwaka
a review, see Fischbach and Fischbach, 2004). and Thomson, 2003), and biological interfer-
For example, mouse ES cell protocols for cul- ence using RNAi (Vallier et al., 2004a). With
ture and genetic manipulation at the single cell respect to reports describing differentiation
level, tetraploid complementation and blasto- into desired lineages, evidence has been re-
cyst injection, and the transfer of mouse ES stricted mostly to morphological and biochem-
cell derivative structures to pseudopregnant fe- ical characterization of cells found, for exam-
males for full-term pregnancy are routine (but ple, within teratomas created form human em-
technically challenging) procedures in many bryonic stem cells following injection under
laboratories (see Thomas and Capecchi, 1987; the kidney capsule of NOD-SCID mice, or fol-
Zimmer and Gruss 1989; Nagy et al., 1993). lowing the application of differentiation pro-
Advances in these disciplines have led to a rel- tocols designed to favor differentiation of par-
atively sophisticated understanding of mouse ticular cell types from an in vitro cultured cell
ES cell definition, developmental molecular colony (see Thomson et al., 1998; Reubinoff
Derivation, genetics and, in association, several techniques et al., 2000; Kaufman et al., 2001; Levenberg
Culture, and for controlling mouse ES cell fate reliably et al., 2002; Mummery et al., 2003; Ramb-
Differentiation
of Embryonic into predicted and functionally relevant pheno- hatla et al., 2003; Conley et al., 2004a). In the
Stem Cells types in vitro and in vivo (see Klug et al., 1996; absence of relevant functional studies and in

23.2.18
Supplement 28 Current Protocols in Cell Biology
agreement with arguments outlined above, ab- cycles severely compromises its cell culture
solute identification as the desired cell type is efficiency. Media should be discarded after 2
difficult to argue. Only recently has a study of weeks as this can influence the differentiation
human ES cell derived functionality appeared of ES cells.
in the literature. According to this study, trans- MEFs are an important and essential part of
plantation of cardiac-like cells derived from the human ES cell culture protocol; it is essen-
human ES cells to the heart of a pig model of tial that the MEFs are not used beyond six pas-
atrioventriclular block reinstated heart rhythm sages, that they grow well, and that they look
in a manner compatible with survival in ∼50% healthy. The strain of mouse used for MEF
of the subject animals (Kehat et al., 2004). derivation can also influence the efficiency of
In addition to the promise of providing human ES cell culture. It is advisable to utilize
tissue for therapeutic replacement strategies, an inbred strain of mouse (e.g., 129sv) and to
ES cell culture facilitates a means to study test each new batch of MEFs for their ability to
several early developmental processes (see support human ES cell culture. When testing
Doetschman et al., 1985). Reports in the lit- MEFs, it is important to propagate human ES
erature suggest that human ES cells possess cells on the new MEFs for a minimum of three
a capacity to produce representatives of both passages to determine their efficiency. Follow-
the trophoblast and the visceral endoderm in ing the first week of culture on new MEFs, ES
vitro when cultured in the presence of growth cell colonies may contain a proportion of dif-
factors or as structures known as embryoid ferentiated cells; however, ES cells may subse-
bodies (EBs; Xu et al., 2002; Conley et al., quently equilibrate on the new MEFs follow-
2004b; Gerami-Naini et al., 2004; Vallier et al., ing repeated transfer and propagate normally
2004b). EB formation has additionally pro- thereafter.
vided an integral first step in many in vitro dif- Human ES cell mechanical transfer must
ferentiation protocols (see Zhang et al., 2001; be delicately performed in the shortest pos-
Levenberg et al., 2002). It is believed that sible time due to reduced cell viability and
research on the differentiation of human ES increased differentiation rates with increased
cells into trophectoderm and visceral endo- dissection times. Additionally, for enzymatic
derm derivatives may provide important infor- digestion of human ES cells, it is important to
mation regarding nutrient and gas exchange stop the reaction prior to dissociation to single
and patterning of the early human embryo. cells as cell viability is severely compromised
otherwise.
Critical Parameters For mouse ES cell derivation, efficiency can
All tissue culture is to be performed in be dependent on the strain of mouse used.
Class 2 biohazard hoods and/or laminar flow Once derived, mouse and human ES cells
hoods using sterile technique. All reagents and are theoretically immortal and can be passaged
media must be sterilized either by autoclaving extensively without loss of differentiation po-
or filtering through a 0.22-µm filter. (Do not tential, although some karyotypic abnormali-
autoclave solutions containing proteins, such ties may arise over time. It is therefore advis-
as media, FBS, amino acids.) Bottles used to able to regularly karyotype ES cells and test
store media should be washed thoroughly to them for appropriate pluripotent markers such
avoid contamination by soaps or detergents, as oct-4, nonog, stage specific embryonic anti-
then placed for 4 hr in a 140◦ C sterilization gen (SSEA) -3 and -4, Tra-1-60, Tra-1-81 and
oven prior to use. GCTM-2 for hES cells, and oct-4, nanog and
Dissection of human ES cell colonies is to SSEA-1 for mES cells. Freezing stocks of cells
be performed using a dissecting microscope at regular passage numbers (e.g., passages 3,
in a laminar flow hood. A microscope with 5, 10, 15, 30, 50) is essential to prevent loss
a dark field function is useful in determining of the cell line. If proper culture conditions
undifferentiated regions. are maintained, the cells should not accumu-
For human ES cell culture, the batch of late karyotypic changes; cells cultured over
FBS utilized is essential for maintenance of 150 passages in the authors’ laboratory still
the undifferentiated state. FBS containing a maintain normal karyotype.
low endotoxin level (<1.5 EU/ml) is pro-
posed to be efficient for human ES cell cul- Troubleshooting
ture. Batch testing is essential prior to culture If human ES cells begin to spontaneously
with human ES cells. FBS should be aliquoted differentiate in culture, it is advisable to imme-
and stored at −20◦ C and thawed only once diately transfer them regardless of the day fol-
Stem Cells
to make medium as repeated freeze/thaw lowing the last transfer. New medium should
23.2.19
Current Protocols in Cell Biology Supplement 28
be prepared and sterilized by filtration through single cells. Subsequent to derivation, passag-
a 0.22-µm filter. A new aliquot of MEFs should ing of MEFs requires 20 to 40 min depending
then be thawed and prepared for transfer of hu- on number of flasks. Mitomycin C inactivation
man ES cells. of MEFs requires ∼3.5 hr (including a 2.5-hr
incubation). Freezing of MEFs or mouse ES
Anticipated Results cells requires ∼20 to 30 min. Thawing cells
MEF derivation is highly efficient and takes 20 to 30 min.
should be successful every time. Each batch
of MEFs, however, may differ in their ability Media
Approximately 1 to 1.5 hr should be al-
to support human ES cell culture and therefore
lowed for media preparation 2 weeks prior to
must be tested for a minimum of three passages
culture.
of human ES cells. Mouse ES cells are gener-
ally more tolerant of MEFs, especially when Mouse ES cells
medium containing LIF is used. Mouse ES cell derivation is laborious and
Mouse ES cell derivation has an efficiency great care is required to ensure their subse-
of 10% to 15% depending on the mouse strain quent growth, due primarily to the low effi-
used. Although inbred strains other than 129sv ciency of derivation. Maintenance of mouse
have been used, success rates for mouse ES cell ES cells is relatively time efficient when using
derivation were significantly reduced. Mouse LIF alone, with simple tissue culture passag-
ES cell cultures tend to contain 5% to 10% ing required; however, time requirements sig-
differentiated cells, this increases upon con- nificantly increase when growing the cells on
fluency, thus passaging prior to confluence is MEFs because of the time-consuming process
important. When mouse ES cells are trans- of cell preparation and mitotic inactivation.
ferred to hanging drop cultures, ∼90% to 95%
of drops are seen to form EBs. At the meniscus Mouse EB formation
of the drop, cells will aggregate and by 1 to 2 The setup for EB formation requires 20 to
days will be seen to form spheres that continue 30 min depending on the number of drops
to grow in the droplet until the desired time needed for hanging drops; it takes 10 to 15
point. When mouse ES cells are transferred to min to set up a suspension culture.
suspension, EB formation is somewhat slower,
with cells forming smaller spheres both with Human ES cells
their progeny (clonal) and by aggregating with Following MEF preparation, human ES cell
other cells. By 2 to 3 days, spheres should be mechanical dissection should take ∼10 min
present, and these spheres should continue to or less per organ culture dish. Medium must
expand. be changed daily until the following transfer.
Human ES cell culture is efficient, provided With prewarmed medium, this can take 5 to
that competent MEFs and sterile medium are 15 min depending on the number of plates.
used and care is taken when transferring them. Medium must be made and MEFs prepared
Upon transfer of clumps of human ES cells, fresh each week for transfers or used to collect
they attach forming colonies that expand in conditioned medium, this is quite a laborious
a regular manner. When freezing/thawing hu- undertaking and can take up to a full day.
man ES cells, expect ∼10% to 30% cell death.
With human EB formation, ∼70% to 80% of Human EB formation
ES cell aggregates placed in suspension will Human EB formation takes approximately
form EBs, with some clumps attaching to the the same time as required for transfers, with
dish. Clumps should become spherical balls clumps cut smaller than those for transfers and
after 2 to 3 days in culture and continue to then placed in suspension.
grow until ∼8 to 12 days when fluid-filled
cysts become obvious and EBs may begin to Literature Cited
form attachments with the dish. Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu,
C.P., Harris, C.P., Waknitz, M.A., Itskovitz-
Eldor, J., and Thomson, J.A. 2000. Clonally de-
Time Considerations rived human embryonic stem cell lines maintain
pluripotency and proliferative potential for pro-
MEFs longed periods of culture. Dev. Biol. 227:271-
Derivation,
Culture, and Dissection of fetuses will depend on the 278.
Differentiation number of fetuses and competence of the Buehr, M. and Smith, A. 2003. Genesis of embry-
of Embryonic operator. Approximately 1 to 1.5 hr should
Stem Cells onic stem cells. Philos. Trans. R. Soc. Lond. B
be spent dissecting and dissolving tissue into Biol. Sci. 358:1397-1402.
23.2.20
Supplement 28 Current Protocols in Cell Biology
Conley, B.J., Young, J.C., Trounson, A.O., and integrity of human embryonic stem cells. Nat.
Mollard, R. 2004a. Derivation, propagation and Biotechnol. 23:19-20.
differentiation of human embryonic stem cells. McDonald, J.W., Liu, X.Z., Qu, Y., Liu, S., Mickey,
Int. J. Biochem. Cell. Biol. 36:555-567. S.K., Turetsky, D., Gottlieb, D.I., and Choi,
Conley, B.J., Trounson, A., and Mollard, R. 2004b. D.W. 1999. Transplanted embryonic stem cells
Human embryonic stem cells form embry- survive, differentiate and promote recovery in
oid bodies containing visceral endoderm-like injured rat spinal cord. Nat. Med. 5:1410-1412.
derivatives. Fetal Diagn. Ther. 193:218-224. Mummery, C., Ward-van Oostwaard, D., Doeven-
Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, dans, P., Spijker, R., van den Brink, S., Hassink,
W., and Kemler, R. 1985. The in vitro devel- R., van der Heyden, M., Opthof, T., Pera, M.,
opment of blastocyst-derived embryonic stem de la Riviere, A.B., Passier, R., and Tertoolen,
cell lines: Formation of visceral yolk sac, blood L. 2003. Differentiation of human embryonic
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153.

Derivation,
Culture, and
Differentiation
of Embryonic
Stem Cells

23.2.22
Supplement 28 Current Protocols in Cell Biology
Maintenance and In Vitro Differentiation UNIT 23.3
of Mouse Embryonic Stem Cells to Form
Blood Vessels
Embryonic stem (ES) cells are pluripotent cells that have been derived from the inner
cell mass of blastocyst-stage embryos. Early studies of the ES differentiation model
demonstrated that vasculogenesis and hematopoiesis were among the earliest develop-
mental processes to occur (Doetschman et al., 1988; Schmitt et al., 1991; Wiles and
Keller, 1991; Wang et al., 1992). Furthermore, the endothelial cells that differentiate
during this model coalesce to form primitive blood vessels that are analogous to the first
vessels that form in the developing embryo and yolk sac (Wang et al., 1992). Vascu-
lar development during mouse ES cell differentiation in vitro occurs by two processes;
vasculogenesis and angiogenesis, similar to embryonic vascular development in vivo
(Poole and Coffin, 1989; Risau, 1997). In vasculogenesis, mesodermally derived pre-
cursor cells, known as angioblasts, differentiate and coalesce to form a primitive blood
vessel. In angiogenesis, endothelial cells from preexisting vessels coordinate cell prolif-
eration and sprouting migration as a basis for expansion of vessels. Primitive erythrocytes
and embryonic macrophages mature alongside vascular endothelial cells during ES cell
differentiation (Wiles and Keller, 1991; Inamdar et al., 1997; Kearney and Bautch, 2003).
Thus this model is ideal for studying molecular and cellular aspects of early vascular
and hematopoietic development. This unit describes protocols for maintaining and dif-
ferentiating mouse embryonic stem cells and methods of analyzing the vasculature via
immunohistochemistry and β-galactosidase staining.

A protocol for the formation of embryoid bodies (EBs) and subsequent in vitro differ-
entiation of mouse ES cells is described (see Basic Protocol). In combination with the
in vitro differentiation protocol, an explanation of how to test different lots of fetal bovine
serum (FBS) for maintaining differentiated cultures has been described (see Support
Protocol 8). Methods for analysis of differentiation are included: testing for optimum
ES cell differentiation via benzidine staining (see Support Protocol 1) and PECAM/
Mac-1 double immunostaining (see Support Protocol 2), antibody immunolocalization
(see Support Protocol 3), and lacZ staining of in vitro differentiated ES cells (see Support
Protocol 4). Other protocols in this unit are designed to provide a method for maintaining
mouse ES cells (see Support Protocol 5). A protocol for harvesting 5637 cell–conditioned
medium, which contains the LIF that prevents differentiation, is explained (see Support
Protocol 6). In addition, a protocol used for testing FBS lots from different manufacturers
for ES Medium for maintenance is described (see Support Protocol 7).

NOTE: The protocols in this unit should be conducted under aseptic conditions using
sterile reagents and equipment. Experiments should be performed in a Class II Biological
Flow Hood.

NOTE: All incubations should be performed in a humidified 37◦ C, 5% CO2 incubator,


unless otherwise indicated.

PROGRAMMED IN VITRO DIFFERENTIATION OF MOUSE EMBRYONIC BASIC


STEM CELLS PROTOCOL
Mouse embryonic stem cells have the distinctive ability to differentiate into numerous cell
types. In vitro ES cell differentiation complements their ability to contribute many tissue
types in vivo, thus providing a unique model system for aspects of early mammalian
Stem Cells
Contributed by Nicholas C. Kappas and Victoria L. Bautch 23.3.1
Current Protocols in Cell Biology (2007) 23.3.1-23.3.20
Copyright 
C 2007 by John Wiley & Sons, Inc.
Supplement 34
development. The conditions that are used initiate a programmed differentiation that
leads to the formation of many of the cell types typically found in the mouse embryo and
yolk sac. The following utilizes an unmanipulated differentiation protocol that involves
the removal of differentiation inhibitory factors, thus allowing the ES cells to undergo a
programmed differentiation to form cells that will in turn provide developmental signals
to each other. In this case, cultures are examined for vasculogenesis and angiogenesis.
Materials
5- to 6-day-old ES cell culture (Support Protocol 5)
1× CMF-PBS (Sigma)
2.4 U/ml dispase, grade II stock (Boehringer-Mannheim; see recipe)
Differentiation medium (see recipe)
50-ml centrifuge tubes (Sarstedt), sterile
10-cm bacteriological petri dishes (do not use TC-treated plates)
24-well tissue culture plates (Costar)
Medidroppers (Fisher Scientific), autoclaved
NOTE: All volumes are given assuming that one 6-cm dish of ES cell colonies is being
used. Double all volumes if using 10-cm dishes.
Collect ES cells
1. Choose the dish that has the best ES cell clumps for differentiation.
ES cell clumps should be round and differentiated on the very edge and tight, shiny, and
undifferentiated in the middle (Figure 23.3.1). ES cell clumps are collected from dishes
after incubation for 5 to 6 days at 37◦ C without feeding after normal passage.
2. Aspirate medium from the ES cell dish. Wash two times, each time with 5 ml of cold
1× CMF-PBS. Aspirate PBS.
3. Add 1 ml of cold 1.2 U/ml dispase (diluted 1:1 with cold CMF-PBS just before use),
and let dish sit at room temperature for 1 to 2 min. Check to see if ES cell clumps
have detached from dish bottom by shaking dish.
If the majority of cells clumps have not detached, let the solution sit longer. Dispase
produces small clumps of cells that are used to start the differentiation.

Figure 23.3.1 ES cells are typically processed for passage after 3 days of growth, when they
Vascular are observed in tight, shiny clumps (A, D). ES cells undergo enzymatic disruption treatment for
Differentiation of
Mouse ES Cells in vitro differentiation on day 5, when the ES clumps are large, but appear differentiated on the
edges (B, E). Day 3 EBs (C, F) prior to plating (Bar = 100 µm).
23.3.2
Supplement 34 Current Protocols in Cell Biology
4. When a majority of the cell clumps have detached from the dish, use a 5-ml pipet to
gently transfer the cells into a 50-ml tube containing 35 ml of room temperature 1×
CMF-PBS. Rinse the dish with 5 ml of 1× CMF-PBS and add the rinse to the 50-ml
tube. Cap tube, and invert the tube once gently to mix.
5. Let the tube sit until the cell clumps have settled to the bottom of the tube.
Wash and plate the clumps
6. Aspirate all but 4 to 5 ml of CMF-PBS, carefully avoiding the ES cell clumps.
7. Add another 35 ml room temperature 1× CMF-PBS gently down the side of the
50-ml tube. Gently swirl the tube to redistribute the cell clumps. Cap tube, and invert
gently to mix.
8. Let the tube sit until the cell clumps have settled to the bottom of the tube.
9. Aspirate the CMF-PBS (leaving 2 to 3 ml CMF-PBS/ES cell clump suspension at
the bottom), and gently add 5 ml prewarmed (37◦ C) differentiation medium down
the side of the tube.
10. Pipet 10 ml of prewarmed differentiation medium into a labeled 10-cm bacterio-
logical petri dish. Using a 25-ml pipet, transfer the contents of the 50-ml tube (cell
clumps/CMF-PBS/medium) to the 10-cm dish.
11. Check the density of cell clumps in each dish (∼100 clumps). Incubate at 37◦ C in a
humidified incubator with 5% CO2 .
The authors attempt to achieve ∼100 clumps/dish. Fewer clumps is not an efficient use of
medium, while more clumps may allow for individual clumps to aggregate together.

Allow formation of embryoid bodies


12. Change the medium at least every other day after the dispase treatment (day 0). To
feed the EBs, remove dish from incubator, place under hood, and gently swirl the
dish in a circular manner so that the EBs migrate to the center of the dish.
13. Aspirate spent medium from the dish.
This is easily done by aspirating along the inside edge of the dish (away from the EBs).

14. Add 10 ml of fresh prewarmed differentiation medium to the dish. Return dish to
incubator.
Differentiate embryoid bodies
15. Set up reattachment cultures on day 3 after the dispase treatment (Fig. 23.3.1). Add
1.5 ml of prewarmed differentiation medium to each well of a 24-well tissue culture
plate that is to be seeded with EBs.
16. Use a sterile medidropper to transfer EBs from the dish to the wells of a 24-well
plate. Generally, dispense between 10 and 20 EBs per well.
Holding the dish up and looking at it from underneath is helpful when determining the
number of EBs in a well.

17. Ensure that the EBs are spread evenly in the well by gently shaking/moving the plate,
or if necessary, use a pipettor with a sterile tip to gently pipet medium up and down
in the well.
18. Place the plate in the incubator. Keep plate level when placed in incubator to ensure
that the EBs do not settle to one side of the well. Incubate at 37◦ C in a humidified
incubator with 5% CO2 .
Attachment generally occurs within a few hours. Stem Cells

23.3.3
Current Protocols in Cell Biology Supplement 34
19. Feed the attached cultures every other day. To feed, aspirate medium, then slowly
add 1.5 to 2 ml fresh prewarmed differentiation medium down the side wall of the
well so as not to disturb the attached cultures.
20. Monitor the cultures for hematopoietic and vascular development (see Support
Protocols 1 to 4).
Using this protocol, the authors typically monitor vascular development in cultures that
have been differentiated for 8 days (day of dispase treatment is day 0). Angioblast
formation is generally observed at day 4 to 6, while vessel formation occurs at day 6 to 8.
All volumes given are for staining cultures from a well of a 24-well plate. Gently
add/remove all solutions from culture dishes, as harsh removal and addition of solu-
tions may result in detachment of cultures.

SUPPORT BENZIDINE STAINING


PROTOCOL 1
Benzidine staining is performed on day-8 differentiated cultures to visualize the
hemoglobin of red blood cells.

Materials
8-day differentiated ES cell cultures (Basic Protocol)
Benzidine solution (see recipe), 4◦ C
Differentiation medium (see recipe)
1× CMF-PBS (Sigma)
1. Remove 8-day differentiated cultures from incubator and add 0.2 ml of cold (4◦ C)
benzidine solution per 2 ml (per well) of differentiation medium. Incubate at room
temperature.
Benzidine solution is added directly to the cultured cells while still in differentiation
medium (i.e., do not rinse cells in CMF-PBS at this time).
The benzidine reaction will occur immediately: upon addition of benzidine solution, the
medium will turn a bluish/brown color, while positive cells will turn blue.
2. Observe/photograph cells within 1 hr, as the stain is not very stable.
It is recommended that wells be rinsed once with 1× CMF-PBS before taking photographs
of stained wells.

SUPPORT DOUBLE STAINING WITH PECAM AND Mac-1


PROTOCOL 2
PECAM/Mac-1 double staining is performed on day 10 differentiated cultures to visualize
both blood vessels (PECAM) and embryonic macrophages (Mac-1).

Materials
Day-10 differentiated cultures (from Basic Protocol)
1× CMF-PBS (Sigma)
4% (w/v) paraformaldehyde fixative (see recipe)
1:500 trypsin solution (see recipe)
100% heat-inactivated fetal bovine serum
Staining medium (see recipe)
Primary and secondary antibodies (see recipe);
Rat anti-mouse CD31 (PECAM-1; Mec 13.3; BD Pharmingen)
FITC-AffinPure F(ab )2 fragment of donkey anti-rat IgG (Jackson
Immunoresearch)
Mac-1-BNHS (biotin coupled to Mac-1 antibody); clone M1/70;
Vascular Rat anti-mouse Mac-1 (BD Pharmingen)
Differentiation of Streptavidin-RPE (Southern Biotechnology Associates)
Mouse ES Cells
Rat serum staining medium (see recipe)
23.3.4
Supplement 34 Current Protocols in Cell Biology
Treat cultures with trypsin
1. Remove day-10 differentiated cultures from incubator and bring plate to room tem-
perature. Remove medium, wash cells twice with 1× CMF-PBS at room temperature,
and fix cells in 4% paraformaldehyde (PFA). Use 1 ml of cold 4% PFA/well for 5 to
10 min at room temperature.
2. Aspirate PFA and add 2 ml room temperature 1× CMF-PBS/well. Allow dish to sit
for 2 min before aspirating CMF-PBS. Repeat wash 1 more time. Either store at 4◦ C
in CMF-PBS until ready to stain, or proceed with the staining protocol.
3. Add 0.2 ml room temperature 1:500 trypsin solution/well of a 24-well plate. Let
1:500 trypsin solution sit for no more than 60 to 75 sec at room temperature.
Incubation times longer than 60 to 75 sec may result in complete detachment of differen-
tiated cultures. This step slightly loosens cells and enhances Mac-1 staining.
4. Stop the trypsin reaction by adding 0.5 ml of room temperature 100% heat-inactivated
FBS to the well. Immediately remove the solution from the well with gentle aspiration
or pipetting.
5. Proceed with immunostaining or store in staining medium at 4◦ C.
Stain with PECAM
6. Gently add 1.5 to 2 ml (per well of a 24-well plate) of staining medium. Incubate for
1 hr at 37◦ C to block nonspecific sites.
7. Remove plate from incubator, aspirate staining medium, and add fresh staining
medium containing Mec13.3 (PECAM antibody) at 1:1000 dilution. Incubate for
1 hr at 37◦ C.
PECAM is expressed by mouse endothelial cells.
Typically, 250 µl of staining medium/antibody is used per well of a 24-well plate.
8. Remove plate from incubator and aspirate PECAM antibody. Wash wells twice, each
time with 2 ml room temperature staining medium.
9. Add fresh staining medium containing FITC-AffinPure F(ab’)2 donkey anti-rat an-
tibody at 1:100 dilution. Incubate for 1 hr at 37◦ C in the dark.
Typically, 250 µl of staining medium/secondary antibody is used per well of a 24-well
plate.
Stain with Mac-1
10. Remove plate from incubator and aspirate FITC reagent. Wash wells twice, each
time with 2 ml staining medium at room temperature.
If desired, the plate can be stored at 4◦ C, in the dark, for later processing. Be sure to
leave staining medium in wells.
11. Add 2 ml/well of rat serum staining medium. Incubate for 30 min at 37◦ C in the dark.
If desired, the plate can be stored at 4◦ C, in the dark, for later processing. Be sure to
leave rat serum staining medium in wells.
12. Remove rat serum staining medium. Add fresh rat serum staining medium containing
Mac-1-BNHS at 1:800. Incubate for 1 hr at 37◦ C, in the dark.
Mac-1 is expressed by embryonic macrophages.
Typically, 250 µl of rat serum staining medium/ antibody is used per well of a 24-well
plate.

Stem Cells

23.3.5
Current Protocols in Cell Biology Supplement 34
13. Remove Mac-1-BNHS and wash well(s) twice with 2 ml staining medium at room
temperature. Add fresh staining medium containing streptavidin-R-PE at 1:800.
Incubate for 1 hr at 37◦ C in the dark.
Typically, 250 µl of staining medium/secondary antibody is used per well of a 24-well
plate.

14. Remove streptavidin-RPE and wash well(s) once with 2 ml staining medium at room
temperature, then aspirate. Next, wash well(s) twice with 2 ml 1× CMF-PBS. Add
2 ml CMF-PBS and store plate at 4◦ C in the dark.
15. Examine the plate using fluorescence microscopy with the appropriate filter sets.

SUPPORT IMMUNOLOCALIZATION
PROTOCOL 3
An important aspect of a model system is that it recapitulates the expression of molecular
markers seen in vivo. Much of the vascular development that is observed in this in vitro
ES differentiation system is analogous to blood island and vascular development that
is observed in the mouse embryo and yolk sac. Fortunately, it is with general ease that
one can observe primitive ES cell–derived mouse blood vessels using several markers
and protocols. There are a number of identified molecular markers for the early mouse
vasculature. Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD-31), VEGF
(vascular endothelial growth factor) receptor 2 (Flk-1), VE-cadherin, and intercellular
adhesion molecule 2 (ICAM-2) have proven particularly useful in visualizing primitive
vessels. It has been shown that PECAM-1 and Flk-1 are expressed on mouse angioblasts
and endothelial cells (Vittet et al., 1996; Redick and Bautch, 1999). Other vascular
markers such as ICAM-2 and VE-cadherin are detectable later, when endothelial cells
coalesce to form vessels. This is a protocol for visualizing expressed genes in the in vitro
ES cell differentiation model system that utilizes antibodies specific for several vascular
markers.

Materials
Differentiated ES cell culture (from Basic Protocol)
1× CMF-PBS (Sigma)
Methanol/actetone fixative (see recipe): 1:1 (v/v) methanol (Fisher
Scientific)/acetone (Mallinckrodt) or 4% (w/v) paraformaldehyde (PFA;
Polysciences; see recipe)
Staining medium (see recipe)
Primary and secondary antibodies (see recipe)
Fluorescent microscope, inverted (equipped with epifluorescence and camera)
NOTE: All volumes given are for staining cultures from a 24-well plate. Gently add and
remove all solutions from culture plates, as harsh removal and addition of solutions may
result in detachment of cultures.

Fix the cells


1. Remove differentiated cultures from incubator, and allow to slightly cool. Aspirate
medium and wash twice with 2 ml/well of 1× CMF-PBS at room temperature.
2. Add 1 ml of cold methanol/acetone fixative/well and incubate for 5 min at room
temperature.
PECAM-1 and ICAM-2 antibodies work in both PFA and methanol/acetone fixatives.
Both fixatives are listed here because some applications (not those described here) favor
one fixative over another. The authors generally use methanol/acetone fixative because it
Vascular is less harsh on the attached cultures.
Differentiation of
Mouse ES Cells

23.3.6
Supplement 34 Current Protocols in Cell Biology
3. Aspirate fixative and add 2 ml of room temperature 1× CMF-PBS/well. Allow plate
to sit for 2 min before aspirating CMF-PBS. Repeat wash two more times. Either
store at 4◦ C in CMF-PBS until ready to stain, or proceed with the staining protocol.
Stain the cells
4. If the plate has been stored at 4◦ C after fixation, allow the plate to come to room
temperature.
5. Aspirate 1× CMF-PBS, and gently add 1.5 to 2 ml of staining medium. Incubate for
45 min to 1 hr at 37◦ C.
6. Remove plate from incubator, aspirate staining medium, and add fresh staining
medium containing the properly diluted primary antibody. Incubate for 1 to 2 hr at
37◦ C.
Typically, 250 µl of staining medium/primary antibody is used per well of a 24-well plate.

7. Remove plate from incubator, aspirate, and wash well(s) two times (2 to 3 min/wash)
with 1.5 to 2 ml staining medium. Add fresh staining medium containing the properly
diluted secondary antibody. Incubate for 1 hr at 37◦ C.
Typically, 250 µl of staining medium/secondary antibody is used per well of a 24-well
plate.

8. Remove plate from incubator, aspirate, and wash well(s) one time for 2 to 3 min
with 1.5 to 2 ml staining medium. Aspirate wash, then wash one to two times (2
min/wash) using 1× CMF-PBS.
9. Aspirate last wash and add 1.5 to 2 ml of 1× CMF-PBS to each well, store in
CMF-PBS at 4◦ C in the dark.
10. To visualize the vasculature, set up an inverted microscope equipped with epifluo-
rescence and a camera.

β -GALACTOSIDASE STAINING SUPPORT


PROTOCOL 4
Another commonly used technique to visualize blood vessels in the in vitro ES cell
differentiation model system relies on the β-galactosidase reporter system. The authors
have mouse ES cells in which expression of the lacZ gene is under the control of a vascular
promoter (Flk-1, Flt-1). The β-galactosidase protein is the gene product of lacZ and is
easily assayed. β-galactosidase catalyzes the hydrolysis of the colorless substrate Xgal,
to form a blue precipitate in β-galactosidase-expressing cells. This protocol describes
the steps involved in β-galactosidase staining of ES cell–derived blood vessels.

Materials
Day-8 differentiated cultures in 24-well plate (Basic Protocol)
0.1 M phosphate buffer, pH 7.3 to 7.5 (see recipe)
Glutaraldehyde fixative solution (see recipe)
Xgal stain (see recipe)
Wash buffer (see recipe)
NOTE: All volumes given are for staining cultures from a 24-well plate. Gently add and
remove all solutions from culture dishes, as harsh removal and addition of solutions may
result in detachment of cultures.

1. Remove differentiated cultures from incubator, and allow to slightly cool. Aspirate
medium and wash well(s) once with 1.5- to 2 ml 0.1 M phosphate buffer (pH 7.3 to
7.5) at room temperature.
Stem Cells

23.3.7
Current Protocols in Cell Biology Supplement 34
2. Aspirate phosphate buffer and add 1 ml glutaraldehyde fixative solution to each well
and fix for 5 min at room temperature.
3. Aspirate fixative. Wash cells three times (5 min/wash), each time with 1.5 to 2 ml
0.1 M phosphate buffer at room temperature.
4. Aspirate phosphate buffer. Add 200 µl Xgal stain to each well. Stain cells for 4 hr to
overnight at 37◦ C depending on level of lacZ activity.
5. After staining, remove Xgal stain, add 1 ml wash buffer to each well, aspirate, then
replace with 1 ml wash buffer to each well.
6. Examine plates for blue-staining structures indicating blood vessels (see
Fig. 23.3.2C).

Figure 23.3.2 The vasculature of differ-


entiated ES cell attached cultures can
be visualized by both immunolocaliza-
tion (PECAM stain) and enzymatic reac-
tion (β-galactosidase stain). (A, B) ES
cell culture differentiated to day 8, fixed
and stained using the PECAM antibody,
depicting a typical vascular pattern by
immunofluorescence (A), and the corre-
sponding phase image (B). (C) ES cell
culture differentiated to day 8, fixed and
stained for β-galactosidase, showing a
vascular pattern (C) in phase.

Vascular
Differentiation of
Mouse ES Cells

23.3.8
Supplement 34 Current Protocols in Cell Biology
CULTURING MOUSE EMBRYONIC STEM CELLS IN THE ABSENCE SUPPORT
OF FEEDER CELLS PROTOCOL 5
There are numerous published protocols describing mouse embryonic stem cell (ES)
maintenance (Bautch, 2002; Kearney and Bautch, 2003). Here the authors describe the
maintenance of mouse ES cells for in vitro differentiation. Leukemia Inhibitory Factor
(LIF) is a leading component that contributes to stem cell maintenance and prevention
of cell differentiation. Traditionally, ES cells are maintained on a layer of feeder cells
(usually mouse embryo fibroblasts or STO cells), which provide LIF to the surrounding
ES cells. Here the authors describe the maintenance of ES cells using medium that has
been conditioned by the 5637 human bladder cancer cell line. This cell line produces
LIF that is subsequently used for ES cell maintenance. The authors prefer the 5637
cell–conditioned medium to commercial LIF because they feel that it best preserves the
undifferentiated morphology of the ES cells grown off of feeder layers.

Materials
3- to 4-day-old ES cell dishes (UNIT 23.2), without feeder layer, passage 3 to 45
1× CMF-PBS (Sigma)
0.25× trypsin/EDTA (see recipe)
Trypsin stop solution (see recipe)
ES cell culture medium (see recipe)
Gelatin-coated dishes (see recipe)
6-cm tissue culture (TC)–treated culture dishes (Corning)
NOTE: All volumes are given assuming that one 6-cm dish of ES cell colonies is being
used. Double all volumes if using 10-cm dishes. Prewarm all solutions to 37◦ C prior to
use.

1. Remove 3- to 4-day-old ES cell dish(es) from 37◦ C incubator. Aspirate medium.


Wash two times, each time with 5 ml 1× CMF-PBS at room temperature and then
remove.
ES cells must be passed frequently to prevent differentiation. The authors typically pass
ES cells every 3 to 4 days. On the day of passage, ES colonies are typically found in tight,
shiny clumps (Fig. 23.3.1). If the colonies are large and flat, the optimal time for passage
has passed.

2. Add 1 ml 0.25× trypsin/EDTA solution to dish. Place the dish in 37◦ C incubator
until a majority of ES cell clumps disassociate upon gentle agitation (1 to 3 min).
This 1-ml volume should just cover the bottom of the dish.

3. Stop trypsinization reaction by adding 4 ml trypsin stop solution to dish. Gently draw
the ES cells/trypsin stop solution up and down with a pipet a few times to break up
the cell clumps.
4. Remove a gelatin-coated 6-cm dish from incubator. Aspirate gelatin solution, and
add 5 ml pre-warmed ES cell culture medium. Add 2 to 3 drops of the cell suspension
into the dish. Observe the sizes of ES cell clumps under a microscope.
ES cells should be in a single-cell suspension, or in clumps of no more than 4 to 6 cells/
clump. If cell clumps are significantly larger, pipet solution to further break apart cell
clumps.

5. Place dish in 37◦ C incubator with 5% CO2 and gently move dish in a back-and-forth
motion in order to evenly disperse ES cells throughout the dish.

Stem Cells

23.3.9
Current Protocols in Cell Biology Supplement 34
SUPPORT PREPARATION OF 5637 CELL–CONDITIONED MEDIUM (CM)
PROTOCOL 6
The conditioned medium of confluent 5637 cells contains LIF (leukemia inhibitory
factor/differentiation inhibiting activity), which is used as a medium component for the
maintenance of ES cell cultures.

Materials
5637 human bladder carcinoma cell line (ATCC #HTB9)
5637 cell growth medium (recipe)
Collection medium (see recipe)
6- and 15-cm tissue culture (TC)–treated culture dishes (Corning)
15- and 50-ml centrifuge tubes (Sarstedt)
200-ml centrifuge tubes, if desired (NUNC)
1-, 3-, 10, and 25-ml disposable pipets
0.22-µm cellulose acetate filter units with bottle (Corning) or Nalgene SFCA
0.2-µm bottle top filter with 33-mm neck to fit standard 500-ml glass bottles
500-ml bottles, autoclaved
NOTE: The following protocol describes the collection of 5637 medium from one 15-cm
plate, however, more plates are typically processed for collection. Adjust volumes as
needed if more plates are to be processed for 5637 collection.

1. Thaw and grow 5637 cells in 5637 cell growth medium to 80% to 90% confluence
(usually 2 to 3 days after thaw) in 15-cm tissue-culture dishes. Grow cells in 37◦ C
incubator.
The authors typically use 25- to 30 ml 5637 cell growth medium/15-cm dish.

2. At cell confluence (2 to 3 days), remove dishes from incubator, aspirate 5637 cell
growth medium and feed cells using collection medium. Use 25 to 30 ml collection
medium/15-cm dish.
3. After 48 hr, remove the dishes from the incubator and carefully transfer the 5637–
conditioned medium from the 15-cm dishes of cells to 50-ml or 200-ml centrifuge
tubes using a 25-ml pipet. Avoid scraping the monolayer of cells. Add 25 to 30 ml
of fresh collection medium and return plate to incubator.
The addition of fresh medium to the dish allows for subsequent collection of additional
5637–conditioned medium.

4. Balance the volumes in the 50-ml tubes and centrifuge 10 min at 770 × g, 4◦ C.
5. In the hood, remove the supernatant from each tube and sterile filter through a
0.22-µm cellulose acetate filter unit.
The supernatant will take a long while to filter.
6. After filtration is complete, place 3 ml of the filtered 5637–conditioned medium in
a 6-cm dish into the incubator and check for contamination after 24 hr.
7. Collect conditioned medium every 2 to 3 days for 5 or 6 collections (30 ml/collection/
plate). Combine/pool the filtered collected media. Store 5637 cell–conditioned
medium up to 1 month at 4◦ C. Freeze medium at −20◦ C for use at a later time
(up to 6 months). Use the conditioned medium in ES cell culture medium.
Collect conditioned medium until the cells look sub-optimal (usually after 5th collection).
Suboptimal cells will typically detach from the dish or have a disrupted cell monolayer.
Vascular
Differentiation of
Mouse ES Cells

23.3.10
Supplement 34 Current Protocols in Cell Biology
ES cell culture medium containing individual pooled collections of conditioned medium
is subsequently used for maintenance of ES cell cultures. It is important for 5637
cell– conditioned medium to maintain ES cells in an undifferentiated state. Overly differ-
entiated ES cells, as evidenced by large, flat cells in colonies and no shiny center, are an
indication of sub-optimal 5637 cell–conditioned medium.

LOT TESTING OF FBS FOR PASSAGE OF ES CELLS SUPPORT


PROTOCOL 7
A number of different lots of FBS (in parallel with the current lot) are tested in ES
medium to ensure the best maintenance and growth of undifferentiated ES cells. To
test different lots of FBS, ES cells are maintained in ES medium containing one lot of
FBS (one lot of FBS/batch ES cell culture medium), for three passages. During these
three passages, ES cells are analyzed for their proper morphology (shiny, tight clumps
of cells) and growth. After this point, ES cells are differentiated to day 8, then analyzed
for a healthy and normal vasculature pattern via PECAM antibody staining. In this way,
ES cells maintained in medium containing different FBS lots during cell passage can
be compared. Typically, three to four lots of FBS are tested independently in batches of
ES cell culture medium.

Materials
ES cell culture medium (see recipe), prepared with different lots of FBS
3- to 4-day-old ES cell culture dish (Support Protocol 5)
6-cm tissue culture–treated culture dishes (Corning)
Additional reagents and equipment on in vitro differentiation of ES cells (Basic
Protocol), passaging of ES cells (Support Protocol 6), and fixing and staining
differentiated cultures (Support Protocol 3)
1. Prepare different batches of ES cell culture medium, with each batch containing a
different lot of FBS including the currently used lot. To test FBS lots, use the same
ES clone to test all lots.
2. Remove 3- to 4-day-old ES cell culture dish from 37◦ C incubator. Passage ES cells
(Support Protocol 6) setting up one to two 6-cm plates of ES cells per lot of FBS to
be tested.
3. Passage ES cells for three passages using the same ES cell culture medium and FBS
lot to be tested to maintain the same group of ES cells for the duration of the three
passages.
One parameter used to assess whether an FBS lot is of good quality is if ES cells
maintain an undifferentiated morphology of tight, shiny clumps for the duration of the
three passages.

4. After completion of the third passage, in vitro differentiate the groups of ES cells
(Basic Protocol).
5. On day 8 of in vitro differentiation, remove cells from incubator. Fix and stain
differentiated cultures with anti-mouse PECAM antibody (Support Protocol 3).
By maintaining/differentiating ES cells and assessing the vasculature (via PECAM anti-
body staining) of ES cells maintained in different lots of FBS, it is possible to evaluate
the ability of each lot of FBS to properly maintain ES cells to form blood vessels.

Stem Cells

23.3.11
Current Protocols in Cell Biology Supplement 34
SUPPORT LOT TESTING OF FETAL BOVINE SERUM FOR IN VITRO
PROTOCOL 8 DIFFERENTIATION OF MOUSE ES CELLS
A number of different lots of FBS are tested in differentiation medium to ensure the
best in vitro differentiation of ES cells into blood vessels. To test different lots of FBS,
differentiated ES cells are maintained in differentiation medium containing a unique test
lot of FBS (one lot of FBS/batch of differentiation medium), until day 8 of differentiation.
Day 8 differentiated cultures are then fixed and stained for vasculature (PECAM stain;
Support Protocol 2), red blood cell differentiation (benzidine stain; Support Protocol 1),
and embryonic macrophages (Mac1 stain; Support Protocol 2). This way, differentiated
cells maintained in medium containing different FBS lots can be compared by the
expression of markers that are observed in differentiated ES cultures.

Typically, three to four lots of FBS, including the lot currently in use, are tested indepen-
dently in batches of differentiation medium.

