SYMPOSIUM: Stem Cells

Neural Subtype Specification from Embryonic Stem Cells

Su-Chun Zhang, MD, PhD

Departments of Anatomy and Neurology, School of Medicine and Public Health, Waisman Center, Wisconsin Stem Cell Research Program, WiCell Institute,
University of Wisconsin, Madison, Wis.

Corresponding author:
Su-Chun Zhang, MD, PhD, Departments of Anatomy and Neurology, School of Medicine and Public Health, Waisman Center, University of Wisconsin, Madison,
1500 Highland Avenue, Madison, WI 53705 (E-mail: Zhang@waisman.wisc.edu)

One of the keys to using embryonic stem cells (ESCs) in brain research and potential (BMPs) and Wnts (54, 66). FGFs may
application in neurological diseases is directed differentiation of neuronal and glial instruct a “pro” neural state at an early
subtypes. This may be achieved by application of developmental principles in guiding stage whereas BMP antagonists may subse-
cell lineage specification from naïve stem cells. Establishment of defined ESC differen- quently solidify the neural identity. Inacti-
tiation models that recapitulate in vivo development, especially from human ESCs, will
vation of Wnts appears to be prerequisite
most likely provide a dynamic tool for dissecting molecular mechanisms underlying
for orchestrating FGF and anti-BMP sig-
early embryonic development that is otherwise not readily obtainable. This is also a
rational and realistic way of producing enriched populations of functional neurons and nals in neuroectodermal specification, and
glia for pathological analyses as well as possible therapeutic applications. the presence of Wnts limits or forms the
boundary of the neural plate. Thus, these
Brain Pathol 2006;16:132–142. signaling pathways play a spatially and
temporarily differential role in specifying
the neuroectoderm. Limited studies using
Embryonic stem cells (ESCs), derived from summarize the success and failure of our mouse (2, 60, 70) and human (19, 26, 43,
the inner cell mass of the early embryo, are ability to direct ESCs to neurons and glial 77) ESC as a model system also indicate
capable of producing all cell types that cells with a focus on a few extensively pur- the involvement of these pathways in
make up an organism. Generation of sued cell types and differentiation systems. mammalian neuroepithelial differentia-
specialized cell types from ESCs in vitro tion. It will be important to apply appro-
as well as in vivo (producing teratoma) DEVELOPMENTAL PRINCIPLES AS priate soluble factors at the right time in
is essentially a recapitulation of early GUIDELINES FOR NEURAL SUBTYPE order to induce a correct type of neuroep-
embryonic developmental processes. The SPECIFICATION FROM ESCS ithelial cells.
ESC differentiation system, especially with
human ESCs, offers a window to mysteries Induction of the neuroectoderm. Neu- Specification of neurons and glia from
underlying early development, such as the rons and glia are born shortly after neuroepithelia. Each neuron in a given
complex brain development that might induction of the neuroectoderm. The location of the brain and spinal cord carries
otherwise be unattainable in other systems. neuroectoderm, in the form of the neural a unique set of transmitter(s) and makes
Similarly, naturally occurring or genetically plate (a sheet of neuroepithelium) and neu- connections with its own target(s). At the
manipulated mutant ESCs may be ral tube, appears at the beginning of the same time, neurons with the same trans-
exploited to unveil developmental disor- second week of mouse embryonic develop- mitter phenotypes reside in different
ders and/or certain pathological processes. ment. In humans, it is easily discernable by regions of the central nervous system
The differentiated progenies such as neural the end of the third week of gestation. The (CNS) exerting distinct functions. It is
cells may also potentially be used for drug biochemical events that lead to neuroecto- thus likely that assignment of the posi-
screening and therapeutic applications in derm differentiation must occur prior to tional identity and specification of the
neurological injuries and diseases. the cellular differentiation, most likely dur- transmitter phenotype is operated by sep-
A key step to unlocking the potential ing or prior to gastrulation, in which cells arate processes. Little is known about how
applications of ESCs is directed differenti- move to form the three primary germ these two parallel pathways are coupled in
ation of functional target cells such as sub- layers. This timeline forms the basis for the specification of neural subtypes. This
classes of neurons and glial cells. How and directing stem cells toward their neural presents a challenge for differentiating
how well are we differentiating functional fate. The molecular mechanism underlying naïve stem cells to neuronal subtypes with
neural cell types from mouse and human neural induction, inferred largely from correct positional and transmitter pheno-
ESCs? Do in vitro differentiation systems studies using xenopus, chick, and other types such as midbrain dopaminergic neu-
thus far devised recapitulate early neural lower vertebrates, appear to involve multi- rons or perhaps the substantia nigra type
development? Are ESC-differentiated neu- ple classic pathways such as activation of of dopaminergic neurons. At present, the
ronal and glial subtypes equivalent to their fibroblast growth factors (FGFs) and/or principle used for directing stem cell dif-
counterparts in the brain? Here I intend to inhibition of bone morphogenetic proteins ferentiation is largely based on the well-

132 Zhang—Neural Subtype Specification from ESCs

© 2006 The Author

Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
characterized positional specification, or
neural patterning.
Because of differential temporal and
spatial exposure to morphogens, the pro-
cess of neuroectoderm formation does not
occur homogenously and simultaneously.
Neuroepithelium forms first in the head
region and gradually extends caudally to
form the entire neural plate. This suggests
that the regional identity of the initially
specified rostral neuroepithelia (or pro-
spective forebrain neuroectoderm) is not
fixed and can be respecified to a more
caudal fate. Meanwhile, the neural plate
begins to fold and fuse dorsally at the
future neck area, which extends both ros-
trally and caudally to form the complete
neural tube. Thus, neuroepithelial cells
are temporally and spatially different from
each other at the time when the neural
plate and neural tube are formed. Accord-
ingly, neuroepithelial or progenitor cells
generated from stem cells in a Petri dish
are likely to be different from each other
depending on the morphogens used. Even
the same morphogen when applied at a
different time or in a different amount will
result in production of different types of
progenitors. Given the differential roles
of these morphogens in neuroepithelial
induction and continued use of these sig-
naling molecules in subsequent specifica-
tion of positional identity and neuronal
and glial fate, one can appreciate the
importance of selecting these soluble fac-
tors for differentiating ESCs to neuroepi-
thelial cells in order to achieve subtype
neural cell differentiation.