Materials
Differentiation medium (see recipe), prepared with different lots of FBS
5-day-old to 6-day-old ES cell culture (Support Protocol 5)
Additional reagents and equipment for in vitro differentiation of ES cells (Basic
Protocol)
1. Make up different batches of differentiation medium, with each batch containing a
different lot of FBS. To test FBS lots, use the same differentiated clone for all test
media.
2. Remove 5-day-old ES cell dishes from incubator. Ensure that there is one 6-cm dish
per batch of differentiation medium (containing a unique FBS lot) to be processed
for in vitro differentiation.
3. In vitro differentiate ES cells (see Basic Protocol).
4. For each group of differentiated ES cells (corresponding to each FBS lot to be tested),
set up attached cultures as described in Basic Protocol (steps 15 to 20). Attach each
group of differentiated cells in four wells in each of two 24-well plates.
The four wells of one 24-well plate will be used for benzidine staining, while the four wells
of the other 24-well plate will be used for double immunostaining with PECAM and Mac-1.

REAGENTS AND SOLUTIONS


Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Antibodies, primary and secondary (and streptavidin-R-PE)


Dilute all antibodies in staining medium (see recipe).

Primary antibodies: Dilute rat anti-mouse PECAM-1 (CD31; Pharmingen) to


1:1000, rat anti-mouse ICAM-2 (3C4; Pharmingen) to 1:500, rat anti-mouse Mac-
1-BNHS (Pharmingen, no. 01711D) to 1:800. Prepare just prior to use.

Secondary antibodies: Dilute reconstituted secondary antibody goat anti-rat Alexa


Fluor 488 (Molecular Probes) to 1:250 (for detecting PECAM-1 and ICAM-2),
FITC-AffinPure F(ab’)2 donkey anti-rat to 1:100 (Jackson Immunoresearch, no.
712-096.150), and streptavidin-R-PE (Southern Biotechnology, no. 7100-09) to
1:800. Store at 4◦ C after reconstitution (according to the manufacturer) for up to
Vascular 6 months.
Differentiation of
Mouse ES Cells
Do not freeze reconstituted antibodies.
23.3.12
Supplement 34 Current Protocols in Cell Biology
Benzidine solution
Stock solution: Prepare a benzidine base stock solution that is 3% (w/v) benzidine
base (Sigma) in 90% (v/v) acetic acid (Fisher Scientific)/10% water. Store up to
6 months at 4◦ C in the dark.

Working solution: To make benzidine solution, add 1 part benzidine base stock,
1 part 30% hydrogen peroxide, and 5 parts water. Prepare fresh for each use.

Cell growth medium, 5637


Heat inactivate FBS (lot tested for ES cell maintenance) at 55◦ C for 1 hr. Store up
to 6 months at −20◦ C for future use.
Prepare growth medium:
DMEM-H containing:
10% (v/v) heat-inactivated FBS
50 µg/ml gentamicin (final concentration)
1× (75 µM) monothioglycerol (MTG; Sigma)
Freeze up to 6 months at −20◦ C. Avoid freeze thaws.
Once thawed, store at 4◦ C and use within 1 month.
Collection medium
Dulbecco’s modified Eagle medium (DMEM-H; Invitrogen)
5% (v/v) heat-inactivated FBS
50 µg/ml gentamicin (final concentration)
1× (75 µM) monothioglycerol (MTG; Sigma)
Freeze up to 3 months at −20◦ C. Avoid freeze thaws.
Once thawed, store at 4◦ C and use within 1 month
Differentiation medium
Add gentamicin (Invitrogen) to a final concentration of 50 µg/ml per 500-ml bottle
of DMEM-H (Invitrogen). Add lot-selected FBS (Support Protocol 8) to 20% (v/v)
and MTG (Sigma) to 2× (150 µM final concentration). Store at 4◦ C. Use within 2
weeks.

Dispase
Dilute dispase, grade II stock (2.4 U/ml) 1:1 in 1× CMF-PBS (1.2 U/ml final). Use
cold. Store at 4◦ C for up to 1 month.

ES cell culture medium


Stock solutions
DMEM-H/gentamicin: To a 500-ml bottle of DMEM-H add 0.5 ml of 50 mg/ml
gentamicin (50 µg/ml final) and invert to mix.

100× MTG: Prepare a 7.5 mM solution of MTG in CMF-PBS. Store at 4◦ C.

FBS: Heat inactivate 100% lot-tested (Support Protocol 7) FBS at 55◦ C for 1 hr.

5637 cell–conditioned medium (Support Protocol 6)

Working solution:
100 ml 5637–conditioned medium (66% v/v final)
26 ml heat-inactivated, lot-tested FBS (17% v/v final)
1.5 ml 100× MTG [1× (75µm) final]
24 ml DMEM-H/gentamicin (16% v/v final)
Stem Cells
Store at 4◦ C. Use within 2 weeks.
23.3.13
Current Protocols in Cell Biology Supplement 34
Gelatin-coated dishes
Add 2 ml of 0.1% (w/v) gelatin (Type A gelatin, porcine, Bloom Factor 200; Difco)
in CMF-PBS (Sigma) per 6-cm dish or 4-ml per 10-cm dish. Ensure that bottom of
dish is gelatin-coated by tilting dish back and forth. Incubate dish for at least 1 hr
at 37◦ C.
Dishes can be prepared up to 1 week in advance, however, to ensure that there is no
bacterial or fungal contamination, check the dish under a microscope.

Glutaraldehyde fixative solution


For 50 ml:
0.4 ml 25% (v/v) glutaraldehyde (Polysciences)
2.5 ml 100 mM EGTA, pH 7.3 (Sigma)
0.1 ml 1 M magnesium chloride (Mallinckrodt)
47 ml 0.1 M phosphate buffer (see recipe)
Prepare fresh for each use
Methanol/acetone fixative
Prepare a 1:1 (v/v) mixture of methanol/acetone fresh on the day of use.
Methanol and acetone can be stored separately at −20◦ C.

Monothioglycerol (MTG)
Prepare 100× MTG stock by adding 32.5 µl of monothioglycerol stock (Sigma no.
M6145) to 50 ml CMF-PBS (7.5 mM final). Store at 4◦ C and use within 2 weeks.

Paraformaldehyde (PFA) fixative, 4% (w/v)


Add 2 g PFA (Polysciences) powder to 50 ml 1× CMF-PBS in a 50-ml conical
centrifuge tube. Heat solution to 60◦ C, shaking occasionally until PFA is in solution.
Cool to room temperature, then filter through a 0.45-µm filter fitted to a syringe.
Divide into 3-ml aliquots and freeze up to 6 months at −20◦ C. Alternatively, store
at 4◦ C in dark and use within 2 weeks.

Phosphate buffer, 0.1 M (pH 7.3 to 7.5)


Prepare 500 ml 0.1 M phosphate buffer solution by combining 115 ml 0.1 M sodium
phosphate monobasic (Mallinckrodt) and 385 ml 0.1 M sodium phosphate dibasic
(Mallinckrodt). The mixture should give a pH between 7.3 and 7.5. Store at room
temperature and use until solution becomes cloudy.

Rat serum staining medium


5% (v/v) rat serum
3% (v/v) FBS, heat inactivated 1 hr at 55◦ C
0.1% (w/v) NaN3 (Fisher Scientific)
1× CMF-PBS
Store up to 6 months at 4◦ C
Staining medium
3% (v/v) FBS, heat inactivated 1 hr at 55◦ C
0.1% (w/v) NaN3 (Fisher Scientific)
1× CMF-PBS
Store up to 6 months at 4◦ C
Vascular
Differentiation of
Mouse ES Cells

23.3.14
Supplement 34 Current Protocols in Cell Biology
Trypsin/EDTA, 0.25×
Add 1 ml 1× trypsin/EDTA stock solution (0.2% (w/v) trypsin/0.53 mM EDTA;
Invitrogen) to 3 ml CMF-PBS. Store at 4◦ C. Use within 2 weeks.
Trypsin solution, 1:500
Dilute 2.5% (w/v) trypsin (no EDTA; Invitrogen) 1:500 using 1× CMF-PBS.
Prepare fresh each time.

Trypsin stop solution


35% (v/v) FBS, heat inactivated 1 hr at 55◦ C
1× CMF-PBS
Store up to 1 month at 4◦ C
Wash buffer
For 200 ml:
0.4 ml 1 M magnesium chloride (Mallinckrodt)
2 ml 2% (v/v) Nonidet P-40 (Sigma)
197.6 ml 0.1 M phosphate buffer (see recipe)
Filter sterilize
Store at room temperature until solution becomes cloudy
Xgal stain
For 50 ml:
2 ml of 25 mg/ml Xgal stock (Bethesda Research Labs) in dimethyl formamide
(Fisher Scientific)
0.106 g potassium ferrocyanide (Sigma)
0.082 g potassium ferricyanide (Sigma)
48 ml wash buffer (see recipe)
Store at 37◦ C for 2 weeks or until cloudy

COMMENTARY
Background Information that first form in the developing embryo and
Embryonic stem (ES) cells are pluripotent yolk sac.
cells that have been derived from the inner An extensive discussion of nonmouse ES
cell mass of developing blastocysts (Evans cell differentiation into endothelial cells and
and Kaufman, 1981). ES cells maintained on vascular structures is beyond the scope of this
embryonic fibroblasts in culture can repopu- unit, but several excellent reviews have re-
late all different cell lineages, including the cently highlighted similarities and differences
germ line, after being injected into host blas- between mouse and human or nonhuman pri-
tocysts (Bradley et al., 1984). Furthermore, in mate ES cells (reviews: Smith, 2001; Pera
vitro studies over the past twenty years have and Trounson, 2004; Hematti et al., 2005).
shown that ES cells can differentiate into a Although human ES cells (hES) differ from
variety of cell lineages under appropriate con- mouse ES cells in requirements to maintain
ditions in culture (Keller, 2005). Many of the their undifferentiated status, hES can be in-
cell types that differentiate under theses condi- duced to differentiate and form embryoid bod-
tions are those that are found in the developing ies similar to their mouse counterparts. Several
embryo and yolk sac, such as hematopoietic reports have characterized endothelial progen-
cells, endoderm, and endothelial cells (Wiles itors, endothelial cells, and vascular structures
and Keller, 1991; Schmitt et al., 1991; Wang derived from hES cells, although the devel-
et al., 1992; Keller et al., 1993). The vascu- opment of hematopoietic cells and vascular
lar endothelial cells that arise during ES cell structures takes longer than with mouse ES
differentiation have the potential to form prim- cell differentiation (Levenberg et al., 2002;
itive blood vessels, comparable to the vessels Gerecht-Nir et al., 2003; Wang et al., 2004).
Stem Cells

23.3.15
Current Protocols in Cell Biology Supplement 34
These recent findings suggest that the basic several days (Wobus et al., 1991; UNIT 23.2).
cell-cell interactions and differentiation pro- Whether forming EBs by enzymatic disrup-
grams are shared among the different species, tion or by hanging drops, differentiation of the
but that the protocols must be modified when EBs can be performed in suspension rather
changing the species source of ES cells. than as attached cultures, as described here.
Mouse ES cells were initially cultured EBs, which contain an inner and outer layer,
on embryonic fibroblast feeder cells (Evans can be expanded to form a large lumen and are
and Kaufman, 1981; Martin, 1981), or with called cystic embryoid bodies (CEBs; Wang
leukemia inhibitory factor (LIF) once it was et al., 1992). CEBs have hemoglobinized ar-
identified as a factor to prevent differentia- eas that are indicative of hematopoietic devel-
tion (Smith et al., 1988; Williams et al., 1988). opment, primitive blood vessels, and areas of
It was subsequently shown that LIF activates beating that indicate differentiation of cardiac
the transcription factor STAT3, and this acti- muscle (Doetschman, et al., 1985; Risau et al.,
vation is critical to ES cells (Matsuda et al., 1988; Wang et al., 1992).
1999). Sources of LIF include: recombinant All methods of ES cell differentiation have
LIF that is commercially available, transient both advantages and disadvantages. The for-
transfection of COS cells with LIF-expressing mation of EBs (either enzymatic disruption or
plasmids, and harvesting medium conditioned hanging drop) has the advantage of providing
by the 5637 human bladder cancer cell line for a cellular structure that enhances cell-cell
(Kearney and Bautch, 2003). The authors pre- interactions. Cells within an EB provide criti-
fer the 5637–conditioned medium because cal developmental cues involved in differenti-
they feel it best preserves the undifferentiated ation of other cell types (review: Keller, 2005).
ES cell morphology, perhaps because other However, EBs have the potential to generate
factors in the medium co-operate with LIF to unknown factors that can complicate analysis
efficiently maintain undifferentiated ES cells. of developmental programs. The advantage of
There are several methods used for initiat- coculturing ES cells with stromal cells is that
ing ES cell differentiation. ES cells are re- stromal cells can provide a beneficial growth
moved from medium containing LIF and cul- environment for hematopoietic development.
tured in differentiation medium in suspension However, undefined factors produced by stro-
where they aggregate to form a colony of par- mal cells may influence the differentiation of
tially differentiated cells known as an embry- ES cells to one cell type over another (review:
oid body (EB; Doetschman et al., 1985; Wang Keller, 2005). Separation of differentiated ES
et al., 1992; review: Keller, 1995). Alterna- cells from the stromal cell layer has also been
tively, ES cells are cultured directly onto stro- found to be a disadvantage of this method.
mal cells to provide an environment conducive The formation of EBs is sometimes preferred
to hematopoietic differentiation (Nakano et al., over the formation of CEBs, because reattach-
1994). A commonly used stromal cell line ment of EBs lends itself to enhanced experi-
for such differentiation is the OP9 cell line, mental manipulations such as in situ localiza-
isolated from CSF-1 deficient op/op mice tion and image analysis (Kearney and Bautch,
(Yoshida et al., 1990). A third method utilizes 2003).
the culture of ES cells in a monolayer on ex- The protocol of ES cell differentiation from
tracellular matrix proteins (Nishikawa et al., EBs that is described here provides a tool for
1998). Also, EBs can be disaggregated into further understanding the process of vascular
single ES cells and plated in methyl cellu- differentiation (vasculogenesis) and vascular
lose, and subsequent analysis of the colonies expansion (angiogenesis) in an in vitro set-
that form shows that EBs contain many of the ting that is analogous to the in vivo setting. A
hematopoietic progenitors found in bone mar- number of markers for the early vasculature
row (Schmitt et al., 1991). More recent studies have been identified and are particularly use-
have shown that single cells disassociated from ful, and protocols for immunolocalization and
EBs and plated in methyl cellulose generate β-galactosidase staining described here are
blast-cell colonies that indicate the existence beneficial tools for analyzing vascular marker
of a common progenitor for hematopoietic and expression.
endothelial cells (Kennedy et al., 1997; Choi
et al., 1998). Critical Parameters
While this protocol discusses the forma- All tissue culture should be performed un-
Vascular tion of EBs by enzymatic disruption of ES cell der sterile conditions in a class II biological
Differentiation of clumps, an alternative method of creating EBs flow hood. All reagents and media should be
Mouse ES Cells
is by culturing ES cells in hanging drops for sterile. Pipet tips and medidroppers should be
23.3.16
Supplement 34 Current Protocols in Cell Biology
sterilized by autoclaving. All bottles should changed every day. Daily feedings are some-
be thoroughly washed and autoclaved before times necessary for densely seeded wells or for
use. All incubations should be performed in later days of a time course (past day 8) when
a humidified 37◦ C, 5% CO2 incubator, unless there are a large number of cells in each well.
otherwise indicated. The lot of FBS that is utilized for proper main-
ES cells should be passed on day 3 or 4 tenance and differentiation of ES cells is also a
after the last passage. As the source of LIF critical parameter and it should be emphasized
is depleted over time, ES cells will become that often different lots are optimal for mainte-
more differentiated, thus passage of ES cells nance versus differentiation. Thus, lot testing
on day 3 is preferred over day 4. Enzymatic of FBS as described in Support Protocol 8 is es-
dissociation of ES cells using trypsin should sential. When performing immunolocalization
be carried out for no longer than 2 to 3 min as experiments, it is important to set up control
described. Longer incubations have the poten- conditions for the experiment. Set up attached
tial to cleave cell surface proteins important cultures that do not receive primary antibody
for cell viability. Once the trypsin reaction is (staining medium alone), but do receive sec-
stopped with trypsin stop solution, it is impor- ondary antibody. This step should help deter-
tant to gently draw cells up and down with a mine if the secondary antibody exhibits non-
pipet for further ES cell dissociation, as harsh specific binding.
handling of the ES cells at this step can result in
decreased cell viability. The authors typically Troubleshooting
culture ES cells for 50 passages, after which, Maintaining undifferentiated ES cells dur-
maintenance of cell viability declines and un- ing ES cell passage is a commonly encoun-
differentiated ES cells are not preserved. tered problem. There are a few potential causes
For ES cell culture, 5637 cell–conditioned for overly differentiated ES cells: (1) The
medium is a critical component for ensuring 5637 cell–conditioned medium does not con-
that ES cells are maintained in their undiffer- tain enough LIF. Thus, it is important to test the
entiated state. Testing of pooled collections of different pools of combined 5637 collections
5637 cell–conditioned medium on ES cells for on ES cells to determine which pool is optimal
proper ES cell maintenance is essential. The at keeping ES cells undifferentiated. (2) The
lot of FBS that is utilized for proper mainte- FBS lot is not efficient in maintaining ES cells.
nance of mouse ES cells is also a critical fac- Testing lots of FBS from different manufactur-
tor in ES cell maintenance. Thus, lot testing of ers is critical for proper ES cell maintenance.
FBS as described in Support Protocols 7 and (3) Cell passage number is too high. The au-
8 is essential. FBS and 5637 cell–conditioned thors typically stop maintaining ES cells once
medium aliquots should be stored at −20◦ C they reach passage 50.
and repeated freeze-thaws should be avoided. If the ES cells begin to differentiate in cul-
When choosing 5- to 6-day-old ES cells ture after 1 or 2 days since passage, it is ad-
for in vitro differentiation, it is important to visable to immediately feed cells with fresh
choose the dish containing ES clumps that ES medium. If ES cells do not adhere to the
are undifferentiated in the middle (tight and gelatin-coated dish, make sure that the gelatin-
shiny appearance), but differentiated along the coated dishes are used within 1 week after
edges (Figure 23.3.1). This morphology in- preparation.
dicates a partial differentiation that promotes When feeding EBs on day 2 after enzymatic
further differentiation. After treating the ES disruption of ES clumps for in vitro differenti-
cells with dispase to generate small clumps ation, many of the EBs stick to the dish bottom.
of cells, it is imperative to then transfer the If this occurs, it may be necessary to transfer
cell clumps to bacteriological dishes to pre- the EBs to a fresh 10-cm bacteriological dish
vent them from sticking to the dish bottom. using a medidropper.
This will ensure that the cell clumps will form When setting up attached cultures for
EBs in suspension culture. Some cell clumps in vitro differentiation, it is best to select
will invariably attach to the dish, but are dis- medium-sized EBs for attachment (Figure
carded upon EB transfer to attachment dishes. 23.3.1). Larger EBs do not adhere as well, and
The authors have found that timely feeding while smaller EBs do attach to the dish, they
of attached cultures with fresh differentiation often do not spread out and differentiate as
medium is important for good differentiation. well. Another factor that can influence ES cell
The pH of medium can be monitored with phe- differentiation is the quality of FBS. Thus, test-
nol red, and if the medium on the cultures is ing lots of FBS from different manufacturers
Stem Cells
light orange or yellow after 24 hr, it should be is critical for optimal ES cell differentiation.
23.3.17
Current Protocols in Cell Biology Supplement 34
A problem that is commonly encountered Preparation of 5637 cell–conditioned
when fixing and staining differentiated ES cul- medium (CM)
tures is detachment of cells from the dish. After 5637 cells are thawed and passed,
As mentioned in Support Protocol 4, the cells are typically grown for 48 to 72 hr (or un-
authors prefer to use methanol/acetone fix- til 80% to 90% confluency in a 15-cm plate. At
ative over PFA when applicable, because this point growth medium is replaced with col-
methanol/acetone fixative is less harsh on the lection medium, and the cells are cultured for a
attached cultures. However, it is recommended further 48 hr. Approximately 45 min should be
that attention be given to treating the cultures allowed to process one 15-cm plate for 5637
as gently as possible during the fixing and cell–conditioned medium. More time should
staining procedures. be added if more than one 15-cm plate is pro-
If background signal problems occur after cessed (typically about 5 min more for every
β-galactosidase staining, try increasing the pH plate added).
of the phosphate buffer. The β-galactosidase
staining procedure works at a pH of 8.5. Enzymatic disruption of ES cells (dispase
treatment)
Approximately 15 min should be allowed to
Anticipated Results prepare all solutions, and 25 to 30 min should
Although maintenance and passage of be allowed to process one 6-cm dish of ES
mouse ES cells with 5637 cell–conditioned cells. More time should be added if more than
medium should result in cultures containing a one 6-cm dish is processed (typically about 2
population of only undifferentiated ES cells, to 3 min for every added dish).
the authors typically do not observe this. In
general, they notice that ∼80% of cultured In vitro differentiation
cells appear to be undifferentiated ES cells on Medium must be changed on day 2 after the
day 3. This percentage goes down significantly dispase treatment. After warming differentia-
if the batch of 5637 cell–conditioned medium tion medium to 37◦ C, ∼3 to 4 min should be al-
is sub-optimal. lowed to aspirate and feed one dish of EBs. The
Attached cultures of EBs are set up on day time required to set up attached cultures varies
3 following the dispase treatment. By day 4 of depending on the number of samples that are
differentiation (1 day after attachment of EBs), to be processed and the number of wells to
most EBs have attached to the bottom of the be plated. It typically takes only a couple of
plastic dishes, and the rest are removed by as- minutes to plate one sample into four wells of
piration. This attachment is strengthened as a 24-well plate. Differentiated samples must
the EBs spread out further on the dish bottom be fed every other day after plating. Generally,
over time. Angioblasts are first observed be- it takes 5 min or less to feed the wells of a
tween days 4 and 6 of differentiation. Vessels 24-well plate.
are routinely visualized between days 6 and 8,
with some expansion of the vasculature after Benzidine staining
Approximately 10 to 15 min should be al-
day 8. A typical time course to observe and
lowed to prepare all of the solutions for the
analyze vascular development would begin on
benzidine staining protocol. Including incu-
day 5 and conclude on day 8. While cultures
bation times, ∼5 min to 1 hr should be al-
are viable past day 8, they begin to deteriorate
lowed for completion of protocol (benzidine
around day 10 of culture under differentiation
stain does not typically last longer than 1 hr).
conditions.
Double immunostaining with PECAM
Time Considerations and Mac-1
Approximately 20 to 30 min should be al-
Passaging mouse ES cells lowed to prepare all of the solutions for this
Approximately 15 min should be allowed protocol. Including incubation times, ∼7 to
to prepare all solutions. Maintaining mouse ES 7.5 hr should be allowed to double immunos-
cells is fairly time efficient. After allowing so- tain 24 wells of a 24-well plate.
lutions to warm to appropriate temperatures (5
to 15 min depending on volumes), a beginner Immunolocalization
can anticipate about 20 to 30 min to process 1 Approximately 10 to 15 min should be al-
to 2 6-cm dishes of ES cells. lowed to prepare all of the solutions for the
Vascular
Differentiation of
Mouse ES Cells

23.3.18
Supplement 34 Current Protocols in Cell Biology
immunolocalization protocol. Including incu- Keller, G. 1995. In vitro differentiation of embry-
bation times, ∼4 to 4.5 hr should be allowed onic stem cells. Curr. Opin. Cell Biol. 7:862-
to stain all 24 wells of a 24-well plate with 869.
antibody. Keller, G. 2005. Embryonic stem cell differentia-
tion: Emergence of a new era in biology and
β-galactosidase staining medicine. Genes Dev. 19:1129-1155.
Approximately 30 to 40 min should be Keller, G., Kennedy, M., Papayannopoulou, T., and
allowed to prepare all of the solutions for Wiles, M. 1993. Hematopoietic commitment
during embryonic stem cell differentiation in
β-galactosidase staining protocol, and ∼30 to
culture. Mol. Cell Biol. 13:473-486.
40 min should be allowed to complete all pro-
Kennedy, M., Firpo, M., Choi, K., Wall, C.,
tocol steps leading up to the addition of Xgal
Robertson, S., Kabrun, N., and Keller, G. 1997.
stain. Cells are incubated with Xgal stain for A common precursor for primitive erythro-
4 hr to overnight depending on the level of poiesis and definitive hematopoiesis. Nature
β-galactosidase activity. 386:488-493.
Levenberg, S., Golub, J., Amit, M., Itskovitz-Eldor,
Literature Cited J, and Langer, R. 2002. Endothelial cells derived
Bautch, V.L. 2002. Embryonic stem cell differen- from human embryonic stem cells. Proc. Natl.
tiation and the vascular lineage. In Methods in Acad. Sci. U.S.A. 99:4391-4396.
Molecular Biology, Vol. 185: Embryonic Stem Martin, G. 1981. Isolation of a pluripotent cell
Cells: Methods and Protocols (K. Turksen, ed.) line from the early mouse embryos cultured in
pp. 117-125. Humana Press. Totawa, N.J.. medium conditioned by teratocarcinoma stem
Bradley, A., Evans, M., Kaufman, M., and cells. Proc. Natl. Acad. Sci. U.S.A. 78:7634-
Robertson, E. 1984. Formation of germ-line 7638.
chimeras from embryo-derived teratocarcinoma Matsuda, T., Nakamura, T., Nakao, K., Arai, T.,
cell lines. Nature 309:255-256. Katsuki, M., Heike, T., and Yokota, T. 1999.
Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, STAT3 activation is sufficient to maintain an
J., and Keller, G. 1998. A common precursor undifferentiated state of mouse embryonic stem
for hematopoietic and endothelial cells. Devel- cells. EMBO J. 18:4261-4269.
opment 125:725-732. Nakano, T., Kodama, H., and Honjo, T. 1994. Gen-
Doetschman, T., Eistetter, H., Katz, M., Schmidt, eration of lymphohematopoietic cells from em-
W., and Kemler, R. 1985. The in vitro devel- bryonic stem cells in culture. Science 265:1098-
opment of blastocyst-derived embryonic stem 1101.
cell lines; formation of visceral yolk sac, blood Nishikawa, S.I, Nishikawa, S., Hirashima, M.,
islands and myocardium. J. Embryol. Exp. Matsuyoshi, N., and Kodama, H. 1998. Progres-
Morphol. 87:27-45. sive lineage analysis by cell sorting and culture
Doetschman, T., Williams, P., and Maeda, N. 1988. identifies FLK+VE-cadherin cells at a diverging
Establishment of hamster blastocyst-derived point of endothelial and hematopoietic lineages.
embryoid stem (ES) cells. Dev. Biol. 127:224- Development 125:1747-1757.
227. Pera, M. and Trounson, A. 2004. Human embryonic
Evans, M. and Kaufman, M. 1981. Establishment stem cells: Prospects for development. Develop-
in culture of pluripotential cells from mouse ment 131:5515-5525.
embryos. Nature 292:154-156. Poole, T. and Coffin, J. 1989. Vasculogenesis
Gerecht-Nir, S., Ziskind, A., Cohen, S., and and angiogenesis: Two distinct morphogenetic
Itskovitz-Eldor, J. 2003. Human embryonic mechanisms establish embryonic vascular
stem cells as an in vitro model for human pattern. J. Exp. Zool. 251:224-231.
vascular development and the induction of Redick, S. and Bautch, V.L. 1999. Developmen-
vascular differentiation. Lab. Invest. 83:1811- tal platelet endothelial cell adhesion molecule
1820. expression suggests multiple roles for a vascu-
Hematti, P., Obrtlikova, P., and Kaufman, D. 2005. lar adhesion molecule. Am. J. Pathol. 154:1137-
Nonhuman primate embryonic stem cells as a 1147.
preclinical model for hemtopoietic and vascular Risau, W. 1997. Mechanisms of angiogenesis.
repair. Exp. Hematol. 33:980-986. Nature 386:671-674.
Inamdar, M., Koch, T., Rapoport, R., Dixon, J.T., Risau, W., Sariola, H., Zerwes, H-G., Sasse,
Probolus, J.A., Cram, E., and Bautch, V.L. 1997. J., Ekblom, P., Kemler, R., and Doetschman,
Yolk-sac-derived murine macrophage cell line T. 1988. Vasculogenesis and angiogenesis in
has a counterpart during ES cell differentiation. embryonic-stem-cell-derived embryoid bodies.
Dev. Dyn. 210:487-497. Development 102:471-478.
Kearney, J. and Bautch, V.L. 2003. In vitro dif- Schmitt, R., Bruyns, E., and Snodgrass, H. 1991.
ferentiation of mouse ES cells: Hematopoietic Hematopoietic development of embryonic stem
and vascular differentiation. Methods Enzymol. cells in vitro: Cytokine and receptor gene ex-
365:83-98. pression. Genes Dev. 5:728-740.
Stem Cells

23.3.19
Current Protocols in Cell Biology Supplement 34
Smith, A. 2001. Embryo-derived stem cells: Of Williams, R., Hilton, D., Pease, S., Willson, T.,
mice and men. Annu. Rev. Cell Dev. Biol. Stewart, C., Gearing, D., Wagner, E., Metcalf,
17:435-462. D., Nicola, N., and Gough, N. 1988. Myeloid
Smith, A., Heath, J., Donaldson, D., Wong, G., leukemia inhibitory factor maintains the de-
Moreau, J., Stahl, M., and Rogers, D. 1988. In- velopmental potential of embryonic stem cells.
hibition of pluripotential embryonic stem cell Nature 336:684-687.
differentiation by purified polypeptides. Nature Wobus, A., Wallukat, G., and Hescheler, J. 1991.
336:688-690. Pluripotent mouse embryonic stem cells are able
Vittet, D., Prandini, M.-H., Berthier, R., Schweitzer, to differentiate into cardiomyocytes express-
A., Martin-Sisteron, H., Uzan, G., and Dejana, ing chronotropic responses to adrenergic and
E. 1996. Embryonic stem cells differentiate in cholinergic agents and Ca2+ channel blockers.
vitro to endothelial cells through successive Differentiation 48:173-182.
maturation steps. Blood 88:3424-3431. Yoshida, H., Hayashi, S., Kunisada, T., Ogawa,
Wang, L., Li, L., Shojaei, F., Lavec, K., Cerdan, M., Nishikawa, S., Okamura, H., Sudo, T.,
C., Menendez, P., Martin, T., Rouleau, A., and and Shultz, L.D. 1990. The murine mutation
Bhatia, M. 2004. Endothelial and hematopoietic osteopetrosis is in the coding region of the
cell fate of human embryonic stem cells origi- macrophage colony stimulating factor gene.
nates from primitive endothelium with heman- Nature 345:442-444.
gioblastic properties. Immunity 21:31-41.
Wang, R., Clark, R., and Bautch, V.L. 1992. Contributed by Nicholas C. Kappas and
Embryonic stem cell-derived cystic embryoid
bodies form vascular channels: An in vitro Victoria L. Bautch
model of blood vessel development. Develop- The University of North Carolina at
ment 114:303-316. Chapel Hill
Wiles, M. and Keller, G. 1991. Multiple hematopoi- Chapel Hill, North Carolina
etic lineages develop from embryonic stem (ES)
cells in culture. Development 111:259-267.

Vascular
Differentiation of
Mouse ES Cells

23.3.20
Supplement 34 Current Protocols in Cell Biology
Differentiation of Mouse Embryonic Stem UNIT 23.4
Cells and of Human Adult Stem Cells into
Adipocytes
Severe obesity is the result of increases in fat cell size and increased fat cell number.
Mature adipocytes do not undergo cell division, and new fat cells arise from a pre-
existing pool of adipose stem cells that are present irrespective of age. The development
of established preadipose cell lines has facilitated the study of different steps leading
to terminal differentiation of preadipocytes into adipocytes. The key events have been
characterized by the identification of transcription factors that play a regulatory role in the
differentiation process. The best characterized transcription factors shown to be important
in the development of mature adipocytes are members of the CCAAT/enhancer binding
proteins (C/EBPs) and peroxisome proliferative-activated receptors (PPARs) families
(Tontonoz et al., 1995; Mandrup and Lane, 1997). However, established preadipose cell
lines are limited for studying early events of differentiation as they represent cells that are
already committed to the adipogenic lineage. Master genes that commit multipotent stem
cells toward adipocytes remain to be identified. In vitro differentiation of multipotent
stem cells to the adipogenic lineage provides an alternative source of adipocytes for study
in tissue culture and offers the possibility to investigate regulation of the first steps of
differentiation and to test effects of drugs on adipose cell development.

This unit includes a protocol for culture conditions in which mouse embryonic stem
(mES) cells can be maintained at an undifferentiated state (Support Protocol 1) or com-
mitted to undergo adipocyte differentiation at a high rate and in a highly reproducible
fashion (Basic Protocol 1). There are also protocols for maintaining (Support Protocol 2)
and differentiating human adult stem cells, isolated form adipose tissue (hMADS cells)
and from bone marrow (hMS cells), into adipocytes (Basic Protocol 2). These culture
systems provide a powerful means for studying the first step of adipose cell development
and the effects of drugs on the biology of adipocytes. There are also protocols for visu-
alization of adipocytes (Support Protocol 3) and analysis of adipocyte gene expression
(Support Protocol 4).

NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37◦ C, 5% CO2


incubator unless otherwise specified.

DIFFERENTIATION OF MOUSE ES CELLS TO ADIPOCYTES BASIC


PROTOCOL 1
Adipocyte differentiation of mES cells is initiated by aggregation of ES cells to form
embryoid bodies (EBs). The authors routinely use the hanging drop method for the
formation of EBs. Two phases can be distinguished in adipogenesis from ES cells.
The first phase, between day 2 and 5 after EB formation, corresponds to the permissive
period for the commitment of ES cells; this phase requires retinoic acid (RA). The second
phase corresponds to the permissive period for terminal differentiation and is influenced
by adipogenic factors (e.g., insulin, triiodothyronine, and rosiglitazone) as previously
shown for the differentiation of cells from preadipose clonal lines. The treatment leads
to 60% to 80% of outgrowths containing adipose cells compared to 2% to 5% in the
absence of RA treatment.

Stem Cells
Contributed by Brigitte Wdziekonski, Phi Villageois, and Christian Dani 23.4.1
Current Protocols in Cell Biology (2007) 23.4.1-23.4.14
Copyright 
C 2007 by John Wiley & Sons, Inc.
Supplement 34
Materials
ES cells
Growth medium for mES cells (see recipe)
Leukemia inhibitory factor (LIF)
CMF-PBS (phosphate-buffered saline, calcium and magnesium free; Cambrex)
Trypsin solution (see recipe)
Fetal bovine serum (FBS, see recipe)
Retinoic acid (RA, see recipe)
Differentiation medium for mES cells (see recipe)
60- and 100-mm bacteriological grade petri dishes (Greiner)
20-ml conical sterile tube
100-mm gelatinized-tissue culture dishes (see recipe)
Additional reagents and equipment for cell counting (UNIT 1.1)
Collect ES cells
ES cells are originally plated at 106 cells per 25-cm2 flask and are grown for 2 or 3 days
until they reach a confluency of 80%. At this point, cells are still maintained from the
original plating in the presence of LIF, in order to keep ES cells in the undifferentiated
state. ES cell cultures may contain a proportion of “differentiated” cells which have lost
their pluripotency. It is crucial to minimize this proportion of differentiated cells. This
is achieved by the addition of LIF in a high-quality culture medium, i.e., an adequate
batch of serum. Identification of pluripotent stem cells is difficult unless one is familiar
with the appropriate cellular morphology. Pluripotent stem cells: (1) are small, (2) have a
large nucleus containing prominent nucleoli structures, and (3) have minimal cytoplasm.
Pluripotent stem cells, in contrast to differentiated cells, grow rapidly.

1. Change medium on ES cells with complete growth medium supplemented with


1000 U/ml LIF 2 hr before subculture.
2. Aspirate medium and wash twice, each time with 5 ml of CMF-PBS.
3. Aspirate the CMF-PBS and add 1 ml of trypsin solution. Incubate 2 to 3 min.
4. Add 5 ml of growth medium to stop trypsinization and suspend the cells by vigorous
pipetting. Transfer the cells to a sterile 20-ml tube and centrifuge 5 min at 250 × g,
room temperature.
5. Aspirate the medium and resuspend the cell pellet in 10 ml of growth medium
without LIF to prepare a single-cell suspension. Count the cells (UNIT 1.1). After cell
counting, adjust the suspension to a concentration of 5 × 104 cells/ml.
Induce EB formation
6. Place aliquots of 20 µl of this suspension onto the lids of 10-cm bacteriological grade
dishes.
This is defined as day 0 of EB formation.
Bacterial grade petri dishes are used to prevent cell attachment to the substrate. EBs have
a tendency to attach to the bottom of the plastic dish. EBs that are firmly attached to the
dish should be eliminated as these EBs seem to have no adipogenic capacity.
7. Invert the lid and place it over the bottom of a bacteriological petri dish filled with
8 ml CMF-PBS containing a few drops of FBS to decrease the surface tension of the
liquid.
Differentiation of It is essential to cover the bottom of the dish with the liquid to prevent the evaporation
Stem Cells into of the hanging drops. When the lid is inverted, each drop hangs and the cells fall to the
Adipocytes
bottom of the drop where they aggregate into a single clump (EB).
23.4.2
Supplement 34 Current Protocols in Cell Biology
Attachment of EBs on petri dishes can be eliminated by coating the dish with poly (2-
hydroxyethylmethylacrylate) supplied as Cellform polymer (ICN Biomedicals). Dishes
are filled with a film of Cellform working solution (0.5 g of Cellform dissolved in 42 ml of
100% ethanol) and left in a culture hood, with the cover of the dish removed, to evaporate
ethanol. Dishes are rinsed with CMF-PBS before use.
8. At a time point 2 days later, remove the lid, invert it and collect drops containing
EBs in a 20-ml conical sterile tube. Let stand for 5 min at room temperature to allow
the aggregates to sediment.
9. Aspirate the supernatant and resuspend the pellet in 4 ml of growth medium sup-
plemented with 10−7 M retinoic acid (RA). Transfer the suspension to 60-mm
bacteriological grade petri dishes.
RA is light-sensitive.

10. Incubate for 3 days in the presence of RA changing the growth medium every day.
Plate EBs on gelatin and differentiate.

Induce differentiation
11. At day 5 after EB formation, change the medium to growth medium without the
addition of RA and plate 2 to 4 EBs per cm2 in 100-mm gelatinized-tissue culture
dishes.

Figure 23.4.1 EB-derived adipocytes. ES cell–derived EBs were allowed to undergo adipocyte
differentiation for 20 days. A large adipocyte colony around the dense center of an EB is visible
under the microscope.

Stem Cells

23.4.3
Current Protocols in Cell Biology Supplement 34
12. The day after plating, change the growth medium to differentiation medium. Change
medium every other day.
The addition of 0.5 µM rosiglitazone, which is a PPARγ activator (Lehmann et al.,
1995), to the differentiation medium dramatically stimulates the terminal differentiation
of RA-treated EBs into adipocytes (Phillips et al., 2003).

13. After 10 to 20 days in the differentiation medium, evaluate the cultures for adipocyte
colonies (see Fig. 23.4.1).
After 10 to 20 days at least 50% to 70% of EB outgrowths should contain adipocyte
colonies.

SUPPORT MAINTENANCE OF MOUSE ES CELLS WITHOUT FEEDER LAYERS


PROTOCOL 1
The conditions outlined below are applicable for the maintenance of feeder layer–
independent ES cell lines. Cells can be grown on gelatin-coated tissue culture flasks
and maintained in a multipotent undifferentiated state by addition of leukemia inhibitory
factor (LIF; Smith, 1992) to the growth medium.

Materials
ES cells in 25-cm2 flasks (up to passage 25)
CMF-PBS (phosphate-buffered saline, calcium and magnesium free; Cambrex)
1× trypsin solution (see recipe)
Growth medium for mES cells (see recipe)
Leukemia inhibitory factor (Chemicon, Invitrogen)
20-ml conical centrifuge tubes, sterile
Gelatinized tissue culture 25-cm2 flasks (see recipe)
Additional reagents and equipment for cell counting (UNIT 1.1)
1. For a 25-cm2 flask containing ES cells, aspirate medium and wash twice each time
with 5 ml CMF-PBS.
2. Aspirate the CMF-PBS and add 1 ml of 1× trypsin solution. Ensure the trypsin
covers the cell monolayer and incubate for 2 to 3 min. Check that the cells are
dissociated in single-cell suspension under an inverted microscope.
It is critical to produce a single-cell suspension for subcultures. This is achieved by tapping
the flask several times to ensure complete dissociation during the trypsin treatment.

3. Add 5 ml of growth medium to stop trypsinization and suspend the cells by vigorous
pipetting.
4. Transfer the cells to a sterile 20-ml tube and centrifuge 5 min at 250 × g, room
temperature.
5. Aspirate the medium and resuspend the cell pellet in 5 ml of growth medium by
pipetting up and down two to three times. Count cells (UNIT 1.1).
6. Add 106 cells to 10 ml of prewarmed growth medium containing 1000 U/ml LIF
then transfer to a freshly gelatinized 25-cm2 flask.
LIF is required to maintain pluripotent ES cells and is omitted to induce the commitment
of ES cells towards the adipogenic lineage.

7. Change growth medium with LIF (1000 U/ml) every day.


8. Trypsinize the cultures 2 days later as in step 2.
Differentiation of
Stem Cells into Cultures should be subcultured before cells have reached confluence.
Adipocytes

23.4.4
Supplement 34 Current Protocols in Cell Biology
DIFFERENTIATION OF HUMAN MULTIPOTENT ADIPOSE-DERIVED BASIC
STEM (hMADS) CELLS TO ADIPOCYTES PROTOCOL 2
Adipocyte differentiation of human multipotent adipose-derived stem (hMADS) cells and
human mesenchymal stem (hMS) cells in vitro does not require pretreatment with RA.
Induction of differentiation of hMADS cells and hMS cells is performed in monolayer
culture. However, in contrast to hMADS cells, differentiation of hMS into adipocyte
requires a differentiation medium supplemented with 10% fetal bovine serum (FBS).