Identification of neural subtypes that are
generated in vitro. Identity crisis is the
major contributor to confusion in the field
of stem cell research. When tissues are dis-
aggregated and placed in a Petri dish, the
easiest and the most important criterion
for defining a cell, the positional identity,
is lost. This leads to the reliance on the
use of molecular markers in defining a cell
type. However, dependence on these mark-
ers is not without caveats. It is particularly
problematic for ESC-derived progenies, as
most molecular markers are not limited to
only neural cells but are also expressed by
nonneural cells. Nestin, for example, is a
good neural progenitor marker. However,
it is expressed by human ESCs as well
as differentiated nonneural cells. It thus
requires a set of markers in combination
with morphological and functional indica-
tors to define a cell type. The absence of
certain markers is equally important. Sub-
cellular localization of the markers is often
indicative of the specificity of the reagents
and in some cases suggestive of functional
attributes. Cells under stress conditions
can exhibit binding to many antibodies
against structurally distinct antigens, thus
giving false positive staining. The charac-
teristic neuronal and glial morphology and
the stereotypic neuronal electrochemical
property such as a typical resting mem-
brane potential and sodium-gated action
potential can significantly aid in the iden-
tification of neurons and glial cells (57,
72).
Determination of neuronal subtypes
can be achieved by expression of home-
odomain transcription factors and/or
secretion of transmitters. Most neurotrans-
mitters are simple amino acids or small
peptides. Most cells possess these essential
amino acids and/or their metabolites or
can simply take them up from the culture
medium. Therefore, the simple presence of
these amino acids or peptides does not nec-
essarily indicate they are transmitters. A
whole package is required to define the
transmitter phenotypes, such as synthesiz-
ing and metabolizing enzymes, transport-
ers, etc.
DIFFERENTIATION OF NEUROEPITHELIAL
CELLS
Differentiation of neuroepithelial cells
from ESCs is the prerequisite step and
gatekeeper toward the generation of neu-
ronal and glial classes. In the developing
embryo, neuroepithelial cells are segre-
gated into rostro-caudal and dorso-ventral
domains by the time of neural tube closure,
where they are fated to different progenitor
pools. This means that naïve neuroepithe-
lial cells in the early neural plate, in
response to locally derived signals, differ-
entiate into region-specific progenitors. In
analogy, neural progenitors differentiated
from ESCs in a Petri dish will adopt a
regional identity depending upon the
presence of morphogens at the time
the precursors are responsive. Hence, the
neuroepithelial cells may represent funda-
mentally different groups of progenitors
even though they can equally produce neu-
rons and glial cells.
Neural Subtype Specification from ESCs—Zhang
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
Position identity and differentiation
potentials of ESC-derived neural
progenitors depend upon
the morphogens used
Forebrain progenitors. In mammals,
especially in primates, the forebrain is the
largest part of the CNS. During develop-
ment, the neuroectoderm at the head
region forms first. Accordingly, one would
expect that neuroepithelial cells with fore-
brain identity can be readily differentiated
from ESCs. Indeed, neuroepithelial cells
differentiated from mouse or human ESCs
initially express forebrain homeodomain
transcription factors such as Otx1/2 and
brain factor 1 (Bf1) but not hox genes that
are generally present in the hindbrain and
spinal cord (26, 65). These cells are likely
primitive neuroepithelial cells, equivalent
to the prospective neuroectodermal cells
that initially form during gastrulation (37,
54). With the presence of mitogens such as
FGF2, which itself can caudalize neuroep-
ithelial cells, it is not surprising that fewer
forebrain progenitors are present in most
of the neural differentiation cultures. This
may explain why few reports on differenti-
ation of forebrain neuroepithelial cells
from ESCs are available and why genera-
tion of forebrain progenitors is often
viewed as a difficult task. It has been sug-
gested that neuroepithelial cells generated
from mouse ESCs after the removal of leu-
kemia inhibitory factor (LIF) and addition
of FGF2 are likely forebrain progenitors
(60), although it was not confirmed
whether these progenitors carry forebrain
but not hindbrain and spinal cord home-
odomain markers. Sasai and colleagues
have recently shown that the proportion of
mouse ESC-derived progenitors with fore-
brain characters, as indicated by the expres-
sion of Bf1, can be increased to 30% when
the Wnt signaling is blocked by dkk1 (62).
Wnts, as well as retinoic acid (RA) and
FGFs, are implicated in the caudalization
of the neuroectoderm during develop-
ment. Ying and colleagues have developed
a simple neural differentiation protocol by
removal of LIF (70). The neuroepithelial
cells generated in this way appear to possess
some of the forebrain progenitor character-
istics and produce gamma-aminobutyric
acid (GABA) neurons (12) (see below). We
have shown that human ESC-derived neu-
roepithelial cells in a chemically defined
condition exhibit almost exclusively the
133
© 2006 The Author
forebrain identity such as expression of
Otx2 and Bf1 but not hox genes, at least
at the primitive neuroepithelial stage (26).
Manipulation of these forebrain precursors
will open an avenue for specification of a
rich array of neuronal subtypes that are
present in the forebrain.
Posterior progenitors. Neural progenitors
differentiated from ESCs by treatment
with RA (0.1–1 M) display hindbrain and
spinal cord phenotypes assessed based on
expression of a host of hox genes (62, 65).
Treatment of ESCs with RA has been the
most commonly used approach for neural
differentiation from mouse ESCs (4, 14,
55) because of its simplicity and high effi-
ciency. RA does not appear to act as a
neural inducer in this system. Instead, it
acts mainly to caudalize the neuroepithelial
cells. This is because the neuroectodermal
fate, based on the expression of the defin-
itive neuroectoderm transcription factor
Sox1 and nestin, is rapidly induced 1–2
days following the removal of the self-
renewing factor LIF from ESCs (60, 70),
whereas RA is usually added to the differ-
entiation culture 2–4 days later (31). Neu-
ral differentiation through treatment with
RA is particularly useful for generation of
motor neurons and other cell types that are
located in the brainstem and the spinal
cord. RA promotes the expression of Hox
genes but suppresses forebrain genes such
as Otx2 and Bf-1 in a dose-dependent
manner (26, 65). By adjusting the concen-
tration of RA, neural progenitors may be
biased toward hindbrain or spinal cord
fate. However, RA alone does not appear
to induce further caudal (thoracic and
lumbar) spinal cord fate. The motor neu-
rons generated from mouse ESCs in the
presence of RA mostly manifest upper cer-
vical spinal cord phenotypes by expressing
HoxC5 (65). Those derived from human
ESCs following FGF2 and subsequent RA
treatment display lower cervical spinal cord
phenotypes by expressing HoxC8 (26).
This is reminiscent of the finding from
embryologic studies that RA promotes the
upper cervical but represses thoracic home-
odomain gene expression (29). Thus, RA
alone will not be sufficient to specify pro-
genitors within all the spinal cord seg-
ments. If cells with more posterior spinal
cord phenotypes are desired, other mor-
phogens such as FGFs, GDF11 or these
134 Neural Subtype Specification from ESCs—Zhang
© 2006 The Author
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
morphogens in combination with RA may
be needed. By the same token, RA may
need to be avoided or its concentration
substantially reduced in the culture if cell
types with forebrain phenotypes are the
differentiated target.
Progenitors with mid/hind brain charac-
ters. The fact that differentiation of pro-
genitors with the seemingly uniform spinal
cord characteristics requires discrete sets of
signaling implies that it will not be trivial
to specify a more restricted mid/hind brain
fate from ESCs. Interestingly, specification
of midbrain progenitors from ESCs has
been a major focus of ESC neural differen-
tiation. It is well established that pattern-
ing of mid/hind brain region is controlled
by signals released in the isthmus area,
mainly FGF8. FGF8a is preferentially
localized to the midbrain region whereas
FGF8b mainly in the hindbrain region.
Both can bind to an FGF receptor but the
affinity of the b isoform is about 100 times
higher than that of the a isoform (21, 27,
28). Treatment of mouse ESCs with FGF8
b, that is commercially available, indeed
promotes the differentiation of progenitors
with mid/hindbrain phenotype, as deter-
mined by the expression of Engrailed 1
(En-1) and Pax2 (5, 25, 44, 68). Given
the expression of En-1 in mid/hind brain
region and Pax2 preferentially in the hind-
brain region, it is conceivable that the
neural progenitors induced by FGF8
treatment may represent more hindbrain
progenitors than midbrain cells. This may
explain why most of the dopamine neurons
differentiated from mouse and human
ESCs using this “widely accepted standard
protocol” do not possess critical midbrain
dopamine neuron phenotypes, such as
expression of Lmx1a/b, Nurr1 and ptx3.
This illustrates the necessity of inducing
correct types of neural progenitors in an
attempt to achieve target neuronal differ-
entiation. It also points to a need to modify
the existing protocols based on the devel-
opmental finding described above in order
to specify ESCs to ventral midbrain pro-
genitors that will ultimately give rise to
dopamine neurons that are lost in Parkin-
son’s disease.
From the above brief account on differ-
entiation of neuroepithelial cells and neu-
ral progenitors from ESCs using various
inducing factors, it becomes obvious that
neural progenitors differ from each other
significantly despite a similarity in mor-
phology, expression of common progenitor
markers, and their ability to differentiate
into neurons and glial cells. RA treatment
tends to generate neural cells that are fated
to hindbrain and spinal cord identities.
FGF8 appears to favor the differentiation
of neural progenitors with mid/hind brain
characteristics, albeit with limited potency
by itself. FGF2-induced neural progenitors
appear to be a mixed population exhibiting
phenotypes ranging from the forebrain to
spinal cord characteristics. Other morpho-
gens such as noggin, an antagonist of trans-
forming growth factor β (TGFβ) family,
have also been shown to efficiently pro-
mote neuroepithelial differentiation (19,
43). Similarly, mouse ESCs readily differ-
entiate to neuroepithelial cells when the
ESCs are dissociated into single cells and
cultured at a low density. This culture sys-
tem is designed to remove the inhibitory
signaling of TGFβ family thus promoting
neural differentiation (60). It is not clear at
present what positional identity these neu-
ral progenitors exhibit.
Dorsal-ventral phenotypes of neural pro-
genitors. I have so far only summarized the
neural precursors that exhibit broad
rostral–caudal positional characteristics. In
order to specify subtypes of neurons and
glia, precursor cells need to be further
restricted to a narrower rostral–caudal
region such as a certain segment of the
spinal cord. This can be potentially
achieved by adjusting the concentrations of
a morphogen or combination of morpho-
gens, as in the case of progenitors of differ-
ent sub-regions of the spinal cord induced
by RA or FGFs and GDF11. Further
restriction by dorsal–ventral morphogens
such as BMPs, Wnts, and sonic hedgehog
(SHH) may limit progenitors to a state
that is equivalent to a specific dorsal ventral
domain of the neural tube. RA and SHH
at an appropriate concentration enrich for
progenitors that express Olig2 (26, 65),
a transcription factor expressed by motor
neuron progenitors at the pMN domain of
the ventral neural tube (58, 79). Similar to
what we have learned from chick embryo
studies, the ventralizing morphogen SHH
activates the transcription of Nkx6.1 and
Nkx2.2 but represses Irx3 and Pax6, which
coordinate to activate Olig2 expression. In
practice, both the activated and repressed
genes may be good indicators of whether
the choice of morphogens is appropriate
for induction of a particular neural progen-
itor. Similarly, FGF8 and SHH, at a cor-
rect isoform and concentration, may favor
the induction of progenitors that are nor-
mally residing in the ventral midbrain or
hindbrain, thus facilitating the generation
of midbrain dopaminergic neurons or
hindbrain serotonin neurons (5). FGF8
and SHH are also required for specifying
ventral forebrain progenitors (15, 47, 49).
This would mean that additional factors
such as timing, space, quantity of the same
factors or other molecules, are needed to
differentiate subtypes of progenitors. In
contrast to SHH, BMP, applied right fol-
lowing neuroepithelial induction, results
in differentiation of neural crest-like cells
(35). Tuning the correct combination of
morphogens at the appropriate concentra-
tion and at the right time is not trivial and
often is only determined via trial and error.
Can subtype progenitors be maintained
and expanded? If the neuroepithelial cells,
especially regionalized neural progenitors,
can be maintained and expanded, a large
population of progenitors with similar
characteristics and differentiation potential
can be obtained. This has significant appli-
cation value as a homogeneous population
of neural stem/progenitors provides a con-
venient tool to study the fundamental biol-
ogy of tissue-specific stem/progenitors. It
also allows production of a homogeneous
population of subclasses of neural cells
from the progenitors over an extended
period. This is particularly useful for neural
transplantation purposes as progenitors are
easier to manipulate and more responsive
to environmental cues for differentiation
and integration than are postmitotic
neurons.