Materials
Adult stem cells: hMADS (for isolation of hMADS cells see Rodriguez et al.,
2005a; Zaragosi et al., 2006)
Growth medium for hMADS cells (see recipe)
Differentiation medium for hMADS cells (see recipe)
Tissue culture 100-mm dishes (Greiner, S.A. Dutscher) or 6-well plates or 12-well
plates
1. Plate adult stem cells at a high density, e.g., 40,000 cells/cm2 in 10 ml, 2 ml, or
1 ml growth medium, on tissue culture 100-mm dishes or 6- or 12-well plates,
respectively.
2. When cells reach confluence (2 or 3 days after plating), change growth medium to
differentiation medium containing 0.5 µM rosiglitazone.
The addition of 0.5 µM rosiglitazone in the differentiation medium is required to differ-
entiate cells into adipocytes.
3. Three days later, change cells to differentiation medium without IBMX and
dexamethasone.

Figure 23.4.2 EB outgrowth fixed and stained with Oil-red O for fat droplets. For color version of
Stem Cells
this figure see http://www.currentprotocols.com.
23.4.5
Current Protocols in Cell Biology Supplement 34
4. Check the cultures for the appearance of adipocytes.
Adipose cells contain lipid droplets which can be easily visualized microscopically, espe-
cially under bright-field illumination. Lipid droplets can also be visualized after staining
with Oil-red O as indicated in Figure 23.4.2.
Adipocytes should appear 5 to 7 days after induction of differentiation.

ALTERNATE DIFFERENTIATION OF HUMAN MS CELLS TO ADIPOCYTES


PROTOCOL
The same growth medium is used for hMADs cells and for hMS cells (see Reagents and
Solutions). In contrast, the differentiation medium is different because hMS cells require
10% FBS to undergo differentiation into adipocytes, whereas adipocyte differentiation
of hMADS cells occurs under a serum-free condition.

Materials
Adult stem cells: hMS cells (for isolation of adult stem cells from bone marrow see
Pittenger et al., 1999)
Growth medium for hMS cells (see recipe)
Differentiation medium for hMS cells (see recipe)
Tissue culture 100-mm dishes or 6-well plates or 12-well plates (Greiner; S.A.
Dutscher)
1. Plate cells at a high density, e.g. 40,000 cells/cm2 in growth medium on tissue culture
100-mm dishes (or 6- or 12-well plates).
2. When cells reach confluence (2 or 3 days after plating), change growth medium to
differentiation medium, containing 0.5 µM rosiglitazone.
The addition of 0.5 µM rosiglitazone in the differentiation medium is required to differ-
entiate cells into adipocytes
3. Three days later, change cells with differentiation medium without IBMX and dex-
amethasone.
4. Check the cultures for the appearance of adipocytes.
Adipose cells contain lipid droplets which can be easily visualized microscopically, espe-
cially under bright-field illumination. Lipid droplets can also be visualized after stained
with Oil-red O as indicated in Figure 23.4.2.
Adipocytes should appear 5 or 7 days after induction of differentiation.

SUPPORT MAINTENANCE OF HUMAN MADS AND HUMAN MS CELLS WITHOUT


PROTOCOL 2 FEEDER LAYER
No feeder layer and no gelatin-coated dishes are required to maintain the proliferation of
hMADs cells and of hMS cells. The same growth medium is used to maintain both cell
types.

Materials
hMADS or hMS cells (for isolation of hMADS see Rodriguez et al., 2005a; for
isolation of hMS cells see Pittenger et al., 1999)
Phosphate-buffered saline, calcium and magnesium free (CMF-PBS, Cambrex)
1× trypsin -EDTA solution (Invitrogen no:25300-054)
Growth medium for hMADS and hMS cells (see recipe)
Tissue culture 100-mm dishes (Greiner; S.A. Dutscher)
Differentiation of
20-ml conical centrifuge tubes, sterile
Stem Cells into Additional reagents and equipment for cell counting (UNIT 1.1)
Adipocytes

23.4.6
Supplement 34 Current Protocols in Cell Biology
1. For a 100-mm dish of hMADS or hMS cells, aspirate medium and wash twice each
time with 10 ml CMF-PBS.
2. Aspirate the CMF-PBS and add 1 ml of 1× trypsin solution. Ensure the trypsin
covers the cell monolayer and incubate for 2 to 3 min. Check that cells are detached
from the bottom of the dish by examining under an inverted microscope.
3. When the cells are detached, add 10 ml of growth medium to stop trypsinization and
suspend the cells by pipetting.
4. Transfer the cells to a 20-ml sterile tube and centrifuge 5 min at 250 × g, room
temperature.
5. Aspirate the medium and resuspend the cell pellet with 5 ml of growth medium by
pipetting up and down 2 to 3 times. Count cells (UNIT 1.1).
6. Add 250 × 103 cells to 10 ml of prewarmed growth medium then transfer to a
100-mm dish.
7. Change medium every other day.
8. Trypsinize the cultures 3 to 4 days later as in step 1.
Cultures should be subcultured before cells have reached confluence.

VISUALIZATION OF ADIPOCYTES SUPPORT


PROTOCOL 3
Adipose cells with lipid droplets are easily visualized microscopically, especially under
bright-field illumination. Nonadipose cells containing structures resembling droplets are
often detectable in untreated- and RA-treated ES cultures. Therefore, it is essential to
identify droplet-like structures as triglyceride droplets. Staining of cultures with Oil-red
O, a specific stain for triglycerides, gives a good indication of adipocyte differentiation.

Materials
Cultures to be examined
CMF-PBS (phosphate-buffered saline, calcium and magnesium free; Cambrex)
Fixation buffer (see recipe)
Oil-red O solution (see recipe)
Storage solution: 70% glycerol (v/v) in H2 O
Oil-red O elution buffer (see recipe)
Spectrophotometer
1. Aspirate medium from cultures and wash cells once with 10 ml CMF-PBS.
2. Fix cells for 15 min in 10, 2, or 1 ml fixation buffer in 100-mm dishes, 6- or 12-well
plates, respectively, at room temperature.
3. Wash twice for 10 min each time with 10 ml Milli-Q water (for a 100-mm dish).
4. Stain with 10 ml Oil-red O solution for 15 min (for a 100-mm dish; stain with 2 ml
or 1 ml Oil-red O for a 6- or 12-well dish, respectively).
5. Wash twice each time with 10 ml Milli-Q water. Cover cells with a film of storage
solution (see Fig. 23.4.2) or elute the bound stain (step 6).
Cells covered with storage solution can be kept at room temperature for several months.

6. Elute bound stain to quantify the formation of adipocytes. Incubate the 100-mm dish
with 10 ml Oil-red O elution buffer (see reicpe) for 30 min at room temperature.
Remove the solution.
Stem Cells

23.4.7
Current Protocols in Cell Biology Supplement 34
7. After elution of the bound stain, measure the amount of bound Oil-red O spectropho-
tometrically at 490 nm.

SUPPORT ANALYSIS OF ADIPOCYTE GENE EXPRESSION


PROTOCOL 4
Finally, expression of a-FABP (adipocyte-fatty acid binding protein) an adipocyte-
specific gene, in 20-day-old ES cell–derived EB or in 10-day-old differentiated human
stem cells can be detected by northern blotting using 20 µg of total RNA. However,
detection of the expression of these genes in early differentiating cells requires a more
sensitive method such as RT-PCR. This protocol gives conditions to reveal mouse and
human a-FABP gene expression.

Materials
TRI Reagent (Molecular Research Center, Euromedex, France)
Chloroform
Isopropanol
100% ethanol
5 M NaCl, sterile
TES buffer (see recipe)
RT-PCR kit (available from several companies)
Primers to reveal mouse a-FABP gene expression:
forward: 5 -GATGCCTTTGTGGGAACCTGG-3
reverse: 5 -TTCATCGAATTCCACGCCCAG-3
Mouse hypoxanthine phosphoribosyltransferase (HPRT, as a standard to balance
the amount of RNA and cDNA used):
forward: 5 -GCTGGTGAAAAGGACCTCT-3
reverse: 5 -CACAGGACTAGAACACCTGC-3
Primers to reveal human a-FABP gene expression:
forward: 5 -GCTTTGCCACCAGGAAAGTG-3
reverse: 5 -ATGACGCATTCCACCACCAG-3
Human β-actin as a housekeeping gene standard:
forward: 5 -AGCCATGTACGTTGCTA-3
reverse: 5 -AGTCCGCCTAGAAGCA-3
RNAs are prepared using TRI Reagent according to the supplier’s protocol.

Days of differentiation are indicated as 20 days for ES cells and 10 days for AS cells.
The authors routinely obtain 100 to 200 µg RNA from one 100-mm tissue culture plate
containing 20-day-old EB outgrowths or 10-day-old hMADS cells and hMS cells.

Using the abovementioned primers to reveal mouse a-FABP gene expression PCR is
performed using an annealing temperature of 56◦ C and 25 cycles of PCR. The size of
the expected cDNA is 213 bp. Mouse hypoxanthine phosphoribosyltransferase (HPRT)
is used as a standard to balance the amount of RNA and cDNA used. With the above-
mentioned primers PCR is performed using an annealing temperature of 60◦ C and 25
cycles of PCR. The expected size of this cDNA is 249 bp.

Using the abovementioned primers to reveal human a-FABP gene expression PCR is
performed using an annealing temperature of 55◦ C and 25 cycles of PCR. The size of the
expected cDNA is 290 bp. Human β-actin is used as a standard. With the abovementioned
primers PCR is performed using an annealing temperature of 55◦ C and 25 cycles of PCR.
The expected size of this cDNA is 656 bp.

Differentiation of PCR products are separated and visualized by electrophoresis on a 2% (w/v) agarose gel
Stem Cells into containing ethidium bromide (see Voytas, 2000).
Adipocytes

23.4.8
Supplement 34 Current Protocols in Cell Biology
Figure 23.4.3 PCR analysis of ex-
pression of an adipocyte-specific gene.
EBs were pretreated from day 2 to day
5 with 0.1% DMSO (1) or 10−7 RA (2).
RNAs were prepared at day 20 after EB
formation and transcripts for a-FABP.
HPRT was used to monitor expression
of a housekeeping gene.

REAGENTS AND SOLUTIONS


Use deionized or distilled water in all recipes and protocols except as noted. For common stock
solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Dexamethasone, 1 mM
Dissolve 3.9 mg dexamethasone (powder; Sigma) in 10 ml ethanol. Store 0.5-ml
aliquots up to 6 months at −20◦ C.
Differentiation medium for hMADS cells
To 25 ml of low-glucose DMEM (Invitrogen) add:
25 ml Ham’s F12 (Cambrex)
0.5 ml 200 mM glutamine
0.5 ml 1 M HEPES-buffered saline (Cambrex)
0.5 ml 5000 IU/ml/5000 µg/ml penicillin/streptomycin (Cambrex)
0.5 ml 1 mg/ml transferrin
0.25 ml 1 mg/ml insulin (see recipe)
0.05 ml 100 mM IBMX (see recipe)
0.05 ml 1 mM dexamethasone (see recipe)
0.005 ml 2 µM T3 (see recipe)
Prepare fresh
The differentiation medium must be prepared just before use. Therefore, prepare only the
volume of medium required for changing cells.

Differentiation medium for hMS cells


To 45 ml low-glucose DMEM (Invitrogen) add:
5 ml FBS
0.5 ml 200 mM glutamine
0.5 ml 1 M HEPES-buffered saline (Cambrex)
0.5 ml 5000 IU/ml/5000 µg/ml penicillin/streptomycin (Cambrex)
0.05 ml 100 mM IBMX (see recipe)
0.05 1 mM dexamethasone (see recipe)
0.25 1 mg/ml insulin (see recipe)
0.005 ml 2 µM T3 (see recipe)
Prepare fresh
The differentiation medium must be prepared just before use. Therefore, prepare only the
volume of medium required for changing cells.

Stem Cells

23.4.9
Current Protocols in Cell Biology Supplement 34
Differentiation medium for mES cells
Supplement growth medium for mES cells with:
0.5 µg/ml insulin (see recipe)
2 nM triiodothyronine (add from 2 µM T3 solution; see recipe)
0.5 µM rosiglitazone (provided by GlaxoSmthKline)
Prepare fresh
The differentiation medium must be prepared just before use. Therefore, prepare only the
volume of medium required for changing cells.

Fetal bovine serum


Fetal bovine serum is tested for maintenance of the undifferentiated state. The au-
thors select a batch of serum that is able to support the growth of stem cells. For that
purpose, plate 106 ES cells/25-cm 2 flask in 10% of each set of FBS, supplemented
with LIF, and subculture the cells every two days for 4 passages. For a high-quality
serum, a flask should yield 5 to 10 × 106 cells at each passage. Furthermore, no
toxicity of the selected serum should be observed at a 30% concentration.
The supplier used for this unit is Dutscher, France, but other suppliers can be used.

Fixation buffer
Dilute 2.5% glutaraldehyde (Sigma) to 0.25% (v/v) in CMF-PBS (Cambrex). Store
up to 1 month at 4◦ C.

Gelatin 0.1% (w/v)


Dilute 2% (w/v) gelatin to 0.1% with CMF-PBS (Cambrex). Store up to 2 weeks
at 4◦ C.

Gelatinized-tissue culture dishes/flasks


Add 5 or 10 ml of 0.1% gelatin (see recipe) per 25-cm2 flask (Corning or Greiner) or
100-mm tissue culture dishes (Corning or Greiner Cell Star), respectively. Incubate
for at least 10 min at room temperature and aspirate gelatin. Prepare fresh.

Growth medium for hMADS and hMS cells


To 500 ml low-glucose DMEM add:
5 ml 200 mM glutamine
5 ml 1 M HEPES -buffered saline (Cambrex)
5 ml 5000 IU/ml-/5000 µg/ml penicillin/streptomycin
50 ml FBS
Store up to 2 weeks at 4◦ C.
Growth medium for mES cell maintenance, 1×
To 440 ml Glasgow MEM/BHK21 medium (Invitrogen) add:
5 ml of 100× nonessential amino acids (stored at 4◦ C)
5 ml of 200 mM glutamine (stored at −20◦ C)
5 ml 100 mM sodium pyruvate (stored at −20◦ C)
0.5 ml 0.1 M 2 -mercaptoethanol (stored at 4◦ C; see recipe)
50 ml of FBS (stored at −20◦ C)
5 ml 5000 IU/ml/5000 µg/ml penicillin/streptomycin (Cambrex)
Store up to 2 weeks at 4◦ C

Differentiation of
Stem Cells into
Adipocytes

23.4.10
Supplement 34 Current Protocols in Cell Biology
IBMX, 100 mM
Prepare IBMX (3-isobutyl-1methylxanthine; Sigma) at 100 mM in water. Dissolve
250 mg IBMX in 9 ml water and 5 to 10 µl of 10 N NaOH. Adjust volume to 11 ml
with water and sterilize by filtration with a 0.22-µm filter. Store 0.1-ml aliquots up
to 2 months at −20◦ C.

Insulin (for mES cells)


Dissolve lypholized insulin, —bovine for mouse cells; recombinant human (Sigma
no. I-9278) for human cells—at 1 mg/ml in cold 0.01 N HCl. Mix gently and
sterilize by filtration with a 0.22-µm filter. Store 0.1-ml aliquots up to 6 months at
−20◦ C. Store thawed aliquots up to 2 weeks at 4◦ C.

2-mercaptoethanol, 0.1 M
100 µl 2-mercaptoethanol
14 ml sterile H2 O
Store up to 3 weeks at 4◦ C
Oil-red O elution buffer
4 M guanidinium thiocyanate (Sigma)
25 mM sodium citrate, pH 7
0.5% (v/v) sarcosyl
20% (v/v) isopropanol
Store up to 1 month at room temperature
Oil-red O solution
Oil-red O stock solution: Dissolve Oil red O (Sigma) at 0.5% (w/v) in isopropanol.
Store up to several months at room temperature.

Oil red O working solution: Mix 6 vol of stock solution with 4 vol water. Mix and
filter using a 0.45-µm filter. Store up to 1 month at room temperature.

Retinoic acid
Dilute all-trans retinoic acid (RA; Sigma) in the dark in dimethyl sulfoxide to
prepare a 10 mM RA stock solution. Store 0.1-ml aliquots up to 1 year at −20◦ C,
protected from the light.

Prepare subsequent dilutions of RA in ethanol and use for one experiment only.

After dilution into the culture medium, the concentration of ethanol should never
exceed 0.1%.

TES buffer
10 mM Tris·Cl, pH 7.4 (APPENDIX 2A)
0.1 mM EDTA
0.1% (w/v) sodium dodecyl sulphate
Sterilize by autoclaving
Store up to several months at room temperature
Transferrin, 1 mg/ml
Dissolve apo-transferrin (human; Sigma) at 1 mg/ml in PBS (APPENDIX 2A) and
sterilize by filtration with a 0.22-µm filter. Store 1-ml aliquots up to several months
at −20◦ C. Store thawed aliquots up to 1 week at 4◦ C.
Stem Cells

23.4.11
Current Protocols in Cell Biology Supplement 34
Triiodothyronine (T3), 2 µM
Dissolve 5 mg T3 (culture tested, powder; Sigma) into 7.43 ml ethanol to prepare
a 1 mM T3 solution. Then, dilute 200 µl of 1 mM solution into 4 ml ethanol to
prepare a 50 µM T3 solution. Finally, dilute 3.2 ml of 50 µM T3 solution into 80 ml
ethanol to prepare a 2 µM T3 solution. Store up to 1 year at −20◦ C.

Trypsin solution, 1× for mES cells


1 ml 2.5% (w/v) trypsin (Invitrogen)
1 ml 100 mM EDTA
1 ml chicken serum
100 ml CMF-PBS (APPENDIX 2A)
Store 10-ml aliquots up to 2 months at −20◦ C
Store thawed aliquots up to 2 weeks at 4◦ C

COMMENTARY
Background Information infant brown adipose tissue (Zilberfarb et al.,
Mouse embryonic (ES) cells have been pre- 1997) or coexpression of human telomerase
viously shown to differentiate spontaneously reverse transcriptase and papilloma E7 onco-
into various lineages in culture (Doetschman protein in SV cells from adult white adipose
et al., 1985). The first morphological observa- tissue (Darimont et al., 2003) led to establish-
tion of adipocyte-like cells derived from ES ment of PAZ6 and Chub-S7 preadipocyte cell
cells was reported by Field et al. (1992). How- lines, respectively. Cells isolated from subcu-
ever, the number of ES cell–derived adipocytes taneous adipose tissue of an infant suffering
was low because spontaneous commitment of from Simpson-Golabi-Behmed syndrome and
ES cells to the adipogenic lineage is rare. In from an adult suffering from liposarcoma were
order to use ES cells to study adipocyte de- used to obtain SGBS preadipocyte cell strain
velopment it was essential to determine condi- and LiSa-2 preadipocyte cell line, respectively
tions of culture that committed ES cells to the (Wabitsch et al., 2000, 2001). Upon differ-
adipogenic lineage at a high rate. This step has entiation, cells from these various lines ex-
been achieved by showing that a prerequisite pressed some of the characteristic markers of
is to treat ES cell–derived embryoid bodies human adipocytes but the lipolytic responses
(EBs) at an early stage of their differentiation specific of human adipocytes and the secre-
with all-trans-retinoic acid (RA) for a short tion of specific adipocytokines were not re-
period of time (Dani et al., 1997; Dani, 1999). ported. Moreover, except for Chub-S7 cells
Adipocytes derived from ES cells display both which were diploid and showed no chromo-
lipogenic and lipolytic activities in response somal alterations, cells from other lines exhib-
to insulin and to β-adrenergic agonists respec- ited chromosomal abnormalities. Recently, the
tively, indicating that mature and functional establishment of multipotent stem cells from
adipocytes are formed. the stromal-vascular fraction of infant adipose
In regard to models of human cells tissue (Rodriguez et al., 2005a) has led the au-
presently available, for primary preadipocytes thors to examine, under serum-free adipogenic
derived from stromal-vascular cells of hu- conditions, whether these human multipotent
man adipose tissue, with increasing pas- adipose-derived stem (hMADS) cells enter the
sage number, the ability to differentiate into adipose lineage and differentiate into cells that
adipocytes under appropriate conditions un- exhibit characteristics of human fat cells. The
dergoes a dramatic decrease before the cells authors have shown that hMADS cells are a
become growth arrested and enter replicative unique cell model in displaying the key fea-
senescence (Ailhaud, 2002). This limitation tures of human adipocytes after differentiation
has been partly circumvented with cells that under adipogenic conditions (Rodriguez et al.,
are immortalized either genetically or sponta- 2004, 2005b). The human adult bone marrow
neously while preserving their ability to un- also contains mesenchymal stem (hMS) cells
dergo adipose conversion. Overexpression of that are able to undergo differentiation into
SV40T and T antigens in human SV cells from adipocytes in vitro (Pittenger et al., 1999).
Differentiation of
Stem Cells into
Adipocytes

23.4.12
Supplement 34 Current Protocols in Cell Biology
Critical Parameters and The level of differentiation may decrease over
Troubleshooting 25 passages of hMADs cells and over 10 pas-
The first critical parameter for the commit- sages of hMS cells.
ment of mES cells into adipocytes is the forma-
tion of EBs. The authors use the hanging drop Time Considerations
method for the formation of EBs. The forma- From mES, adipocytes can be observed
tion of EBs in mass culture by maintaining ES 10 days after EB formation. However, 20 days
cells in suspension at a high density in non- in differentiation medium are required to com-
tissue culture grade plastic is rapid and gives plete adipocyte differentiation. Additions of
rise to a high number of EBs formed. However PPARγ activator dramatically increase the ki-
in the hands of the authors, this method leads netics and the level of adipocyte differentiation
subsequently to a low number of outgrowths of ES cells. From human adult stem cells, adi-
containing adipocyte colonies. pogenesis is completed within 2 weeks. In that
The, treatment of EBs with RA between case PPARγ activator is required.
day 2 and 5 after EB formation is prerequi-
site. The authors have observed that treatment Literature Cited
of EBs with RA between days 2 and 5 after Ailhaud, G. 2002. Autocrine/paracrine effectors of
EB formation or between days 3 and 6 gives adipogenesis. Ann. Endocrinol. (Paris) 63:83-
similar results. Owing to the high instability 85.
of RA, the concentration of RA able to com- Dani, C. 1999. Embryonic stem cell-derived
mit ES cells into the adipogenic lineage at a adipogenesis. Cells Tissues Organs 165:173-
high rate should be determined for each new 180.
preparation of RA (try 10−8 to 10−6 M). RA Dani, C., Smith, A., Dessolin, S., Leroy, P., Staccini,
is light-sensitive. L., Villageois, P., Darimont, C., and Ailhaud, G.
1997. Differentiation of embryonic stem cells
Adipocyte differentiation of hMADS cells into adipocytes in vitro. J. Cell Sci. 110:1279-
and hMS cells in vitro does not require pre- 1285.
treatment with RA. However, activators of Darimont, C., Zbinden, I., Avanti, O., Leone-
PPARγ such as rosiglitazone, is required for Vautravers, P., Giusti, V., Burckhardt, P., Pfeifer,
the terminal differentiation of these stem cells A.M., and Mace, K. 2003. Reconstitution of
into adipocytes. telomerase activity combined with HPV-E7 ex-
hMS cells can be expanded for several pas- pression allow human preadipocytes to preserve
their differentiation capacity after immortaliza-
sages in vitro. However, in the hands of the au- tion. Cell Death Differ. 10:1025-1031.
thors these cells lose their capacity to undergo
Doetschman, T.C., Eistetter, H., Katz, M., Schmidt,
differentiation after 15 passages. In contrast, W., and Kemler, R. 1985. The in vitro devel-
hMADS cells can be maintained for 30 pas- opment of blastocyst-derived embryonic stem
sages with no loss of their ability to undergo cell lines: Formation of visceral yolk sac, blood
adipocyte differentiation. islands and myocardium. J. Embryol. Exp.
Morphol. 87:27-45.
Field, S.J., Johnson, R.S., Mortensen, R.M.,
Anticipated Results Papaioannou, V.E., Spiegelman, B.M., and
The authors usually pool 2-day-old EBs Greenberg, M.E. 1992. Growth and differenti-
from four lids of 100-mm bacteriological petri ation of embryonic stem cells that lack an in-
tact c-fos gene. Proc. Natl. Acad. Sci. U.S.A.
dishes into one 60-mm bacteriological grade 89:9306-9310.
petri dish. After RA treatment, EBs contained
Lehmann, J.M., Moore, L.B., Smith, O.T.,
in one 60-mm dish are plated into one 100-mm Wilkison, W.O., Willson, T.M., and Kliewer,
tissue culture dish. Under these conditions de- S.A. 1995. An antidiabetic thiazolidinedione is a
velopment of more than 100 EBs can be ob- high affinity ligand for peroxisome proliferator-
served. Twenty days after EB formation, 50% activated receptor gamma (PPAR gamma).
to 70% of EBs contain adipocyte colonies. J. Biol. Chem. 270:12953-12956.
Over 25 passages, adipocyte differentiation Mandrup, S. and Lane, M.D. 1997. Regulating adi-
may decrease. In regard to the differentiation pogenesis. J. Biol. Chem. 272:5367-5370.
of adult stem cells, the authors usually perform Phillips, B.W., Vernochet, C., and Dani, C. 2003.
differentiation in 24-well plates when mon- Differentiation of embryonic stem cells for
pharmacological studies on adipose cells.
itoring differentiation by Oil-red O staining, Pharmacol. Res. 47:263-268.
and into 60-mm plates, or larger plates, for
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal,
RNA preparation. Ten days after induction of R.K., Douglas, R., Mosca, J.D., Moorman,
differentiation, 50% to 90% of hMADS and M.A., Simonetti, D.W., Craig, S., and Marshak,
hMS cells are differentiated into adipocytes. D.R. 1999. Multilineage potential of adult Stem Cells

23.4.13
Current Protocols in Cell Biology Supplement 34
human mesenchymal stem cells. Science Wabitsch, M., Bruderlein, S., Melzner, I., Braun,
284:143-147. M., Mechtersheimer, G., and Moller, P. 2000.
Rodriguez, A.-M., Elabd, C., Delteil, F., Astier, LiSa-2, a novel human liposarcoma cell line with
J., Vernochet, C., Saint-Marc, P., Guesnet, J., a high capacity for terminal adipose differentia-
Guezennec, A., Amri, E.-Z., Dani, C., and tion. Int. J. Cancer 88:889-894.
Ailhaud, G. 2004. Adipocyte differentiation of Wabitsch, M., Brenner, R.E., Melzner, I., Braun,
multipotent cells established from human adi- M., Moller, P., Heinze, E., Debatin, K.M., and
pose tissue. Biochem. Biophys. Res. Commun. Hauner, H. 2001. Characterization of a hu-
315:255-263. man preadipocyte cell strain with high capacity
Rodriguez, A.-M., Pisani, D., Dechesne, C.A., for adipose differentiation. Int. J. Obes. Relat.
Turc-Carel, C., Kurzenne, J.-Y., Wdziekonski, Metab. Disord. 25:8-15.
B., Villageois, A., Bagnis, C., Breittmayer, J.-P., Zaragosi, L.E., Ailhaud, G., and Dani, C. 2006.
Groux, H., Ailhoud, G., and Dani, C. 2005a. Autocrine fibroblast growth factor 2 signaling
Transplantation of a multipotent cell popula- is critical for self-renewal of human multipotent
tion from human adipose tissue induces dys- adipose-derived stem cells. Stem Cells 24:2412-
trophin expression in the immunocompetent 2419.
mdx mouse. J. Exp. Med. 201:1397-1405. Zilberfarb, V., Pietri-Rouxel, F., Jockers, R., Krief,
Rodriguez, A.-M., Elabd, C., Amri, E.Z., Ailhaud, S., Delouis, C., Issad, T., and Strosberg, A.D.
G., and Dani, C. 2005b. The human adipose 1997. Human immortalized brown adipocytes
tissue is a source of multipotent stem cells. express functional beta3-adrenoceptor coupled
Biochimie 87:125-128. to lipolysis. J. Cell Sci. 110:801-807.
Smith, A.G. 1992. Mouse embryo stem cells: Their
identification, propagation and manipulation.
Semin. Cell Biol. 3:385-399. Contributed by Brigitte Wdziekonski, Phi
Tontonoz, P., Hu, E., Devine, J., Beale, E.G., Villageois, and Christian Dani
and Spiegelman, B.M. 1995. PPAR gamma 2 CNRS Université de Nice Sophia
regulates adipose expression of the phospho- Antipolis
enolpyruvate carboxykinase gene. Mol. Cell Nice, France
Biol. 15:351-357.
Voytas, D. 2000. Agarose gel electrophoresis. Curr.
Protoc. Mol. Biol. 51:2.5A.1-2.5A.9.

Differentiation of
Stem Cells into
Adipocytes

23.4.14
Supplement 34 Current Protocols in Cell Biology
Induction of ES Cell–Derived Cartilage UNIT 23.5
Formation
Embryonic stem (ES) cells are generated from the inner cell mass of the blastocyst (Evans
and Kaufman, 1981; Martin, 1981). Therefore, these cells are pluripotent, and if they
are cultivated in vitro as cellular aggregates—so called embryoid bodies (EBs)—they
spontaneously differentiate into various cell types of all three germ layers (Rathjen et al.,
1998). The ES cell model system of in vitro differentiation is useful to analyze cell
differentiation from a pluripotent stem cell to progenitor cells, and, finally, to terminally
differentiated cell types (for review, see Guan et al., 1999; Rohwedel et al., 2001). This
unit contains protocols for ES cell differentiation to chondrocytes and osteocytes (Basic
Protocol 1 and Alternate Protocol) and for characterization of the differentiated cell types
(Kramer et al., 2000; Hegert et al., 2002).

The authors analyze chondrogenic and osteogenic differentiation in EBs by histochem-


ical staining (Support Protocols 1 to 6), immunostaining (Support Protocol 7), mRNA
in situ hybridization (Support Protocol 8), RT-PCR techniques (Support Protocol 9),
and electron microscopy (Support Protocol 10; Kramer et al., 2000; 2005a,b). Chondro-
cytes are organized in cartilage nodules, which develop from aggregates of mesenchymal
prechondrogenic cells (Basic Protocol 2). Later, chondrogenic cells within these nod-
ules become hypertrophic and finally show characteristics of osteogenic differentiation
(Hegert et al., 2002). In contrast to other mesodermal cell types, such as cardiac muscle
cells, the amount of cartilage nodules is relatively low in EBs derived from ES cell line D3
(Kramer et al., 2005a). However, cartilage nodule formation can be induced, for example
by growth factors of the TGF-β family (Kramer et al., 2000). Another important factor
involved in modulating ES cell–derived cartilage formation is the basal medium and its
basic supplementations (see Commentary).

For regenerative strategies, ES cell–derived chondrocytes have to be selected from EBs


to enrich chondrogenic cells and to avoid teratoma formation by undifferentiated ES
cells. For example, ES cells formed teratomas and destroyed the ambient tissue if they
were injected in an undifferentiated stage into knee joints of mice (Wakitani et al.,
2003). The authors of this unit isolated chondrocytes from cartilage nodules using mi-
crodissection (Basic Protocol 2) and collagenase treatment (Basic Protocol 3; Hegert
et al., 2002) and analyzed their differentiation behavior in culture. These single ES
cell–derived chondrocytes possess a high potential for regeneration, indicated by a rapid
redifferentiation after an initial dedifferentiation. However, transdifferentiation into other
mesodermal cell types was observed in the chondrocyte single-cell cultures (Hegert et al.,
2002). Therefore, it may be necessary to improve the selection strategies to obtain de-
fined cell populations before ES cell–derived chondrocytes can be used for therapeutic
applications.

The model system of ES cell–derived chondrogenesis described in this unit can also be
used to analyze the influence of different parameters—e.g., signaling molecules, growth
factors, and extracellular matrix molecules—on cellular chondrogenic development. In
particular, the authors have studied growth factor modulation of ES cell–derived chon-
drocyte differentiation in vitro (Kramer et al., 2000, 2003; Hegert et al., 2002).

In vivo, murine ES cells are used to generate knockout mice, which enable study of
the phenotypic consequences of a gene mutation that has been established in vitro in
ES cells via gene targeting. This is possible because genetically modified ES cells take

Stem Cells
Contributed by Jan Kramer, Peter Schlenke, and Jürgen Rohwedel 23.5.1
Current Protocols in Cell Biology (2007) 23.5.1-23.5.33
Copyright 
C 2007 by John Wiley & Sons, Inc.
Supplement 34
part in the development of all somatic tissues and most importantly, germ cells, after
injection into blastocysts (Thomas and Capecchi, 1987). However, frequently, embryos
carrying a homozygous knockout mutation die shortly after implantation. In this case,
in vitro differentiation of homozygous knockout ES cells is an excellent alternative
approach to analyze the consequences of a loss-of-function mutation at the cellular level.
Therefore, the model system of ES cell–derived chondrogenesis in vitro can be used
to characterize the function of specific genes during cartilage formation. For example,
the authors investigated the consequence of a knockout mutation of the transcription
factor Sox9 on ES cell–derived chondrogenesis in vitro (Hargus et al., submitted). In
vivo studies have shown that Sox9 plays an important role during chondrogenesis (Bi
et al., 1999; Akiyama et al., 2002). The authors tested EBs generated from homozygous
Sox9-deficient ES cells for their cartilage differentiation in vitro and found that Sox9−/−
cells failed to develop into cartilage nodules (Hargus et al., submitted). This result is in
line with the previous in vivo studies.

Taken together, the ES cell in vitro model system is an alternative approach to in vivo stud-
ies with embryos and can be used to characterize cellular events of chondrogenic differ-
entiation. In this unit, some basic techniques for ES cell cultivation and for differentiation
into the chondrogenic direction in vitro are provided, along with protocols to characterize
chondrogenic differentiation of ES cells by histochemical staining, immunofluorescence
methods, mRNA-in situ hybridization, RT-PCR technique, and electron microscopy.
Moreover, the authors of this unit review how ES cell–derived chondrogenic differentia-
tion can be modulated by growth factors and discuss studies demonstrating chondrogenic
differentiation of ES cells.

STRATEGIC PLANNING
Successful ES cell cultivation requires knowledge and experience with basic cell culture
techniques like thawing, trypsinization, and freezing of cells. For isolation and ultra-
structural analysis of chondrogenic aggregates from EBs, microdissection and electron
microscopy techniques have to be established. For differentiation of ES cells, two prin-
cipal methods can be used: (1) ES cells can simply be cultivated in bacteriological petri
dishes where they form EBs spontaneously, or (2) ES cells can be cultivated via the
hanging-drop method to obtain EBs. The advantage of the latter technique is that EBs of
the same size are obtained, which enables a more reproducible differentiation process.
Therefore, this unit provides detailed protocols for differentiation of ES cells via hanging
drops (Basic Protocol 1). ES cell differentiation also requires advanced development of
a time schedule (see Table 23.5.1) for the specific study and calculation of the number of
EBs needed. The hanging-drop cultivation is performed for 2 days (days 0 to 2) to obtain
the EBs, followed by cultivation of EBs in suspension for 3 days (days 2 to 5) and plating
of EBs on the fifth day of differentiation (day 5). After plating, the EBs grow out and are
subsequently cultivated for up to 35 days (day 5 plus 35 days). Initially, before plating of
EBs (days 0 to 5), samples of 25 EBs are taken every day for RT-PCR analysis (a total
of 125 EBs). Later, after plating of EBs, samples are analyzed every second to fifth day
by RT-PCR, histochemical staining, immunostaining, or mRNA in situ hybridization
(ten EBs per sample and per method). For isolation of chondrogenic cells from EBs,
it is necessary to obtain sufficient amounts of chondrogenic aggregates in the EBs for
microdissection. Ten 60-mm tissue culture plates, each with ten EBs, have to be prepared
for every sample. Initially, every hanging drop contains 800 ES cells resulting in one
single EB. These details have to be considered in advance to calculate the quantity of
undifferentiated ES cells that have to be generated before the onset of the differentiation
experiment. The standard ES cell protocol is based on the assumption that a 60-mm tissue
ES Cell–Derived
Cartilage culture plate with undifferentiated ES cells contains around 1 × 106 cells after 2 days of
Formation cultivation. However, to achieve this quantity of cells, the size and morphology of the ES
23.5.2
Supplement 34 Current Protocols in Cell Biology
Table 23.5.1 A Time Schedule Useful for an ES Cell Differentiation Experimenta

Monday Tuesday Wednesday Thursday Friday Saturday Sunday

1st week Thaw feeder Split feeder Inactivate feeder


nd
2 week Thaw ES cells 0 days R R 2 days R R R
rd
3 week 5 days R 5+2 days R,H I 5+4 days R,H
th
4 week 5+8 days R,I,H 5+10 days R,H I 5+12 days R,H
th
5 week 5+15 days R,I,H 5+17 days R,H I 5+19 days R,H
th
6 week 5+22 days R,I,H 5+24 days R,H I 5+26 days R,H
th
7 week 5+29 days R,I,H 5+31 days R,H I 5+33 days R,H
th
8 week 5+36 days R,I,H
a Hanging-drop cultivation of ES cells (Basic Protocol 1) is performed to generate embryoid bodies (EBs) in vitro. In general, 2 days after thawing,
a sufficient number of ES cells for one differentiation experiment is obtained. The hanging-drop cultivation is performed for 2 days (days 0 to 2) to
generate the EBs, followed by cultivation of EBs in suspension for 3 days (days 2 to 5) and plating of EBs on the fifth day of differentiation (day 5).
After plating, the EBs grow out and are cultivated subsequently for up to 35 days (day 5 plus 35 days). Initially, RNA probes (R) for RT-PCR analysis
are collected every day (days 0 to 5). Later, specimens for RT-PCR analysis and histochemical stainings (H) are taken approximately every second
day. Indirect immunostaining and in situ hybridization (I) are performed twice a week, for example on Monday and Thursday. Samples for electron
microscopy are collected at day 5 plus 10 days (mesenchymal condensations) and at day 5 plus 22 days (cartilage nodules), respectively.

cell colonies has to be controlled carefully by light microscopy (see Critical Parameters).
A total of 1 × 106 ES cells can be used to generate 1250 EBs. The murine ES cell lines
BLC6 (Wobus et al., 1988), D3 (Doetschman et al., 1985), E14 (Hooper et al., 1987), and
R1 (Nagy et al., 1993) have been successfully used by the authors. Line BLC6 is most
effective with respect to cartilage nodule formation (Kramer et al., 2005a). In general, all
analyzed ES cell lines showed spontaneous chondrogenic differentiation. However, an
important consideration with respect to the differentiation efficiency is the use of growth
factors to enhance chondrogenic differentiation. For example, chondrogenic differentia-
tion of murine ES cells can be enhanced by the addition of BMB-2 and/or BMP-4 to the
differentiation medium (for details, see Commentary).

NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37◦ C, 5% CO2


incubator unless otherwise specified.

IN VITRO DIFFERENTIATION OF EMBRYONIC STEM CELLS FROM BASIC


EMBRYOID BODIES PROTOCOL 1
Pluripotent ES cells differentiate spontaneously into cells of all three primary germ layers.
ES cells can be differentiated into chondrogenic and osteogenic cell types by cultivation
as cellular aggregates, so called embryoid bodies (EBs), generated by hanging drops.
The major steps of ES cell differentiation are: (1) generation of EBs by hanging-drop
cultivation of ES cells for 2 days (days 0 to 2); (2) cultivation of the EBs in suspension
for 3 days (days 2 to 5); and (3) plating of the EBs at the fifth day of differentiation
(day 5), followed by further cultivation up to 35 days (day 5 plus 35 days; Table 23.5.1).

Materials
ES cell lines BLC6 (Wobus et al., 1988), D3 (Doetschman et al., 1985), E14
(Hooper et al., 1987), and/or R1 (Nagy et al., 1993) growing (Support
Protocol 12) on feeder layers (Support Protocol 11) in Medium 2 (see recipe)
and Medium 4 (see recipe)
Stem Cells

23.5.3
Current Protocols in Cell Biology Supplement 34
Phosphate-buffered saline (PBS; see recipe)
Trypsin/EDTA solution (see recipe)
Medium 2 (see recipe) and Medium 4 (see recipe)
Low-FBS medium (see recipe)
100-mm bacteriological petri dishes (Greiner), uncoated
100-µl sterile pipet tips with aerosol-filter barriers (Eppendorf)
5-ml glass pipets
Gelatin-coated (see recipe) 60-mm tissue culture dishes (Nunc), chamber slides
(Falcon CultureSlides from BD Biosciences), and 24-well tissue culture plates
Additional reagents and equipment for basic cell culture techniques including
counting cells (UNIT 1.1)
Cultivate ES cell in hanging drops
For the differentiation analysis described in Table 23.5.1, 475 EBs with 800 cells/EB
must be prepared. In addition, ∼40,000 ES cells are used for RNA isolation (at day 0).
In total, 420,000 ES cells are necessary for the analysis. However, one should not use
all cultivated ES cells for a single differentiation experiment, but should leave some that
can be further cultivated in the undifferentiated state for freezing (see Support Protocol
12). A 60-mm dish contains ∼1 × 106 ES cells (if carefully cultivated), as mentioned in
Strategic Planning. The authors thus suggest that two 60-mm dishes be prepared: one for
the differentiation analysis and one for further cultivation.

1. Wash cells in dish once with 4 ml PBS. Trypsinize undifferentiated ES cells cultivated
on MEF feeder layer in medium 2 in 60-mm dishes by adding 2 ml trypsin/EDTA
solution. When the cells are dissociated pipet up and down and transfer the cells to
a 15-ml centrifuge tube with 10 ml medium.
The undifferentiated ES cells are grown, subcultivated, and cropreserved as in Support
Protocol 12; feeder layers are produced as in Support Protocol 11.

2. Centrifuge 5 min at 180 × g, room temperature. Remove and discard supernatant.


3. Resuspend cells in 2 ml of Medium 4 and determine the cell number (UNIT 1.1).
The authors use 800 ES cells per EB for differentiation into chondrogenic and osteogenic
cells.

4. Dilute cells in Medium 4 to obtain a cell suspension with 4 × 104 cells/ml.


For cell dilution, it is helpful to use a small sterile beaker with a diameter of 4 cm
for preparing the cell suspension. The 20-µl drop samples are then transferred with a
micropipettor under sterile conditions.

5. Place ∼50 20-µl aliquots of the cell suspension on the inner side of the lid of a
100-mm uncoated bacteriological petri dish, using a micropipettor with sterile
100-µl tips containing aerosol-filter barriers (see Fig. 23.5.1).
If using gloves for cell culture handling, the electrostatic charge of the petri dish lid can
cause the drops to flow together. Therefore, avoid touching the lids outside on the top.

6. Place 10 ml PBS into the bottom of the petri dish (to avoid evaporation of the
hanging-drop medium during cultivation). Carefully invert the lid containing the
drops of ES suspension onto the bottom of the petri dish to create the hanging drops.
Culture the hanging drops for 2 days at 37◦ C (day 0 to day 2).
Cultivate EBs in suspension (day 2 to day 5)
7. On the second day of hanging-drop culture, collect the drops containing the EBs
ES Cell–Derived
Cartilage from two petri dishes (containing ∼50 drops per lid) by flooding the inverted lid
Formation

23.5.4
Supplement 34 Current Protocols in Cell Biology
Figure 23.5.1 Schematic illustrating Basic Protocol 1 (left) and Alternate Protocol (right) for
“hanging drop” cultivation of embryonic stem cells as embryoid bodies (EBs). The Alternate
Protocol may provide the advantage of easier handling.

containing the drops with 2 ml of Medium 4 and then aspirating with a 5-ml glass
pipet.
Do not do this if following the Alternate Protocol; also see Figure 23.5.1.

8. Place 8 ml of fresh Medium 4 into the bottom of an uncoated 100-mm bacteriological


petri dish. Pool the EBs collected from the lids of the two dishes in step 7 in this dish.
It is important to select a batch of bacteriological petri dishes that enable suspension
culture. With some batches, the EBs attach, even if the dishes are not coated with gelatin.
If this happens, it is sometimes possible to resuspend the EBs again by carefully rinsing
the dishes with Medium 4.

9. Cultivate the EBs in suspension for 3 days (day 2 to day 5).


Plate EBs (day 5) and analyze
10. On the fifth day of differentiation (day 5), plate the appropriate number of EBs
(see Table 23.5.2) onto gelatin-coated 60-mm tissue culture dishes (for Alcian
blue staining, RT-PCR, microdissection, and electron microscopy), gelatin-coated
chamber slides (for immunostaining, mRNA in situ hybridization, and most Stem Cells
histochemical staining), and gelatin-coated 24-well plates (for morphological
23.5.5
Current Protocols in Cell Biology Supplement 34
Table 23.5.2 Conditions for Setting Up EB Cultures

Culture container Number of EBs Volume medium Used for


(ml)

60-mm petri dish 10 4 Alcian Blue staining, RT-PCR,


microdissection, electron microscopy
2-chambered slide 5/chamber 2.5/chamber Immunostaining, mRNA in situ
hybridization, histochemistry
24-well plate 1/well 1/well Morphological analysis

Figure 23.5.2 (A) Alcian blue staining demonstrates ES cell–derived cartilage nodule formation.
(B) Cultivation of embryoid bodies (EBs) under low-serum conditions (0.2% FBS) after EB plating
significantly enhanced the number of Alcian blue–positive cartilage nodules per EB. Bar = 100 µm.
For the color version of this figure go to http://www.currentprotocols.com.

analysis) containing the appropriate volume of Medium 4 as listed in Table 23.5.2.


Transfer EBs using a micropipettor with sterile 100-µl aerosol-filter barrier tips into
the Medium 4 and handle the dishes very carefully during the first 2 days of culture
ES Cell–Derived to allow the EBs to attach.
Cartilage
Formation

23.5.6
Supplement 34 Current Protocols in Cell Biology
11. Initially, change the medium after 4 and 8 days.
In currently running studies the authors use Medium 4 with low FBS (0.2%) for chondro-
genic differentiation of ES cells. This low-FBS medium (see recipe) is applied from 2 days
after plating (day 5 plus 2 days) up to the end of EB cultivation. Medium is changed every
4 days. Up to the first 2 days after plating, Medium 4 (with 20% FBS) is used. Low-FBS
conditions then induce cartilage nodule formation (see Fig. 23.5.2).

12. During further cultivation steps, up to 35 days after plating (day 5 plus 35 days)
renew the medium about every second day.
13. To study the differentiation process, analyze samples every second to fifth day
(see Table 23.5.1).

ALTERNATIVE PLATING METHOD FOR GENERATING EBs OF THE ALTERNATE


SAME SIZE PROTOCOL
This Alternate Protocol simplifies the “hanging drop” method (Basic Protocol 1), which is
used as a standard protocol to obtain same-sized EBs. The Alternate Protocol is presented
as an optional way to generate EBs of the same size.

For materials, see Basic Protocol 1.

1. Prepare cell suspension for hanging-drop-cultivation (see Basic Protocol 1, steps 1


to 4).
2. Place ∼50 20-µl aliquots of the cell suspension on the inner side of the bottom of a
100-mm bacteriological petri dish (not the lid, in contrast to Basic Protocol 1),
using a micropipettor with sterile 100-µl tips containing aerosol-filter barriers
(see Fig. 23.5.1).
3. Place an open 60-mm culture dish filled with 4 ml PBS onto the lid of the bacteri-
ological petri dish to avoid evaporation of the drops. Carefully invert the bottom of
the petri dish containing the drops of ES onto the lid to create the hanging drops.
Culture the hanging drops for 2 days at 37◦ C (day 0 to day 2).
4. Remove the PBS-filled 60-mm culture dish at the second day of hanging drop-culture,
then turn the bacterial petri dish over to its normal configuration (see Fig. 23.5.1).
5. Add 8 ml Medium 4 to each 100-mm bacteriological petri dishes with the drops of
EBs that are now at the bottom.
6. Cultivate EBs in suspension for 3 days (day 2 to day 5).
7. Follow Basic Protocol 1, steps 10 through 13, for plating of EBs.

MICRODISSECTION AND CHARACTERIZATION OF MESENCHYMAL BASIC


CONDENSATIONS AND CARTILAGE NODULES FROM EBs PROTOCOL 2
Mesenchymal condensations containing chondrogenic precursors are isolated around
10 days after EB plating (day 5 plus 10 days), and cartilage nodules with mature chon-
drocytes are isolated around 20 days after plating (day 5 plus 20 days) using either a
microdissector or a microscalpel. Condensations and nodules can be easily detected by
their specific morphology (Kramer et al., 2005b). EBs are cultivated as in Basic Protocol
1 (steps 1 through 11) or the Alternate Protocol (steps 1 through 7). To characterize the
differentiation stage of the cells, samples of these isolated chondrogenic aggregates are in-
vestigated for expression of collagen marker proteins by immunostaining of cryosections
(Hegert et al., 2002) and analyzed by ultrastructural analysis using electron microscopy.

Stem Cells

23.5.7
Current Protocols in Cell Biology Supplement 34
Materials
EB cultures, (day 5 plus 10 days or day 5 plus 20 days; see Basic Protocol 1 or
Alternate Protocol)
Tissue-Tek OCT compound (Sakura Finetek)
Acetone
Phosphate-buffered saline (PBS; see recipe)
Microscalpel or microdissector (Eppendorf)
Tissue-Tek Cryomold 10 × 10 × 5–mm (Sakura Finetek;
http://www.sakuraus.com/)
Cryostat (Leica)
Vectabond-coated glass microscope slides (Vector)
Additional reagents and equipment for indirect immunostaining (Support Protocol 7)
1. Select nodules from EB cultures (day 5 plus 10 days or day 5 plus 20 days).
2. Microdissect the nodules and place them in ∼0.5 ml Tissue-Tek OCT compound in
a Tissue-Tek Cryomold for embedding.
Microdissection is performed using a commercially available microdissector (Eppendorf).
The procedure to isolate cartilage nodules using this device is outlined in detail in Eppen-
dorf Applications No. 48 (follow the “Applications” link at http://www.eppendorf.com).

3. Freeze the samples at −20◦ C.


4. Prepare 10-µm cryosections using a cryostat and plate onto Vectabond-coated slides.
5. Dry the sections in air.
6. Fix in acetone for 10 min at −20◦ C.
7. Perform indirect immunostaining (see Support Protocol 7, steps 3 to 10).

BASIC ISOLATION OF CELLS FROM SELECTED CARTILAGE NODULES BY


PROTOCOL 3 COLLAGENASE TREATMENT
Chondrogenic cells can be isolated from cartilage nodules microdissected from EBs to
analyze their behavior in single-cell cultures.

Materials
Microdissected nodules from EB cultures (Basic Protocol 2, steps 1 to 2)
0.1% (w/v) collagenase in Medium 4 (see recipe for Medium 4)
Medium 4 (see recipe)
Gelatin-coated (see recipe) or collagen II-coated 60-mm tissue culture dishes or
chamber slides
Shaking water bath or incubator
15-ml centrifuge tubes
Centrifuge
Additional reagents and equipment for basic cell culture techniques including
counting cells (UNIT 1.1)
1. Incubate ten microdissected nodules in 5 ml of 0.1% collagenase solution for 50 min
at 37◦ C, with moderate shaking, to obtain a single-cell suspension.
2. Transfer the cells to a 15-ml centrifuge tube. Centrifuge the cells for 5 min at
180 × g, room temperature. Discard the supernatant.
ES Cell–Derived
Cartilage
Formation

23.5.8
Supplement 34 Current Protocols in Cell Biology
3. Resuspend the cell pellet in 2 ml Medium 4 and count the cells (UNIT 1.1). Plate the
cells at high density onto gelatin- or collagen II–coated 60-mm tissue culture dishes
(1–2 × 105 cells per 60-mm dish) or onto chamber slides (2.1 × 104 cells/well).
Use 60-mm dishes for total RNA isolation or chamber slides for immunostaining and
histochemical staining.

4. Culture cells at 37◦ C in Medium 4.


The cellular processes of de-, re-, and transdifferentiation can be investigated using the
isolated ES cell–derived chondrocytes (for details see Hegert et al., 2002).

HISTOCHEMICAL STAINING OF EB
Histochemical staining of EBs is performed after plating of EBs from day 5 plus 2 days
up to about day 5 plus 35 days at regular intervals of about 2 days. Alcian blue staining
(Support Protocol 2) is used as a fast screen for detection of cartilage nodules (see
Figure 23.5.2A). Alcian blue selectively stains muco-substances, in particular cartilage-
specific proteoglycans. Alizarin red (Support Protocol 3) forms chelate conjugates with
bivalent cations (mainly with calcium), and is therefore used to detect bone nodule
formation. In addition, von Kossa staining (Support Protocol 4) is performed to visualize
bone nodule formation due to ion exchange (calcium ions are replaced by silver), which
depends on the influence of daylight. ES cell–derived adipogenic differentiation can be
demonstrated by cell staining with the vital dye Sudan III (Support Protocol 5), which
can be tracked directly by light microscopy.

Fixation for Histochemical Staining SUPPORT


PROTOCOL 1
The specimens must be fixed before histochemical staining (except for Sudan III and AP
staining).

Materials
Cultures with differentiating EBs (Basic Protocol 1), day 5 plus 2 days to day 5
plus 35 days, in chamber slides or tissue culture dishes depending on staining
technique to be used (see Support Protocols below)
Phosphate-buffered saline (PBS; see recipe)
3.7% (v/v) formaldehyde in distilled H2 O
1. Remove medium from the culture and wash twice, each time for 30 sec with 1.5 ml
PBS (for chamber slides) or 4 ml PBS (for 60-mm dishes).
2. Add 3.7% formaldehyde solution to the cultures (1.5 for chamber slides or 4 ml for
60-mm dishes) and incubate for 30 min at room temperature.
3. Remove formaldehyde and wash three times with PBS using the time and volume
specified in step 1, then once with water using the time and volume specified in
step 1.
4. Proceed to staining (Support Protocols 2 to 4).

Alcian Blue Staining SUPPORT


PROTOCOL 2
Alcian blue staining is a quick screen for cartilage nodules. The dye stains muco-
molecules, especially cartilage proteoglycans.

Materials
Fixed EB cultures at the desired interval after plating on day 5 (Support Protocol 1)
in 60-mm culture dishes
Stem Cells
Alcian blue working solution (see recipe)
23.5.9
Current Protocols in Cell Biology Supplement 34
Phosphate-buffered saline (PBS; see recipe)
5% (v/v) acetic acid solution
1. To fixed EBs cultivated on 60-mm culture dishes, add 4 ml Alcian blue working
solution.
2. Incubate the samples overnight at room temperature.
3. Remove the staining solution and wash for 30 sec once with 4 ml PBS and once with
4 ml 5% acetic acid.
4. Wash one more time with 4 ml PBS, then add ∼5 ml PBS to avoid dehydration of
the specimens.
5. View by light microscopy.
Deeply blue stained nodules should be visible (see Fig. 23.5.2A).

SUPPORT Alizarin Red Staining


PROTOCOL 3
Alizarin red forms chelate-conjugates with bivalent cations (mainly with calcium) and is
therefore used to detect bone nodule formation.

Materials
Fixed EB cultures at the desired interval after day 5 (Support Protocol 1) in
chamber slides
100 mM Tris·Cl, pH 9 (APPENDIX 2A)
Alizarin red staining solution 1: 5% (w/v) Alizarin red S (Sigma) in H2 O; adjust to
pH 9 with sodium hydroxide
Alizarin red staining solution 2: 0.5% (w/v) Alizarin red S (Sigma) in H2 O; adjust
to pH 9 with sodium hydroxide
Vectashield mounting medium (Vector)
Clear nail polish
Coverslips
1. Incubate fixed EBs cultured on chamber slides in 100 mM Tris·Cl, pH 9, for 30 sec
at room temperature.
The reagents are added to the chambers on the slides.

2. Incubate the samples in 1.5 ml Alizarin red staining solution 1 for 1 hr.
3. Remove the staining solution and rinse two times with 1.5 ml PBS, each time for
30 sec.
4. Incubate the samples in 1.5 ml Alizarin red staining solution 2 per chamber for 5 min.
5. Remove the staining solution and rinse two times with 1.5 ml PBS, each time for
30 sec.
6. Remove chambers from slide and apply Vectashield mounting medium. Cover the
slide with a coverslip and seal with clear nail polish.
7. View by light microscopy.
Red-stained bone nodules should be visible.

ES Cell–Derived
Cartilage
Formation

23.5.10
Supplement 34 Current Protocols in Cell Biology
Von Kossa Staining SUPPORT
PROTOCOL 4
Von Kossa staining allows visualization of bone nodule formation due to ion exchange
(calcium ions are replaced by silver), which depends on the influence of daylight.

Materials
Fixed EB cultures at the desired interval after plating on day 5 (Support Protocol 1)
in chamber slides
Von Kossa silver stain: 500 mg silver nitrate (Fluka) in 10 ml distilled water
(5% w/v AgNO3 )
Von Kossa fixation solution: 1 g anhydrous sodium thiosulfate (Merck) in 20 ml
distilled water (5% w/v sodium thiosulfate)
Phosphate-buffered saline (PBS; see recipe)
Vectashield mounting medium (Vector)
Clear nail polish
Coverslips
1. Treat EBs cultivated for the desired interval in chambers of chamber slides for 10 to
20 min with Von Kossa silver stain.
2. Wash carefully twice, each time with 1.5 ml distilled water for 15 sec.
3. Fix the resulting stained sample with Von Kossa fixation solution for 2 min.
4. Wash fixed EBs in chambers twice, each time with 1.5 ml PBS. Remove chambers
from slide and immediately add Vectashield mounting medium, cover the slide with
a coverslip, and seal with clear nail polish.
5. View by light microscopy.

Sudan III Staining SUPPORT


PROTOCOL 5
ES cell–derived adipogenic differentiation can be demonstrated by staining with the vital
stain Sudan III (Support Protocol 5; also see UNIT 23.4), which can be tracked directly by
light microscopy.

Materials
Unfixed EBs cultured in chamber slides for the desired interval after plating on day
5 (Basic Protocol 1 or Alternate Protocol)
Phosphate-buffered saline (PBS; see recipe)
Sudan III staining solution (see recipe)
Vectashield mounting medium (Vector)
Clear nail polish
Coverslips
1. Wash the EBs with PBS for 15 sec.
The reagents are added to the chambers on the slides.

2. Add 1.5 ml Sudan III staining solution and incubate for 3 min at room temperature.
3. Wash three times, each time with 1.5 ml PBS.
4. Remove chambers from slide and immediately add Vectashield mounting medium,
cover the slide with a coverslip, and seal with clear nail polish.
5. View by light microscopy.
Lipid droplets in cells are stained red. Adipogenic cells contain large amounts of such
droplets. Stem Cells

23.5.11
Current Protocols in Cell Biology Supplement 34
SUPPORT Alkaline Phosphatase (AP) Staining
PROTOCOL 6
Osteogenic cells show alkaline phosphatase activity.

Materials
Unfixed EBs cultured in chamber slides for the desired interval after plating on day
5 (Basic Protocol 1 or Alternate Protocol)
Phosphate-buffered saline (PBS; see recipe)
Leukocyte Alkaline Phosphatase staining kit (Sigma, cat. no. 86-R) containing:
Citrate solution
FRV-alkaline solution
Hematoxylin solution, Gill No. 3
Naphthol AS-BI alkaline solution
Sodium nitrite solution
Acetone
37% (w/v) formaldehyde
Vectashield mounting medium (Vector)
Clear nail polish
15-ml conical polypropylene centrifuge tubes
Coverslips
1. Wash the EB cultures twice, each time with 1.5 ml PBS for 15 sec.
The reagents are added to the chambers on the slides.

2. Combine 2.5 ml citrate solution (from the Sigma Leukocyte Alkaline Phosphatase
staining kit) with 6.5 ml acetone and 0.8 ml of 37% formaldehyde to prepare the
fixing solution. Add 1.5 ml fixing solution to the EBs and incubate 30 sec at room
temperature to fix.
3. Remove the fixing solution and rinse the samples with 1.5 ml distilled water for
45 sec.
4. Prepare the AP staining solution by combining 125 µl FRV-alkaline solution and
125 µl sodium nitrate solution (both included in the staining kit) in a 15-ml tube.
Mix and incubate 2 min at room temperature. Add 5.63 ml distilled water and 125 µl
naphthol AS-BI alkaline solution (from kit), and mix. Add 1.5 ml of this solution to
each chamber and incubate for 15 min at room temperature in the dark.
5. Wash once for 2 min in 1.5 ml distilled water. Counterstain with 1.5 ml hematoxylin
solution (from kit) for 2 min according to the manufacturer’s instructions. Again
wash once for 2 min in 1.5 ml distilled water.
6. Remove the chambers and rinse slide thoroughly in tap water. Immediately add
Vectashield mounting medium, cover slide with coverslip, and seal with clear nail
polish.
7. View by light microscopy.
Alkaline phosphatase–expressing cells are stained light red, whereas all other cells are
stained a dark purple color.

SUPPORT INDIRECT IMMUNOSTAINING OF EBs


PROTOCOL 7
Whole EBs can be analyzed for specific specialized cell types by indirect immunostaining.
EB cultures are set up and maintained on chamber slides. Immunostainings are performed
ES Cell–Derived at intervals of about 2 to 5 days starting 2 days after plating (day 5 plus 2 days) up to about
Cartilage 35 days after plating (day 5 plus 35 days; see Table 23.5.1). As an example, immunos-
Formation
taining for collagen II demonstrates ES cell-derived cartilage formation (Fig. 23.5.3).
23.5.12
Supplement 34 Current Protocols in Cell Biology
Figure 23.5.3 Immunostaining for collagen II demonstrates ES cell–derived cartilage forma-
tion. (A) The extracellular matrix of nodules consists of collagen II. (B) In addition, formation
of collagen II fibrils could also be observed outside the nodules. (C) Chondrogenic cells within
the cartilage nodules showed a typical round-shaped phenotype and the nodules were compact
and clearly seperated from the surrounding tissue. (D) In comparison, the single cells express-
ing the network of collagen II fibrils outside the nodules do not form such distinct structures.
(E, F) Cell nuclei are stained with DAPI. Bar = 100 µm. For the color version of this figure go to
http://www.currentprotocols.com.

Materials
Unfixed EBs cultured on chamber slides for the desired interval after plating on day
5 (Basic Protocol 1 or Alternate Protocol)
Phosphate-buffered saline (PBS; APPENDIX 2A)
7:3 (v/v) methanol/acetone (e.g., 42 ml methanol/18 ml acetone), −20◦ C
10% (v/v) goat serum (Dianova, http://www.dianova.de) in PBS (see recipe)
Antibodies for immunostaining, primary and fluorophore-labeled secondary,
appropriately diluted (see recipe)
Vectashield mounting medium (Vector)
Clear nail polish
Humidified chamber (15-cm covered glass dish with wet piece of filter paper)
Coverslips Stem Cells
Fluorescence microscope (e.g., AXIOSKOP; Zeiss)
23.5.13
Current Protocols in Cell Biology Supplement 34
Wash and fix EBs
1. Wash the cultures in the chamber slides twice, each time with 1.5 ml PBS for 30 sec.
2. Fix the cells for 5 min with 1.5 ml of 7:3 methanol/acetone at −20◦ C.
3. Rinse three times each time with 1.5 ml PBS for 30 sec.
Perform blocking and immunostaining
4. Move the sample in the chamber slide to a humidified chamber. Incubate the cells
for 15 min in 10% goat serum at room temperature to block nonspecific antibody
binding.
5. Incubate the specimens with 300 µl appropriately diluted primary antibodies for 1 hr
at 37◦ C in the humidified chamber.
6. Rinse three times, each time with 1.5 ml PBS for 30 sec.
7. Incubate the slides for 45 min at 37◦ C in the humidified chamber with 300 µl of
appropriately diluted fluorophore-labeled secondary antibody.
Choice of species for the secondary antibody depends on the origin of the primary
antibody (see Reagents and Solutions).

8. Wash the slides three times, each time in 1.5 ml PBS for 30 sec, then once in 1.5 ml
distilled water for 30 sec.
Mount, coverslip, and examine slides
9. Remove the chambers, immediately add Vectashield mounting medium, cover the
slides with a coverslip, and seal with clear nail polish. Store the slides up to 1 month
in the dark at 4◦ C.
10. Examine slides with fluorescence microscope.
Differentiated cell types are detected by fluorescence.

SUPPORT WHOLE-MOUNT FLUORESCENCE IN SITU HYBRIDIZATION FOR mRNA


PROTOCOL 8 OF A GENE OF INTEREST COUPLED WITH IMMUNOSTAINING FOR
COLLAGEN II
For a combination of fluorescence mRNA–in situ hybridization (ISH) and indirect
immunofluorescence (IF) staining, a modified whole-mount procedure for EBs
(Yamada et al., 1994) is used. This combination allows the simultaneous detection of a
tissue-specific protein and mRNA expression. Samples of five EBs plated on chamber
slides are analyzed at different developmental stages up to 35 days after plating (day 5
plus 35 days).

Materials
16-day post-coitum (p.c.) mouse embryo limb buds
TOPO II cloning kit (Invitrogen,)
pCR-BluntII-TOPO vector (Invitrogen)
DIG RNA labeling mix kit including Polymerases SP6 and T7 (Boehringer
Mannheim; cat. no. 1175025).
EB cultures plated on chamber slides at day 5 (Basic Protocol 1 or Alternate
Protocol)
Phosphate-buffered saline (PBS; see recipe)
4% (w/v) paraformaldehyde/4% (w/v) sucrose in PBS
2× SSC, 0.2× SSC, and 0.1× SSC (see recipe for 20× SSC)
ES Cell–Derived 50%, 70%, 95%, and 100% ethanol series
Cartilage
Formation Prehybridization buffer (see recipe) with and without salmon sperm DNA

23.5.14
Supplement 34 Current Protocols in Cell Biology
Monoclonal antibody II-II6B3 against collagen II (diluted 1:20 in PBS; see recipe
for Antibodies for immunostaining)
FITC-conjugated sheep F(ab) fragments against digoxigenin (Roche, cat. no.
1207741).
Cy3-conjugated goat anti-mouse secondary antibodies (Dianova; cat. no.
115-165-062; http://www.dianova.de) for indirect detection of collagen II, both
diluted 1:800 in PBS.
Vectashield mounting medium (Vector; cat. no. H-1000).
Clear nail polish
Humidified chamber (15-cm covered glass dish with wet piece of filter paper)
45◦ and 70◦ C incubators
Coverslips
Fluorescence microscope with FITC and Cy3 filters
Additional reagents and equipment for RNA isolation and cDNA synthesis via
RT-PCR (Support Protocol 9), and DNA sequencing (Chapter 7 in Ausubel
et al., 2007)
Generate hybridization probes
1. Isolate RNA from 16-day p.c. mouse embryo limb buds and synthesize cDNA
following the RT-PCR protocol (Support Protocol 9) using the primers for the gene
of interest listed in Table 23.5.3.
2. Blunt-end and clone the fragment into the plasmid vector pCR-BluntII-TOPO us-
ing the TOPO II cloning kit according to the manufacturer’s protocol. Verify the
sequences by sequencing (Chapter 7 in Ausubel et al., 2007).
3. Label RNA probes of sense and antisense orientation with digoxigenin using DIG
RNA labeling mix kit. Synthesize the probes from linearized plasmids of the cloned
cDNA fragments by in vitro transcription using the T7 or SP6 RNA polymerase and
the DIG RNA labeling mix following the protocol supplied by the manufacturer.
Final concentration of labeled probes should be 1 µg/ml.

Fix the cell-containing slides


4. Rinse EB cultures on chamber slides twice, each time with 1.5 ml PBS for 30 sec.
5. Fix the EBs with 4% (w/v) paraformaldehyde/4% (w/v) sucrose in PBS for 20 min
at room temperature.
6. Wash the cells twice, each time in 1.5 ml PBS for 5 min.
7. Incubate the specimens with 1.5 ml of 2× SSC for 15 min in a humidified chamber
inside a 70◦ C incubator.
8. Wash with 1.5 ml PBS followed by 1.5 ml 2× SSC, each time for 3 min.
9. Repeat steps 5 through 8.
Prepare the cell-containing slides for hybridization
10. Dehydrate the cells through an ethanol series at room temperature by adding 1.5 ml
of the indicated concentration of ethanol directly to each chamber:

2 min in 50% ethanol


2 min in 70% ethanol
2 min in 95% ethanol
2 min in 100% ethanol
2 min in 100% ethanol.
Stem Cells

23.5.15
Current Protocols in Cell Biology Supplement 34
Table 23.5.3 Primer Pairs Used to Analyze Chondrogenic and Osteogenic Differentiation of Murine ES Cellsa

Annealing
Fragment
Gene Antisense primer Sense primer temperature Reference
length (bp)
(◦ C)

Pre-cartilage
Chordin-like 1 5 -ACA ATG CCA AAT 5 -TGC GAA TAC AAT 68 506 Nakayama
GCT CGT AGA T-3 GGA ACC ACT TA-3 et al. (2003)
Pax-1 5 -TTC TCG GTG TTT 5 -GAT GGA AGA CTG 60 318 Kramer
GAA GGT CAT TGC GGC GGG TGT GAA-3 et al. (2000)
CG-3
Scleraxis 5 -GTG GAC CCT CCT 5 -GAC CGC ACC AAC 63 375 Kramer
CCT TCT AAT TCG-3 AGC GTG AA-3 et al. (2000)
Sox9 5 -TCT TTC TTG TGC 5 -TGG CAG ACC AGT 57 135 Kramer
TGC ACG CGC-3 ACC CGC ATC T-3 et al. (2000)
Mature cartilage
Aggrecan 5 -TCC TCT CCG GTG 5 -CCA AGT TCC AGG 60 270 Kramer
GCA AAG AAG TTG-3 GTC ACT GTTACCG-3 et al. (2000)
Biglykan 5 -CAT GAC AAC CGT 5 -ATT CCC GCC CAT 60 —a zur Nieden
ATC CGC AA-3 CTC AAT G-3 et al. (2005)
Collagen II 5 -AGG GGTACC AGG 5 -CTG CTC ATC GCC 60 432b 225 c Kramer
TTC TCC ATC-3 GCG GTC CTA-3 et al. (2000)
COMP 5 -GCT GCC CGG TCT 5 -GTT GCG ACA CGA 68 410 Nakayama
CAC ACT CAT T-3 GGT CAA GGA G-3 et al. (2003)
Decorin 5 -ATG ACC CTG ACA 5 -CCC AGA TCA GAA 60 —a zur Nieden
ATC CCC TG-3 CAC TGC ACC-3 et al. (2005)
Link protein 5 -TTC TGG GCT ATG 5 -AGC GCC TTC TTG 60 —a zur Nieden
ACC GCT G-3 GTC GAG A-3 et al. (2005)
Hypertrophic cartilage
Collagen X 5 -ATG CCT TGT TCT 5 -CTT TCT GCT GCT 61 164 Hegert et al.
CCT CTTACT GGA-3 AAT GTT CTT GAC (2002)
C-3
MMP13 5 -CGT GTG CCA GAA 5 -CAG TTG ACA GGC 57 372 Kawaguchi
GAC CAG AA-3 TCC GAG AA-3 et al. (2005)
Bone
Alkaline 5 -TCT GGT GGC ATC 5 -CCT GAA AAC TCC 57 465 Kawaguchi
phosphatase TCG TTA ATC-3 AAA AGC TC-3 et al. (2005)
Cbfa-1/Runx2 5 -ATC CAT CCA CTC 5 -AAG GGT CCA CTC 63 371 Kramer
CAC CAC GC-3 TGG CTT TGG-3 et al. (2000)
Osteocalcin 5 -ATG CTA CTG GAC 5 -GCG GTC TTC AAG 64 330 Hegert et al.
GCT GGA GGG T-3 CCA TAC TGG TC-3 (2002)
Osteoprotegerin 5 -ACT CCT GCT TCA 5 -TGC TCC TGG CAC 57 158 Kawaguchi
CGG ACT G-3 CTA CCT A-3 et al. (2005)
Osterix 5 -TGC CTG GAC CTG 5 -CCT CTG CGG GAC 57 355 Kawaguchi
GTG AGA TG-3 TCA ACA AC-3 et al. (2005)
continued

ES Cell–Derived
Cartilage
Formation

23.5.16
Supplement 34 Current Protocols in Cell Biology
Table 23.5.3 Primer Pairs Used to Analyze Chondrogenic and Osteogenic Differentiation of Murine ES Cellsa , continued

Annealing
Fragment
Gene Antisense primer Sense primer temperature Reference
length (bp)
(◦ C)

PTHR1 5 -ACA GTC CCT CCA 5 -ACT ACA GCG ACT 57 417 Kawaguchi
CCA GAA TC-3 GCC TCA AG-3 et al. (2005)
RANKL 5 -AGT ACG TCG CAT 5 -GGA AGC GTA CCT 57 234 Kawaguchi
CTT GAT CC-3 ACA GAC TA-3 et al. (2005)
Dedifferentiated cartilage
Collagen I 5 -GGC GGT TAT GAC 5 -GGC ATG TTG CTA 60 702 —d
TTC AGC TTC -3 GGC ACG AAG-3
Internal standard
β-Tubulin 5 -GGA ACA TAG CCG 5 -TCA CTG TGC CTG 54 317 Kramer
TAA ACT GC-3 AAC TTA CC-3 et al. (2000)
HPRT 5 -GCC TGT ATC CAA 5 -AGC GTC GTG ATT 63 507 Kramer
CAC TTC G-3 AGC GAT G-3 et al. (2000)
a Not given.
b Juvenile splice variant.
c Adult splice variant.
d Not published.

11. Prehybridize 3 hr in 1.5 ml prehybridization buffer in a humidified chamber in an


incubator at 45◦ C.
12. Perform hybridization with 500 µl/well of 1 ng/µl digoxigenin-labeled sense and
antisense probes (prepared in step 3) in prehybridization buffer without salmon sperm
DNA at 45◦ C in a humidified chamber overnight.
13. The next day, wash the slide twice with 2× SSC for 15 min, once with 0.2× SSC
for 15 min and twice with 0.1× SSC for 15 min, all at 45◦ C. Rinse in 1.5 ml PBS
for 30 min.
Immunolabel the protein
14. Apply 300 µl monoclonal antibody II-II6B3 against collagen II (diluted 1:20 in PBS)
to the cells and incubate in a humidified chamber for 1 hr at 37◦ C.
15. Wash the cells three times each time with 1.5 ml PBS at room temperature for
30 sec.
16. Add 300 µl FITC-conjugated sheep F(ab) fragments against digoxigenin to detect the
RNA and 300 µl Cy3-conjugated goat anti-mouse secondary antibodies for indirect
detection of collagen II, both diluted 1:800 in PBS as a cocktail. Incubate for 1 hr at
37◦ C.
17. Wash the slides three times, each time in 1.5 ml PBS for 30 sec, then once in 1.5 ml
distilled water for 30 sec.
18. Mount in Vectashield mounting medium, cover the slides with coverslips, and seal
with clear nail polish.
19. Analyze using a fluorescence microscope.
Differentiated cell types are detected by ISH and IF.

Stem Cells

23.5.17
Current Protocols in Cell Biology Supplement 34
SUPPORT RT-PCR ANALYSIS TO DETECT CARTILAGE-SPECIFIC GENE
PROTOCOL 9 EXPRESSION IN EBs
The expression of tissue-specific genes is analyzed during EB cultivation from days 1 to
5 days before plating, and up to 35 days after plating (day 5 plus 35 days). Total RNA is
isolated from 25 EBs at the stages before plating (days 0 to day 5). After plating, samples
of ten EBs are used (from one 60-mm tissue culture dish). Distilled water and no-RT
reactions are always included as negative controls. RNA from limb buds or limbs of 10
and 16 day p.c. mouse embryos are used as positive controls.

Materials
Cultures of EBs at the desired time points (Basic Protocol 1 or Alternate Protocol)
Phosphate-buffered saline (PBS; see recipe)
RNeasy Mini Kit (Qiagen)
RNase-free DNase set (Qiagen)
Oligo-dT primer (Life Technologies)
Superscript II reverse transcriptase (Life Technologies)
Taq DNA polymerase (Roche)
Primer (see Table 23.5.3)
2% (w/v) agarose gel (Voytas, 2000)
Additional reagents and equipment for quantification of nucleic acids (APPENDIX 3D),
isolation of total RNA (Ausubel et al. 2007, Chapter 4), RT-PCR (Beverley,
2001), agarose gel electrophoresis (Voytas, 2000), and densitometry (UNIT 6.3)
1. Wash EB cultures twice, each time with 4 ml PBS for 30 sec.
2. Isolate total RNA using the RNeasy Mini Kit along with the RNase-free DNase
set (to avoid DNA contamination) according the manufacturers’ protocols (also see
Chapter 4 in Ausubel et al., 2007).
3. Determine the RNA concentrations by measuring the absorbance at 260 nm
(APPENDIX 3D).
4. Reverse transcribe samples of 500 ng RNA using oligo-dT primer and Superscript
II reverse transcriptase according to the manufacturer’s protocols (also see Beverley,
2001).
5. Use 1-µl aliquots from the RT reactions for amplification of transcripts with primer
(see Table 23.5.3) specific for the analyzed genes and Taq DNA polymerase according
to the manufacturer’s instructions. Use the following program:

1 cycle: 2 min 95◦ C (initial denaturation)


35 to 45 cycles: 40 sec 95◦ C (denaturation)
40 sec primer-specific (annealing)
temperature
50 sec 72◦ C (elongation).

Cycle numbers have to be determined using positive controls of RNA isolated from limb
buds of 18-day-old embryos. The optimal cycle number depends upon the PCR machine
used; further information may be found in the references listed in Table 23.5.3.
Primer-specific temperatures are listed in Table 23.5.3. For primer pairs established by
other groups please also compare to the original reference given in Table 23.5.3.
For RT-PCR analysis of scleraxis, an established method (Wong et al., 1994; Wobus et al.,
ES Cell–Derived 1997) including the oligonucleotide primer for HPRT and scleraxis in the same reaction,
Cartilage can be used.
Formation
6. Separate PCR products electrophoretically on 2% agarose gels (Voytas, 2000).
23.5.18
Supplement 34 Current Protocols in Cell Biology
7. Analyze the fragments by computer-assisted densitometry (UNIT 6.3) in relation to
GAPDH, HPRT, or β-tubulin gene expression.
It has been suggested that GAPDH is a more suitable candidate to act as an internal
RNA standard, while both HPRT and β-tubulin appear to be inappropriate (Murphy and
Polak, 2002).

ULTRACTRUCTURAL ANALYSIS OF ES CELL-DERIVED MESENCHYMAL SUPPORT


CONDENSATIONS AND CARTILAGE NODULES BY ELECTRON PROTOCOL 10
MICROSCOPY
ES cell–derived mesenchymal condensations and cartilage nodules exhibit a typical
morphology and can be identified in EB outgrowths by light microscopy. The following
protocol prepares such structures for further electron-microscopic analysis.

Materials
ES cell–derived mesenchymal condensations and cartilage nodules (see Basic
Protocol 2)
5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4
1% (w/v) OsO4
50%, 70%, 95% and 100% ethanol
Araldite embedding kit (e.g., Fluka)
Ultracut E (Leica Microsystems Nussloch, http://www.leica-microsystems.com/)
LKB Bromma Ultrostainer Carlsberg System for uranyl acetate and lead citrate
staining
CAUTION: Glutaraldehyde and OsO4 are potentially hazardous reagents. Work should
be performed under a hood and protective clothing and gloves must be used during the
procedure.

1. Fix the ES cell–derived outgrowths with 1 ml 5% glutaraldehyde in 0.1 M sodium


cacodylate, pH 7.4, for ∼1 hr at 4◦ C.
2. Treat specimens with 1 ml 1% OsO4 for 2 hr at room temperature.
3. Dehydrate the EB outgrowths in graded ethanol series:

50% ethanol
70% ethanol
95% ethanol
100% ethanol.

4. Embed the specimens in Araldite using kit according to manufacturer’s instructions.


5. Cut ultrathin sections of the EBs on an Ultracut E.
6. Stain the sections with uranyl acetate and lead citrate using the LKB Bromma
Ultrostainer Carlsberg System according to manufacturer’s instructions.
7. Examine the sections for chondrogenic cell types with an electron microscope.

DERIVATION, CULTIVATION, AND INACTIVATION OF EMBRYONIC SUPPORT


FIBROBLASTS PROTOCOL 11
To grow them in an undifferentiated state, ES cells are cocultivated with a feeder layer of
murine embryonic fibroblasts. Soluble factors produced by the feeder layer cells prevent
the ES cells from differentiation (Evans and Kaufman, 1981). Embryonic fibroblasts
used as feeder layers for ES cell cultivation are prepared and cultivated according to an
established method (Wobus et al., 1984). Stem Cells

23.5.19
Current Protocols in Cell Biology Supplement 34
Materials
Day 14 p.c. pregnant mice
Phosphate-buffered saline (PBS; APPENDIX 2A)
Trypsin/EDTA solution (see recipe)
Medium 1 (see recipe) for cultivation of embryonic fibroblasts
1 µg/ml MMC working solution (see recipe)
Medium 3 (see recipe)
100 mm-bacteriological petri dishes (Greiner), uncoated
Sterile dissection instruments: scissors, forceps
100-ml Erlenmeyer flask with a stir bar, sterile
∼4-mm-diameter glass beads
Sieve (e.g., autoclavable metal tea filter) with pore diameter of ∼0.5 mm
15- and 50-ml conical polypropylene centrifuge tubes
Gelatin-coated (see recipe) 100-mm and 60-mm dishes
2 ml-cryopreservation vials (Nunc)
Dewar flasks with liquid nitrogen
Additional reagents and equipment for counting cells (UNIT 1.1)
Derive embryonic fibroblasts
1. Remove embryos at day 14 p.c. from uteri of pregnant mice and place under sterile
conditions in PBS in a 100-mm bacteriological petri dish.
Mouse embryos 14 days p.c. are optimal for isolation of embryonic fibroblasts used as a
feeder layer. However, embryos up to 16 days p.c. can be used.

2. In a separate dish containing PBS, cut off the placenta and fetal membranes and
remove head and liver using sharp sterile scissors.
3. Transfer the remaining carcasses first into a new petri dish with PBS for 30 sec and
then into a dish with 5 ml trypsin/EDTA solution.
4. Mince the carcasses in the trypsin/EDTA solution at room temperature.
5. Transfer the 5 ml solution to a sterile 100-ml Erlenmeyer flask with fifty 4-mm glass
beads and stir for 25 up to 45 min until most of the tissue pieces are dissociated, but
not longer than 45 min.
6. Filter the resulting cell suspension through a sterile sieve and flush into a 15-ml
conical centrifuge tube with 10 ml medium 1.
The authors use a metal tea filter that can be autoclaved for a sieve.

7. Centrifuge the cells 5 min at 180 × g, room temperature. Remove the supernatant
very carefully by aspiration and resuspend the pellet in 3 ml Medium 1.
8. Plate ∼2 × 106 cells, isolated from about two embryos, onto a gelatin-coated
100-mm tissue culture dish filled with 10 ml Medium 1. Culture at 37◦ C in a
humidified 5% CO2 incubator overnight before freezing (at 1 × 106 cells/ml in
Medium 3 in 2-ml cryovial) or MMC treatment (steps 9 to 12).
These feeder layer cells can be cultivated for 1 to 2 days and either trypsinized and frozen
in medium 3 (1 dish per freezing vial) or split 1:3 onto gelatin-coated 100-mm tissue
culture dishes and cultivated for one more day in medium 1 for further use.

Inactivate embryonic fibroblasts


9. Incubate the cells with 6 ml MMC solution for ∼2.5 hr at 37◦ C.
ES Cell–Derived
Cartilage CAUTION: Mitomycin C (MMC) is a carcinogen, and for this reason at least in these
Formation steps disposable material/pipets are used.
23.5.20
Supplement 34 Current Protocols in Cell Biology
MMC is used for growth inactivation of the subcultured or frozen feeder cells before
cocultivation with ES cells.
Frozen feeder layer cells are thawed rapidly, plated onto two gelatin-coated 100 mm-
tissue culture dishes and cultivated overnight before MMC treatment as described in
step 8.

10. Aspirate MMC solution. Wash the cells three times each time with 15 ml PBS for
30 sec.
11. Add 2 ml trypsin/EDTA solution to the cells and incubate 30 to 60 sec at room
temperature, observing the detachment of the cells under the microscope. Suspend
the cells in the trypsin/EDTA solution by pipetting up and down several times and
immediately transfer them to a centrifuge tube with 10 ml Medium 1. Centrifuge
5 min at 180 × g. Determine the cell number (UNIT 1.1).
12. Plate ∼1.5 × 105 cells onto gelatin-coated 60 mm-culture dishes with 4 ml Medium
1 to create a confluent monolayer. Use as feeder cells in Support Protocol 12.
Cells can be used immediately or stored up to 5 days in the incubator before use. They
should not be frozen after MMC treatment.

PROLIFERATION AND SUBCULTIVATION OF ES CELLS SUPPORT


PROTOCOL 12
Murine ES cells are maintained in the undifferentiated state by cultivation on feeder
layer cells (Support Protocol 11) and under the influence of leukemia inhibitory factor
(LIF; Smith et al., 1988; Williams et al., 1988). LIF, also produced by feeder layer cells,
binds to a heterodimer of the LIF receptor and gp130 that activates JAK/Stat3 signaling,
and activated Stat3 is sufficient to sustain undifferentiated proliferation of mouse ES
cells cultured in serum (Hocke, 1995). It is critical to subculture ES cells carefully for
maintaining the undifferentiated ES cells in the pluripotent state. Therefore, ES cells
should be subcultivated every 24 to a maximum of 48 hr.