Neural stem/progenitor cells have been
expanded in culture in the presence of
mitogens such as epidermal growth factor
(EGF) and/or FGF2 (63). However, the
expansion is accompanied by diminished
potential of these progenitors to generate
neurons over glial cells (59). This trend is
in general agreement with the shift from
neurogenesis to gliogenesis during normal
development, suggesting the preservation
of intrinsic cellular program in governing
cell fate in vitro. In this regard, a recent
report by Conti et al was quite interesting,
in that mouse ESC-derived neural stem
cells were expanded for over 100 passages
in the presence of FGF2 and EGF yet
retained the capability to produce a large
proportion of βIII-tubulin positive neurons
(12). The expanded neuroepithelial cells
display a bipolar morphology and express
Sox2, Pax6, nestin, and RC2, suggestive of
a radial glia-like phenotype. During devel-
opment, radial glia functions as neural
stem cells (16, 39). The authors postulate
that the maintenance of radial glial prop-
erty endows the cells with a “niche” that
traps the cells in a state of symmetric cell
division. How a simple treatment with
FGF2 and EGF creates the special niche
remains peculiar. FGF2 and EGF have
been widely used for expanding neural
stem/progenitors isolated from rodent and
human brain and, with no exception, fail
to preserve a high degree of neurogenesis
over a long period. Temple and colleagues
have shown that factors released from vas-
cular endothelial cells can prolong the
neurogenic potential of neuroepithelial
cells (50), suggesting the importance of a
neurogenic niche in maintaining the stem/
progenitor cell state. The suggestion that
the ESC-derived neuroepithelial popula-
tion contains endothelia-like niche cells is
also plausible given the tendency of ESCs
to give rise to many cell types other than
the neural lineage. On the other hand, it is
peculiar why the neuronal cells, differenti-
ated from expanded progenitors, regardless
of whether they are derived from cortex,
striatum, or ESCs, exhibit an almost uni-
form GABAergic phenotype (12). If the
neural stem/progenitors can differentiate
to neurons with multiple transmitter phe-
notypes (other than GABAergic) before
expansion, it would suggest that FGF2 and
EGF selectively promote the proliferation
of GABAergic progenitors. Alternatively,
prolonged expansion with the growth fac-
tors may alter the phenotypes of progeni-
tors, thus influencing their differentiation
potential.
It is encouraging that the neurogenic
potential of neural stem cells can be main-
tained after extensive expansion, even
though there are many unanswered ques-
tions. A related question is whether the
positional identity and/or differentiation
potential of subtype neural progenitors can
be maintained after in vitro expansion.
Neural Subtype Specification from ESCs—Zhang
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
Limited studies indicate that expanded
neural stem/progenitors retain positional
information associated with their origins of
brain regions (17, 40). This, however, does
not imply the maintenance of the differ-
entiation potential. Embryonic ventral
mesencephalic progenitors, which produce
robust dopaminergic neurons at the time
of isolation, lose their dopaminergic
potential shortly after expansion in the
presence of FGF2 (56). Similarly, human
ESC-derived neural progenitors retain
their positional identity, based on home-
odomain transcription factor expression,
and a high degree of neurogenic potential
even after months of expansion (73,
Zhang). However, the potential to pro-
duce large projection neurons such as mid-
brain dopamine neurons and spinal cord
motor neurons fades in two to four pas-
sages and is replaced by other neuronal
populations. Nevertheless, neuroepithelial
cells expanded in the form of neural tube-
like rosettes continuously generate new
dopaminergic neurons for a much longer
period (Zhang, unpub. obs.). This suggests
that neural tube-like rosettes may act as an
embryonic niche to maintain the progeni-
tor state, giving hopes for extending the
neurogenic potential of a subtype neuronal
progenitor.
SPECIFICATION OF NEURONAL SUBTYPES
FROM ESCS
The birth of a neuronal type is the con-
sequence of the interplay between intrinsic
program of precursor cells and extracellular
signals at a given time and place (20, 30,
41). At an early stage, extracellular factors
appear to dictate the fate choice of precur-
sors whereas following specification of a
neuronal progenitor, extrinsic factors have
little influence on lineage choice as the pro-
genitors essentially mature following their
intrinsic process. Therefore, the key to
making the right type of neurons is to spec-
ify a correct type of neuronal progenitor.
Although extrinsic morphogens play a
major role in specifying stem cells to neu-
ronal progenitors, intrinsic cellular pro-
grams are always in play. Neurons are born
first, followed by glial cells. Within the
neuronal lineage, large projection neurons
appear prior to smaller interneurons. Thus,
steps for specifying large projection neu-
rons from ESCs are relatively straight-
forward as compared with generation of
135
© 2006 The Author
interneurons and glial cells. This, however,
is not the case for neural stem/progenitors
expanded from embryonic brain tissues,
because the intrinsic clock for the genera-
tion of large projection neurons has
elapsed. Large projection neurons are often
targets of neurodegenerative diseases such
as striatal projection GABA neurons that
are affected in Huntington’s disease, mid-
brain dopamine neurons that are degener-
ated in Parkinson’s patients and motor
neurons that are lost in amyotrophic lateral
sclerosis (ALS) patients. Developmentally,
molecular pathways that lead to the speci-
fication of neuronal types in the structur-
ally relatively simple spinal cord region are
better understood than those governing
cell birth in more complex structures such
as the brainstem and particularly the fore-
brain. Consequently, differentiation of spi-
nal cord cells such as motor neurons is
better achieved than those of brainstem
and forebrain.
and HB9. This molecular pathway learned
from chick embryo studies has now been
largely recapitulated using mouse ESCs. By
treatment of mouse ESCs with RA and
SHH, Wichterle and colleagues have suc-
cessfully differentiated stem cells to spinal
motor neurons with up to 20% efficiency
(65). The effect of RA and SHH is trans-
duced through the regulation of home-
odomain transcription factors that are
necessary for the birth of spinal cord motor
neurons. The mouse ESC-derived motor
neurons can innervate muscles following
transplantation into chick embryos.
Using the same principle, we have estab-
lished a chemically defined system for
efficient differentiation of spinal motor
neurons from human ESCs. Human ESCs
are first differentiated to neuroepithelial
cells in the presence of FGF2 (77) followed
by treatment of the enriched neuroepithe-
lial cells with RA and SHH (26). In this
study, we have identified two sequential
developmental stages during neuroepithe-
lial differentiation, an early primitive neu-
roepithelial stage that is characterized by
columnar epithelial cells expressing most of
the neuroectodermal transcription factors
but not the definitive neuroectodermal
transcription factor Sox1, and a later defin-
itive neuroepithelial stage that is character-
ized by Sox1-expressing columnar epithelial
cells forming neural tube-like rosettes
(Figure 1A). The primitive neuroepithelial
cells can be readily caudalized by RA and
these posteriorized progenitors can be effi-
ciently differentiated into HB9-expressing
motor neurons in the presence of SHH,

Coupling the intrinsic cellular program

with extrinsic factors is critical for specify-
ing neuronal subtypes. Specification of
spinal cord motor neurons during develop-
ment is one of the best-characterized path-
ways among neural cells. Not only have the
transcriptional codes been elucidated for
progenitors and postmitotic motor neu-
rons but the inductive extracellular mole-
cules are also defined. Specification of
motor neuron progenitors requires activa-
tion of class II (induced by SHH, eg,
Nkx6.1 and Nkx2.2) and suppression of
class I (inactivated by SHH, eg, Pax6 and
Irx3) homeodomain transcription factors.