Materials
Feeder layer cells: 60-mm dishes of MMC-treated embryo fibroblasts (Support
Protocol 11)
Medium 2 (see recipe)
Frozen vial of ES cells
Phosphate-buffered saline (PBS; see recipe)
Trypsin/EDTA solution (see recipe)
Medium 3 for freezing the ES cells
15- and 50-ml conical polypropylene centrifuge tubes
2-ml glass pipet
2-ml cryopreservation vials (Nunc)
Dewar flasks with liquid nitrogen
Thaw undifferentiated ES cells
1. At a time point 2 hr before thawing ES cells, replace the Medium 1 in the dishes
containing the MMC-treated feeder layer with 4 ml Medium 2. Return the cells to
the incubator.
2. Thaw the vial with ES cells rapidly at 37◦ C.
3. Dilute the cold cell suspension 1:10 with Medium 2 in a 15-ml centrifuge tube and
centrifuge 5 min at 180 × g, room temperature.

Stem Cells

23.5.21
Current Protocols in Cell Biology Supplement 34
4. Resuspend the cell pellet in 2 ml Medium 2 and transfer to the prepared feeder layer.
Incubate 1 to 2 days.
The cell suspension is divided on to two 60-mm feeder layer dishes at 1 ml per plate. This
corresponds to ∼0.5 × 106 cells per plate.

Subcultivate undifferentiated ES cells


For cultivation of ES cells in the pluripotent state it is important that the cells grow in
distinct colonies with a clear contour and a limited size.

5. Replace Medium 1 with 4 ml Medium 2 on fresh feeder layer cultures 1 to 2 hr


before subcultivation.
6. At time of subcultivation, remove Medium 2 from the ES cells and wash the cells
once with 4 ml PBS.
7. Add 2 ml trypsin/EDTA solution to the ES cells and incubate for 30 to 60 sec at
room temperature. Monitor the detachment of the cells under the microscope, then
suspend the cells in trypsin/EDTA solution by pipetting up and down several times.
8. Immediately transfer the cells to a 15-ml conical centrifuge tube with 10 ml Medium
2, then centrifuge 5 min at 180 × g, room temperature. Remove supernatant.
9. Resuspend the cell pellet thoroughly in 1.5 ml Medium 2 using a 2-ml glass pipet.
10. Check by microscopy that the suspension is a single-cell suspension, which is
essential for efficient cultivation.
11. Transfer the single-cell suspension, splitting it 1:3 into 60 mm-tissue culture dishes
with MMC-inactivated feeder layers.
Freeze undifferentiated ES cells
12. Trypsinize the cells and resuspend in 10 ml Medium 2.
13. Centrifuge for 5 min at 180 × g, room temperature. Resuspend the cells in 1.5 ml
Medium 3.
14. Transfer the single-cell suspension to a 2-ml freezing vial.
15. Freeze the cells slowly at −80◦ C overnight.
To avoid freezing the cells too rapidly, a simple styrofoam box or a freezing container
(Cryo 1◦ C freezing container; Nalgene) can be used.

16. After 7 to 10 days, transfer the vials into liquid nitrogen.

REAGENTS AND SOLUTIONS


Use deionized or distilled water in all recipes and protocols. For common stock solutions,
see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Alcian blue working solution, 0.05%, pH 1.5


Dissolve the following in 500 ml of 3% (v/v) acetic acid:
0.25 g Alcian blue (Sigma; cat. no. A-3157)
4.5 g sodium chloride
6.4 g magnesium chloride
Stir 2 to 3 hr
Clear by filtration through filter paper
Store up to 6 months at room temperature
ES Cell–Derived
Cartilage
Formation

23.5.22
Supplement 34 Current Protocols in Cell Biology
Antibodies for immunostaining, primary and secondary
Primary antibodies: the following monoclonal antibodies (MAbs) can be ob-
tained from the Developmental Studies Hybridoma Bank, Iowa City, U.S.A.
(http://www.uiowa.edu/∼dshbwww/). The designation of the MAb, the dilution
(in PBS, see recipe) and a reference are given in brackets:

Collagen II [II-II6B3; 1:20; Linsenmayer and Hendrix (1980)]


Osteopontin [MPIIIB101 ; 1:10; Dorheim et al. (1993)]
Collagen X [X-AC9; 1:20; Schmid and Linsenmayer (1985)]
Bone sialoprotein I and II [WVID1(9C5); 1:10; Dorheim et al. (1993)]
Immunostaining for cartilage oligomeric matrix protein (COMP) and collagen I
(CoI) are performed using polyclonal antisera. Again, designation of Ab, dilution
in PBS and a reference are given in brackets:

[COMP; 1:20; Hedbom et al., (1992); a kind gift of M. Paulsson, Köln, Germany]
[CoI; 1:100; Chemicon cat. no. AB765P)]
In addition, the MAbs for cytokeratin (cytokeratin 1, 4, 5, 6, 8, 10, 13, 18, 19;
1:100; Sigma; cat. no. C-2562) and sarcomeric actinin (EA-53; 1:200; Sigma; cat.
no. A-7811) are used to characterize transdifferentiation of isolated chondrocytes.

Secondary antibodies (dilution in PBS; see recipe): FITC (1:200)– or Cy3 (1:400)–
labeled anti-mouse IgG (Dianova; http://www.dianova.com; cat. no. 111-015-144,
115-165-062)

2-Mercaptoethanol, 5 mM
Add 7 µl of 2-mercaptoethanol (2-ME; Serva) to 10 ml PBS (see recipe) to prepare
the 5 mM 2-ME-stock solutio. Sterilize by filtration through a 0.2-µm filter.

2-ME is used in media at a concentration of 50 µM (1 ml 2-ME-stock solution per


100 ml medium).

Denhardt’s solution, 50×


5 g Ficoll 400
5 g polyvinylpyrrolidone
5 g bovine serum albumin (BSA, Fraction V)
500 ml distilled H2 O
Store up to 1 year at −20◦ C
Fetal bovine serum (FBS)
Heat inactivation: Thaw FBS (Sigma) at room temperature and incubate at 54◦ C
for 30 min for heat inactivation.

To select an appropriate FBS batch for supplementation of media (see individual


recipes): ES cells (line BLC6 and D3) are differentiated via EBs using Medium 4
(see recipe; see Basic Protocol 1) supplemented with different FBS batches and
analyzed for their chondrogenic differentiation efficiency by Alcian blue staining
(see Support Protocol 2).

Gelatin-coated culture ware


Prepare a 1% (w/v) stock solution of gelatin (Fluka, cat. no. 48720) in PBS (see
recipe) and autoclave. At a time point 1 day before cell or EB plating dilute the stock
solution 1:10 with PBS for a final concentration of 0.1%, fill the dishes, multiwell Stem Cells

23.5.23
Current Protocols in Cell Biology Supplement 34
plates, or chamber slides with the 0.1% gelatin solution, and incubate overnight at
4◦ C. Aspirate the gelatin solution immediately before use.

Low-FBS medium
Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with:
0.2% (v/v) heat-inactivated FBS (see recipe)
100 U penicillin (add from 10,000 U/ml penicillin/10,000 µg/ml streptomycin
stock; Invitrogen)
100 µg/ml streptomycin (add from 10,000 U/ml penicillin/10,000 µg/ml strepto-
mycin stock; Invitrogen)
50 µM 2-mercaptoethanol (add from 5 mM 2-ME stock; see recipe)
20 µM L-glutamine (add from 2 mM stock; Invitrogen)
1× nonessential amino acids (add from 100× stock; Invitrogen)
Store up to 1 week at 4◦ C
Medium 1
Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with:
15% (v/v) heat-inactivated FBS (see recipe)
100 U penicillin (add from 10,000 U/ml penicillin/10,000 µg/ml streptomycin
stock; Invitrogen)
100 µg/ml streptomycin (add from 10,000 U/ml penicillin/10,000 µg/ml strepto-
mycin stock; Invitrogen)
50 µM 2-mercaptoethanol (add from 5 mM 2-ME stock; see recipe)
20 µM L-glutamine (add from 2 mM stock; Invitrogen)
1× nonessential amino acids (add from 100× stock; Invitrogen)
Store up to 1 week at 4◦ C
Medium 2
Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with:
15% (v/v) heat-inactivated FBS (see recipe)
100 U penicillin (add from 10,000 U/ml penicillin/10,000 µg/ml streptomycin
stock; Invitrogen)
100 µg/ml streptomycin (add from 10,000 U/ml penicillin/10,000 µg/ml strepto-
mycin stock; Invitrogen)
50 µM 2-mercaptoethanol (add from 5 mM 2-ME stock; see recipe)
20 µM L-glutamine (add from 2 mM stock; Invitrogen)
1× nonessential amino acids (add from 100× stock; Invitrogen)
5 ng/ml leukemia inhibitory factor (LIF; Chemicon, cat. no. LIF2005)
Store up to 1 week at 4◦ C
Medium 3
Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with:
20% (v/v) heat-inactivated FBS (see recipe)
100 U penicillin (add from 10,000 U/ml penicillin/10,000 µg/ml streptomycin
stock; Invitrogen)
100 µg/ml streptomycin (add from 10,000 U/ml penicillin/10,000 µg/ml strepto-
mycin stock; Invitrogen)
50 µM 2-mercaptoethanol (add from 5 mM 2-ME stock; see recipe)
20 µM L-glutamine (add from 2 mM stock; Invitrogen)
1× nonessential amino acids (add from 100× stock; Invitrogen)
8% (v/v) dimethylsulfoxide
ES Cell–Derived Store up to 2 days at 4◦ C
Cartilage
Formation

23.5.24
Supplement 34 Current Protocols in Cell Biology
Medium 4
Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with:
20% (v/v) heat-inactivated FBS (see recipe)
100 U penicillin (add from 10,000 U/ml penicillin/10,000 µg/ml streptomycin
stock; Invitrogen)
100 µg/ml streptomycin (add from 10,000 U/ml penicillin/10,000 µg/ml strepto-
mycin stock; Invitrogen)
50 µM 2-mercaptoethanol (add from 5 mM 2-ME stock; see recipe)
20 µM L-glutamine (add from 2 mM stock; Invitrogen)
1× nonessential amino acids (add from 100× stock; Invitrogen)
Store up to 1 week at 4◦ C
Mitomycin C (MMC) working solution, 10 µg/ml
Dissolve 2 mg mitomycin C (MMC; Serva) in 10 ml PBS (see recipe) to prepare
the MMC stock solution. Sterilize by filtration through a 0.2-µm filter and store up
to 6 months at −20◦ C. Dilute 300 µl of MMC stock solution in 6 ml of Medium 1
(see recipe) to obtain a 1 µg/ml MMC working solution.

Phosphate buffered saline (PBS)


10 g/liter NaCl
0.25 g/liter KCl
1.44 g/liter Na2 HPO4 ·H2 O
0.25 g/liter KH2 PO4
Adjust to pH 7.2 using 0.1 N NaOH
Sterilize by autoclaving
Store up to 6 months at room temperature
Prehybridization buffer
5× SSC (see recipe for 20×)
5× Denhardt’s solution (see recipe)
50% (v/v) formamide
250 µg/ml yeast tRNA (Sigma; cat. no. R-8759)
250 µg/ml denatured salmon sperm DNA (Life Technologies; omit as indicated in
protocol)
4 mM EDTA
Store up to 3 months at −20◦ C
SSC, 20×
175.3 g NaCl
88.2 g sodium citrate dihydrate
Adjust pH to 7.0
Add H2 O to 1000 ml
Store up to 6 months at room temperature
Sudan III staining solution
Prepare 0.2% to 0.3% (w/v) Sudan III (Sigma, cat. no. S-4136) in 70% ethanol.
Heat the solution for 15 min at 60◦ C and filter through filter paper. Store up to
3 months at room temperature.
Trypsin/EDTA solution
Combine the following in a 1:1 ratio:
0.2 % (w/v) trypsin in PBS (see recipe for PBS)
0.02 % (w/v) disodium EDTA in PBS (see recipe for PBS)
Stem Cells
Store the trypsin and EDTA stock solutions up to 1 year at −20◦ C
23.5.25
Current Protocols in Cell Biology Supplement 34
COMMENTARY
Background Information trast to elastic cartilage, hyaline cartilage lacks
elastin.
Main stages of chondrogenesis are The in vitro ES cell model system has
recapitulated during ES cell differentiation been established to study cartilage cell dif-
in vitro ferentiation. The authors have demonstrated
Skeleton formation during embryogenesis that pluripotent mouse ES cells cultivated as
is an orchestrated multistep process that is cellular aggregates, so-called embryoid bodies
still incompletely understood (for review see (EBs), differentiate spontaneously into chon-
Sandell and Adler, 1999; Cancedda et al., drogenic cell types in vitro (Kramer et al.,
2000; Provot and Schipani, 2005). Bones of 2000; Hegert et al., 2002). Cellular events dur-
the vertebral column, pelvis, and upper and ing chondrogenesis can be recapitulated from
lower limbs are formed on an initial carti- the undifferentiated stem cell to mesenchymal
laginous template during vertebrate skeletal and chondrogenic progenitor cells, through
development. This process of endochondral mature and hypertrophic chondrocytes result-
ossification is represented by a complex cas- ing in osteogenic cells. In line with develop-
cade of cellular events including proliferation, mental processes in vivo, the mesenchymal
hypertrophy, and apoptosis of chondrogenic cells form condensations in the EBs expressing
cells. Initially, mesenchymal cells condense mesenchymal marker molecules such as the
and form mesenchymal aggregations at the transcription factors scleraxis (Kramer et al.,
locations where later skeletal elements occur. 2000) and Sox5 and Sox6 (Kramer et al.,
These mesenchymal cells are chondrogenic 2005b), as well as the cell adhesion molecule
progenitor cells differentiating into early N-cadherin (H.G. Hargus, unpub. observ.).
chondroblasts, producing the extracellular Furthermore, these cells bind peanut agglu-
matrix of the cartilage anlagen. Finally, tinin. Later on, these cellular condensations ac-
the cartilage template is replaced by bone. quire a compacted phenotype, and collagen II
However, in a few skeletal elements such as expression can be detected at the stage of car-
the lateral parts of the clavicle and parts of tilage nodule formation (Fig. 23.5.3). Alcian
the skull, the mesenchymal cells bypass the blue staining (see Fig. 23.5.2A) demonstrates
chondrogenic stage and differentiate directly that cartilage-specific proteoglycans can be
into osteoblast cells via intramembranous found in the ES cell–derived cartilage nodules.
ossification. Moreover, different types of Indeed, large amounts of proteoglycans as well
chondrocytes form the hyaline joint (Mitrovic, as collagen fibrils are detectable by electron
1977), the tracheal and nasal cartilage (Pavlov microscopy within the nodules (Kramer et al.,
et al., 2003), and elastic cartilage of the 2005b). Immunostaining against elastin re-
external ear (Moskalewski, 1976). veals that this marker of elastic cartilage is not
The specific patterns of marker-molecule detectable within the nodules. However, some
expression can be used to define different single collagen II positive cells within the EB-
stages of chondrogenesis and cellular subtypes outgrowths outside the nodules express elastin.
of cartilage. Sox5 and Sox6 expression re- Moreover, groups of single cells express col-
veal the onset of differentiation into the chon- lagen II fibrils, as revealed by immunostain-
drogenic lineage after the formation of con- ing (see Fig. 23.5.3). During later cultivation
densations by mesenchymal cells (Lefebvre stages, collagen II expression in the ES cell–
et al., 1998). These chondrogenic precursors derived cartilage nodules is down-regulated,
differentiate into chondroblasts expressing and increasing collagen X expression indicates
cartilage marker molecules such as the pro- that cells are becoming hypertrophic. At this
teoglycan aggrecan and the main protein of stage, collagen II fibrils outside the nodules
mature cartilage, collagen II. Collagen X ex- are still detectable. Finally, the nodules are no
pression indicates that the chondrocytes have longer stainable with Alcian blue, and expres-
become hypertrophic, and, finally, these cells sion of bone marker molecules such as os-
undergo apoptosis (Zenmyo et al., 1996) or teopontin, bone sialoprotein, and osteocalcin
differentiate into osteoblasts (Bianco et al., can be detected. Moreover, cells within these
1998), depending on their microenvironment late nodules produce alkaline phosphatase, and
(Riminucci et al., 1998). To distinguish be- the nodules can be stained by Von Kossa sil-
tween elastic and hyaline cartilage, the ex- ver stain and Alizarin red due to osteomin-
ES Cell–Derived pression analysis of elastic fibrils, e.g., elastin
Cartilage eralization. Furthermore, direct differentiation
Formation
(Moskalewski, 1976), can be used. In con- of precursors into osteoblasts has rarely been

23.5.26
Supplement 34 Current Protocols in Cell Biology
detected in EB outgrowths independent of car- spect to transdifferentiation. Differentiation of
tilage formation (Hegert et al., 2002). other cell types, in addition to dedifferenti-
Single-cell analysis of ES cell–derived ating and redifferentiating chondrocytes, was
chondrocytes demonstrates that these cells ex- observed after prolonged cultivation of the iso-
hibit a plasticity of differentiation (Hegert lated cells. Adipocytes were detected by Sudan
et al., 2002). Chondrogenic cells expressing III staining; muscle cell and epithelial cell dif-
collagen II are isolated from cartilage nodules ferentiation was revealed by immunostaining
within the EBs. These nodules show a dis- for sarcomeric α-actinin and pan-cytokeratin,
tinct phenotype and can easily be detected by respectively. Analysis revealed that the addi-
light microscopy. After collagenase treatment, tional cell types formed colonies. The num-
the cells are plated in low-density culture and bers of these colonies and of additional cells
initially show dedifferentiation during further were relatively low compared to the number
single-cell cultivation, as indicated by a de- of chondrogenic cells. The possibility that the
creasing expression of collagen II and X and observed additional cell types originated from
increasing expression of collagen I. Moreover, contaminating precursor cells was excluded by
isolated cells cultivated in single-cell culture repeated clonal analysis (Hegert et al., 2002).
initially showed a fibroblastoid phenotype,
but the cells later redifferentiated into chon- Modulation of ES cell–derived cartilage
drogenic cells expressing collagen II and X formation
again, but not collagen I. Interestingly the red- In general, different exogenous factors in-
ifferentiated mature chondrocytes aggregate fluence the differentiation of ES cells into the
again into cartilage nodules. Because TGF-β3 chondrogenic lineage (Fig. 23.5.4).
promotes chondrogenic differentiation in cul- In general, cell density plays an important
tures of mesenchymal stem cells (Mackay role in determining the degree of cell-to-cell
et al., 1998), an investigation was performed contact mediating intercellular signals for cel-
with application of TGF-β3 (10 ng/ml) or (50 lular differentiation. In fact, the number of ES
ng/ml) to the culture medium of chondro- cells used for EB formation via hanging-drop
genic cells isolated from EBs (Hegert et al., cultivation was found to be decisive for the for-
2002). Analysis of the data showed that the mation of cartilage nodules. EBs derived from
process of redifferentiation was completely in- the ES cell line D3 were prepared from 200,
hibited by TGF-β3 , independently of the con- 500, and 800 cells, and the highest number
centration used. Moreover, mature chondro- of nodules was detected in EBs derived from
cytes isolated from ES cell–derived cartilage 800 cells. Moreover, pellet culture and high-
nodules exhibit a distinct plasticity with re- cell-density micromass culture, which allow

Figure 23.5.4 Different factors and signals influence chondrogenic differentiation of embryonic
Stem Cells
stem cells.
23.5.27
Current Protocols in Cell Biology Supplement 34
cell-cell interactions, are favorable for chon- conditions do not significantly alter the differ-
drogenic differentiation of ES cells, but algi- entiation efficiency of ES cells (Gissel et al.,
nate encapsulation of EBs does not increase 2005). Actually, EB cultivation in low FBS
cartilage formation in comparison to plating induces ES cell–derived cartilage nodule for-
of EBs (Tanaka et al., 2004). Even the use of mation (J. Kramer and J. Rohwedel, unpub.
different ES cell lines for generation of EBs observ.; Fig. 23.5.2B). Another important fac-
revealed differences regarding the efficiency tor for modulation of a specific differentiation
of chondrogenic differentiation (Kramer et al., process is the presence of other cell types in the
2005a). For example, in EBs derived from ES cellular environment. For example, it has been
cell line BLC6, the number of cartilage nod- demonstrated that pluripotent murine ES cells
ules was ∼5-fold higher ∼3 weeks after plat- can be directed to differentiate into chondro-
ing, compared to those derived from ES cell cytes by coculture with progenitor cells from
line D3. Other basic parameters playing an the limb buds of the developing embryo (Sui
important role on the differentiation potential et al., 2003).
of ES cells are the basic cultivation medium, Growth factors and signaling molecules are
the batch of serum, the time of EB cultiva- useful to influence the efficiency of differen-
tion in suspension, and the day of EB plating tiation (Fig. 23.5.5). Different cytokines and
(Kramer et al., 2003). For example, nodule growth factors have been described to promote
formation was enhanced in outgrowths of EBs stem cell–derived chondrogenesis (for review
cultivated in DMEM compared to EBs culti- see Heng et al., 2004). ES cell–derived chon-
vated in Iscove’s modified Dulbecco’s medium drogenesis is modulated by growth factors of
(J. Kramer, unpub. observ.). Previous studies the TGF-β-family (Kramer et al., 2000). For
showed that optimal differentiation in the mes- example, a slightly reduced ES cell–derived
enchymal direction could be obtained by cul- chondrogenic differentiation was detected af-
tivation in suspension for 3 days (days 2 to 5) ter treatment of EBs with 2 ng/ml TGF-β1
and plating at the fifth day of differentiation for the whole time of differentiation (from
(Rohwedel et al., 1994, 1998a,b). To estab- day 1 up to day 5 plus 35 days). Applica-
lish a defined culture milieu, it is favorable to tion of FGF-2 also resulted in a minor de-
avoid the application of FBS. However, previ- crease of cartilage nodule formation in EBs
ous studies showed a reduced ES cell–derived (Kramer et al., 2003). However, if BMP-2 (at
mesodermal differentiation in the absence of a concentration of 2 ng/ml) or BMP-4 (at a
FBS. It has been demonstrated that low FBS concentration of 10 ng/ml) was applied to the

ES Cell–Derived
Cartilage Figure 23.5.5 Overview on some growth factors and signaling molecules influencing mesenchy-
Formation mal ES cell differentiation in vitro.

23.5.28
Supplement 34 Current Protocols in Cell Biology
EBs during the entire cultivation time (from depending on supplementary cofactors (zur
day 1 up to day 5 plus 35 days) the develop- Nieden et al., 2005). In contrast to findings ob-
ment of chondrogenic cells organized in nod- tained by the authors of this unit, in that study,
ules increased ∼2.5 fold (Kramer et al., 2000). TGF-β1 induced chondrogenic differentiation
No obvious differences in the size or growth in ES cell line D3-derived EBs, and TGF-β1
rate of EBs cultivated in the presence or ab- and BMP-2 worked synergistically. Prolonged
sence of growth factors were observed. Fur- treatment of EB cultures with BMP-2 induced
thermore, the modulation of ES cell–derived hypertrophy of ES cell-derived chondrocytes
cartilage formation by BMP-2 was found to and resulted in alteration of the marker
depend on a specific stage of EB differentia- molecule expression towards a profile typical
tion. The effect of BMP-2 to enhance chon- for osteoblasts, as well as an induced forma-
drogenic differentiation was limited to a time tion of bone nodules in EBs (Phillips et al.,
window corresponding to the cultivation step 2001; zur Nieden et al., 2005). Compactin,
of the EBs in suspension (days 2 to 5). In line a member of the statin family of HMG-CoA
with these findings, previous studies revealed reductase inhibitors, promoted an increase of
the sensitivity of this period for the influence BMP-2 expression that resulted in an induction
of signaling molecules, such as retinoic acid of osteogenic differentiation in EB outgrowths
(RA), on other mesodermal cell types, e.g., (Phillips et al., 2001). Application of miner-
cardiogenic, skeletal muscle, and adipogenic alization factors such as β-glycerophosphate
cells (Wobus et al., 1994; Dani et al., 1997). and vitamin D3 to the culture medium has
In addition, it is known that this early stage of been found to enhance osteogenic differenti-
ES cell differentiation is a decisive period of ation (zur Nieden et al., 2003, 2005). It has
early mesodermal development (Yamada et al., also been shown that differentiation of murine
1994; Rohwedel et al., 1998a). RA treatment ES cells towards the osteoblast lineage can be
during EB cultivation in suspension seems to increased by supplementing media with ascor-
play a pivotal role in induction of the mesoder- bic acid, β-glycerophosphate, and/or dexam-
mal pathway of ES cell differentiation (Dani ethasone/retinoic acid, or by coculture with
et al., 1997; Kawaguchi et al., 2005). For fetal murine osteoblasts (Buttery et al., 2001).
example, Sox9 expression was found to be up- However, dexamethasone alone, which is also
regulated in RA-treated EBs (Kawaguchi et al., known as an inducer of chondrogenic dif-
2005). Sox9 is known to play an important ferentiation (Zimmermann and Cristea, 1993;
role during development of pre-cartilage mes- Poliard et al., 1995), did not stimulate ES cell–
enchymal condensations. In fact, RA treatment derived chondrogenesis (Tanaka et al., 2004;
of EBs followed by application of TGF-β3 is zur Nieden et al., 2005).
sufficient to induce cartilage nodule formation Extracellular matrix (ECM) molecules, as
in EB outgrowths (Kawaguchi et al., 2005). well as soluble growth factors associated or
Because RA treatment resulted in both an up- connected to the matrix, modulate prolifer-
regulation of the mesenchymal cell markers ation and differentiation of stem cells. The
Runx2 and Ptprv and the appearance of neu- molecular composition of the ECM mediates
roectodermal neural crest cells in EBs, ei- cellular differentiation processes via cell ma-
ther a mesodermal cell type or an ectomes- trix receptors, called integrins (Fässler et al.,
enchymal cell type may be the source of 1996; Rohwedel et al., 1998a; Czyz and
the mesenchymal cartilage progenitor cells Wobus, 2001). However, the role of the matrix
(Kawaguchi et al., 2005). Another two-step material composition and structure in promot-
model for induction of cartilage formation in ing ES cell growth and differentiation is only
EBs was demonstrated by analysis of differ- poorly understood (for example, Battista et al.,
entiation of ES cell–derived mesodermal pro- 2005). Three-dimensional cultivation proce-
genitor cells into the chondrogenic direction dures such as EB encapsulation in polyethy-
(Nakayama et al., 2003). This study sug- lene glycol–based hydrogels (Hwang et al.,
gested that chondrogenesis of ES cell-derived 2005) or alginate (Tanaka et al., 2004), as well
mesodermal cells requires the influence of as collagen matrices (Chen et al., 2003), influ-
TGF-β3 followed by application of BMP-4. ence ES cell differentiation.
Moreover, a synergy of TGF-β with PDGF- Finally, biophysical parameters such as hy-
BB regarding the induction of chondrogen- drostatic pressure, shear, oxygen tension, tem-
esis was observed in a serum-free medium perature, and electromagnetic forces have been
(Nakayama et al., 2003). In addition to induc- found to influence chondrogenic differentia-
tion of cartilage formation, BMP-2 can drive tion (for review Malda et al., 2003; Smith
Stem Cells
ES cells to the osteoblast or adipogenic fate et al., 2004; Fini et al., 2005). For example,
23.5.29
Current Protocols in Cell Biology Supplement 34
enhanced ES cell–derived bone nodule forma- Time Considerations
tion could recently be demonstrated after treat- Table 23.5.3 provides a time schedule for
ment of ES cell cultures with soluble ions re- an ES cell differentiation experiment.
leased from bioactive glasses undergoing dis-
solution in vitro (Bielby et al., 2005). Acknowledgements
The skilful technical assistance of A.
Critical Parameters and Eirich, M. Dose, and M. Hell is gratefully
Troubleshooting acknowledged. The work was supported by
1. Heat inactivation of FBS is performed at the Deutsche Forschungsgemeinschaft DFG
54◦ C for 30 min after thawing the FBS at room Ro2108, the University of Lübeck, and In-
temperature. termed Service GmbH & CoKG, Geesthacht,
2. It is important to select a specific FBS Germany.
batch for the chondrogenic differentiation ex-
periments. Perform a differentiation experi-
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Stem Cells

23.5.33
Current Protocols in Cell Biology Supplement 34
Hematoendothelial Differentiation UNIT 23.6
of Human Embryonic Stem Cells
Maxim A. Vodyanik1 and Igor I. Slukvin1,2
1
University of Wisconsin, Madison, Wisconsin
2
WiCell Research Institute, Madison, Wisconsin

ABSTRACT
Human embryonic stem cells (hESCs) represent a unique population of cells capable of
self-renewal and differentiation into all types of somatic cells, including hematopoietic
and endothelial cells. Since the pattern of hematopoietic and endothelial development
observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs
can be used as a model for studies of specification and diversification of hematoendothe-
lial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and
endothelial cells for in vitro studies. This unit describes a method for efficient differen-
tiation of hESCs into hematopoietic progenitors and endothelial cells through coculture
with mouse OP9 bone marrow stromal cells, as well as an approach for their analysis
and isolation. Support protocols are provided for culture of mouse embryonic fibrob-
lasts, evaluation of hematopoietic and endothelial differentiation by flow cytometry and
colony-forming assay, removal of OP9 cells, and propagation of hESC-derived endothe-
lial cells. Curr. Protoc. Cell Biol. 36:23.6.1-23.6.28.  C 2007 by John Wiley & Sons,

Inc.
Keywords: human embryonic stem cells r hematopoietic development r in vitro
differentiation r hematopoietic progenitors r endothelial cells

INTRODUCTION
Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating
into all types of cells found in the body, including hematopoietic cells. Since the pattern
of hematopoietic development observed in the embryo can be reproduced using ESCs
differentiated in culture, hESCs provide a unique opportunity for elucidating mechanisms
of early hematopoietic commitment, lineage specification, and maturation in humans
(Keller et al., 1993; Nakano et al., 1996; Robertson et al., 1999; Lensch and Daley,
2004). Moreover, genetically modified hESCs provide an unprecedented opportunity for
studying the role of human genes in hematopoietic development.

There are two major approaches for inducing hematopoietic differentiation of ESCs. One
approach allows ESCs to form embryoid bodies after the withdrawal of factors that main-
tain ESCs in undifferentiated state. Various lineages of cells, including hematopoietic
cells, develop inside embryoid bodies. Another approach is coculture of ESCs with bone
marrow stromal cell lines, which provides an inductive environment for directed hESC
differentiation to hematopoietic lineage.

This unit describes procedures for differentiating hESCs into hematopoietic cells by
coculturing with mouse bone marrow stromal cell line OP9 (Basic Protocol 1), as well as
methods for analyzing the efficiency of hematopoietic (and endothelial) differentiation by
flow cytometry (Support Protocol 1) and a methylcellulose-based colony-forming assay
(Support Protocol 2). Basic Protocol 2 describes isolation of hESC-derived hematopoietic
progenitors. Since hematopoietic and endothelial lineages develop simultaneously in OP9
coculture, these protocols can also be used for generation and isolation of hESC-derived
Stem Cells

Current Protocols in Cell Biology 23.6.1-23.6.28, September 2007 23.6.1


Published online September 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471143030.cb2306s36 Supplement 36
Copyright C 2007 John Wiley & Sons, Inc.
endothelial cells. Additional supporting protocols describe the removal of OP9 cells from
hESC/OP9 coculture (Support Protocol 3), maintenance of human embryonic stem cells
on mouse embryonic fibroblasts (MEFs; Support Protocol 4), methods for culture of
mouse embryonic fibroblasts (Support Protocol 5), techniques for OP9 culture (Support
Protocol 6), and propagation of hESC-derived endothelial cells (Support Protocol 7).

NOTE: All procedures described in this unit—tissue culture, reagent preparation, and
cell sorting—require sterilization facilities and a standard cell culture workplace, i.e., a
laminar flow hood equipped with pipet-aids and an adjustable pipet set, sterile disposable
items (tips, glass Pasteur pipets, serological pipets, polypropylene centrifuge tubes),
and a vacuum-driven aspiration unit as well as a CO2 incubator, 37◦ C water bath,
temperature controlled swinging-bucket centrifuge, inverted phase-contrast microscope,
and cell counting device. All experiments should be performed under sterile conditions
in Class II biological safety cabinets.

NOTE: All incubations should be performed in a humidified 37◦ C, 5% CO2 incubator


unless otherwise indicated.

NOTE: All solutions and equipment coming into contact with cells must be sterile, and
proper aseptic technique should be used accordingly.

BASIC HEMATOENDOTHELIAL DIFFERENTIATION OF hESCs IN OP9


PROTOCOL 1 COCULTURE
This protocol describes the optimal conditions for induction of hematoendothelial dif-
ferentiation of hESCs through coculture with a mouse OP9 bone marrow stromal cell
line. Hematopoietic differentiation of hESCs in coculture with OP9 cells proceeds very
rapidly, and the first hematopoietic progenitors can already be detected after 4 to 5 days
of coculture. Using this method, efficient hematopoietic differentiation can be achieved
without the addition of cytokines or growth factors. The number of endothelial cells
and hematopoietic progenitors peak on day 7 to 8 of hESC/OP9 coculture and decrease
thereafter due to excessive cell density and overall decline in cell viability.

Materials
6-well plates with undifferentiated hESCs on day 5 to 6 of culture (Support
Protocol 4)
1× phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO)
Differentiation medium (see recipe), warmed to 37◦ C
10-cm dishes with OP9 cells on day 8 to 12 after plating (Support Protocol 6)
Collagenase IV solution (see recipe)
0.05% (w/v) trypsin/0.5 mM EDTA solution (1×; GIBCO)
Cell washing/MACS buffer (see recipe)
15- and 50-ml polypropylene centrifuge tubes
Temperature controlled centrifuge
70-µm nylon filters (cell strainers; Falcon)
Additional reagents and equipment for counting cells (UNIT 1.1)
Count hESCs
1. From one well of a 6-well hESC plate, aspirate growth medium and wash the well
with 2 ml PBS. Add 1 ml trypsin/EDTA solution and incubate 5 min at 37◦ C.
As for regular hESC passage, hESCs used for OP9 coculture must be in aggregates and
Hematoendothelial therefore, cannot be counted directly. To determine the absolute number of hESCs within
Differentiation of aggregates, a separate well of 6-well hESC plate should be treated with trypsin to obtain
hESCs a single-cell suspension for counting.
23.6.2
Supplement 36 Current Protocols in Cell Biology
2. Dissociate hESC colonies by pipetting. Transfer the single-cell suspension to a 15-ml
tube containing 2 ml differentiation medium. Centrifuge 5 min at 300 × g, room
temperature, and resuspend in 1 ml differentiation medium.
3. Count the number of cells in 1 ml (see UNIT 1.1).
The total number of cells in 1 ml will be equal to the absolute number of hESCs in
aggregates collected from single well.

Plate hESCs on OP9 dishes


4. Remove OP9 dishes prepared for differentiation (i.e., overgrown) from the CO2 in-
cubator. Aspirate growth medium completely and add 10 ml differentiation medium.
Return the dishes to the CO2 incubator.
It is important to remove OP9 medium completely before hESC plating. Adding hESCs
directly to an OP9 monolayer with conditioned medium suppresses hematopoietic differ-
entiation.

5. Prepare undifferentiated hESCs as a suspension of small aggregates (described in


Support Protocol 4), but perform the washing step using differentiation medium and
resuspend the cell pellet in differentiation medium to a concentration of 1.5 × 106
cells/ml.
The total resuspension volume can be calculated by formula (n × x)/1.5, where n is the
number of hESC wells collected for differentiation, and x is the absolute number of hESCs
per single well determined by a counting of trypsin-dissociated cells (steps 1 to 3).
Small clumps (not single hESCs) should be plated for differentiation. Single hESCs in
suspension will not survive in the OP9 coculture.
hESCs should be plated on OP9 cells at optimal density to ensure optimal growth and
differentiation during 8 days of coculture. In the authors’ experience, optimal plating
density is 1–1.5 × 106 hESCs (lines H1 and H9) per one 10-cm dish. Since each well
of a 6-well hESC plate contains 2–3 × 106 cells, hESCs collected from one well are
routinely plated on two 10-cm OP9 dishes. However, before such routine plating, control
cell counting should be performed in preliminary experiments.
6. Remove OP9 dishes (prefilled with 10 ml differentiation medium in step 4) from the
CO2 incubator. Add 1 ml hESC suspension to each OP9 dish. Place the dishes on
the shelf in CO2 incubator and move forth-and-back and side-to-side to distribute
the hESC aggregates evenly throughout the dishes. Incubate overnight.
Avoid rotating motions as this will lead to accumulation and attachment of hESC clumps
in the middle of dish and in such instances the experiment can not be continued.

Feed hESC/OP9 cocultures


7. On day 1 after hESC plating, gently agitate the hESC/OP9 coculture dishes, aspirate
the medium, and fill the dishes with 20 ml fresh, prewarmed differentiation medium.
The 20-ml incubation volume and less frequent feeding are important for successful
hematoendothelial differentiation. Conversely, a standard 10-ml incubation volume and
more frequent feeding favor generation of mesenchymal-like cells.

8. On culture days 4 and 6, change half of the medium by removing 10 ml of medium


from hESC/OP9 dishes by pipet and adding 10 ml of fresh, prewarmed differentiation
medium.
Optimized feeding is critical for efficient hematopoietic differentiation. Any changes in
feeding may change differentiation significantly. The authors recommend a full change of
medium on the first day after hESC plating (step 7) because it removes all nonattached
(dead) cells and suppresses initial growth of undifferentiated colonies. As a result, a
more synchronous differentiation can be achieved. In the following days, only half of
the medium should be changed. The feeding regimen is one approach for manipulating Stem Cells
differentiation, and variations can be tried to improve differentiation efficiency.
23.6.3
Current Protocols in Cell Biology Supplement 36
Collect cells
9. Warm the collagenase IV and trypsin/EDTA solutions in a 37◦ C water bath.
10. Remove hESC/OP9 dishes from CO2 incubator. Aspirate the medium and wash the
cell monolayer with 10 ml PBS.
11. Add 5 ml collagenase IV solution/dish and incubate 20 min in the CO2 incubator.
12. Remove the collagenase solution using a pipet and transfer to a 15- or 50-ml collection
tube. Reserve this tube for subsequent collection of trypsin-digested cells.
13. Add 5 ml trypsin/EDTA solution/dish and incubate for an additional 15 to 20 min in
the CO2 incubator.
After 5 days of differentiation, hESC/OP9 cocultures form a collagen-rich matrix that can-
not be efficiently digested by trypsin treatment. Therefore, a pretreatment of hESC/OP9
monolayers with collagenase is used to facilitate subsequent cell dissociation with trypsin.
Successive collagenase-trypsin treatment is especially important to maximize cell recov-
ery from day 7 to 9 hESC/OP9 cocultures.

14. Suspend the cells by pipetting and transfer the cell suspension to the collection tube.
Close the tube, mix the cells gently by inverting tube, and centrifuge 5 min at 400 ×
g, 4◦ C.
15. Wash cells three times with cell washing/MACS buffer. Use at least a 10-ml wash vol-
ume for cells collected from one hESC/OP9 dish, and centrifuge the cell suspension
5 min at 400 × g, 4◦ C.
16. At the last washing step, resuspend the cells in 10 ml cell washing/MACS buffer,
filter through a 70-µm nylon filter, and centrifuge as in step 15.
17. Resuspend the cells in 1 ml cell washing/MACS buffer or medium for each
hESC/OP9 dish collected. Place the tube on ice.
Cells are now ready to analysis (Support Protocols 1 and 2) and separations (Basic
Protocol 2 and Support Protocol 3).

18. Count the cells (see UNIT 1.1).


The cell yield from one 10-cm hESC/OP9 dish should be >107 , typically in range of 1.5–
2 × 107 cells.

SUPPORT ASSESSMENT OF HEMATOENDOTHELIAL DIFFERENTIATION


PROTOCOL 1 BY FLOW CYTOMETRY
The hESC-derived endothelial cells and hematopoietic progenitors can be identified by
the expression of a common hematoendothelial marker, CD31 (PECAM-1). Hematopoi-
etic progenitors can be distinguished from endothelial cells by CD43 (leukosialin) ex-
pression; flow cytometric enumeration of CD31+ CD43− and CD31+ CD43+ cells can
be used to evaluate the efficiency of endothelial and hematopoietic differentiation in
hESC/OP9 cocultures, respectively (Vodyanik et al., 2006). Based on expression of
CD235a (glycophorin A), multipotent hematopoietic progenitors (CD43+ CD235a− ) can
be separated from precommitted CD43+ CD235a+ erythro-megakaryocytic progenitors,
which also express CD41a (Vodyanik et al., 2006). The marker of the most primitive
somatic hematopoietic stem cells/progenitors, CD34, is expressed on hESC-derived en-
dothelial cells and a majority of hematopoietic progenitors. However, in contrast to bone
marrow or cord blood, hESC-derived CD34+ cells are more heterogeneous and include a
significant proportion of mesenchymal cells (Vodyanik et al., 2006). Thus, the presence
Hematoendothelial of CD34+ cells does not necessarily reflect hematoendothelial differentiation in hESCs.
Differentiation of
hESCs Moreover, some erythroid progenitors are lacking CD34 expression, but express CD43

23.6.4
Supplement 36 Current Protocols in Cell Biology
and CD31. The first CD34+ , CD31+ , and CD43+ cells are detectable on day 3 to 4 of
hESC/OP9 coculture. Cells collected on day 7 to 8 of hESC/OP9 coculture are optimal for
assessment of hematoendothelial differentiation, because they contain a maximal number
of endothelial cells and all types of hematopoietic progenitors. Total hESC-derived cells
in OP9 coculture can be identified using TRA-1-85 monoclonal antibodies (mAb), which
detect the OKa blood group antigen expressed by virtually all human cells (Williams
et al., 1988). Absolute numbers of cell populations can be calculated by knowing the
total number of cells harvested from one 10-cm hESC/OP9 dish, the percentage of total
hESC-derived (TRA-1-85+ ) cells, and the percentage of the cell population of interest.

Materials
Cells collected from hESC/OP9 coculture (Basic Protocol 1)
FACS buffer with and without fetal bovine serum (FBS; see recipe)
Mouse anti-human CD31-PE mAb (clone WM59; BD Pharmingen)
Mouse anti-human CD43-FITC mAb (clone 1G10; BD Pharmingen)
Mouse anti-human TRA-1-85-APC mAb (clone TRA-1-85; R&D Systems)
Mouse anti-human CD235a-PE mAb (clone CLB-ery-1; Caltag)
Mouse anti-human CD45-APC mAb (clone HI30; BD Pharmingen)
Mouse anti-human CD29-FITC/PE mAb (clone MEM-101A; Caltag)
Mouse anti-human CD34-APC mAb (clone 581; BD Pharmingen), optional
Mouse IgG1 FITC control mAb (clone MOPC-21; BD Pharmingen)
Mouse IgG1 PE control mAb (clone MOPC-21; BD Pharmingen)
Mouse IgG1 APC control mAb (clone MOPC-21; BD Pharmingen)
7-aminoactinomycin D (7AAD) staining solution (Via-Probe; BD Pharmingen)
5-ml polystyrene test tubes (Falcon)
Temperature-controlled centrifuge
Flow cytometer (FACSCalibur; BD Immunocytometry Systems)
FlowJo flow cytometry analysis software (Tree Star)
Stain cells
1. Suspend cells collected from hESC/OP9 coculture (Basic Protocol 1, step 17) in
FACS buffer with FBS at 5 × 106 cells/ml (5 × 105 cells/100 µl per test).
2. Add monoclonal antibodies (mAbs) on the bottoms of 5-ml polystyrene test tubes
according to the following plan (also see Table 23.6.1):

Tubes S1 to S3 for setting up instrument compensation controls


Tubes E1 to E2 for analysis of total hematopoietic (CD31+ CD43+ ) and
endothelial (CD31+ CD43− ) cells within TRA-1-85+ human cell population
Tubes E3 to E4 for analysis of multipotent (CD43+ CD235a− CD45± ) and
erythro-megakaryocytic (CD43+ CD235a+ CD45− ) progenitors within total
CD43+ hematopoietic population
Tubes E5 to E6 (optional) for analysis of CD34+ subpopulations of endothelial
(CD31+ CD43− ), hematopoietic (CD31+ CD43+ ), and mesenchymal
(CD31− CD43− ) cells.

3. Add 100 µl cell suspension to each tube. Incubate 30 to 40 min at 4◦ C, mixing the
cells by agitation at least once during the incubation.
4. Add 4 ml FACS buffer with FBS to the tubes and centrifuge 5 min at 400 × g, 4◦ C.
5. Discard the supernatant by inverting the tubes and resuspend cells in 400 µl FACS
buffer without FBS.
Samples can be stored up to 4 hr at 4◦ C. Stem Cells

23.6.5
Current Protocols in Cell Biology Supplement 36
Table 23.6.1 Monoclonal Antibody Combinations for Flow Cytometric Analysis

FL1-FITC FL2-PE FL3-7AADb FL4-APC


Tube
(488/519)a (488/578)a (488/647)a (633/660)a

Instrument setup samples


S1 IgG1 control IgG1 control - IgG1 control
S2 CD29c IgG1 control 7AAD IgG1 control
S3 IgG1 control CD29c - TRA-1-85
Experimental samples
E1 IgG1 control IgG1 control 7AAD TRA-1-85
E2 CD43 CD31 7AAD TRA-1-85
E3 CD43 IgG1 control 7AAD IgG1 control
E4 CD43 CD235ac 7AAD CD45
E5 (optional) IgG1 control IgG1 control 7AAD CD34
E6 (optional) CD43 CD31 7AAD CD34
a Fluorochrome excitation/emission wavelengths (nm).
b 7AAD is a DNA staining dye (Via-Probe, BD) for dead cell exclusion. It should be added to mAb-stained cells
immediately before analysis (10 µl/sample).
c These monoclonal antibodies (mAbs) are used at 2 µl/test; all other mAbs are used at 5 µl/test.

6. Before analysis, add 10 µl 7AAD staining solution to each tube.


7AAD staining is used for dead cell exclusion. S1 negative sample and S2 and S3 samples
stained with nonoverlapping fluorochromes (FITC/7AAD and PE/APC) are used for
setting up instrument compensation.

Analyze samples
7. Perform analysis of cell samples on the flow cytometer and save a minimum of
50,000 (E1 to E2) and 100,000 (E3 to E4) acquisition events.
Analysis of all samples begins from selection (gating) of 7AAD-negative viable cells. In
E1 to E2 samples, total TRA-1-85+ hESC-derived cells are gated for analysis of en-
dothelial (CD31+ CD43− ) and total hematopoietic (CD31+ CD43+ ) cells. In E3 to E4
samples, total CD43+ cells are gated for analysis of multipotent (CD235a− CD45− and
CD235a− CD45+ ) and erythro-megakaryocytic (CD235a+ CD45− ) hematopoietic pro-
genitors. See Figure 23.6.1 for examples.

8. Open listmode files in FlowJo analysis software.


9. Start analysis with E1 or E3 isotype control samples. Set a gate on all 7AAD-negative
(viable) cells using FL3-7AAD/FCS dot plot.
10. Open the 7AAD− gate in FL4-TRA-1-85-APC/SSC (E1) or FL1-CD43-FITC/SSC
(E3) dot-plots. Set a gate on all TRA-1-85+ cells (E1) or all CD43+ cells (E3).
11. Open the TRA-1-85+ gate in FL1-IgG1-FITC/FL2-IgG1-PE (E1) or CD43+ gate
in FL2-IgG1-PE/FL4-IgG1-APC (E3) dot-plots. Set thresholds for positive staining
using quadrant statistics.
12. Transfer (drag-and-drop) all gates to the samples stained with specific mAbs: E1
gates to E2 sample or E3 gates to E4 sample.
13. Determine percentage (%) values.
E2 sample: % cell viability (7AAD− ), % hESC-derived cells (TRA-1-85+ ), %
Hematoendothelial
Differentiation of CD31+ CD43− cells (endothelial), and % CD31+ CD43+ (hematopoietic)
hESCs cells (Fig. 23.6.1A).
23.6.6
Supplement 36 Current Protocols in Cell Biology
Figure 23.6.1 Flow cytometric analysis of hematopoietic progenitors and endothelial cells gen-
erated in hESC/OP9 coculture. (A) Representative flow cytometric analysis of hematopoietic
(CD31+ CD43+ ) and endothelial (CD31+ CD43− cells on day 7 of H1/OP9 coculture. Total H1-
derived cells are brightly stained with TRA-1-85-APC mAb (left dot-plot). TRA-1-85+ cells are
selected (gated) for analysis of CD31 and CD43 expression (right dot-plot). (B) Representative
flow cytometric analysis of CD43+ hematopoietic progenitors on day 8 of H1/OP9 coculture. By low
side scatter profile, total CD43-FITC-stained cells can be identified as compact cell population (left
dot-plot). Selected (gated) total CD43+ cells are then analyzed for CD235a and CD45 expression.
In the right dot-plot, three distinct populations of CD43+ hematopoietic progenitors can be defined:
CD235a+ CD45− , CD235a− CD45− and CD235a− CD45+ . Percentage values are shown for
respective gates/quadrants.

E4 sample: % CD43+ multipotent progenitors (CD235a− CD45− and


CD235a− CD45+ ), and % CD43+ erythro-megakaryocytic progenitors
(CD235a+ ) (Fig. 23.6.1B).

CD31− CD43+ hematopoietic cells can be found in hESC/OP9 coculture after 8 days of
differentiation. These cells represent erythroid cells, which down-regulate CD31 expres-
sion along with maturation.

14. Calculate absolute numbers of endothelial (CD31+ CD43− ) and hematopoietic


(CD31+ CD43+ ) cells recovered from hESC/OP9 coculture by the formula:
number of cells/cm2 = (N × V × H × S)/78.56
where
N is the number in millions of cells collected from one 10-cm dish
V is % cell viability
H is % human cells (TRA-1-85+ )
S is % cell subpopulation (CD31+ CD43− or CD31+ CD43+ ) Stem Cells

23.6.7
Current Protocols in Cell Biology Supplement 36
78.56 is surface area (cm2 ) of a standard 10-cm dish (use 9.62 for one well of
6-well plate).

For example, if 20 × 106 collected cells contain 95% 7AAD− viable cells, 90%
TRA-1-85+ human cells and 5% CD31+ CD43+ hematopoietic progenitors, the number
of CD31+ CD43+ cells = (20 × 95 × 90 × 5)/78.56 = 10883 cells per cm2 of hESC/OP9
coculture.

SUPPORT HEMATOPOIETIC PRECURSOR COLONY-FORMING ASSAY


PROTOCOL 2
In addition to phenotypic analysis, hematopoietic progenitors can be identified using
functional assays. A short-term test such as a colony forming assay is widely used
to identify hematopoietic precursors at the intermediate stage between hematopoietic
stem cells and cells with morphologically distinct features of differentiation. This assay
is standardized and easy to perform. When grown in semisolid methylcellulose-based
media in the presence of cytokines, hematopoietic progenitors form colonies with spe-
cific appearances and cell compositions. These colonies are formed by colony-forming
units (CFUs) or colony-forming cells (CFCs) and can be morphologically discriminated
into erythroid (E-CFC); granulocyte, erythroid, macrophage, megakaryocyte (GEMM-
CFC); granulocyte-macrophage (GM-CFC); and macrophage (M-CFC) cells (Eaves and
Lambie, 1995). Complete ready-to-use MethoCult H4435 GF+ methylcellulose medium
with added FBS and cytokines (SCF, G-CSF, GM-CSF, IL3, IL6) can be obtained from
Stem Cell Technologies. This is a high-quality medium optimized for detection of very
early hematopoietic progenitors, and it has minimal lot-to-lot variation. The cells pre-
pared from day 7 to 8 hESC/OP9 cocultures are optimal for the colony-forming assay
because they contain all CFC types. In the authors’ experience, the presence of OP9 cells
in the cell samples does not interfere with the CFC assay. However, if desired, the OP9
cells can be removed from the cell suspension by magnetic sorting (Support Protocol 3).

Materials
MethoCult H4435 GF+ complete medium (Stem Cell Technologies): thaw, divide
into 3-ml single-test aliquots (using a 10-ml syringe and blunt-end needle), and
refreeze; store up to 6 months at −20◦ C
6 × 104 cells (Basic Protocol 1, step 17)/0.3 ml differentiation medium
Differentiation medium (see recipe)
Sterile distilled water
Blunt-end needles (Stem Cell Technologies) and 5- and 10-ml syringes, optional
37◦ C water bath
Disposable 5-ml serological pipet
3.5-cm low-adherence CFU assay dishes (Stem Cell Technologies)
3.5-cm regular plastic tissue culture dishes
10-cm regular plastic tissue culture dishes
Inverted microscope
Gridded scoring dishes (Stem Cell Technologies)
Prepare culture plates
1. Remove tubes with 3 ml MethoCult medium from the freezer and thaw in a 37◦ C
water bath.
If acquired in the bulk format (100 ml), MethoCult medium should be thawed and divided
into single-test aliquots (3 ml per 15-ml polypropylene tubes) using a 10-ml syringe and
blunt-end needles. Details on handling of methylcellulose medium can be found in the
documentation accompanying the product.
Hematoendothelial
Differentiation of
hESCs

23.6.8
Supplement 36 Current Protocols in Cell Biology
2. Add 6 × 104 cells in 0.3 ml (or less) differentiation medium to a tube containing
3 ml MethoCult medium.
The final concentration for plating is 2 × 104 cells/ml. The cell suspension should be
prepared in differentiation medium and added in volume less than or equal to 0.3 ml.

3. Vortex the tube vigorously to thoroughly mix the cells and place in a 37◦ C water
bath for 10 to 15 min, allowing bubbles to dissipate.
4. Slowly collect the suspended cells in a 5-ml pipet and dispense equal volumes
(∼1 ml/dish) to duplicate 3.5-cm CFU assay dishes. Rotate the dishes until semisolid
medium covers entire plastic surface.
5. Place the two CFU assay dishes, along with an uncovered 3.5-cm dish filled with
4 ml sterile distilled water, into a 10-cm dish and cover.
6. Incubate 14 to 16 days in the CO2 incubator.
Score colonies
7. Score different types of colonies (E, GEMM, GM, and M) according to their mor-
phology (see Eaves and Lambie, 1995) using an inverted microscope and gridded
scoring dishes.
8. Calculate the means for the different colony types from duplicate dishes and multiply
by 5 (the dilution factor) to express CFC frequency per 105 plated cells.

REMOVAL OF OP9 CELLS FROM hESC/OP9 COCULTURE SUPPORT


PROTOCOL 3
As a xenogeneic system, hESC/OP9 coculture provides the advantage of easy discrim-
ination between hESC-derived cells and feeder cells. Most monoclonal antibodies di-
rected to human antigens do not cross-react with mouse homologs, and therefore, can
be used for specific detection and isolation of hESC-derived cells. However, in several
functional assays or gene expression studies, OP9 cells may interfere with results and
should be removed. For such instances, this protocol describes removal of OP9 cells
by magnet-activated cell sorting (MACS) using monoclonal antibodies (mAb) against
mouse β1-integrin (CD29), which does not recognize human CD29. Depletion of OP9
cells using antibody-carrying magnetic microbeads results in 99.9% purity of human
cells (TRA-1-85+ ). This method can also be used for purification of human cells from
other human-mouse cell coculture systems because CD29 is expressed in high density
by virtually all adherent cell types. This protocol is optimized for one-column processing
of up to 50 × 106 cells collected from day 7 to 8 hESC/OP9 cocultures. If using more
cells or earlier days of coculture, the sample should be divided and processed through
additional magnetic columns.

Materials
Cell suspension (Basic Protocol 1), hold on ice
Cell washing/MACS buffer (see recipe), degassed under vacuum and ice cold
Hamster anti-mouse CD29-PE mAb (clone HM beta 1-1; Serotec)
Anti-PE magnetic microbeads (Miltenyi Biotec)
Mouse anti-human TRA-1-85-APC mAb (clone TRA-1-85; R&D Systems)
7-aminoactinomycin D (7AAD) staining solution (Via-Probe; BD Pharmingen)
15-ml graduated polypropylene tubes
Temperature controlled centrifuge, 4◦ C
MACS rotation mixer (Miltenyi Biotec)
35-µm preseparation filters (Miltenyi Biotec)
Midi-MACS magnet/stand (Miltenyi Biotec) Stem Cells
LD (depletion) columns (Miltenyi Biotec)
23.6.9
Current Protocols in Cell Biology Supplement 36
Additional reagents and equipment for counting cells (UNIT 1.1) and analyzing cells
by flow cytometry (Support Protocol 1)
NOTE: Cells and cell washing/MACS buffer must be kept on ice during the separation
procedure.

Prepare cells
1. Add the suspension of cells (up to 5 × 107 cells) isolated from hESC/OP9 coculture
(Basic Protocol 1) to a 15-ml graduated polypropylene tube.
2. Centrifuge 5 min at 400 × g, 4◦ C.
3. Aspirate the supernatant. Note the volume of the cell pellet and add an equal volume
of cell washing/MACS buffer.
The resulting total volume is referred to as the labeling volume.

Label mouse cells


4. Resuspend the cells thoroughly by gentle pipetting and add 1/10 vol hamster anti-
mouse CD29-PE mAb.
For example, if the cell pellet is ∼0.2 ml, add 0.2 ml cell washing/MACS buffer (labeling
volume is 0.4 ml), resuspend cells, and add 40 µl (1/10 vol) CD29-PE mAb.

5. Set up tube on the MACS mixer. Place the MACS mixer with the tube in the
refrigerator (4◦ C). Incubate 15 min at the lowest rotation speed.
Add beads for separation
6. Wash cells once with 14 ml ice-cold cell washing/MACS buffer. Resuspend cells in
cell washing/MACS buffer to the original labeling volume and add 1/10 vol anti-PE
microbeads.
7. Incubate cells on MACS mixer 20 min at 4◦ C and lowest rotation speed.
8. Wash cells once with 14 ml ice-cold cell washing/MACS buffer. Resuspend cell
pellet in 1 ml cell washing/MACS buffer.
9. Place a 35-µm preseparation filter on the top of an empty 15-ml tube. Apply the cell
suspension on the filter and allow the cells to pass through filter completely. Wash
the filter with 0.5 ml cell washing/MACS buffer.
It is important to filter cells prior to application onto magnetic columns. Even very small
cell aggregates may block the magnetic columns and adversely affect separation.

10. Place the tube with filtered cells on ice and reserve a 10- to 20-µl aliquot for FACS
analysis of presort cells in step 16.
Separate mouse cells
11. Assemble the MACS-LD separation unit (midi-MACS magnet with LD column and
15-ml collection tube). Rinse the column with 2 ml MACS buffer and discard the
tube with eluate. Place an empty collection tube under column.
There are two types of magnetic columns optimized for positive (LS) and negative (LD)
MACS separation, and they differ in flow-through rate. High flow rate LS columns provide
a high purity of column-retained cell fractions (>95%), whereas low flow rate LD columns
provide maximal recovery (depletion) of magnetically labeled cells (>99%).

12. Apply the cell suspension to the LD column and allow cells to pass through the
column completely. Wash column with 2 ml cell washing/MACS buffer.
Hematoendothelial
Differentiation of 13. Remove collection tube with unlabeled (human) cell fraction. Centrifuge 5 min at
hESCs 300 × g, 4◦ C.
23.6.10
Supplement 36 Current Protocols in Cell Biology
Figure 23.6.2 Major steps in establishing hESC differentiation cultures on OP9 cells and obtaining feeder-
free differentiated human cells. (A) Morphology of OP9 cells on the day after plating. (B) Day 4 confluent
OP9 cultures ready for passage. (C) Day 8 overgrown OP9 cultures prepared for hESC differentiation.
(D) Differentiated “mesodermal” hESC colony on day 4 of H1/OP9 coculture. (E) Representative flow
cytometric analysis of OP9 (mouse CD29-PE+ ) and total hESC-derived (TRA-1-85-APC+ ) cells on day 3
of H1/OP9 coculture before (left dot-plot) and after (right dot-plot) OP9 removal by magnetic sorting using
depletion (LD) columns and anti-PE microbeads. Percentage values are shown in respective quadrants.
Scale bar = 100 µm for all photographs.

14. Discard the supernatant and resuspend the cells in 1 ml cell washing/MACS buffer
or medium. Reserve a 10- to 20-µl aliquot for FACS analysis.
15. Count the cells (see UNIT 1.1) and take an aliquot (∼2 × 104 cells/test) for FACS
analysis.
16. Analyze the cells by flow cytometry, labeling them with TRA-1-85-APC mAb and
using 7AAD staining to exclude dead cells (see Support Protocol 1).
At analysis, mouse CD29-PE positive cells (OP9) should be negligible if detectable
(<0.02%), whereas TRA-1-85+ human cells should comprise >99.5% (Fig. 23.6.2E).

ISOLATION OF hESC-DERIVED HEMATOPOIETIC PROGENITORS BASIC


AND ENDOTHELIAL CELLS PROTOCOL 2
This protocol describes the isolation of hESC-derived hematopoietic progenitors and
endothelial cells. As a first step, cells harvested on day 7 to 8 of hESC/OP9 co-
culture (Basic Protocol 1) are processed to isolate all hematopoietic progenitors by
positive selection of CD43+ cells using magnet-activated cell sorting (MACS) and
depletion (LD) columns designed for efficient recovery of labeled cells. MACS-
enriched CD43+ cells labeled with CD235a (glycophorin A) and CD45 mono-
clonal antibodies (mAbs) are subsequently subjected to a FACS sorting procedure
to isolate three types of hematopoietic progenitors: CD43+ CD235a+ CD45− erythro-
megakaryocytic and multipotent CD43+ CD235a− CD45− and CD43+ CD235a− CD45+
Stem Cells
progenitors. Both CD43+ CD235a− CD45− and CD43+ 235a− CD45+ cells are negative
23.6.11
Current Protocols in Cell Biology Supplement 36
for lineage-specific markers and represent multipotent hematopoietic progenitors; how-
ever, CD43+ CD235a− CD45+ cells are enriched in myeloid progenitors and limited in
lymphoid potential (Vodyanik et al., 2006). Endothelial cells can be subsequently iso-
lated from the CD43− cell fraction by positive MACS selection of CD31+ cells. CD43+
subsets can be used for functional studies as well as a source of hematopoietic progenitors
for directed differentiation towards specific hematopoietic lineages. Endothelial cells can
be expanded in vitro for several passages in serum-free conditions (Support Protocol 7).

Materials
Cell suspension (Basic Protocol 1), hold on ice
Cell washing/MACS buffer (see recipe), degassed under vacuum and ice cold
Mouse anti-human CD43-FITC mAb (clone 1G10; BD Pharmingen)
Anti-FITC magnetic microbeads, (Miltenyi Biotec)
Basic (nonconjugated) microbeads (Miltenyi Biotec), optional
Mouse anti-human CD31-PE (clone WM59; BD Pharmingen)
Anti-PE magnetic microbeads, (Miltenyi Biotec)
FACS buffer without FBS (see recipe)
7-aminoactinomycin D (7AAD) solution (Via-Probe; BD Pharmingen)
Mouse IgG1-PE isotype control mAb (clone MOPC-21; BD Pharmingen)
Mouse IgG1-APC isotype control mAb (clone MOPC-21; BD Pharmingen)
Mouse anti-human CD235a-PE (clone CLB-ery-1; Caltag)
Mouse anti-human CD45-APC (clone HI30; BD Pharmingen)
FBS (GIBCO), heat inactivated
LD (depletion) columns, (Miltenyi Biotec)
15-ml graduated polypropylene tubes
Temperature-controlled centrifuge, 4◦ C
MACS rotation mixer, (Miltenyi Biotec)
35-µm preseparation filters (Miltenyi Biotec)
LS (positive selection) columns (Miltenyi Biotec)
Midi-MACS magnet/stand (Miltenyi Biotec)
Cell sorter (FACS Vantage SE; BD Immunocytometry Systems)
5-ml polypropylene tubes (Falcon)
Additional reagents and equipment for counting cells (UNIT 1.1) and performing
FACS (Support Protocol 1)
NOTE: Cells and cell washing/MACS buffer must be kept on ice during the separation
procedure.

Prepare cells
1. Add the suspension of cells (up to 108 cells) isolated from hESC/OP9 coculture
(Basic Protocol 1) to a 15-ml graduated polypropylene tube.
2. Centrifuge 5 min at 400 × g, 4◦ C.
3. Aspirate the supernatant. Note the volume of cell pellet and add an equal volume of
cell washing/MACS buffer.
This resulting total volume is referred as labeling volume.
Remember to keep cells and cell washing/MACS buffer on ice during separation
procedure.

4. Resuspend cells thoroughly by gentle pipetting and add 1/10 vol CD43-FITC mAb.
Hematoendothelial For example, if the cell pellet is ∼0.2 ml, add 0.2 ml cell washing/MACS buffer (labeling
Differentiation of volume is 0.4 ml), resuspend cells, and add 40 µl (1/10 vol) of CD43-FITC mAb.
hESCs

23.6.12
Supplement 36 Current Protocols in Cell Biology
5. Set up the tube on a MACS mixer. Place the MACS mixer with tube in refrigerator
(4◦ C). Incubate 20 min at the lowest rotation speed.
Add magnetic beads
6. Wash the cells once by adding 14 ml ice-cold cell washing/MACS buffer and cen-
trifuging 5 min at 400 × g, 4◦ C. Discard the supernatant and resuspend the cells in
MACS buffer to original labeling volume and add 1/10 vol anti-FITC microbeads.
7. Incubate the cell suspension on MACS mixer 20 min at 4◦ C and low rotation speed.
8. Wash cell once by adding 14 ml ice-cold cell washing/MACS buffer and centrifuging
5 min at 400 × g, 4◦ C. Discard the supernatant and resuspend cell pellet in 1 ml cell
washing/MACS buffer.
9. Place a 35-µm preseparation filter on an empty 15-ml tube. Apply the suspension
to the filter and allow to completely pass through. Wash the filter with 0.5 ml cell
washing/MACS buffer. Place tube with the filtered cells on ice.
Select CD43+ cells
10. Assemble the MACS-LD separation unit (midi-MACS magnet with LD column and
15-ml collection tube). Rinse the column with 2 ml cell washing/MACS buffer and
discard the tube with eluate. Place an empty collection tube under column.
11. Apply the suspension to the LD column and allow to completely pass through. Wash
the column with 2 ml cell washing/MACS buffer.
12. Remove the collection tube containing the CD43− cell fraction and place on ice.
Save the CD43− cells for endothelial cell isolation.
13. Remove the LD column from magnet and insert in an empty 15-ml tube. Add 5 ml
cell washing/MACS buffer to the column funnel. Using a plunger (supplied with
column), flush the CD43+ cells out of the column. Discard the emptied column.
14. Centrifuge the tube containing the eluted CD43+ cells 5 min at 300 × g, 4◦ C.
15. Discard the supernatant and resuspend the cells in 0.2 ml cell washing/MACS buffer
and place tube on ice
Typically, up to 10% of the cells are recovered in MACS-enriched CD43+ cell fraction.
For example, if the column were loaded with 108 cells, one should expect to recover 107
cells in CD43+ fraction. This fraction usually contains 40% to 60% CD43+ cells and
can be used for subsequent sorting of CD43+ subsets by FACS (step 23). If a higher
purity of MACS-enriched CD43+ cells is desired, all potential contaminants that bind
nonspecifically to the magnetic beads and column can be removed prior to selection
of CD43+ cells. For this, cells (step 1) should be incubated with basic (unconjugated)
microbeads (as in steps 6 to 9) and passed through an LD column.
MACS magnetic microbeads are biodegradable microparticles (∼50 nm) that do not
interfere with subsequent FACS analysis, sorting, or functional assays. There is no need
to release them from the cells after the separation procedure.

Select endothelial cells


16. Label the CD43− cell fraction (collected in step 12) with CD31-PE mAb and anti-PE
microbeads as described in steps 2 to 9, but prepare the 35-µm filtered cell suspension
(step 9) in 3 ml cell washing/MACS buffer.
The cell filtrate should be prepared in 3 ml because this is the application volume for
LS columns. This can be accomplished by doubling filtration volumes: resuspending the
cells in 1 ml buffer, filtering, and washing the filter with 2 ml buffer to give a total volume
of 3 ml or by adding 1.5 ml to the 1.5 ml cell filtrate obtained in step 9.
Stem Cells

23.6.13
Current Protocols in Cell Biology Supplement 36
17. Assemble the MACS-LS separation unit (midi-MACS magnet with LS column and
15-ml collection tube). Rinse the column with 5 ml cell washing/MACS buffer and
discard the tube with eluate. Place an empty collection tube under column.
For a high purity (>98%) of isolated endothelial cells, positive selection (LS) columns
must be used.
There are two types of magnetic columns optimized for positive (LS) and negative (LD)
MACS separation, and they differ in flow-through rate. High flow rate LS columns provide
a high purity of column-retained cell fractions (>95%), whereas low flow rate LD columns
provide maximal recovery (depletion) of magnetically labeled cells (>99%).

18. Apply the suspension to the LS column and allow to pass completely through. Wash
the column three times with 3 ml cell washing/MACS buffer.
19. Discard the collection tube with the CD31− cell fraction.
Collect CD31+ cells
20. Remove the LS column from the magnet and insert an empty 15-ml tube. Add 5 to
7 ml cell washing/MACS buffer to column funnel. Using a plunger (supplied with
column), flush the CD31+ cells out of column.
21. Discard emptied column. Centrifuge tube with eluted CD31+ cells 5 min at 300 × g,
4◦ C.
22. Resuspend the cells in 1 ml cell washing/MACS buffer or medium for endothelial
cell expansion (see Support Protocol 7). Count the cells (UNIT 1.1).
23. For FACS analysis, add 10 to 20 µl cell suspension to 0.4 ml FACS buffer (without
FBS). Add 10 µl 7AAD solution for dead cell exclusion. Examine the cells by FACS
(Support Protocol 1).
CD43-FITC positive cells should not be detectable, whereas CD31-PE positive cells
should comprise >98%.

Sort CD43+ subsets by FACS


24. Add 5 µl mouse IgG1-PE and 5µl IgG1-APC isotype control mAbs to one 5-ml
polypropylene tube, and add 10 µl CD235a-PE and 40 µl CD45-APC mAbs to
another 5-ml tube.
25. Label MACS-enriched CD43+ cells (from step 15, up to 107 cells in 0.2 ml) with
isotype control and specific mAb combinations by adding 20 to 50 µl of the CD43+
cells to the tube with isotype control mAbs and the rest of CD43+ cells (150 to
180 µl) to the tube with CD235a-PE and CD45-APC mAbs.
26. Mix the cells gently and incubate samples 30 min at 4◦ C, with occasional agitation.
27. Wash cells twice with 5 ml cell washing/MACS buffer and resuspend in 0.5 ml cell
washing/MACS buffer. Keep tubes on ice until sorting.
28. Before running on the cell sorter, pass cell samples through a 35-µm filter.
29. Using the control sample, setup the following gates:

Live cells, using an FSC-A/SSC-A dot-plot (live CD43+ cells form a compact
population with low FSC/SSC profile)
Single cells, using an SSC-A/FCS-W dot plot (exclude cell doublets)
Total CD43+ cells, using an FL1-CD43-FITC/SSC-A dot plot (verify
instrument compensation).
Hematoendothelial
Differentiation of
hESCs CD43-FITC-stained cells should not overlap the FL2 channel.

23.6.14
Supplement 36 Current Protocols in Cell Biology
30. Open finally gated CD43+ cells in an FL2-IgG1-PE/FL4-IgG1-APC dot plot. Set
thresholds for positive staining.
31. Remove the control sample and insert a tube with CD43+ cells stained with CD235a
and CD45 mAbs. Identify three main CD43+ subsets:

CD235a+ CD45−
CD235a− CD45−
CD235a− CD45+

on an FL2-CD235a− PE/FL4-CD45− APC dot plot (Fig. 23.6.2B).


32. Set sorting gates on these populations and start sorting into 5-ml polypropylene tubes
prefilled with 0.3 ml FBS. Sort at low pressure, using a wide 100-µm nozzle tip.
33. Remove tubes with sorted cells. Fill tubes with 4 ml cell washing/MACS buffer or
medium and mix well by inverting tube several times. Centrifuge 5 min at 300 × g,
4◦ C.
34. Resuspend the cells in 0.5 ml medium and determine the number of sorted cells by
manual counting (see UNIT 1.1).
During the FACS sorting procedure, cells become electrically charged and may adhere to
the tube walls. Therefore, the real number of sorted cells recovered after centrifugation
is usually lower than known cell counts deposited by the sorter. For use in the following
tests, sorted cells should be counted manually.

PROPAGATION OF hESCs ON MOUSE EMBRYONIC FIBROBLASTS SUPPORT


PROTOCOL 4
This protocol describes the maintenance of hESCs for in vitro hematopoietic differ-
entiation based on the procedures described in Amit et al. (2000) and Thomson et al.
(1998), with slight modifications. The hESCs are cultured on mouse embryonic fibrob-
lasts (MEFs) and maintain pluripotency during an extended period of culture (Amit et al.,
2000). They are less likely to produce chromosomal abnormalities, compared to methods
that use feeder-free conditions (Draper et al., 2004). Undifferentiated hESCs grow in tight
colonies attached to the MEF feeder. Passaging hESC cultures includes (1) detachment
of hESC colonies by collagenase treatment, (2) fragmentation of hESC colonies into
small clumps by pipetting, and (3) plating of hESC clumps to the newly prepared MEF
feeder plate.

Materials
hESC cultures (H1 or H9 hESC lines; National Stem Cell Bank; see Internet
Resources)
6-well plates with pre-plated irradiated MEFs (Support Protocol 5)
hESC growth medium (see recipe)
1× phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO),
room temperature
Collagenase IV solution (see recipe)
37◦ C water bath
5-ml glass pipet, sterile
Detach hESC colonies from plates
1. Warm the collagenase IV solution and hESC growth medium 15 to 20 min in a 37◦ C
water bath.
2. Remove an MEF feeder plate from CO2 incubator. Aspirate the MEF growth medium
Stem Cells
and wash wells quickly with 2 ml PBS at room temperature.
23.6.15
Current Protocols in Cell Biology Supplement 36
3. Add hESC growth medium at 1.5 ml/well and keep MEF feeder plates in the CO2
incubator until the hESC are ready for plating.
4. Remove the hESC plate from the CO2 incubator. Aspirate the growth medium. Wash
wells quickly with 2 ml PBS at room temperature and add 2 ml/well collagenase IV
solution.
5. Incubate the hESC plate 10 to 15 min in the CO2 incubator until the edges of the
hESC colonies detach from plate.
6. Using a 5-ml glass pipet, dislodge the hESC colonies by washing the plastic surface
with the collagenase solution. Use gentle scraping to dislodge colonies that may still
be attached.
After collagenase treatment, hESC colonies should be dislodged easily by washing or gen-
tle scraping. Forceful scraping should be avoided to prevent cell damage and collection of
the firmly attached colonies, which may contain differentiated cells. Excessive mechanical
damage of hESCs during passage may also provoke their spontaneous differentiation.
7. Transfer the suspension to a 15-ml tube and centrifuge 5 min at 200 × g, room
temperature.
Disperse colonies and plate
8. Aspirate the medium and resuspend the cells in 4 ml hESC growth medium. Break
up the hESC colonies into a fine suspension of small cell aggregates by pipetting
cells up and down against the bottom of tube. Centrifuge 5 min at 200 × g, room
temperature.
9. Aspirate the medium, resuspend the cells in 6 ml hESC growth medium, and dispense
the suspension in a 6-well MEF feeder plate (1 ml/well), using a vertically positioned
glass serological pipet.
10. Place the plate on the shelf in the CO2 incubator and move it forth-to-back and
side-to-side to distribute hESC clumps evenly throughout the plate.
Avoid rotating motions because this will lead to accumulation of cell clumps in the center
of wells.
11. Allow the hESC clumps to attach to the MEFs for 24 hr. Do not disturb the plate
during this time.
12. Feed the hESC cultures daily by aspirating 2.5 to 3 ml medium/well and adding the
same amount of fresh prewarmed hESC growth medium. Passage on day 6 to 7.
It is imperative to avoid excessive density of hESC in maintenance cultures because it
may lead to hESC growth retardation, long-lasting spontaneous differentiation, genetic
abnormalities, and eventually poor differentiation efficiency in OP9 coculture. As a rule,
hESC cultures must be split when evenly positioned single hESC colonies begin to reach
confluence. The authors typically split H1 and H9 hESC cultures every 6 to 7 days at a
1:6 split ratio. The number of hESCs on the day of passage is ∼3 × 106 cells/ well of a
6-well plate.

SUPPORT PREPARATION OF MEF FEEDER PLATES


PROTOCOL 5
MEF feeder cells support attachment and undifferentiated growth of hESC colonies.
MEF derivation and propagation can be performed according to established protocols
from the suppliers—the National Stem Cell Bank (see Internet Resources) or commercial
sources (e.g., ATCC, GlobalStem). The authors use an MEF stock frozen at passage 3
Hematoendothelial for weekly preparation of MEF feeder plates. After thawing, MEFs are propagated
Differentiation of during one adaptation passage, inactivated by irradiation, and plated to 6-well plates in
hESCs
semiconfluent density.
23.6.16
Supplement 36 Current Protocols in Cell Biology
Materials
Gelatin-coated 10-cm dishes and 6-well plates (see recipe)
CF-1 strain MEFs (e.g., National Stem Cell Bank or ATCC: frozen stock at passage
3; 2–3 × 106 cells/vial)
MEF growth medium (see recipe)
1× phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO)
0.05% (w/v) trypsin/0.5 mM EDTA solution (1×; GIBCO)
15-ml polypropylene tubes
Gamma irradiator
Additional reagents and equipment for counting cells (UNIT 1.1)
Initiate MEF culture
1. Prepare one 15-ml tube and two gelatin-coated 10-cm dishes for each vial of MEFs
to be thawed.
2. Thaw an MEF vial in a 37◦ C water bath and transfer the cells to a 15-ml tube.
3. Add 10 ml of cold (4◦ C) MEF growth medium to the cells in the tube and centrifuge
5 min at 300 × g, 4◦ C.
4. Aspirate the supernatant, resuspend the cells in 2 ml MEF growth medium, and add
1 ml to each gelatin-coated 10-cm dishes containing 10 ml MEF growth medium.
Mix the cells by agitation and place the dishes in the CO2 incubator.
5. Grow MEFs to confluence (3 to 4 days).
Collect MEFs
6. Remove the MEF dishes from the CO2 incubator. Wash the cell monolayer once with
10 ml PBS and add 4 ml/dish prewarmed trypsin/EDTA solution. Incubate dishes 2
to 3 min in the CO2 incubator.
Do not incubate longer because the MEF monolayer disaggregates quickly.

7. Transfer the cell suspension to a 15-ml tube containing 5 ml MEF growth medium.
Centrifuge tube 5 min at 300 × g, room temperature.
8. Aspirate and discard the supernatant and resuspend the cells in 2 ml MEF growth
medium.
Inactivate MEFs
9. Irradiate tube with 5000 rads of gamma irradiation.
10. Centrifuge the cells 2 to 3 min at 300 × g, room temperature.
11. Resuspend the cells in 1 ml MEF growth medium.
12. Count the cells (UNIT 1.1) and adjust the MEF suspension to 2 × 105 cells/ml MEF
growth medium.
Plate MEFs
13. Fill gelatin-coated 6-well plates with 2 ml/well MEF growth medium. Add 1 ml
MEF suspension per well, mix, and incubate the plates in the CO2 incubator at least
24 hr before hESC plating. Use MEF feeder plates within 1 week.

CULTURE OF OP9 CELLS SUPPORT


PROTOCOL 6
Originally isolated from macrophage colony–stimulating factor (M-CSF) deficient os-
teopetrotic (op/op) mice, OP9 cells have been identified as efficient inducers of hema-
Stem Cells
toendothelial differentiation in mouse ESCs (Kodama et al., 1994; Nakano et al., 1994;
23.6.17
Current Protocols in Cell Biology Supplement 36
Zhang et al., 2005). While a direct application of mouse ESC/OP9 protocol for hESC re-
sulted in relatively poor performance, modifications of OP9 culture have enabled efficient
hematopoietic differentiation from hESCs (Vodyanik et al., 2005). These modifications
include the use of overgrown OP9 cultures for hESC differentiation and the maintenance
of OP9 cells on gelatin-coated dishes to minimize spontaneous adipogenesis and focal
outgrowth during extended post-confluence culture.

After confluence, OP9 cells are prone to spontaneous adipogenic differentiation espe-
cially when cultured in adipogenic FBS. The supplier and particular lot of FBS should
be selected by the ability of FBS to support efficient OP9 growth with minimal (if
noticeable) adipogenesis during maintenance culture and after feeding (half medium
change) and prolonged culture of confluent OP9 cells for 4 to 6 days. A high-quality FBS
is mandatory. The authors use a “defined” type of FBS (a commercial description for
high-quality, biochemically characterized FBS with minimal lot-to-lot variation) from
HyClone without heat inactivation, which does not benefit culture but usually results in a
higher adipogenic effect. Basal αMEM (standard formulation without nucleosides) must
be prepared from powder. The authors split OP9 cultures on the next day after conflu-
ence, typically every 4th day at up to a 1:10 split ratio. On the day of passage, 2–3 ×
106 OP9 cells can be recovered from one 10-cm dish.

Materials
OP9 growth medium (see recipe).
0.05% (w/v) trypsin/0.5 mM EDTA solution (1×; GIBCO)
OP9 cells (ATCC #CRL-2749)
1× phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO)
Gelatin-coated 10-cm dishes (see recipe)
15-ml polypropylene tubes
Inverted microscope
Passage OP9 cells
1. Warm OP9 growth medium and trypsin/EDTA solution in a 37◦ C water bath.
2. Remove the OP9 dishes from the CO2 incubator. Aspirate and discard the medium
and wash the cell monolayer with 10 ml PBS.
3. Add 5 ml trypsin/EDTA solution and incubate 5 to 10 min in the CO2 incubator.
4. Suspend the trypsinized OP9 cells by pipetting and add the cell suspension to a
15-ml tube containing 5 ml OP9 growth medium. Centrifuge 5 min at 300 × g, room
temperature.
5. Discard the supernatant and resuspend the cell pellet in 1 ml OP9 growth medium.
6. Add 1/7 to 1/10 vol of the cell suspension to each gelatin-coated 10-cm dish con-
taining 10 ml OP9 growth medium. Mix cells by agitation and place dishes in the
CO2 incubator.
It is essential to use gelatin-coated dishes for OP9 propagation to prevent spontaneous
adipogenesis. Because the intensity of OP9 growth is largely influenced by FBS, a split
ratio should be adjusted with each new FBS lot to ensure formation of confluent OP9
cultures during 3 to 4 days. Feeding of OP9 cells between passages and splitting of
semiconfluent cultures should be avoided.

7. Grow OP9 cells to confluence and on the day after reaching confluence (typically
4 days), split again as described above.
Hematoendothelial
Differentiation of
hESCs

23.6.18
Supplement 36 Current Protocols in Cell Biology
Prepare OP9 dishes for hESC differentiation
8. Plate OP9 cells on gelatin-coated 10-cm dishes as for a regular OP9 passage (steps 1
to 6).
9. On day 4, remove 5 ml of the medium using a pipet and add 5 ml fresh prewarmed
OP9 medium. Incubate OP9 dishes for an additional 4 days.
OP9 cultures used for hESC differentiation must be 8 to 10 days old, fed with fresh medium
at confluence (day 4) and incubated for extra 4 to 6 days.

10. Observe cultures on day 8 under an inverted microscope.


A typical overgrown OP9 monolayer should be formed (Fig. 23.6.2C) indicating that cells
are ready for hESC plating.
Overgrown OP9 cells do not appear as multilayered overcrowding cells, but form a regular
dense monolayer embedded in extracellular matrix (Fig. 23.6.2C). Scattered adipocytes
may appear in overgrown OP9 cultures, but no areas of extensive adipogenesis or focal
outgrowth should be detectable.
It is possible to incubate OP9 dishes for an additional 4 days without feeding until hESCs
will be available for differentiation. However, the authors usually do not use OP9 dishes
beyond total 12 days of culture.

PROPAGATION OF ENDOTHELIAL CELLS SUPPORT


PROTOCOL 7
Endothelial cells isolated on day 7 to 8 of hESC/OP9 coculture can be expanded for 4
to 5 passages in serum-free medium in the presence of fibroblast growth factor (FGF).
Since a >100× cell expansion can be achieved, hESC/OP9 cocultures may be used as a
source of hESC-derived endothelial cells for a variety of functional studies.