Activation of Nkx6.1 and suppression of
Irx3 defines the dorsal boarder of the
motor neuron progenitor domain. Activa-
tion of Nkx2.2 and suppression of Pax6
defines the ventral edge of the progenitor
domain. Together, the combinatorial
actions of these mutual repressive home-
odomain proteins restrict the motor neu-
ron progenitor domain, resulting in the Figure 1. Stereotypic neuronal subtype specification from human embryonic stem cells (ESCs). A. Human
appearance of Olig2-expressing motor ESCs were differentiated to primitive neuroepithelial cells (NE) at around day 10 and then NE that exhibit
neuron progenitors (20). Remarkably, such neural tube-like rosettes in 14–17 days of differentiation in a chemically defined neural medium (77)
without the presence of morphogens. B. Treatment of the primitive NE (green arrows) with retinoic acid
a complicated transcription network can
(RA) and sonic hedgehog (SHH) resulted in efficient generation of HB9+ spinal motor neurons. Similarly,
be orchestrated by a rather simple soluble FGF8 and SHH treatment at this stage resulted in differentiation of TH+ dopamine neurons some of
factor SHH at a specific concentration. which also expressed the midbrain transcription factor engrailed 1 (En1). C. Treatment of more advanced
With the continued action of RA, the NE (red arrows) with RA and SHH resulted in few HB9+ motor neurons although similar proportion of
Isl1+ neurons, mostly likely spinal interneurons. Similarly, FGF8 and SHH treatment at a later stage
motor neuron progenitors become postmi- resulted in differentiation of TH+ dopamine neurons, many of which expressed Bf1, a transcription factor
totic motor neurons by expressing Lim expressed by forebrain cells. Bar = 50 µm. (B) and (C) are reproduced partially from Li et al (26) and Yan
transcription factors such as Lim3, Isl1/2, et al (68), respectively, with permission.

136 Neural Subtype Specification from ESCs—Zhang

© 2006 The Author

Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
with about 20%–50% of the total differ-
entiated progenies. In contrast, FGF2-
induced definitive neuroepithelial cells,
although they can be caudalized by RA,
generate fewer motor neurons (<5%) under
the same culture condition (Figure 1B).
Analyses of homeodomain transcription
factors regulated by the morphogen treat-
ments at each of the developmental stages
indicate that RA (at 0.1–1 µM) eliminates
rostral homeodomain transcription factor
Otx2 and elicits hox gene expression in the
primitive neuroepithelial cells in a dose-
dependent manner. SHH subsequently
coordinates with the caudalized neuroepi-
thelial cells in regulating the expression of
Pax6, Nkx6.1, and Irx3, which activates the
transcription of Olig2 and subsequently
HB9 (26). In contrast, RA has less caudal-
izing effect on FGF2-induced definitive
neuroepithelial cells. Addition of SHH does
not create a transcription network that is
necessary for activating Olig2 and HB9
(Figure 1B). Thus, specification of spinal
motor neurons from human ESCs requires
sequential activation/inhibition of tran-
scription factors similar to those learned
from chick embryo studies. Importantly,
activation of the motor neuron specifica-
tion machinery is contingent upon the spe-
cific neuroectoderm induction process, i.e.,
RA is required before neuroepithelial fate
is determined. This translates into an early
application of RA between primitive and
definitive neuroepithelial cells in the differ-
entiation cultures. Similarly, early applica-
tion of FGF8 appears to induce midbrain
type of dopaminergic neurons more effi-
ciently, whereas later application of FGF8
has little effect even though tyrosine
hydroxylase (TH)-expressing dopamine
neurons can be readily differentiated under
both conditions (68) (Figure 1C). There-
fore, it is crucial to couple the intrinsic
program of precursors at a particular devel-
opmental stage with a specific set of extrin-
sic morphogens in order to efficiently
differentiate ESCs to a subclass of neurons.
Coordination of positional patterning
and transmitter specification may be nec-
essary for generation of functional neuron
subtypes. Although RA-induced mouse
and human motor neurons acquire cholin-
ergic phenotypes along differentiation, it
does not necessarily mean that regionally
patterned progenitors will automatically
acquire a correct type of transmitter phe-
notypes or vice versa. This debatable con-
cept appears to manifest in an effort to
induce ESCs to various subtypes of neu-
rons that are present in the mid/hind brain
region. The brainstem is structurally simi-
lar to the spinal cord and is also segmented
during development, although changes
from one segment to another in the brain-
stem are more drastic than those in the
spinal cord. This would imply that each
segment of the brainstem may need to be
patterned somewhat independently. Sig-
naling in the mid/hind brain boundary, eg,
by FGFs, plays a key role in patterning the
mid/hind brain (18, 21, 47). If explants
taken from the anterior neural plate region
are cultured in the presence of FGF8 and
SHH, a large number of progenitors will
adopt a dopaminergic fate (69). This has
been interpreted as the requirement of
FGF8 and SHH for specification of mid-
brain dopamine neurons.
By treatment of mouse ESC-derived
neuroepithelial cells with FGF8 and SHH,
a large proportion of TH+ dopamine neu-
rons (~30% of total differentiated proge-
nies) can be differentiated (5, 25). These
cells are indeed dopamine neurons as they
secrete dopamine in an activity-dependent
manner and reverse locomotive functional
deficit in Parkinson’s rodents following
transplantation into the lesion-side stria-
tum. Thus, FGF8 and SHH treatment
appears to activate the dopaminergic path-
way. Many of these dopamine neurons
express a mid/hind brain transcription fac-
tor En1, when FGF8 is added at an early
stage (5) or even after the expansion of
neuroepithelial cells (25), suggesting that
FGF8 can promote the patterning of mid/
hind brain cells. For human ESCs, we
found that a simple shift of the timing of
FGF8 application significantly alters the
phenotype of dopamine neurons (68).
Treatment of human ESC-derived primi-
tive neuroepithelial cells with FGF8 and
SHH results in differentiation of dopam-
ine neurons, a significant population of
which express En1. Addition of FGF8
and SHH to definitive neuroepithelial cells
results in generation of TH neurons, most
of which do not coexpress the midbrain
transcription factor En1. Instead, many of
them coexpress Bf1 (Figure 1C) or GABA,
suggesting that many of these dopamine
neurons may represent forebrain/olfactory
Neural Subtype Specification from ESCs—Zhang
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
dopamine neurons. Some do not express
Bf-1 or En-1, resembling those in the
hypothalamus (80). Thus, FGF8 and SHH
can promote the differentiation of dopam-
ine neurons but may not be sufficient to
restrict cells to the midbrain dopamine
neuronal fate, particularly in humans.