Materials
MACS-isolated endothelial (CD31+ CD43− ) cells on day 7 to 8 of hESC/OP9
coculture (Basic Protocol 2, step 22)
Fibronectin-coated 10-cm tissue culture dishes (see recipe)
Endothelial cell growth medium (ECGM; see recipe)
1× phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO)
HyQ-Tase cell detachment solution (HyClone)
Endothelial serum-free medium (ESFM; GIBCO)
Temperature controlled centrifuge, 4◦ C

1. Plate 106 MACS-isolated endothelial (CD31+ CD43− ) cells on a fibronectin-coated


10-cm dishes containing 10 ml ECGM (106 cells/dish). Incubate in a CO2 incubator
4 to 6 days until cells reach confluence.
After a 1 to 2 day lag period, cells start proliferation and reach confluence in 3 to 4 days.

2. Aspirate the medium and wash the cell monolayer with 10 ml PBS.
3. Add 2 ml PBS and 2 ml HyQ-Tase solution. Mix the solutions by gently agitating
and incubate 5 min in a CO2 incubator.
4. Suspend the detached cells by pipetting and transfer the cell suspension to a 15-ml
tube containing 5 ml ESFM. Centrifuge 5 min at 300 × g, room temperature.
5. Resuspend the cell pellet in 1 ml ESFM, add 1/5 vol to the freshly prepared
fibronectin-coated 10-cm dish containing 10 ml ECGM. Mix the cells by agitation
and place the dishes in the CO2 incubator.
6. Grow the endothelial cells until they reach confluence (typically 4 days) and split as Stem Cells
in steps 2 to 5.
23.6.19
Current Protocols in Cell Biology Supplement 36
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Cell washing/MACS buffer


Phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO) supple-
mented with 5% (v/v) heat-inactivated FBS (GIBCO) and 2 mM EDTA. Sterilize
by passing through a 0.22-µm filter under vacuum. Allow the solution to remain
under vacuum 15 to 20 min for degassing. Store up to 6 months at 4◦ C.
Keep the buffer on ice during a MACS procedure.

Collagenase IV solution
Dissolve 50 mg collagenase IV (GIBCO) in 50 ml DMEM/F12 basal medium
(1 mg/ml final concentration). Sterilize by passing through a 0.22-µm filter. Store
up to 2 weeks at 4◦ C.
Differentiation medium
Prepare the following components:

αMEM basal medium prepared from powder and sterilized by passing through a
0.22-µm filter. Store up to 2 months at 4◦ C.

50-ml aliquots of non-heat-inactivated defined FBS (HyClone). Store up to 1 year


at −20◦ C.

1000× (100 mM) monothioglycerol (MTG) solution. Add 87 µl MTG (Sigma) to


10 ml water. Mix well and dispense into 0.5-ml aliquots. Store up to 6 months at
−20◦ C.

1000× (50 mg/ml) ascorbic acid solution. Dissolve 500 mg ascorbic acid (Sigma)
in 10 ml water. Dispense into 0.5-ml aliquots and store up to 6 months at −20◦ C.

To prepare differentiation medium:

Combine 450 ml αMEM basal medium (90%), 50 ml FBS (10%), 0.5 ml MTG
(1×; 100 µM), and 0.5 ml ascorbic acid (1×; 50 µg/ml) in the upper chamber of a
500-ml bottle-top 0.22-µm filter unit (Nalgene). Sterilize by vacuum filtration and
store up to 1 month at 4◦ C.
Endothelial cell growth medium
Supplement endothelial serum-free medium (ESFM; GIBCO) with 10 ng/ml basic
fibroblast growth factor (bFGF; PeproTech) and 1/100 vol endothelial cell growth
factor (acidic FGF and heparin; Sigma E9640). Prepare immediately before use.
Do not store medium with cytokines.

FACS buffer, with and without FBS


Phosphate-buffered saline, without calcium and magnesium (PBS; GIBCO) sup-
plemented with 2% (v/v) heat-inactivated fetal bovine serum (FBS; GIBCO), if
required; 2 mM EDTA; and 0.05% (w/v) sodium azide. Sterilize by passing through
a 0.22- or 0.45-µm filter under vacuum. Store up to 6 months at 4◦ C.
Fibronectin-coated dishes
Dissolve 5 mg human fibronectin (GIBCO) in 5 ml sterile endotoxin-free water
Hematoendothelial
Differentiation of (Millipore). Store in 0.1-ml aliquots up to 6 months at −20◦ C. For plastic coating,
hESCs dilute fibronectin solution 1/200 in phosphate-buffered saline (PBS; GIBCO; final
continued
23.6.20
Supplement 36 Current Protocols in Cell Biology
concentration 5 µg/ml) and add 5 ml to each 10-cm tissue-culture dish (Falcon).
Incubate dishes overnight at 4◦ C. Before use, aspirate the fibronectin solution and
add the required amount of an appropriate growth medium.
Gelatin-coated dishes/plates
Prepare a 0.1% (w/v) gelatin solution: To a borosilicate glass bottle with auto-
clavable cap, add 500 mg Gelatin type A powder (Sigma) to 500 ml endotoxin-free
reagent-grade water (Millipore). Mix well and autoclave the gelatin slurry 45 min
at 121◦ C (gelatin will dissolve during autoclaving). Store up to 6 months at 4◦ C.
Add 6 to 7 ml gelatin solution to each 10-cm tissue-culture dish (Falcon) or 2 ml
to each well of a 6-well tissue-culture plate (Falcon). Allow the gelatin solution to
cover the entire plastic surface. Incubate dishes/plates in a CO2 incubator at least
overnight. Store up to 1 week in a CO2 incubator. Before use, aspirate the gelatin
solution and add the required amount of an appropriate medium.
hESC growth medium
Prepare the following components:

Prepare DMEM/F12 medium from powder (GIBCO). Sterilize by passing through


a 0.22-µm filter. Store up to 2 months at 4◦ C.

100× L-glutamine/2-mercaptoethanol supplement. Dissolve 146 mg L-glutamine


(GIBCO) in 10 ml phosphate-buffered saline, without calcium and magnesium
(PBS; GIBCO) and add 7 µl of 2-mercaptoethanol (Sigma). Store up to 1 week at
4◦ C.

500× basic fibroblast growth factor (bFGF) solution. Dissolve 50 µg bFGF (Pepro-
Tech) in 25 ml PBS supplemented with 2 mg/ml bovine serum albumin V (GIBCO).
Store up to 6 months at −80◦ C in 0.5-ml aliquots.
Dispense 50 ml aliquots of Knockout Serum Replacer (KSR; GIBCO). Store up to
1 year at −20◦ C.
To prepare complete hESC growth medium:

Combine 200 ml DMEM/F12 basal medium (80%), 50 ml Knockout Serum Re-


placer (20%), 2.5 ml 10 mM (100×) nonessential amino acid solution (GIBCO)
(1×), 2.5 ml L-glutamine/2-mercapthoethanol (1×), and 0.5 ml bFGF (4 ng/ml) in
the upper chamber of a 250-ml bottle-top 0.22-µm filter unit (Nalgene) and sterilize
by passing through the filter under vacuum. Store up to 2 weeks at 4◦ C.
Final concentrations of components are given in parentheses.

MEF growth medium


Prepare the following components:

Sterile-filtered DMEM medium prepared from powder (GIBCO). Store up to


2 months at 4◦ C.

50-ml aliquots of heat-inactivated FBS (GIBCO). Store up to 1 year at −20◦ C.


To prepare MEF growth medium:

Combine 450 ml DMEM basal medium (90%), 50 ml FBS (10%), and 5 ml 10 mM


(100×) nonessential amino acid solution (GIBCO) (1×) in upper chamber of
500-ml bottle-top 0.22-µm filter unit (Nalgene). Sterilize by passing through the
filter under vacuum. Store up to 1 month at 4◦ C. Stem Cells
Final concentrations of components are given in parentheses.
23.6.21
Current Protocols in Cell Biology Supplement 36
OP9 growth medium
Prepare the following components:

Prepare αMEM medium from powder (GIBCO). Sterilize by passing through a


0.22-µm filter. Store up to 2 months at 4◦ C.

Dispense 50-ml aliquots of non-heat-inactivated defined FBS (HyClone). Store up


to 1 year at −20◦ C.
To prepare OP9 growth medium:

Combine 400 ml αMEM basal medium (80%), and 100 ml FBS (20%) in upper
chamber of 500-ml bottle-top 0.22-µm filter unit (Nalgene). Sterilize by passing
through the filter under vacuum. Store up to 1 month at 4◦ C.
Final concentrations of components are given in parentheses.

COMMENTARY
Background Information et al., 1991; Schmitt et al., 1991; Keller et al.,
ESCs represent a unique population of 1993; Zambidis et al., 2005). The embryoid
cells capable of self-renewal and differenti- body method can be adapted to serum-free
ation. hESCs give rise to tissues from all conditions (Ng et al., 2005), making it possi-
three germ layers upon injection into im- ble to exclude a variability related to different
munodeficient mice or when induced to form lots of the serum and identification of factors
embryoid bodies in vitro (Thomson et al., required for lineage-specific differentiation.
1998; Schuldiner et al., 2000). Systems for However, it is difficult to induce lymphoid dif-
hematopoietic differentiation of hESCs pro- ferentiation under normal culture conditions
vide a unique opportunity to study mech- using an embryoid body method (Potocnik
anisms regulating lineage commitment and et al., 1994; Nakano, 2003).
maturation, identification of novel hematopoi- Another approach to inducing hematopoi-
etic precursors, and evaluation of the function etic differentiation is coculture of ESCs with
of genes critical for hematopoietic develop- bone marrow stromal cell lines. Several stro-
ment. The ability of mouse ESCs to differenti- mal cell lines, e.g., mouse bone marrow
ate into hematopoietic cells was demonstrated stromal cell lines S17, RP.0.10, and OP9
in 1985 (Doetschman et al., 1985). In 2001, (Gutierrez-Ramos and Palacios, 1992; Nakano
hematopoietic progenitors were obtained from et al., 1994; Kaufman et al., 2001; Vodyanik
hESCs cells (Kaufman et al., 2001). Since that et al., 2005), mouse yolk sac C166 cell line
time almost all blood lineages have been suc- (Kaufman et al., 2001), and human fetal liver
cessfully generated from mouse and human FH-B-hTERT cell line (Qiu et al., 2005),
ESCs, including red blood cells (Nakano et al., have been employed to induce hematopoi-
1996; Qiu et al., 2005), neutrophils (Lieber etic differentiation of ESCs. The most com-
et al., 2004; Vodyanik et al., 2005), megakary- monly used stromal cell line for hematopoi-
ocytes (Eto et al., 2002; Gaur et al., 2006), lym- etic differentiation studies is OP9, which
phocytes (Cho et al., 1999; Schmitt et al., 2004; was derived from M-CSF-deficient op/op os-
Vodyanik et al., 2005; Woll et al., 2005; Galic teopetrotic mice (Yoshida et al., 1990; Kodama
et al., 2006), and dendritic cells (Fairchild et al., 1994). The OP9 coculture was used to
et al., 2000; Senju et al., 2003; Slukvin et al., obtain multilineage hematopoietic progenitors
2006). as well as mature hematopoietic cells such as
Two major approaches are used to induce lymphocytes and megakaryocytes, which are
hematopoietic differentiation of ESCs. One difficult to obtain using the embryoid body
approach allows ESCs to form embryoid bod- method (Nakano et al., 1994; Eto et al., 2002;
ies after withdrawal of factors that keep ESCs Vodyanik et al., 2005; Gaur et al., 2006).
in an undifferentiated state. Various lineages of An important advantage of OP9 coculture
cells, including hematopoietic cells, develop is that efficient hematopoietic differentiation
inside embryoid bodies. The hematopoietic from hESCs can be achieved within a short
Hematoendothelial development within embryoid bodies is very period of time and without added cytokines.
Differentiation of similar to that found in the yolk sac (Burkert In addition, in contrast to the embryoid body
hESCs

23.6.22
Supplement 36 Current Protocols in Cell Biology
method, OP9 coculture allows one to obtain itors and usually require 3 to 5 weeks of cul-
the earliest lymphohematopoietic progenitors ture in the presence of bone marrow-derived
without applying excessive mechanical disag- stromal cells. These assays identify cells that
gregation, which results in substantial viability retain CFC potential after prolonged culture
loss. It should be noted, that differentiation of (long-term culture initiating cells; LTC-ICs)
ESCs in a monolayer on extracellular matrix as well lymphoid progenitors (Coulombel,
proteins has also been described (Nishikawa 2004). However, long-term in vitro assays are
et al., 1998; Zhang et al., 2006), but it is not laborious and subject to great variability due
widely used. to differences between laboratories in the use
The hematopoietic system comprises a hi- of media, growth factors, serum, and feeder
erarchy of cells at different stages of mat- cells for performing assays. It should also be
uration, which can be identified using mor- noted that all in vitro assays do not reflect the
phologic and phenotypic criteria as well as presence of cells with stem cell potential in a
functional assays. The hematopoietic stem cell sample.
(HSC) is defined as a cell able to gener- Phenotypic analysis represents a valuable
ate all types of hematopoietic cells and ca- tool for the identification of different types of
pable of self-renewal throughout the lifetime hematopoietic cells. The phenotype of adult
of an individual. While isolation of CD34+ hematopoietic progenitors is well character-
cells from bone marrow, unbilical cord, or pe- ized. However, it is important to emphasize
ripheral blood highly enriches for HSCs, true that somatic and hESC-derived hematopoi-
HSCs can only be identified retrospectively etic progenitors may demonstrate substan-
using an in vivo repopulation assay. In this as- tial phenotypic differences. While CD34 is
say, HSCs are injected into immunocompro- present on most hESC-derived hematopoi-
mised mice (e.g., NOD/SCID), and the pres- etic cells, it is also expressed on endothe-
ence of hematopoietic progeny is evaluated 8 lial and mesenchymal cells. Flow cytomet-
to 10 weeks after transplantation. While sev- ric analysis using CD34, CD43, and KDR
eral investigators have reported successful en- (FLK1) or CD31 antibodies is useful for
graftment of mouse and human ESC-derived discriminating these subsets of CD34 cells
hematopoietic progenitors (Hole et al., 1996; in hESC/OP9 coculture (Vodyanik et al.,
Potocnik et al., 1997; Burt et al., 2004; Wang 2006). The commonly used pan-hematopoietic
et al., 2005a), these results have not been marker CD45 is not expressed on the ear-
widely reproduced, and it is generally accepted liest hESC-derived multipotent hematopoi-
that engraftment by ESC-derived hematopoi- etic progenitors. Another pan-hematopoietic
etic progenitors is inefficient at the least marker (CD43) is present on all hematopoi-
(Daley, 2003; Nakano, 2003; Keller, 2005). etic progeny from hESCs. This molecule is
This failure to engraft is at least partially due useful for evaluation of hESC hematopoi-
to an abnormal expression profile of Hox genes etic differentiation as well as for isolation
and could be corrected through forced expres- of hematopoietic progenitors from hESC/OP9
sion of HoxB4 and Cdx4 genes (Kyba et al., cocultures (Vodyanik et al., 2006). The first-
2002; Wang et al., 2005b). appearing CD43+ CD235a+ CD41a+/− CD45−
Hematopoietic progenitors represent cells hematopoietic progenitors in hESC/OP9
that may be multipotent, oligopotent, or unipo- coculture represent precommitted erythro-
tent, but they lack significant self-renewal ca- megakaryocytic progenitors.
pacity. Several types of in vitro assays have The primary hESC progeny with mul-
been developed to characterize hematopoi- tilineage hematopoietic potential has
etic progenitors at different stages of matu- CD34+ CD43+ CD45− Lin− phenotype. These
ration. The colony-forming cell (CFC) assay cells are capable of differentiation toward
in semisolid media is the most widely used all blood lineages as well as B lymphoid
and well-standardized assay for detection of cells. Acquisition of CD45 expression by
progenitors at intermediate stages of develop- CD34+ CD43+ CD45− Lin− cells is associated
ment. This relatively simple assay requires a with progressive myeloid commitment and
short period of time to perform and allows a decrease of lymphoid potential (Vodyanik
identification of several types of hematopoi- et al., 2006). Isolation of these progenitors
etic progenitors in one dish. The CFC as- and subsequent studies of their differentiation
say remains an essential tool for characteriz- potential may provide important information
ing hematopoietic progenitors generated from regarding regulatory signals governing the de-
hESCs. Long-term in vitro assays are designed velopment of specific hematopoietic lineages,
Stem Cells
to identify less mature hematopoietic progen- and will eventually facilitate development of
23.6.23
Current Protocols in Cell Biology Supplement 36
bioreactor-based technology for large-scale plating density of 1.5 × 106 cells per 10-cm
production of blood cells for transfusion and OP9 dish is acceptable for H1 or H9 hESC
cellular therapy. lines, although H9 cells differentiate more
efficiently when plated at a lower density
Critical Parameters and (106 cells/OP9 dish). Optimal plating density
Troubleshooting for other hESC lines should be determined
It is essential to use semiconfluent mono- in preliminary experiments using an initial
layers of MEFs for hESC propagation. Over- range of 0.5–2.5 × 106 cells/OP9 dish with 0.5
crowded MEFs suppress growth of undiffer- intervals.
entiated hESC colonies and may cause their One problem that can be encountered
spontaneous differentiation. Different batches is poor viability and cell loss following
of MEFs prepared using different lots of FBS preparation of a single-cell suspension from
and plastic may vary in cell size, and, there- hESC/OP9 cocultures, especially after 8 days
fore, MEF plating density should be adjusted of differentiation. This may happen because of
accordingly to ensure a semiconfluent feeder excessive production of an extracellular matrix
layer for hESC culture. that withstands collagenase and trypsin treat-
A high-density overgrown OP9 monolayer ment. As a result, many cells may be lost due
is the most critical parameter for success- to clumping and subsequent mechanical dam-
ful hematopoietic differentiation of hESCs. age during pipetting. Longer incubation with
However, OP9 cells are prone to spontaneous collagenase and trypsin (up to 30 min each)
adipogenic differentiation in postconfluence should be used first to improve cell recovery.
cultures. To minimize adipogenesis in high- In addition, collagenase solution can be sup-
density overgrown cultures, OP9 cells should plemented with 0.1 mg/ml hyaluronidase to
be cultured on gelatin-coated dishes using further facilitate cell dissociation.
preselected nonadipogenic lots of FBS. OP9 While the function of the CD43 molecule
cells obtained from different sources (directly on early hematopoietic progenitors is largely
from Dr. Nakano or ATCC) efficiently support unknown, it has been shown that cross-linking
hematopoietic hESC differentiation. However, of CD43 on hematopoietic progenitors may in-
the authors recommend adaptation of OP9 duce apoptosis (Bazil et al., 1995). Therefore,
cells for growth on gelatin-coated dishes for it is critical to use antibodies that minimally
at least five passages before the cells are used affect the cell viability and function of iso-
for hESC differentiation. lated CD43+ cells. Procedures in this unit use
A high-quality basal αMEM medium is es- clone 1G10 anti-CD43 mAbs, which detect
sential for maintenance of OP9 cells as well CD43+ cells at maximal frequency compared
as hematopoietic differentiation in hESC/OP9 to other anti-CD43 mAb clones (L10, 290111)
cocultures. Freshly prepared αMEM from and enable successful isolation of functional
powder formulation is superior to commercial CD43+ cells. It is important to note, that some
ready-to-use liquid medium. The αMEM anti-CD235a mAbs may cause cell agglutina-
formulation supplemented with nucleosides tion, affecting flow cytometric detection and
increases proliferation of OP9 cells in mainte- isolation of erythroid progenitors. Such cell
nance cultures, but has little effect on hema- clumping may be prevented by using a lower
toendothelial differentiation in hESC/OP9 concentration of agglutinating mAb for cell
coculture. Addition of 100 µM (final con- labeling. Anti-CD235a mAb included in the
centration) SH-agent MTG to differentiation present protocols are non-agglutinating.
medium significantly increases the yield of
total differentiated cells in hESC/OP9 cocul- Anticipated Results
tures including hematopoietic and endothe- On day 1 of hESC/OP9 coculture, only con-
lial cells, however at >200 µM concentra- densed hESC clumps attached to OP9 mono-
tion, hematoendothelial differentiation may be layer can be observed. Differentiation begins
suppressed. While αMEM already contains a on day 2 when outgrowing hESCs immerse
high concentration of ascorbate, the authors into OP9 monolayer. At this time, the first
use additional supplementation of differenti- differentiating hESC colonies are readily de-
ation medium with 50 µg/ml ascorbic acid to tectable. On day 3, the number and size of
specifically improve hematoendothelial differ- differentiated colonies increase dramatically.
entiation. Colonies acquire the characteristic appearance
Hematoendothelial Optimal hESC plating density in OP9 co- of “mesodermal” colonies with an elevated
Differentiation of cultures is important for efficient differentia- central portion composed of tightly packed
hESCs
tion and may vary for different hESC lines. A rounded cells (Fig. 23.6.2D). On day 4, almost
23.6.24
Supplement 36 Current Protocols in Cell Biology
all colonies have a typical “mesodermal” mor- endothelial (CD31+ CD43− ) or hematopoietic
phology and reach maximal size. On day 5, (CD31+ CD43+ ) cells, one to two H1/OP9
colonies begin the lateral growth that leads to dishes is usually enough, but for FACS isola-
their decomposition. Actively growing differ- tion of CD43+ subsets, the number of H1/OP9
entiated cells displace the OP9 cells and form dishes should be scaled up as follows: for
confluent cultures on day 7. During 8 to 9 days, 106 CD43+ CD235a+ erythro-megakaryocytic
cultures do not change significantly, although progenitors, three to four dishes; for 106 total
ongoing differentiation can be observed by the CD43+ CD235a− CD45+/− multipotent pro-
emergence of proliferating cell clusters and genitors, eight to ten dishes. To isolate 106
vascular tubes. Due to high cell density, cul- FACS-sorted CD45− and CD45+ multipotent
tures begin to deteriorate after 10 to 12 days. progenitors on day 8, at least twenty H1/OP9
The efficiency of hematopoietic differen- dishes should be processed.
tiation may vary for different hESC lines.
Using H1 and H9 hESC lines, consistently Time Considerations
higher differentiation efficiency in H1 cells Setting up of hESC/OP9 cocultures re-
is observed, especially regarding hematopoi- quires coordinated maintenance of undifferen-
etic cell generation. Although H9 cells repro- tiated hESC, MEF, and OP9 cell cultures. One
duce the kinetic profile of hematoendothelial passage hESC culture takes 6 to 7 days. On the
differentiation observed in H1 cells, H9 dif- next day after hESC passage, new MEF cul-
ferentiation is usually delayed for 1 day and ture should be initiated to ensure availability
results in 1.5 to 2× lower numbers of CD43+ of MEF feeder plates for the next hESC pas-
hematopoietic cells. A description of typical sage. From 2–3 × 106 initially thawed MEFs,
results for H1 cells (assuming that efficiency three to four feeder plates can be prepared. At
with other hESC lines may be different) fol- splitting of a 6-well hESC plate, two wells will
lows: On day 7 to 8 of H1/OP9 coculture, H1- be used for hESC passage and counting, and
derived cells (TRA-1-85+ ) constitute ∼90% four wells can be used for differentiation. Up
of total cells. Hematopoietic and endothelial to 8–12 × 106 hESCs can be collected from
cells (CD31+ ) comprise 8% to 15% of to- four wells, and that is sufficient for setting up
tal H1-derived cells. CD34+ cells are always six to eight hESC/OP9 dishes. At splitting of
detectable in higher frequency (12% to 25%), one 10-cm OP9 dish, 1/7 to 1/10 portions will
due to the presence of CD34+ mesenchy- be used for passage, and remaining cells can
mal cells. The CD31+ population is typically be plated on six to nine dishes for hESC dif-
composed of 60% to 70% endothelial cells ferentiation. hESCs should be plated on OP9
(CD31+ CD43− ) and 30% to 40% hematopoi- dishes within 8 to 12 days after OP9 plating.
etic progenitors (CD31+ CD43+ ) that corre- During this time interval, actively growing un-
sponds to 5% to 10% and 3% to 5% of differentiated hESCs (at 5 to 7 days of culture)
total H1-derived cells, respectively. In the should be available for differentiation.
CD43+ population, CD43+ CD235a+ erythro- All standard cell culture procedures with
megakaryocytic progenitors comprise 50% to hESC, MEF, and OP9 maintenance cultures
70% of total CD43+ cells or ∼3% of to- are time efficient and can be performed within
tal H1-derived cells. Multipotent progenitors 1 hr or less. One experiment for hESC dif-
found within the CD43+ CD235a− population ferentiation can be completed within 15 to 20
(30% to 50% of total CD43+ or ∼2% of to- days; it requires 8 to 12 days for OP9 prepara-
tal H1-derived cells) undergo transition from tion and 7 to 8 days for hESC/OP9 coculture.
CD45− to CD45+ stage during 7 to 8 days of Depending on the volume of hESC differenti-
H1/OP9 coculture; on day 7, CD45− cells pre- ation cultures (typically up to ten hESC/OP9
dominate (∼80%), whereas on day 8, CD45− dishes in one experiment) each of the follow-
and CD45+ cells comprise nearly equal ing procedures may take 2 to 4 hr: harvesting
populations. of hESC/OP9 cocultures, analysis of differen-
The yield of endothelial and hematopoi- tiated cells by flow cytometry and cell plating
etic cells in isolation procedures is primar- for the methylcellulose CFC assay, single pa-
ily dependent on the efficiency of hESC dif- rameter MACS sorting, and FACS sorting.
ferentiation and the absolute number of cells
generated in hESC/OP9 cocultures. The lat-
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Stem Cells
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the myeloid pathway. J. Immunol. 176:2924- Provides a critical overview of in vivo and in vitro
2932. functional assays used for analysis hematopoietic
stem cells and progenitors.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S.,
Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Vodyanik, M.A., Thomson, J.A., and Slukvin, I.I.
and Jones, J.M. 1998. Embryonic stem cell 2006. See above.
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Vodyanik, M.A., Bork, J.A., Thomson, J.A., and
Slukvin, I.I. 2005. Human embryonic stem cell- Zambidis, E.T., Peault, B., Park, T.S., Bunz, F., and
derived CD34+ cells: Efficient production in the Civin, C.I. 2005. See above.
coculture with OP9 stromal cells and analysis of Describes procedures for and pathways of
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626. embryoid body method.
Stem Cells

23.6.27
Current Protocols in Cell Biology Supplement 36
Internet Resources
http://www.nationalstemcellbank.org
National Stem Cell Bank. Distributes hESC lines
and provides technical support to the hESC re-
search community and technical training in the cul-
ture of hESCs. In addition, information regarding
karyotype and HLA genotype of distributed cells is
provided. Protocols for culture, freezing, and thaw-
ing of hESCs as well as for derivation, culture,
and propagation of mouse embryonic fibroblasts
are also available.
http://www.stemcell.com
Stem Cell Technologies. Protocols for hematopoi-
etic stem cell/progenitor research.

Hematoendothelial
Differentiation of
hESCs

23.6.28
Supplement 36 Current Protocols in Cell Biology
Neural Differentiation of Human ES Cells UNIT 23.7
1 1 1
Malkiel A. Cohen, Pavey Itsykson, and Benjamin E. Reubinoff
1
Hadassah University Medical Center, Ein-Kerem, Jerusalem, Israel

ABSTRACT
Human embryonic stem cells (hESCs) may be converted into highly enriched cultures of
neural precursors under defined culture conditions. The neural precursors can proliferate
in culture for prolonged periods of time, and can differentiate in vitro into mature
neurons, astrocytes, and oligodendrocytes. The neurons are functional and have normal
electrophysiological properties. After transplantation to the developing rodent brain, the
neural precursors migrate extensively into the host brain parenchyma, respond to host
brain signals, and differentiate in a region-specific manner to progeny of the three neural
lineages. The establishment of neuroectodermal precursors from hESCs allows the study
of human neurogenesis in vitro and is an aid in drug discovery. In addition, the neural
precursors may potentially serve as a platform for the development of specific functional
neural cells for transplantation and gene therapy of neurological disorders. In this unit, we
introduce methods for the derivation, propagation and characterization of hESC-derived
neural precursors. Curr. Protoc. Cell Biol. 36:23.7.1-23.7.20.  C 2007 by John Wiley &

Sons, Inc.
Keywords: neural induction r human embryonic stem cells r neural precursors r
noggin r neural differentiation

INTRODUCTION
Embryonic stem (ES) cells can differentiate into all cell types of the body including
neural cells, and thus offer an in vitro model for tracing early cell lineages in mammals.
Derivation of neural precursors (NPs) from human ES cells (hESCs) may be invaluable
for the study of early human neurogenesis and for the utilization of hESCs as an unlimited
source of neural cells for transplantation in human neurodegenerative disorders. Neural
cells may be generated from hESCs following spontaneous differentiation in vitro, either
in high-density cultures or through embryoid body (EB) formation. However, the neural
cells that are generated from these cultures are within a mixture of other types of
differentiated cells. Protocols for the controlled derivation of cultures highly enriched
for proliferating, developmentally competent NPs were recently reported.

Here, a protocol is described for the derivation of NPs from hESCs in chemically defined
serum-free culture conditions (Basic Protocol). This protocol enables the controlled
differentiation of hESCs into NPs by two major steps. In the first step, clusters of
hESCs are enzymatically removed from the feeder cells to chemically defined serum-free
suspension culture conditions. Contamination of the hESC-clusters by residual feeder
cells is avoided. In the second step, controlled efficient differentiation of the hESCs to
an NP fate is achieved by supplementation with the growth and differentiation–inducing
factors noggin [bone morphogenic protein (BMP) antagonist] and basic fibroblast growth
factor (bFGF), mimicking neural induction in vivo. Methods are also described for the
characterization of the NPs by immunocytochemistry (Support Protocol 1) and flow
cytometry (Support Protocol 2), and for induction of their spontaneous differentiation
(Support Protocol 3). Finally, a protocol for culturing human embryonic stem cells is
provided (Support Protocol 4).

Stem Cells

Current Protocols in Cell Biology 23.7.1-23.7.20, September 2007 23.7.1


Published online September 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471143030.cb2307s36 Supplement 36
Copyright C 2007 John Wiley & Sons, Inc.
NOTE: All incubations are performed in 5% CO2 , humidified 37◦ C incubators unless
otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile,
and aseptic technique should be used accordingly.

NOTE: All procedures describing the use of phosphate-buffered saline (PBS) in this unit
refer to calcium- and magnesium-free PBS (e.g., Invitrogen, cat. no. 14190) unless PBS
with calcium and magnesium (e.g., Invitrogen, cat. no. 14040) is specified.

BASIC CONTROLLED DERIVATION OF NEURAL PRECURSORS FROM hESCS


PROTOCOL
Controlled neural differentiation of hESCs enables the enrichment of NPs in the cell cul-
ture under serum-free suspension culture conditions. hESCs are cultured in a chemically
defined medium supplemented with noggin and bFGF to promote neural differentiation,
and then in a medium supplemented with bFGF to further propagate the hESC-derived
NPs.

Materials
0.1% (w/v) low melting temperature (LMT) agarose (see recipe)
hESCs cultured on a feeder layer (see Support Protocol 4) in 6-well plates
1 mg/ml collagenase type IV (see recipe)
Neural precursor medium (NPM; see recipe)
20 µg/ml bFGF (see recipe)
100 µg/ml noggin (see recipe)
NPM (see recipe) containing 20 ng/ml bFGF (added from 20 µg/ml bFGF stock;
see recipe) and 500 ng/ml noggin (add from 100 µg/ml noggin stock; see recipe)
NPM (see recipe) containing 20 ng/ml bFGF (added from 20 µg/ml bFGF stock;
see recipe)
50-ml conical polypropylene centrifuge tubes
Centrifuge
24-well tissue culture plates
50-ml centrifuge tubes
20-G surgical blades
Additional reagents and equipment for culturing hESCs (Support Protocol 4)
Collect hESC clusters from culture
1. To prevent adhesion of hESC clusters to the culture dishes, cover the well bottoms
of a 24-well plate with 0.5 ml of 0.1% low-melting temperature agarose for at least
30 min. Aspirate the agarose suspension and dry the plate at room temperature.
Alternatively, one can use a low-cell-attachment plate.

2. Aspirate the medium from a 6-well plate containing hESC-colonies cultured on


feeders, 6 to 7 days after their passage with EDTA as described in Support Protocol 4.
Use the EDTA passage method (Support Protocol 4) for at least the last passage before
hESC-cluster formation, to achieve hESC colonies homogenous in size (Fig. 23.7.1A).

3. Apply 1 ml of 1 mg/ml (∼200 U) collagenase type IV to each well and incubate for
30 to 60 min.
After this incubation, the edges of the hESC colonies should detach from the feeder layer
(Fig. 23.7.1B).
Neural
Differentiation of
Human ES Cells

23.7.2
Supplement 36 Current Protocols in Cell Biology
Figure 23.7.1 Derivation of NPs from undifferentiated hESCs. (A) An undifferentiated hESC colony cultured on human
foreskin fibroblasts, 1 week after passaging with the aid of an EDTA dissociation solution (phase-contrast image; 40×
magnification). (B) hESC colony after 30 min of collagenase treatment (phase-contrast image; 40× magnification).
(C) hESC-derived neural spheres 3 weeks after derivation and culture in defined medium (NPM) supplemented with
bFGF and noggin (dark-field stereomicroscopic image; 2× magnification).

4. Tap the plate to gently dislodge the clusters of hESCs from the feeder cells.
The feeders should remain intact on the bottom of the well and the hESC clusters should
be free of contaminating feeder cells.
5. Collect the supernatant medium with the hESC clusters from all wells and transfer
to a 50-ml tube. Gently centrifuge 5 min at ∼40 × g, room temperature.
Transfer hESC clusters to cultures with NPM
6. Aspirate the supernatant from the cell clusters and resuspend them in neural precursor
medium (NPM) at a density of up to 80 × 103 cells/ml (∼40 clumps/ml).
7. Add 1 µl of 20 µg/ml bFGF to each 1 ml of cluster suspension, to reach a final
concentration of 20 ng/ml.
8. Add 5 µl of 100 µg/ml noggin to each 1 ml of cluster suspension, to reach a final
concentration of 500 ng/ml.
9. Transfer the clusters at 1 ml per well to a 24-well culture dish pretreated with 0.1%
LMT agarose (see step 1).
Upon transfer of the hESC clusters into NPM, significant cell death should be observed
during the first 2 to 3 days, which should be followed by a gradual increase in the sizes
of the floating clusters, which acquire the form of spheres (Fig. 23.7.1C).

Propagate the neural cultures


10. Culture the clusters for the first 3 weeks in NPM containing 20 ng/ml bFGF and 500
ng/ml noggin.
11. Change the medium every 2 days as follows. Swirl the plate to centralize the spheres
in the middle of the well, collect the spheres into a microcentrifuge tube, let the tube
stand for 3 min to allow the spheres to sink, remove the supernatant, add 1 ml of
fresh prewarmed NPM containing 20 ng/ml bFGF and 500 ng/ml noggin, and replate
the sphere suspension in the well.
12. After 3 weeks of culture, change the medium to NPM supplemented with 20 ng/ml
bFGF. Refresh the medium every 2 days, using the technique described in step 11.
Stem Cells

23.7.3
Current Protocols in Cell Biology Supplement 36
13. Check the cultures weekly and dissect any spheres or aggregates of spheres whose
diameters exceed 0.5 mm into small clusters with two 20-G surgical blades that are
used like scissors, or by trituration. For trituration, use a pipettor with a 200-µl tip,
and repeatedly draw the sphere suspension up and down to dissociate the spheres.
At a time point 4 weeks after transfer of the hESC clusters into NPM, the spheres are
highly enriched for NPs that can be used for further differentiation protocols.

SUPPORT CHARACTERIZATION OF THE NEURAL PRECURSORS BY


PROTOCOL 1 IMMUNOSTAINING
The phenotype of the pluripotent cells and the NPs derived from them may be analyzed
by immunostaining. Staining of both pluripotent and NP markers and their analysis at
sequential time points makes it possible to track the progression of neural differentiation
over time.

Materials
10 µg/ml poly-D-lysine (see recipe)
Tissue culture–grade distilled H2 O
4 µg/ml laminin (see recipe)
NPs derived from hESCs (see Basic Protocol)
0.008% (w/v) trypsin/2.4 mM EDTA (see recipe)
Phosphate-buffered saline (PBS; containing calcium and magnesium; Invitrogen,
cat. no. 14040)
2.35 mg/ml DNase (see recipe)
4% (w/v) paraformaldehyde (see recipe)
FACS buffer (see recipe)
0.2% (v/v) Triton X-100 (see recipe)
Blocking solution (see recipe)
Appropriate primary antibody (Table 23.7.1)
Secondary antibody: fluorophore-conjugated antibody against IgG of species from
which primary antibody was derived
Mounting medium with 4 -6-diamidino-2-phenylindole (DAPI; e.g., Vectashield
from Vector Laboratories)
16-mm-diameter round glass coverslips, sterile (Paul Marienfeld & Co.;
http://www.marienfeld-superior.com)
Center-well organ culture dish (Falcon)
Glass microscope slides
Coat coverslips
1. Place a glass coverslip in the center of a center-well organ culture dish. Cover the
glass coverslip with 500 µl of 10 µg/ml poly-D-lysine and incubate 1 hr at room
temperature.
The staining procedure is done in organ culture dishes; coverslips must be maintained
in the organ culture dishes until the fixation step (step 8) and can then be transferred to
12-well plates.

2. Aspirate the poly-D-lysine solution and wash the glass coverslip with 1 ml of tissue
culture–grade distilled water for 1 min. Aspirate the water and cover the glass
coverslip with 500 µl of 4 µg/ml laminin. Incubate overnight at 4◦ C or for 2 hr at
room temperature.
Laminin-coated glass coverslips can be kept up to 1 month at 4◦ C in a sealed chamber.
Neural
Differentiation of
Human ES Cells

23.7.4
Supplement 36 Current Protocols in Cell Biology
Plate single-cell suspensions of NPs
The NPs can be plated on the glass coverslips as cell clusters or as single cells. Cell
clusters should be plated on precoated glass coverslips with 1 ml of NPM for 24 hr. For
single-cell plating, follow the steps described below.

3. Collect the clusters of NPs (Basic Protocol) into a microcentrifuge tube and micro-
centrifuge for a few seconds at 240 × g, room temperature.
4. Aspirate the supernatant and resuspend the clusters in 500 µl of 0.008% trypsin/2.4
mM EDTA. Incubate at 37◦ C for 15 min.
5. Pipet the clusters up and down to dissociate them into a single-cell suspension.
Microcentrifuge the cells for a few seconds at 240 × g.
A white cloudy pellet should appear.
6. Gently remove the supernatant and add 500 µl of PBS (with Ca and Mg) and 5 µl
of 2.35 mg/m DNase, then incubate for an additional 10 min. Pipet the pellet gently
up and down to obtain a single-cell suspension. Microcentrifuge the cells for a few
seconds at 240 × g, room temperature. Remove the supernatant and resuspend the
cells in 500 µl NPM.
7. Plate the cell suspension on glass coverslips pre-coated with poly-D-lysine and
laminin (from step 2) at a density of 5–10 × 103 cells/ml, and incubate for 1 to 2 hr
at 37◦ C to allow the cells to attach to the coverslips.
Fix the cells
8. Fix the cells by flooding them with 2 ml of 4% paraformaldehyde and incubating 20
min at room temperature.
9. Aspirate the supernatant from the coverslip and wash three times, each time for 1
min with 1 ml of FACS buffer.
The cells can be kept under the FACS buffer for up to 1 week at 4◦ C.

10. For immunostaining of intracellular markers, permeabilize cell membranes by flood-


ing the cells with 1 ml of 0.2% Triton X-100 and incubating 5 min at room temper-
ature.
11. Incubate the cells with 500 µl blocking solution for 30 to 60 min at room temperature.
Immunostain the cells
12. Aspirate the supernatant from the coverslip and incubate the cells with the desired
primary antibody (Table 23.7.1), diluted in FACS buffer as described in Table 23.7.1,
for 1 to 2 hr at room temperature.
Proper control staining for the primary and secondary antibodies should be conducted
to rule out nonspecific staining or antibody cross-reactivity.

13. Aspirate the supernatant from the coverslip and wash three times, each time for 1
min with 1 ml of FACS buffer.
14. For primary antibody localization, incubate the cells with an appropriate fluorophore-
conjugated secondary antibody, diluted in FACS buffer according to the manufac-
turer’s recommendations, for 1 to 2 hr at room temperature.
15. Aspirate the supernatant from the coverslip and wash three times, each time for 1
min with 1 ml of FACS buffer.
16. Thoroughly aspirate the FACS buffer from the glass coverslip and mount it on a glass
microscope slide using mounting medium with DAPI. Stem Cells

23.7.5
Current Protocols in Cell Biology Supplement 36
Table 23.7.1 Primary Antibodies for In Vitro Immunostaining and Flow Cytometrya

Antigen/antibody Species and type Clonality Purpose Dilution Source

ESC Oct-4 Mouse IgG Monoclonal Staining 1:100 Santa Cruz


pluripotency FACS 1:20 Biotech.
SSEA4 Mouse IgG Monoclonal Staining 1:100 DSHB
FACS 1:100
SSEA3 Rat IgM Monoclonal Staining 1:100 Chemicon
FACS
Tra-1-60 Mouse IgM Monoclonal Staining 1:20-50 Chemicon
FACS 1:100
Tra-1-81 Mouse IgM Monoclonal Staining 1:10 Chemicon
FACS 1:100

Neural NCAM Mouse IgG Monoclonal Staining 1:10 Dako


precursors
PSA-NCAM Mouse IgM Monoclonal Staining 1:200 Chemicon
FACS 1:250
Nestin Rabbit Polyclonal Staining 1:200 Chemicon
Pax6 Mouse IgG Monoclonal Staining 1:100 DSHB
Sox1 Chicken Polyclonal Staining 1:1000 Chemicon
Musashi Rabbit Polyclonal Staining 1:100 Chemicon

Neurons β-tubulin III Mouse IgG Monoclonal Staining 1:2000 Sigma


MAP2ab Rabbit Polyclonal Staining 1:500 Chemicon
NF 70 kDa Mouse IgG Monoclonal Staining 1:100 Dako
NF 160 kDa Mouse IgG Monoclonal Staining 1:50 Chemicon
NF 200 kDa Rabbit Polyclonal Staining 1:5000 Sigma
NeuN Mouse IgG Monoclonal Staining 1:100 Chemicon
Neuron-specific Rabbit Polyclonal Staining 1:200 Zymed
enolase (NSE)
Synaptophysin Mouse IgG Monoclonal Staining 1:50 Dako
GABA Rabbit Polyclonal Staining 1:1000 Sigma
Glutamate Rabbit Polyclonal Staining 1:1000 Sigma
Serotonin Rabbit Polyclonal Staining 1:1000 Sigma
Tyrosine Mouse IgG Monoclonal Staining 1:500 Sigma
hydroxylase (TH)

Glia GFAP Rabbit Polyclonal Staining 1:200 Dako


O4 Mouse IgM Monoclonal Staining 1:30 Chemicon

a Abbreviations: DSHB, Developmental Studies Hybridoma Bank, University of Iowa (http://dshb.biology.uiowa.edu/); GABA, γ-aminobutyric acid;
GFAP, glial fibrillary acid protein; NCAM, neural cell adhesion molecule; PSA-NCAM, polysialylated form of NCAM; MAP2, microtubule-associated
protein 2; NF, neurofilament; NeuN, neuronal nuclei.