Additional factors must come into play in
order to further restrict precursors to the
midbrain fate.
Coculture with mesoderm-derived stro-
mal cells, such as PA6 cells (9, 22, 35) and
MS5 cells (5, 44), is at present the most
widely used approach for differentiating
mouse and human ESCs to dopaminergic
neurons. Although FGF8 and SHH have
been added to the culture system, the
(dopaminergic) neural inducing effect
comes from direct contact with stromal
cells (22, 44) and the inducing activity
remains even after the PA6 cells are fixed
with formaldehyde (22). This suggests that
factors other than FGF8 and SHH play
a critical role in midbrain dopaminergic
neuron specification. It has also been
shown that neural progenitors that overex-
press Nurr1 can differentiate to dopamine
neurons when cocultured with mesenceph-
alic astrocytes (61). One of the signals asso-
ciated with astrocytes may be Wnts (10).
Wnt1 has been found to be critical for the
morphogenesis of the midbrain and the
genesis of dopaminergic neurons (45),
partly by maintaining expression of En1, a
midbrain transcription factor (13). Null
mutation in Wnt1 results in loss of major
parts of the midbrain and enlargement
of the forebrain (32). Overexpression of
Wnt1, however, increases the size of the
midbrain (42). Other Wnts, including
Wnt3a, and Wnt5a are involved in the
proliferation, survival and maturation of
midbrain dopamine neurons (11). Thus,
more sophisticated approaches such as
coordinated action of FGF8, SHH, and
Wnts, are required for generation of mid-
brain dopamine neurons from ESCs, par-
ticularly those of human origin.
Are the dopamine neurons generated
from mouse and human ESCs with com-
binations of FGF8/SHH or by treatment
with stromal signals the midbrain dopam-
inergic neurons? By reevaluating the
phenotypes of dopaminergic neurons
generated from mouse and human ESCs
using the so far published FGF/SHH or
stromal cell inducing protocols, we found
137
© 2006 The Author
that only a small fraction (10%–30%) of
mouse ESC-derived and hardly any of the
human ESC-derived dopamine neurons
express Lmx1b and ptx3 (Zhang, unpub-
lished), the latter of which is exclusively
expressed by midbrain dopamine neurons
and is required for the survival of postmi-
totic dopamine neurons (52, 53). Using a
green fluorescent protein (GFP) reporter in
the ptx3 locus, Li and colleagues had a
similar finding (78). The dopaminergic
transmitter phenotype appears to be effi-
ciently specified whereas the midbrain
specification is much less effective. By
expressing Lmx1a, a transcription factor
expressed by ventral midbrain precursors as
well as a much larger population of dorsal
progenitors (34, 34), under the control of
the nestin enhancer Ericson, Perlmann
and colleagues can efficiently differentiate
mouse ESCs into dopamine neurons that
exhibit correct phenotypes of midbrain
dopaminergic neurons (1). These in vitro
produced mouse dopamine neurons pos-
sess the whole set of genes in the same TH-
expressing neurons, including Lmx1a,
Lmx1b, En-1, Nurr1, ptx3, and dopamine
transporter. This will be a “gold standard”
for defining the identity of midbrain
dopaminergic neurons that are produced
in a Petri dish. This study also points to the
necessity of combining SHH/FGF8 with
Lmx1a when inducing midbrain dopamine
neurons. Thus, the molecular pathways for
specification of transmitter phenotypes
and for ventral midbrain positional iden-
tity need to be coupled in order to produce
functional midbrain dopamine neurons.
The above discussion also points to an
important technical issue in characterizing
neuronal subtypes generated in vitro. Most
of the analyses are limited to the demon-
stration of TH expression and dopamine
release. Expression of midbrain dopamine
neuron phenotypes is generally performed
using RT-PCR. As neural progenitors with
various regional specificities and trans-
mitter properties are present in most dif-
ferentiation culture conditions, the mere
expression of midbrain neural transcrip-
tion factor mRNA in bulk cultures is not
sufficient to demonstrate that the TH+
neurons are the midbrain type of dopam-
ine neurons. Colocalization of TH with
nuclear midbrain transcription factors will
be necessary to confirm the midbrain
dopamine neuron identity.
138 Neural Subtype Specification from ESCs—Zhang
© 2006 The Author
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
Specification of forebrain neuronal sub-
types is hardly explored. The forebrain is
evolutionarily the newest part of the CNS
and contains the richest array of subtypes
of neurons that organize into the most
complex structure. The patterning of the
forebrain is barely understood and spe-
cification of neuronal subtypes in the
forebrain is largely unknown. The poor
understanding of the molecular mecha-
nisms underlying forebrain development is
one of the main reasons why hardly any
report is presented on efficient differentia-
tion of a subtype of forebrain neurons.
Technically, commonly used mitogens
such as FGF2 often possess caudalizing
effect on neuroepithelial cells, especially at
an early stage. Although preventing caudal-
ization by dkk1 increases the pool of fore-
brain neural progenitors derived from
mouse ESCs (62), the progenitor popula-
tion is still too small to produce a mean-
ingful proportion of subtype forebrain
neurons unless the differentiated progenies
can be significantly enriched or purified.
Neuroepithelial cells differentiated from
human ESCs initially are almost exclusively
of forebrain identity (26). This indicates a
possible strategy for generating forebrain
neural cells by simply preventing caudaliza-
tion of the primitive forebrain neuroepithe-
lial cells. By using factors other than FGF2 in
our culture system, these primitive forebrain
neuroepithelial cells can be maintained to a
large extent (Li and Zhang, unpublished).
This has laid down a solid foundation for the
generation of forebrain neuronal subtypes.
Still, neuronal subtypes are usually located in
a small anterior-posterior forebrain domain
and at a particular cortical layer or a subcor-
tical area, suggesting that a delicate pattern-
ing would be necessary. An added difficulty
is the almost total lack of molecular markers
for progenitors of forebrain neuronal sub-
types. Careful and creative strategies will be
needed in order to direct ESCs to the vast
array of neuronal subtypes that are harbored
in the primate forebrain.
SPECIFICATION OF GLIAL SUBTYPES
FROM ESCS
Understanding the molecular switch
from neurogenesis to gliogenesis may be a
key to specification of oligodendroglial lin-
eage. During embryonic development,
glial cells, including astrocytes and myeli-
nating oligodendrocytes, are generated fol-
lowing the birth of major neuronal types.
The same neurogenesis to gliogenesis
sequence is preserved when early neuroec-
toderm is cultured (46) or ESCs are differ-
entiated along the neural lineage (71). In
our chemically defined culture system, for
example, human ESCs generate neuroepi-
thelial cells in 2–3 weeks. These neuroepi-
thelial cells differentiate predominantly
into βIII-tubulin+ neurons in the next
2–3 weeks. Glial fibrillary acidic protein
(GFAP+) astrocytes appear thereafter, 6–9
weeks after human ESCs are differentiated.
O4+ oligodendrocytes arise in a much
longer period of differentiation (77). This
temporal sequence of neuronal and glial
differentiation roughly corresponds to the
timeline observed from limited samples of
fetal tissues (51). Thus, the intrinsic pro-
gram governing neuronal and glial lineage
development is retained in vitro.
Myelinating oligodendrocytes have been
efficiently generated from mouse ESCs (6,
8, 31). The general procedure is to expand
the ESC-derived neuroepithelial cells with
FGF2, EGF, or both until the gliogenic
phase. The gliogenic progenitors are then
expanded in a glial restricted medium in
the presence of FGF2 and platelet-derived
growth factor (PDGF), both of which are
known to promote proliferation of oligo-
dendrocyte progenitors (33). Oligoden-
drocytes generated in this way are bona
fide myelinating cells as they produce mye-
lin sheaths following transplantation into
the dysmyelinating or injured rat spinal
cord (8, 31). Nevertheless, these proce-
dures do not tell us how oligodendrocytes
are specified from ESCs as the large pro-
portion of oligodendrocytes is mainly
achieved through expansion of the progen-
itors that had spontaneously differentiated.
Rodent oligodendrocytes can be cultured
to near pure populations through expan-
sion of their progenitors by growth factors
such as FGF2 and PDGF or conditioned
medium from the B104 neuroblastoma
cells (3, 75, 76). We have devised a proto-
col for oligodendrocyte specification from
mouse ESCs based on the understanding
that oligodendrocytes are derived from a
progenitor that expresses the transcription
factor Olig2 (48). Using a similar proce-
dure described for generating motor neu-
rons (see previous section), we are able to
induce mouse ESCs to Olig2-expressing
neural progenitors. In the absence of RA
but presence of SHH, these Olig2 progen-
itors differentiated mainly to oligodendro-
cyte progenitors during the gliogenic phase
(Du and Zhang, in preparation). Thus,
oligodendrocytes can be specified from
mouse ESCs by SHH.
Differentiation of oligodendrocytes
from human ESCs has been observed (19,
77). However, the proportion of oligoden-
drocytes among the differentiated progeny
is generally very low. Attempts to expand
the progenitors with known growth factors
that are effective for rodent oligodendro-
glial progenitors are not successful, similar
to what is seen using brain-derived progen-
itors (67, 74). Keirstead and colleagues
have recently reported the generation of
pure cultures of oligodendrocytes using a
rather simple “expansion protocol” (38).
Human ESCs are cultured in a “glial
restricted medium,” similar to the Sato
medium (7), with the presence of 2–4 ng/
mL of FGF2 and 20 ng/mL of EGF for
6 weeks. The culture is exposed to RA
(10 µM) for 8 days after ESCs are differ-
entiated for 2 days, which results in the
formation of “yellow spheres.” Upon
removal of EGF, these cells become multi-
polar cells with branched processes (38).
Oligodendrocytes generated in this way,
however, do not exhibit typical morphol-
ogy that is generally unambiguous regard-
less whether they are cultured from brain
tissues or from mouse and human ESCs
(Figure 2A). It will be important that such
unprecedented oligodendrocyte differenti-
ation efficiency from human ESCs be
reproduced by independent laboratories.
We have shown that the Olig2 neural
progenitors can be readily differentiated
from human ESCs in response to RA
and SHH (26). These Olig2 progenitors
generate mostly motor neurons during
the neurogenic period. However, Olig2
progenitors persist after neurogenesis.
Oligodendrocytes with typical morphol-
ogy and sequential myelin protein expres-
sion appear during the gliogenic phase
several weeks later (Zhang, Figure 2B,C).
This suggests that the Olig2 progenitors
may differentiate into oligodendrocytes.
Attempts to promote the proliferation of
the Olig2 progenitors with similar strate-
gies used for mouse Olig2 progenitors have
not yet been successful, again suggesting a
potentially different molecular mechanism
underlying the expansion of human
oligodendroglial progenitors. The key to
the production of oligodendrocytes from
human ESCs is perhaps the signaling path-
way that switches the neurogenic potential
of Olig2 progenitors to the gliogenic
capability. Unraveling the molecular switch
from neurogenesis to oligodendrogliogen-
esis in human cells will be a challenging
task given the extremely long stretch
between neurogenesis and gliogenesis in
humans. Manipulating the switch might
also speed up gliogenesis.
Astrocytes: too common to be ignored?
Astrocytes are the most abundant cell type
and are distributed throughout the brain
and spinal cord. Unlike their cousin oligo-
dendrocytes, which have a unique myeli-
nating capability, astrocytes participate in
almost every aspect of physiology and
pathology of the CNS. The generation of
astrocytes is poorly understood, partly
because of lack of markers for their progen-
itors (48, 72). Neural progenitors isolated
from brain tissues or derived from ESCs
generate mainly astrocytes during gliogenic
stages without special treatment. A recent
finding by Rowitch and colleagues, how-
ever, indicates that some astrocytes may be
specified from a specific progenitor pool in
a particular CNS location through a simi-
lar molecular mechanism as for generation
of neurons and oligodendrocytes. In the
spinal cord, progenitors in a small domain
just dorsal to the oligodendrocyte progen-
itor domain express a transcription factor
stem cell leukemia (scl), which appears
to be critical for the specification of
astrocytes. Knockout of scl results in the
enlargement of the ventral oligodendrocyte
progenitor domain, suggesting that the
specification of the two major types of glia
may be adjusted through mutual repres-
sion at the transcription level (36). How
this finding may be applied to astrocyte
differentiation from ESCs remains to be
seen. This finding may also help generation
of oligodendrocytes from stem cells.
Although astrocytes appear to distribute
uniformly throughout the CNS, astrocytes
in different parts of the brain and spinal
cord possess differential functional
attributes. Astrocytes in the ventral mesen-
cephalon, but not the dorsal mesencepha-
lon or other parts of the brain, can
promote the specification of neural pro-
Neural Subtype Specification from ESCs—Zhang
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
Figure 2. Oligodendrocytes from human neural
stem/progenitors and human embryonic stem cells
(ESCs). A. Neurospheres, generated from embry-
onic human (18–20 gestation weeks) cerebral
tissues, were differentiated for 7 days. Immun-
ostaining with O4 revealed differentiating oligo-
dendroglial cells with typical tripolar to multipolar
morphologies. “NS” indicates neurosphere. B.