Neural
Differentiation of
Human ES Cells

23.7.6
Supplement 36 Current Protocols in Cell Biology
CHARACTERIZATION OF NEURAL DIFFERENTIATION BY FLOW SUPPORT
CYTOMETRIC ANALYSIS PROTOCOL 2
The percentage of cells expressing markers of NPs or pluripotent cells and the intensity
of expression of the markers should be analyzed by FACS. Analysis at sequential time
points enables the characterization of the progression of neural differentiation over time.
This protocol describes the characterization of both undifferentiated hESCs and NPs.
The initial preparation of the two types of cells is described separately.

Materials
hESCs cultured on a feeder layer (see Support Protocol 4) or NPs derived from
hESCs (see Basic Protocol)
0.05% (w/v) disodium EDTA
0.008% (w/v) trypsin/2.4 mM EDTA (see recipe)
Phosphate-buffered saline (PBS; Invitrogen) containing calcium and magnesium
(Invitrogen, cat. no. 14040)
2.35 mg/ml DNase (see recipe)
FACS buffer (see recipe)
100% ethanol, –20◦ C
Permeabilization buffer (see recipe)
Appropriate primary antibody (Table 23.7.1)
Goat anti–mouse immunoglobulin conjugated with fluorescein isothiocyanate
(FITC; Dako)
2 µg/ml propidium iodide (see recipe)
35-µm nylon mesh
15-ml centrifuge tubes
5-ml polystyrene round-bottom tubes (Falcon)
Refrigerated centrifuge
Flow cytometer (also see Robinson et al., 2007)
Additional reagents and equipment for counting cells (UNIT 1.1) and flow cytometry
(Robinson et al., 2007)
Prepare cells
To prepare hESCs
1a. Dissociate the hESC colonies into a single-cell suspension by adding 2 ml of 0.05%
EDTA per well and incubating 10 min at room temperature.
2a. With the aid of a pipettor, repeatedly blow 1 ml of the EDTA solution onto the hESCs
to dislodge them from the feeders.
The feeders should remain intact and adherent to the culture dish.

3a. Filter the hESC suspension through a 35-µm nylon mesh to remove clusters.
4a. Transfer the cells to a 15-ml tube and centrifuge 5 min at ∼240 × g, room temperature.

Prepare neural precursor clusters


1b. Collect the NP clusters into a 15-ml centrifuge tube and centrifuge 5 min at ∼40 ×
g for 5 min, room temperature.
2b. Aspirate the supernatant and resuspend the NP clusters in 2 ml of 0.008% trypsin/2.4
mM EDTA. Incubate at 37◦ C for 15 min.
3b. Pipet the clusters up and down to dissociate them into a single-cell suspension.
Centrifuge the NPs 5 min at ∼40 × g, room temperature.
A white cloudy pellet should appear. Stem Cells

23.7.7
Current Protocols in Cell Biology Supplement 36
4b. Gently remove the supernatant and add 1 ml of PBS (with Ca and Mg) and 10 µl of
2.35 mg/ml DNase. Incubate an additional 10 min, and pipet the pellet gently up and
down to obtain a single-cell suspension. Filter the cell suspension through a 35-µm
nylon mesh and centrifuge 5 min at ∼240 × g, room temperature.
Prepare cells for staining
The following steps should be done on ice. Centrifugation should be carried out in a
refrigerated centrifuge appropriate for the centrifugation of 5-ml tubes.

5. Resuspend the cells in 1 ml cold FACS buffer. Count the cells and split them into
aliquots of 100–150 × 103 cells in 5-ml round-bottom tubes.
6. To detect nuclear proteins: Fix the dissociated cells with 3 ml precooled 100%
ethanol at –20◦ C for 15 min. Centrifuge the fixed cells 5 min at ∼460 × g, 4◦ C,
and remove the supernatant. Permeabilize the cells by adding 3 ml permeabilization
buffer and incubating 15 min at 4◦ C.
7. Centrifuge cells (from step 5 or 6) 5 min at ∼460 × g, 4◦ C.
8. Wash the cells by adding 2 ml of cold FACS buffer, then centrifuging 5 min at ∼460
× g, 4◦ C.
9. Remove the supernatant by turning the tubes upside down.
The cells should stay on the bottom of the tubes with ∼100 µl of remaining fluid.

Immunostain cells
10. Incubate the cells 30 min on ice with the desired primary antibody (Table 23.7.1),
diluted in FACS buffer as described in Table 23.7.1.
11. Wash the cells by adding 2 ml of cold FACS buffer and centrifuge for 5 min at ∼460
× g, 4◦ C. Spill out the supernatant as described in step 9.
12. Detect primary antibodies by incubating the cells on ice for 30 min with goat anti-
mouse FITC conjugated immunoglobulins diluted 1:100 with FACS buffer.
13. Wash the cells again by adding 2 ml of cold FACS buffer and centrifuging for 5 min
at ∼460 × g at 4◦ C. Spill out the supernatant as described in step 9.
14. Add 300 µl of 2 µg/ml propidium iodide solution for gating of viable cells.
15. Acquire 1–2 × 104 cells for each sample and analyze by flow cytometry with
appropriate software.
Robinson et al. (2007) provides detailed protocols for flow cytometry.

SUPPORT SPONTANEOUS DIFFERENTIATION OF hESC-DERIVED NPS


PROTOCOL 3
The characterization of the NPs should include an analysis of their potential to give rise
to mature neurons and glia cells. Here, the methodologies for induction of differentiation
in vitro and for the immunophenotyping of differentiated progeny are described.
Materials
10 µg/ml poly-D-lysine (see recipe)
Tissue culture–grade distilled H2 O
4 µg/ml laminin (see recipe)
NPs derived from hESCs (see Basic Protocol)
Neural precursor medium (NPM; see recipe)
Neural 16-mm-diameter round glass coverslips, sterile (Paul Marienfeld & Co.;
Differentiation of
Human ES Cells http://www.marienfeld-superior.com)
Center-well organ culture dish (Falcon)
23.7.8
Supplement 36 Current Protocols in Cell Biology
1. Place a glass coverslip in the center of a center-well organ culture dish. Cover the
glass coverslip with 500 µl of 10 µg/ml poly-D-lysine and incubate 1 hr at room
temperature.
2. Aspirate the poly-D-lysine solution and wash the glass coverslip with 1 ml of tissue
culture–grade distilled water for 1 min. Aspirate the water and cover the glass
coverslip with 500 µl of 4 µg/ml laminin. Incubate overnight at 4◦ C or for 2 hr at
room temperature.
Poly-D-lysine/laminin-coated glass coverslips can be kept up to 1 month at 4◦ C in a sealed
chamber.

3. Collect the NP clusters (Basic Protocol) into a microcentrifuge tube and microcen-
trifuge for a few seconds at ∼240 × g, room temperature. Aspirate the supernatant
and add 1 ml NPM.
4. Partially dissociate the cluster by pipetting the clumps gently up and down several
times.
5. Plate the cell suspension on a glass coverslip precoated with poly-D-lysine and
laminin (from step 2), bring the volume to 1 ml with NPM (not supplemented with
mitogens), and incubate the cells at 37◦ C for 7 to 21 days.
6. Replenish the culture medium (NPM) every 3 to 4 days.
To evaluate the developmental potential of the NPs, their capability to differentiate into
progeny representing the three major neural lineages should be characterized by im-
munostaining for neuronal, astrocytic, and oligodendrocytic markers. The methods for
fixation and immunostaining are described in Support Protocol 1.

CULTURING HUMAN EMBRYONIC STEM CELLS SUPPORT


PROTOCOL 4
hESCs with a normal karyotype may be maintained on mouse embryonic fibroblasts, on
the extracellular matrix substrate Matrigel (see UNIT 23.2) or on human foreskin fibrob-
lasts. The authors’ NP-derivation protocol is well established for hESCs maintained on
mitomycin C–treated human foreskin fibroblasts.

Materials
Cultures of human foreskin fibroblasts (ATCC # SCRC-1041)
Phosphate-buffered saline (PBS; Invitrogen, cat. no. 14190), prewarmed to 37◦ C
0.04% (w/v) trypsin/0.16 mM EDTA (see recipe)
Feeder cell medium (see recipe)
2 mg/ml mitomycin C (see recipe)
0.1% (w/v) gelatin (see recipe)
Cultures of hESCs
0.05% (w/v) disodium EDTA
KnockOut (KO) medium (see recipe)
1 mg/ml collagenase type IV (see recipe)
175-cm2 tissue culture flasks with 2-µm vent caps
Inverted microscope
15- and 50-ml conical polypropylene centrifuge
6-well tissue culture plates
Additional reagents and equipment for counting cells (UNIT 1.1)
Culture human foreskin fibroblasts
1. Culture human foreskin fibroblasts as a monolayer in a 175-cm2 tissue culture flask
in 40 ml feeder cell medium.
Stem Cells

23.7.9
Current Protocols in Cell Biology Supplement 36
2. When the cells appear confluent, aspirate the medium. Rinse twice with 10 ml
prewarmed PBS, each time for 1 min.
3. Add 2 ml of 0.04% trypsin/0.16 mM EDTA to the 175-cm2 flask. Ensure that the
entire cell surface is covered for 1 to 2 min.
4. Tap the flask to remove the cells. Observe the flask under an inverted microscope
to ensure that trypsinization has been effective and the detachment of fibroblasts is
complete.
5. Inactivate trypsin by adding 10 ml of prewarmed feeder cell medium and pipet the
medium gently several times, blowing it onto the culture surface of the flask to
remove remnants of attached fibroblasts and to disaggregate clusters of fibroblasts.
Transfer the cells to a 50-ml tube.
6. Centrifuge the cells 5 min at ∼240 × g, room temperature. Remove the supernatant
and resuspend the cells in 10 ml feeder cell medium.
7. Count cells in a 10-µl aliquot using a hemacytometer (UNIT 1.1) and calculate the
total cell number. Plate the cells in new 175-cm2 flasks (3–3.4 × 106 cells per flask
for a 3-day incubation period, or 2–2.6 × 106 cells per flask for a 4-day incubation
period). Add prewarmed feeder cell medium to a total volume of 40 ml per flask.
8. Swirl the flasks gently. Incubate the flasks in a 37◦ C 5% CO2 incubator for 3 to 4
days. When the cells are confluent, repeat the procedure.
Prepare feeder layers
9. Incubate a 175-cm2 flask of confluent human foreskin fibroblasts with 20 ml of
feeder cell medium supplemented with 125 µl of 2 mg/ml mitomycin C solution for
2.5 hr.
10. Meanwhile, cover the bottom of wells of a 6-well plate with 0.1% gelatin for a
minimum time period of 30 min. Aspirate the gelatin solution and allow the bottom
of the wells to dry.

11. Aspirate the mitomycin C–containing feeder cell medium from the 175-cm2 flask of
human foreskin fibroblasts and replace it with 20 ml prewarmed feeder cell medium
without mitomycin C.
12. Trypsinize the fibroblasts as described in steps 1 to 5.
13. Count cells in a 10-µl aliquot using a hemacytometer (UNIT 1.1) and calculate the
total cell number.
14. Plate 3 × 105 of the mitomycin C–treated fibroblasts in 2 ml of feeder cell medium
per each gelatin-precoated well of the plates prepared in step 10. Incubate plate,
without changing the medium, for at least 24 hr and up to 5 days before plating the
hESCs.
Culture hESCs
Colonies of hESCs are cultured on feeders in KO medium and are passaged to a new
feeder layer every 6 to 7 days. Passage is performed before the hESC colonies attach one
to another and before the cells in their center start piling up, which may be associated
with unwanted differentiation. Passage can be done using EDTA (Fig. 23.7.1A.) or using
collagenase.

Neural
Differentiation of
Human ES Cells

23.7.10
Supplement 36 Current Protocols in Cell Biology
To passage cells using EDTA
15a. Dissociate the hESCs into a single-cell suspension by incubation with 2 ml per well
of 0.05% EDTA for 10 min. With the aid of a pipettor, repeatedly blow 1 ml of the
EDTA solution on the hESCs colonies to dislodge them from the feeders.
The feeders should remain intact and adherent to the culture dish.

16a. Transfer the cells to a 15-ml tube and centrifuge 5 min at ∼240 × g, room temper-
ature.
17a. Resuspend the cells in KO medium, count them (UNIT 1.1), and plate 4–6 × 104 cells
per well of a 6-well plate on fresh mitomycin C–treated foreskin feeders generated
as described above.
18a. Replenish the culture medium every day.
Note that, due to the high frequency of chromosomal abnormalities observed after
extended passaging of hESCs as single cells, it is recommended that the number of
passages performed with the aid of EDTA be limited (Mitalipova et al., 2005).

To passage cells using collagenase


15b. Dissociate the hESCs into small clusters by incubating for 1 to 2 hr with 1 ml per
well of 1 mg/ml (∼200 U/ mg) collagenase type IV.
16b. Tap the plate to gently dislodge the clusters or gently blow medium onto the hESC
colonies to remove them from the feeders.
The feeders should remain intact on the bottom of the well and the hESC clusters should
be free of contaminating feeder cells.
17b. Transfer the cell clusters to a 15-ml centrifuge tube and centrifuge the clusters 5
min at ∼40 × g, room temperature.
18b. Resuspend the clusters in KO medium, pipet them gently up and down to disassem-
ble the clumps, and plate the hESC-clusters on fresh, mitomycin C–treated foreskin
feeder layers, splitting at 1:3 ratio (one well to three new wells).
19b. Continue incubation, replenishing culture medium every day.

REAGENTS AND SOLUTIONS


Use tissue culture–grade, distilled water in all recipes and protocol steps. All procedures describ-
ing the use of phosphate-buffered saline (PBS) in this unit refer to calcium- and magnesium-free
PBS (e.g. Invitrogen, cat. no. 14190) unless PBS with calcium and magnesium is specified (e.g.,
Invitrogen, cat. no. 14040). For common stock solutions, see APPENDIX 2A; for suppliers, see
SUPPLIERS APPENDIX.

bFGF, 20 µg/ml
Dissolve 50 µg of recombinant human bFGF (R&D Systems) by adding 250 µl of
5 mM Tris·Cl, pH 7.6 (APPENDIX 2A) and 2250 µl of sterile 0.1% (w/v) BSA (Sigma)
in PBS (Invitrogen, cat. no. 14190). Divide into aliquots and store up to 3 months
at −20◦ C.

Blocking solution
Supplement FACS buffer (see recipe) with 5% (v/v) donkey or goat serum (Sigma).
Prepare fresh.

Collagenase IV, 1 mg/ml


Add 6 mg (∼1200 U) of collagenase IV (Invitrogen) to 6 ml of KO medium (see
recipe) for each 6-well plate to be used. Filter through a 0.22-µm filter. Prepare Stem Cells
fresh as needed.
23.7.11
Current Protocols in Cell Biology Supplement 36
DNase, 2.35 mg/ml
Dissolve 2.35 mg (∼4700 U) of DNase (Sigma) in 1 ml of PBS (Invitrogen, cat.
no. 14190), aliquot and store up to 2 months at −20◦ C.

FACS buffer
PBS (Invitrogen, cat. no. 14190) supplemented with:
0.1% (w/v) BSA (Sigma)
0.05% (w/v) sodium azide (Na N3 )
Store up to 3 months at −20◦ C
Feeder cell medium
High-glucose DMEM (Invitrogen) supplemented with:
10% (v/v) fetal bovine serum (FBS; Hyclone)
2 mM L- glutamine (Invitrogen)
50 U/ml penicillin/50 µg/ml streptomycin (Invitrogen)
Store up to 2 weeks at 4◦ C
Gelatin, 0.1% (w/v)
Stock solution: (1% gelatin): Dissolve 0.25 g gelatin powder (Sigma) in 25 ml
distilled water in a 50-ml tube (Falcon) to obtain a 1% (w/v) gelatin stock. Autoclave
and store as 25-ml aliquots up to 1 year at 4◦ C.

Working solution (0.1% gelatin): Dilute 25 ml of 1% (w/v) gelatin in 225 ml distilled


water. Store up to 4 weeks at 4◦ C.

KnockOut (KO) medium


KnockOut DMEM (Invitrogen) supplemented with:
14% (v/v) KnockOut Serum Replacement (Invitrogen)
2 mM L-glutamine (Invitrogen)
1% (v/v) nonessential amino acids (Invitrogen)
50 U/ml penicillin/50 µg/ml streptomycin (Invitrogen)
4 ng/ml bFGF: add 1 µl of 20 µg/ml bFGF (see recipe) per 5 ml KO medium to be
prepared
Store up 2 weeks at 4◦ C
Batch testing of KnockOut serum replacement is imperative since there is variability,
in the author’s experience, with regard to the potential of different batches to support
undifferentiated proliferation of hESCs.

Laminin, 4 µg/ml
Stock solution (1 mg/ml laminin): Prepare 1 mg/ml laminin (Sigma). Divide into
40-µl aliquots and store up to 1 year at −20◦ C.

Working solution (4 µg/ml laminin): Dilute 40 µl of 1 mg/ml laminin stock with


10 ml of PBS (Invitrogen, cat. no. 14190), for a final concentration of 4 µg/ml.
Prepare fresh.

Low-melting-temperature (LMT) agarose, 0.1% (w/v)


Dissolve 0.2 g low-melting-temperature agarose (FMC BioProducts) in 200 ml
distilled water. Store up to 3 months at room temperature. Boil the solution in a
microwave oven for 1 to 2 min before each usage.

Neural Culture dishes are pretreated with LMT agarose to prevent adhesion of cell clusters to their
Differentiation of surfaces.
Human ES Cells

23.7.12
Supplement 36 Current Protocols in Cell Biology
Mitomycin C, 2 mg/ml
Dissolve a 2 mg ampule of mitomycin C (Sigma) in 1 ml of PBS (Invitrogen, cat.
no. 14190). Inject the PBS into the mitomycin C ampule using a syringe connected
to a 23-G needle. Use a second needle to vent the ampule. Remove the solution and
store in a 15-ml tube up to 1 week at 4◦ C protected from light.

CAUTION: Mitomycin C is a toxic substance. See manufacturer’s MSDS for han-


dling instructions. Preparation of mitomycin C solutions should be conducted within
an appropriate chemical fume hood, given the toxicity of this reagent.

Neural precursor medium (NPM)


DMEM/F12 (1:1) (Invitrogen) supplemented with:
2% (v/v) B27 supplement (Invitrogen, cat. no. 17504)
2 mM L-glutamine (Invitrogen)
50 U/ml penicillin/50 µg/ml streptomycin (Invitrogen)
Store up to 2 weeks at 4◦ C
B27 supplement includes a low concentration of retinol. Since retinoic acid may restrict
the developmental potential of NPs, B27 supplement that does not contain retinol (B27
supplement minus vitamin A; Invitrogen, cat. no., 12587) may be substituted.

Paraformaldehyde, 4% (w/v)
Dissolve 4 g of paraformaldehyde powder (Fluka) in 100 ml of PBS (Invitrogen,
cat. no. 14190) at 60◦ C. Calibrate the pH to 7.4, filter, divide into aliquots, and store
in a −20◦ C freezer for up to 6 months.

Permeabilization buffer
Supplement FACS buffer (see recipe) with 0.1% (v/v) Triton X-100 (Sigma).
Prepare fresh.

Poly-D-lysine, 10 µg/ml
Stock solution (1 mg/ml poly-D-lysine): Dissolve 1 mg of poly-D-lysine (30 to 70
kDa; Sigma) in 1 ml of PBS (Invitrogen, cat. no. 14190), divide into 100-µl aliquots
and store up to 2 months at −20◦ C.

Working solution (10 µg/ml poly-D-lysine) Dilute 100 µl of 1 mg/ml poly-D-lysine


with 10 ml of PBS to a final concentration of 10 µg/ml. Prepare fresh.

Propidium iodide solution, 2 µg/ml


Dilute 1 volume of 1 mg/ml propidium iodide (Sigma) in 500 volumes of FACS
buffer (see recipe) and store at 4◦ C up to 3 months.

Noggin stock, 100 µg/ml


Dissolve 50 µg of recombinant mouse noggin (R&D Systems) by adding 500 µl of
sterile 0.1% (w/v) BSA (Sigma) in PBS (Invitrogen, cat. no. 14190). Divide into
aliquots and store up to 3 months at −20◦ C.

Triton X-100, 0.2% (v/v)


Dilute 1 volume of Triton X-100 (Sigma) in 49 volumes of PBS (Invitrogen, cat.
no. 14190) and store at room temperature up to 6 months.

Stem Cells

23.7.13
Current Protocols in Cell Biology Supplement 36
Trypsin/EDTA solutions
0.04% (w/v) trypsin/0.16 mM EDTA: Dilute 1 volume of 0.25% (w/v) trypsin/EDTA
(Invitrogen) in 5 volumes of PBS (Invitrogen, cat. no. 14190). Store at 4◦ C for up
to 1 week.

0.008% (w/v) trypsin/2.4 mM EDTA: Dilute 1 volume of 0.04% (w/v) trypsin/


0.16 mM EDTA (prepared as described above) in 4 volumes of 3 mM disodium
EDTA in PBS. Keep at 4◦ C for up to 1 week.

COMMENTARY
Background Information In addition to methods involving initial
Controlled differentiation of ES cells into spontaneous differentiation within EBs, meth-
neural precursors (NPs) is required for the ex- ods for directed differentiation of ES cells cul-
perimental dissection of the molecular events tured as individual cells or in a monolayer
that occur during early development of the ner- have been developed by several investiga-
vous system. Moreover, the generation of pure tors. These approaches use specific mesoderm-
populations of hESC-derived neural progeny, derived feeder cells or conditioned media to
rather than mixed populations of differenti- induce neural differentiation. The rationale be-
ated cells, is one of the major requirements hind coculturing with mesodermal cells is that
for transplantation therapy (Li et al., 1998; signals from the mesoderm are required to in-
Stavridis and Smith, 2003). duce neural specification of the ectoderm in
vivo. Coculture of undifferentiated mouse ES
Derivation of NPs from mouse embryonic cells with the bone marrow–derived stromal
stem cells cell line PA6, under serum-free conditions or
During almost three decades of mouse ES as a suspension culture in medium conditioned
cell research, several methods have been pro- by the human hepatocellular carcinoma cell
posed for the conversion of ES cells into line HepG2, induces neural differentiation in
NPs. The most commonly used approach for a high percentage of the colonies (Kawasaki
induction of neural differentiation of mouse et al., 2000; Rathjen et al., 2002). However, the
ES cells includes initial spontaneous differ- neuralizing agents exerted by PA6 or HepG2
entiation within embryoid bodies (EBs) fol- lines remain unidentified as yet.
lowed by treatment with retinoic acid (RA). Given the limitations of most approaches
EBs are formed when ES cells are cultured as for directing the differentiation of ES cells into
free-floating clusters in the absence of feeder a homogeneous population of NPs, comple-
cells and anti-differentiation agents such as mentary strategies to select neural cells from
leukemia inhibitory factor (LIF). Under these a heterogeneous population of differentiated
conditions, spontaneous differentiation oc- cells have been developed. Enrichment for
curs, partially mimicking early processes of NPs may be accomplished in the mouse ES
differentiation in the embryo, and therefore cell system by incorporating selective culture
these clusters are termed EBs. Spontaneous conditions. In this approach, ES cells are first
differentiation of mouse ES cells within EBs cultured as aggregates to initiate spontaneous
yields a relatively small fraction of neural- differentiation. They are then plated and cul-
lineage cells. To promote neural differentia- tured on an adhesive substrate in a serum-free
tion, ES cell aggregates are cultured first in medium. Under these selective conditions, the
the regular ES cell medium without LIF for 4 majority of cells die while NPs survive. The
days and are then exposed to RA for another 4 medium is then supplemented with bFGF to
days. Hence, this method is often regarded as induce proliferation. After 6 to 8 days of se-
a "4–/4+" protocol (Bain et al., 1995). While lection and expansion, the NP cells are en-
RA treatment of EBs promotes neural differ- riched to ∼80% (Okabe et al., 1996; Brustle
entiation, the neural progeny that are formed et al., 1997). Withdrawal of bFGF induces
are of a wide range of developmental stages spontaneous differentiation into various sub-
and have a restricted developmental potential. types of neurons and glia cells (Okabe et al.,
This is supported by the study of Renoncourt 1996; Brustle et al., 1999). The neurons are
et al. (1998), which showed that EBs treated mature and electrophysiologically functional,
Neural with RA selectively differentiated into neu-
Differentiation of and can generate both excitatory and inhibitory
Human ES Cells ronal cell types characteristic of ventral CNS. synaptic connections. In contrast to the RA

23.7.14
Supplement 36 Current Protocols in Cell Biology
approach, NPs that are derived with the bFGF Recently, pure expandable cultures of neu-
protocol are more synchronized with regard to ral stem cells were derived from mouse ES
their stage of differentiation, and their devel- cells. Adherent monolayer cultures of ES cells
opmental potential is less restricted. In an alter- initially differentiated into the neural lineage
native approach, neural cells have been sorted under defined culture conditions, which lack
from heterogeneous populations of differenti- inductive signals for non-neural fates (Ying
ated cells based on the expression of neural et al., 2003). Basal culture medium supple-
lineage-specific cell surface markers (Mujtaba mented with epidermal growth factor (EGF)
et al., 1999), or by genetic selection. In the lat- and bFGF promoted the selection and pro-
ter approach, a selectable marker was inserted longed propagation of pure adherent cultures
into the open reading frame of genes encod- of neural stem cells. This defined monoculture
ing neural lineage-specific transcription fac- system can allow, for the first time, sustained
tors, allowing genetic selection of NPs either robust expansion of neural stem cells, closely
by fluorescence-activated cell sorting (FACS) related to a radial glia lineage, liberated from
or drug selection (Li et al., 1998; Ying et al., any requirement for a specific cellular niche
2003). (Conti et al., 2005).
The inductive exocrine signals, such as
those that are obtained from coculture or con- Derivation of NPs from human embryonic
ditioned medium, are probably not required stem cells
for mouse ES cells to commit themselves ef- While hESCs are similar to their mouse
ficiently to the neural fate. Multicellular ag- counterparts, they do differ in many aspects
gregates (Wiles and Johansson, 1999) or ad- (Thomson and Odorico, 2000; Ginis et al.,
herent monolayer cultures of ES cells (Ying 2004). Therefore, the protocols for neural in-
et al., 2003) readily differentiated into neu- duction of hESCs share many of the princi-
ral cells when LIF and inductive signals for ples that apply to mouse ES cells, though there
non-neural fates were eliminated. In the latter are aspects that are specific to the human sys-
report, it was shown that the neural differen- tem. In the last 5 years, various approaches
tiation was not a simple default pathway but have been developed for the derivation of en-
was dependent on autocrine fibroblast growth riched cultures of proliferating developmen-
factor (FGF) signaling. This specific culture tally competent NPs from hESCs. Due to the
system could induce neural differentiation in poor survival of hESCs in single-cell suspen-
∼60% of the ES cells, but could not give rise to sion culture conditions, direct neural differen-
a highly enriched preparation of NPs. A sim- tiation procedures from disaggregated individ-
ilar result was demonstrated when mouse ES ual hESCs, similar to those described above for
cells were disaggregated into individual cells mouse ES cells, have not been reported thus
and were cultured at low density, under defined far.
serum-free factor-free culture conditions. In The initial protocols for the derivation of
the absence of non-neural inductive signals, NPs from hESCs involved, as a first step,
the surviving individual cells acquired a neu- spontaneous disorganized differentiation that
ral identity, and gave rise to primitive neural was induced by prolonged culture of hESC
stem cells (Tropepe et al., 2001; Smukler et al., colonies at high density or by the forma-
2006). Differentiation towards neural fate was tion of floating three-dimensional EB-like ag-
more efficient in the presence of the BMP an- gregates. This initial differentiation was fol-
tagonist noggin. Neuralization in these reports lowed by neural lineage selection by various
was independent of FGF signaling, which is in approaches and culture under conditions that
line with a default mechanism of neural spec- promoted the proliferation of NPs (Carpenter
ification (Wilson and Edlund, 2001; Munoz- et al., 2001; Reubinoff et al., 2001; Zhang
Sanjuan and Brivanlou, 2002). Nevertheless, et al., 2001). Two research groups induced
the efficiency of this approach is relatively initial uncontrolled spontaneous differentia-
low (Tropepe et al., 2001) and in the pres- tion of the hESCs through the formation
ence of survival factors [N-acetyl-L-cysteine of EBs, and subsequently plated the cells
(NAC) and cAMP], ∼20% of the starting pop- on appropriate substrates in defined medium
ulation of undifferentiated ES cells will differ- containing mitogens (Carpenter et al., 2001;
entiate into neural stem cells (Smukler et al., Zhang et al., 2001). Carpenter et al. (2001)
2006). These chemically defined neural dif- used the traditional mouse ES cell differen-
ferentiation systems facilitate the dissection of tiation protocol by RA treatment to derive
the molecular mechanisms of early neural dif- NPs from hESC. The resulting hESC EBs
Stem Cells
ferentiation. were treated with RA in serum-containing
23.7.15
Current Protocols in Cell Biology Supplement 36
medium for 4 days to induce initial differ- petent NPs. Upon transplantation into the
entiation to NPs. NPs were then selected brain ventricles of newborn mice, the NPs
by immunosorting, based on the expression migrated extensively and differentiated in a
of neural-specific cell-surface markers. This region-specific manner to progeny represent-
method yielded heterogeneous neural cell pop- ing the three major neural lineages (Reubi-
ulations similar to those observed in mouse noff et al., 2001). In further studies, the au-
ES cells. The neural cells generated by this thors have developed an alternative simple
approach displayed a wide range of devel- one-step approach for directed controlled dif-
opmental stages, from nestin-expressing and ferentiation of hESCs into NPs. In this proto-
polysialylated neural cell adhesion molecule col, small hESC colonies are removed from the
(PSA-NCAM)–expressing precursors to β- feeder layer and cultured in defined serum-free
tubulin III+ neurons. Zhang and colleagues medium supplemented with noggin and bFGF.
used a process that initiated uncontrolled spon- The hESC clusters differentiate almost uni-
taneous differentiation of hESC within EBs formly into NPs. The authors have shown that
cultured in serum-free medium for 4 days. noggin, which inhibited BMP signaling, sig-
The EBs were then plated onto an adhesive nificantly enhanced the level of enrichment for
culture plate for 7 days’ culture in a serum- NPs within the hESC clusters and suppressed
free medium supplemented with bFGF, which the expression of transcripts of markers of
supported the cultivation of NPs. Cells in the non-neural lineages. Hence, it was suggested
center of the plated EBs transformed initially that noggin-mediated blockage of endogenous
into small columnar cells, whereas those in the BMP signaling, within the hESC clusters, sup-
periphery of the outgrowth gradually became pressed the differentiation into lineages other
flattened. The small columnar cell population than the neural one (Itsykson et al., 2005). The
expanded in the presence of FGF2 and orga- potential of noggin to promote neural differen-
nized into rosette formations by 7 to 10 days af- tiation was also demonstrated by others with
ter plating the aggregates. The neural tube–like adherent cultures of hESCs rather than free-
structures were isolated by selective enzymatic floating clusters (Gerrard et al., 2005).
digestion followed by further purification on In addition to noggin, the authors’ protocol
the basis of differential adhesion (Zhang et al., included the use of bFGF. FGF signaling is
2001; Li and Zhang, 2006). After transplanta- essential for neural specification in planarian
tion into the neonatal mouse brain, the prim- (Cebria et al., 2002), frog (Launay et al., 1996),
itive neuroectodermal cells differentiated in a and avian embryos (Streit et al., 2000; Wilson
region-specific manner into neurons and glia et al., 2000). In the mouse ES cell system,
cells. (Zhang et al., 2001; Guillaume et al., autocrine FGF signaling was shown to have
2006). Recently, this group further demon- a role in neural l specification (Ying et al.,
strated the potential to direct the differentiation 2003). However more recent data suggest that
of the NPs in vitro into dopaminergic neurons neural differentiation of mouse ES cells oc-
with midbrain properties and into motor neu- curs in the absence of FGF signaling (Smukler
rons with spinal cord characteristics (Li et al., et al., 2006). While FGF signaling induced the
2005; Yan et al., 2005). proliferation of the hESC-derived NPs (Itsyk-
The authors of this unit have reported an son et al., 2005), its role in neural induction of
alternative approach where initial spontaneous hESCs is unclear at present.
differentiation was induced by prolonged cul- Similar to mouse ES cells, coculture of
ture of the hESC colonies to high density with- hESCs with mouse stromal cells can effec-
out replenishment of the feeders. Within the tively induce neural differentiation (Muotri
large colonies that were formed, there were a et al., 2005; Tabar et al., 2005). The factor
variety of differentiated cells, including dis- or factors that exert the stromal cell–derived
tinct areas comprised of small, piled, tightly- inducing activity (commonly termed SDIA)
packed early precursor cells that were destined have not yet been identified. Recently, a simi-
to give rise to neural progenitors when trans- lar effect was demonstrated when both mouse
ferred to serum-free media. Clusters of ∼150 and human ES cells were cultured on the ma-
cells were mechanically dissected from these trix components of the human amniotic mem-
distinct areas and were plated in serum-free brane (amniotic membrane matrix-based ES
defined medium supplemented with mitogens cell differentiation; AMED; Ueno et al., 2006).
(FGF2 and EGF). The aggregates gradually In contrast to the SDIA method, which uses an-
Neural turned into round spheres that were highly en- imal cells, the AMED culture uses a noncellu-
Differentiation of riched for proliferating, developmentally com- lar inductive material derived from an easily
Human ES Cells

23.7.16
Supplement 36 Current Protocols in Cell Biology
available human tissue; therefore, AMED These encouraging results suggest that hESC-
should provide a more suitable and versa- derived NPs may eventually be applicable to
tile system for generating a variety of neural cell and gene therapy of human neurological
tissues for future clinical applications (Ueno disorders. Long-term studies are required to
et al., 2006). Lastly, the role of the Notch sig- determine the safety of hESC-derived neural
naling system in promoting the entry into the progeny transplantation and to rule out po-
neural lineage was recently demonstrated. Co- tential hazards such as tumor formation (Roy
culturing of mouse and human ES cells with et al., 2006) or the development of cells from
genetically modified stromal cells expressing other lineages. These pioneer transplantation
Notch ligand stimulated neural specification studies highlight the potential of hESCs to
(Lowell et al., 2006). serve in the future as an unlimited donor source
The major advantages of the Basic Protocol of neural cells for transplantation.
in this unit are the use of a chemically defined
culture system as opposed to serum-containing Critical Parameters
systems or coculture with unknown factors The starting hESCs should be passaged two
generated by stromal, amniotic, or feeder cells to three times on feeder cells after thawing, be-
(Shin et al., 2006). Neural differentiation is fore being used for NP derivation. The hESC
obtained in one controlled step, which con- cultures should exhibit a minimal level of
sistently gives rise to highly enriched popula- background differentiation. During those pas-
tions of expandable, developmentally compe- sages, assess the number of cells that should
tent NPs within spheres. The authors’ system, be plated per well at the last passage prior to
as well as those that induce neural differentia- the derivation of the NPs (4–6 × 105 cells per
tion of hESCs in monolayer cultures (Gerrard well), so that the hESC colonies, after 6 to 7
et al., 2005; Shin et al., 2006; Ueno et al., days of culturing, would be of sufficient size
2006), are invaluable for the dissection of the to enable their survival as clusters, but not so
molecular mechanisms of early human neural large as to adhere to one another.
specification and differentiation.

Potential applications of hESC-derived Troubleshooting


neural precursors If differentiating hESC clusters attach to
The establishment of neuroectodermal pre- the plastic bottoms of the wells, it is advis-
cursors from hESCs provides real advantages able to transfer them to new wells precoated
for basic and applied studies of human neural with 0.1% gelatin. Gently blow medium from a
development and diseases. Directing hESCs pipet on top of the hESC clusters to detach the
to differentiate to the neural lineage and the clusters from the plastic surface, and transfer
establishment of NPs and their differentiated them to a fresh well precoated with gelatin.
progeny enable a complete in vitro study of If hESC clusters begin to aggregate one to
human neurogenesis. This approach allows ac- the other, gently disperse them by gentle pipet-
cess to hitherto unexplored territories of gene ting or dissect them with surgical blades.
expression for modern genomics data mining,
and will provide a platform for the discov- Anticipated Results
ery of polypeptide growth and differentiation The derivation of NPs from hESCs with the
factors which might find application in neu- protocol described in this unit is very efficient
ral tissue regeneration. In vitro human models and reproducible. A significant amount of cell
of neurodegenerative diseases may be created death is expected during the first 2 to 3 days of
for basic research and drug discovery. New culturing of the hESC clusters in suspension.
assays for toxicology and high-throughput However, after an additional few days, an in-
screens for neuroprotective compounds may crease in size of the clusters is observed due to
be developed. cell proliferation.
Generation of NPs from hESCs in vitro may After 4 weeks, 97% of the cells within the
serve as a platform for further manipulations cultures should express NP markers such as
with growth and differentiating factors that Nestin and NCAM, and <2% of the cells
may eventually enable the derivation of spe- should express markers of pluripotent cells
cific functional neural cells for transplantation such as Oct-4, Tra-1-81, and SSEA-3.
therapy. The proof of principle of this poten- The NPs can differentiate into neurons and
tial application was demonstrated both with glial cells both in vitro under various culture
mouse (Brustle et al., 1999; Kim et al., 2002) conditions, as well as in vivo after transplan-
and human (Roy et al., 2006) ES cell systems. tation. Stem Cells

23.7.17
Current Protocols in Cell Biology Supplement 36
While the NPs differentiate mainly into Brustle, O., Jones, K.N., Learish, R.D., Karram, K.,
neurons at early culture stages, during pro- Choudhary, K., Wiestler, O.D., Duncan, I.D.,
and McKay, R.D. 1999. Embryonic stem cell-
longed propagation (25 weeks), a gradual shift
derived glial precursors: A source of myelinat-
in differentiation from a neuronal to a predom- ing transplants. Science 285:754-756.
inantly glial fate occurs (Itsykson et al., 2005).
Carpenter, M.K., Inokuma, M.S., Denham, J.,
This finding is consistent with the concept of a Mujtaba, T., Chiu, C.P., and Rao, M.S. 2001.
gradual shift from predominantly neuronogen- Enrichment of neurons and neural precursors
esis to gliogenesis during neural development from human embryonic stem cells. Exp. Neurol.
in vivo (Nakai and Fujita, 1994; Encha-Razavi 172:383-397.
and Sonigo, 2003; Fujita, 2003). Cebria, F., Kobayashi, C., Umesono, Y., Nakazawa,
M., Mineta, K., Ikeo, K., Gojobori, T., Itoh, M.,
Time Considerations Taira, M., Sanchez Alvarado, A., and Agata, K.
Approximately 0.5 to 1 hr should be al- 2002. FGFR-related gene nou-darake restricts
brain tissues to the head region of planarians.
lowed for the preparation of the media and
Nature 419:620-624.
various other factors and reagents. This may
Conti, L., Pollard, S.M., Gorba, T., Reitano, E.,
be performed up to 2 weeks before the protocol
Toselli, M., Biella, G., Sun, Y., Sanzone, S.,
is initiated. Ying, Q.L., Cattaneo, E., and Smith, A. 2005.
The time period for the whole process from Niche-independent symmetrical self-renewal of
the establishment of feeders through deriva- a mammalian tissue stem cell. PloS Biol 3:283.
tion of the NPs and their differentiation in vitro Encha-Razavi, F. and Sonigo, P. 2003. Features of
is 8 weeks. the developing brain. Child’s Nerv. Syst. 19:426-
Foreskin feeders are propagated 3 to 4 days 428.
before their inactivation by mitomycin C. Fujita, S. 2003. The discovery of the matrix cell,
About 30 min should be allowed for the human the identification of the multipotent neural stem
cell and the development of the central nervous
foreskin fibroblasts passaging procedure.
system, Cell Struct. Funct. 28:205-228.
Preparation of mitomycin C inactivated
Gerrard, L., Rodgers, L., and Cui, W. 2005. Dif-
feeder layers takes ∼3 hr. The hESCs are
ferentiation of human embryonic stem cells to
plated on the feeders ∼1 to 4 days after their neural lineages in adherent culture by block-
preparation. About 0.5 to 2.5 hr should be al- ing bone morphogenetic protein signaling. Stem
lowed for hESC passaging. Cells 23:1234-1241.
The hESCs are cocultured with the feeders Ginis, I., Luo, Y., Miura, T., Thies, S.,
6 to 7 days, until they are removed as clusters Brandenberger, R., Gerecht-Nir, S., Amit, M.,
for induction of neural differentiation. During Hoke, A., Carpenter, M.K., Itskovitz Eldor,
J., and Rao, M.S., 2004. Differences between
this week, medium must be changed every day.
human and mouse embryonic stem cells. Dev.
Changing the medium can take between 5 and Biol. 269:360-380.
20 min, depending on the number of plates.
Guillaume, D.J., Johnson, M.A., Li, X.J., and
Removing the hESC colonies from the Zhang, S.C. 2006. Human embryonic stem cell-
feeders should take ∼1 to 3 hr. derived neural precursors develop into neurons
Neural induction of the hESC clusters into and integrate into the host brain. J. Neurosci.
neural spheres highly enriched for NPs takes 4 Res. 84:1165-1176.
weeks. Spontaneous differentiation of the NPs Itsykson, P., Ilouz, N., Turetsky, T., Goldstein,
into neurons and glial cells occurs within an R.S., Pera, M.F., Fishbein, I., Segal, M., and
Reubinoff, B.E. 2005. Derivation of neural pre-
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Neural
Differentiation of
Human ES Cells

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Supplement 36 Current Protocols in Cell Biology