Human ESCs were differentiated to Olig2 progen-
itors in 4 weeks according to Li et al (26) and then
differentiated to neurons and glial cells for addi-
tional 8 weeks. Oligodendrocytes with a multipo-
lar morphology appeared. Immunostaining with
O4 at this stage revealed characteristic membrane
and process staining (C). Blue: Hoechst labeled
nuclei. Bar = 50 µm. (A) is reproduced partially
from Zhang et al (74), with permission.
genitors to adopt the midbrain dopamin-
ergic neuronal fate (61). Such functional
difference may be attributed to the molec-
ular profiles produced by region-specific
astrocytes. Ventral mesencephalic astro-
cytes express a high level of Wnts (10)
which has been shown to be important in
the specification of midbrain dopamine
neurons (11). Can region-specific astro-
cytes be differentiated from naïve ESCs?
More importantly, will in vitro produced
astrocytes possess the functional property
associated with region-specific astrocytes?
If the answers are yes, the implication
including therapeutic application will
likely be far reaching.
ARE WE THERE YET?
Neural differentiation from both mouse
and human ESCs is generally robust judg-
ing from differentiated cells that possess
neuronal morphology and express general
neural markers. This picture is less bright
if one is to ask whether a subclass of neu-
rons or glial cells can be selectively differ-
139
© 2006 The Author
entiated. Hence, directed differentiation of
neural subtypes from ESCs will remain a
major focus of efforts in the next few years
toward applying ESC technology in studies
of developmental neurobiology, neurode-
generation, and regeneration. Along these
efforts, we may find answers to many of
the questions we have been trying to
answer.
Are ESC-derived neuronal subtypes
equivalent to their counterparts in the
brain? This question goes back to the heart
of how a neural subtype can be directed
from ESCs. Motor neurons differentiated
from mouse and human ESCs based on
developmental principles correspond to
the cervical somatic spinal motor neurons
judging from series of molecular markers
and their interactions with target muscles.
The identity of ESC-generated dopamin-
ergic neurons is less clear as limited molec-
ular markers have been employed, which is
not sufficient to define dopaminergic sub-
types [except (1)]. Most of the cultures
including ours comprise many different
subtypes of dopamine neurons. This is
because a simple application of FGF8 and
SHH or in combination with stroma sig-
nals is not sufficient to restrict most ESC-
derived neuroepithelia to a ventral mid-
brain fate, at least for primate cells. More
sophisticated procedures will be required
to guide differentiation of dopamine neu-
ronal subtypes. One may ask further which
subtype of midbrain dopamine neurons,
substantia nigra vs. ventral tegmental area,
is produced from ESCs in response to a
specific set of signals. Similarly, which pool
of upper limb muscles are target of the
cervical spinal motor neurons? Informa-
tion concerning the development of some
of these neuronal subtypes is available,
which may be used to guide neuronal dif-
ferentiation from ESCs. More importantly,
ESC neural differentiation system provides
a dynamic model to tease out molecular
mechanisms underlying decision-making
process along each individual lineage
branch.
Does the positional identity of neuronal
subtypes matter? Much of the discussion
points to the critical role of positional
information precursor cells possess in order
to make a fate decision. The final position
of a neuron also links to its function.
140 Neural Subtype Specification from ESCs—Zhang
© 2006 The Author
Journal Compilation © 2006 International Society of Neuropathology • Brain Pathology
Dopaminergic neurons in the substantia
nigra and ventral tegmental area in the
ventral midbrain possess distinct molecular
characteristics, project to diverse brain
regions, and execute unique functions.
Developmentally, motor neuron progeni-
tors always innervate the correct target
muscles and do not connect to mis-
matched muscles even when a segment of
spinal cord is deleted or is rotated upside
down (23, 24). Whether this matters from
a therapeutic standpoint is debatable.
Transplanted neural progenitors into
ectopic brain regions can mature and form
functional synapses even though these
grafted cells retain their donor regional
identity (64). Of course, it is not known
whether such synapses will yield appropri-
ate function. This issue will become clearer
when transplant studies in both developing
and diseased environments are carried out.
Can neural differentiation from human
ESCs be simply scaled up from mouse
ESCs? For structures such as the spinal
cord that are evolutionally conserved, fun-
damental principles and even techniques
learned from mouse ESCs may be readily
applied to humans. A good example is the
differentiation of cervical/brachial spinal
motor neurons in which a similar set of
morphogens at equivalent concentrations
induces comparable populations of motor
neurons from mouse and human ESCs
with similar characteristics (26, 65). Even
under such a condition, nuances exist,
which involves significant trial and error
(26). Differentiation of midbrain dopam-
ine neurons can be readily achieved from
mouse ESCs with a simple application of
FGF8 and SHH at the correct time in
an appropriate concentration (Zhang).
This is achieved through induction of
endogenous factors such as Wnts, which
act in concert with the exogenous factors
in a relatively short developmental window
to specify a cell fate. In human ESCs, a
simple FGF8/SHH treatment has much
less effect in inducing a ventral midbrain
fate. Additional steps are required to sup-
plement the deficit in the long stretched
developmental period in order to ulti-
mately activate the midbrain dopaminergic
pathway. The forebrain is quite different
between rodents and humans. The motor
cortex, for example, is located in the fore-
front of the forebrain in rodents and birds
whereas in humans, it is a narrow band in
the middle part of the cerebral hemisphere.
This would suggest that even the initial
patterning of cortical motor neuron pro-
genitors may well be significantly different
between rodents and humans. Not only
neuronal types, but also glial cells may be
generated through different mechanisms.
Myelinating oligodendrocytes can be
readily differentiated and expanded from
mouse ESCs, which is not so in human
ESCs. We do not even have a strategy for
expanding isolated human oligodendro-
cyte progenitors in culture yet, although
we have known appropriate procedures for
mouse cells for over two decades. Thus, we
may have to learn and uncover mysteries
of human cell biology by working on
human cells directly. The dismissive atti-
tude toward the originality of human
cell studies discourages investigators from
exploring basic biological questions that
are unique to human cells and fuels the
unrealistic rush for clinical application.
The net consequence is in fact slowing
down the arrival of stem cell application in
therapy.
ACKNOWLEDGMENT
Studies in my laboratory have been sup-
ported by the NIH-NCRR (RR016588),
NIH-NINDS (NS045926, NS046587),
the Michael J. Fox Foundation, the ALS
Association, the National Multiple Sclero-
sis Society, the Myelin Project, Bryon
Riesch Paralysis Foundation, and Heck-
rodt Fund.
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