Multiple layers of molecular controls modulate self-renewal and neuronal lineage specification of embryonic stem cells
Elucidating the molecular changes that arise during neural differentiation and fate specification is crucial for building accurate in vitro models of neurodegenerative diseases using human embryonic stem cells (hESCs). Here we review the importance of hESCs and derived progenitors in treating and modeling neurological diseases, and summarize the current efforts for the differentiation of hESCs into neural progenitors and defined neurons. We recapitulate the recent findings and discuss open questions on aspects of molecular control of gene expression by chromatin modification and methylation, posttranscriptional regulation by alternative splicing and the action of microRNAs, and protein modification. An integrative view of the different levels will hopefully provide much needed insight into understanding stem cell biology.
Multiple layers of molecular controls modulate
self-renewal and neuronal lineage speciﬁcation
of embryonic stem cells
Gene W. Yeo
, Nicole Coufal
, Stefan Aigner
, Beate Winner
, Jonathan A. Scolnick
Maria C.N. Marchetto
, Alysson R. Muotri
, Christian Carson
and Fred H. Gage
Laboratory of Genetics and,
Crick-Jacobs Center for Computational and Theoretical Biology, Salk Institute, 10010
North Torrey Pines Road, La Jolla, CA 92037, USA and
BD Biosciences, 11077 North Torrey Pines Road, La Jolla,
CA 92037, USA
Received February 13, 2008; Revised February 13, 2008; Accepted February 28, 2008
Elucidating the molecular changes that arise during neural differentiation and fate speciﬁcation is crucial for
building accurate in vitro models of neurodegenerative diseases using human embryonic stem cells (hESCs).
Here we review the importance of hESCs and derived progenitors in treating and modeling neurological dis-
eases, and summarize the current efforts for the differentiation of hESCs into neural progenitors and deﬁned
neurons. We recapitulate the recent ﬁndings and discuss open questions on aspects of molecular control of
gene expression by chromatin modiﬁcation and methylation, posttran scription al regulation by alternative
splicing and the action of microRNAs, and protein modiﬁcati on. An integrative view o f the different levels
will hopefully provide much needed insight into understanding stem cell biology.
THE IMPORTANCE OF STEM CELLS
IN NEUROLOGICAL DISEASES
Human neurological diseases are complex and often difﬁcult
to model in vitro, and rodent models are frequently genetically
irrelevant. The hallmarks of several neurodegenerative dis-
orders, such as Parkinson’s disease (PD), Huntington’s
disease (HD) and Alzheimer’s disease (AD), are slow pro-
gressive losses of speciﬁc neuronal populations in the brain.
So far only symptomatic therapeutic approaches are available,
making these diseases potential candidates for restorative
therapeutic approaches. Therefore, major research efforts
have focused on cell transplantation of hESCs, derived
neural progenitors and/or neural stem cells (NSCs) to restore
depleted diseased cells.
For instance, motor deﬁcits in PD are due to the progressive
degeneration of the dopaminergic neurons (DAs) of the sub-
stantia nigra pars compacta resulting in decreased dopamine
release into the striatum. Transplantation strategies aiming
toward rectifying the striatal dopamine deﬁcit conducted in
PD patients provided the proof of principle that transplanted
neurons can elicit beneﬁcial effects. However, difﬁculty in
obtaining cells suitable for transplantation reduces the utility
of the transplantation approach. More recently, there has
been an intense effort to use human embryonic stem cell
(hESC)-derived neural progenitor cells (NPCs) for transplan-
tation. These cells can be grown in large numbers from a
single source to help control for variations in source tissue.
It may also be possible to differentiate the cells in vitro in
such a way as to avoid the problems of fetal cell transplants
(e.g. uncontrolled dopamine release, ethical concerns).
However, many barriers still need to be overcome before
hESC-derived neural progenitors can be considered safe for
transplantation into humans. For example, Roy et al. showed
that hESC-derived neural progenitors may be tumorigenic in
vivo (1). In fact, hESC-derived neurons will be more beneﬁcial
as an in vitro model system in which diseases such as PD can
Induced plu ripotent stem cells (iPSCs) offer a potentially
new direction for the study and treatment of neurodegenerative
diseases. Several groups recently demonstrated that overex-
pression of a combination of several genes, including OCT4,
SOX2, and some combination of Klf4, Lin28 and c-Myc in
human ﬁbroblasts leads to the creation of pluripotent embryonic
These authors contributed equally to this work.
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stem-like cells that can contribute to all cell lineages (2,3). In
principle, Human iPSCs can be generated from an individual
affected by a neurodegenerative disease, allowing researchers
to produce neurons in vitro that contain the same genetic
makeup as the patient. These patient-speciﬁc neurons will
provide a useful resource for understanding the genetic-basis
for sporadic cases of neurodegeneration.
CURRENT MODELS FOR NEURONAL
DIFFERENTIATION OF HESCS
Accurate differentiation of hESCs (or iPSCs) into NSCs will
be crucial for in vitro modeling and treatment. Figure 1 sum-
marizes the current and future efforts in neural speciﬁcation
and enrichment from hESCs. There are three major
approaches for differentiating hESCs into NSCs: co-culturing
hESCs with a stromal feeder layer such as PA6 (4) and MS5
(5); directly differentiating hESC colonies in culture by
addition of the bone morphogenetic protein (BMP) antagonist
Noggin (6,7); and isolating NSC-containing neuroectoderm
from embryoid bodies (EBs) (8). Researchers have developed
methods to prom ote the neuroectoderm population in EBs
including incubating EBs in a deﬁned neural promoting
media (8), in conditioned media from stromal HepG2 cells
(9) or in the presence of Noggin (7,10–12). With the excep-
tion of complete neural differentiation by co-culture on PA6
cells, the majority of these methods converge at the formation
of neural rosettes (4). Rosettes consist of radial arrangements
of columnar cells that express many markers also found in the
developing neural tube. Rosettes can be manually dissected
and plated to form NSCs, which can be propagated as neuro-
pheres (8) or as adherent cultures (12) for multiple passages
before further differentiation into neurons and glia. Moreover,
engraftment of hESC-derived NSCs into rodents can result in
functional neuronal integration (8).
Directed differentiation of hESCs into speciﬁc functional
neuronal subtypes is more complex than speciﬁcation of
NSCs alone and requires mimicking the in vivo development
of each unique neuronal subtype. Many laboratories are focus-
ing on generating two speciﬁc neuronal subtypes, DAs, which
could be useful for furthering the understanding and treatment
of PD, and motor neurons expressing choline acetyltransfer-
ase, which could be used in the study and treatment of amylo-
trophic lateral sclerosis (also known as Lou Gehrig’s disease)
and spinal cord injury. Both differentiation schemes employ
deﬁned signaling molecules to speciﬁcally pattern the cells.
Sonic Hedgehog (SHH) and FGF8 (13) or Noggin (14,15)
have been used to pattern cells toward the dopaminergic
subtype, and a recent study employed the co-culture of an
immortalized human fetal midbrain astrocyte cell line, in
addition to signaling molecules to signiﬁcantly enhance the
differentiation (1). Similarly, SHH and retinoic acid (RA)
have been used to promote motor neuron differentiation
(16,17). Importantly, many groups have demonstrated that
hESC-derived DA neurons and motor neurons are functional
both in vitro and in vivo. Differentiated DA neurons have
been shown to ﬁre action potentials and release dopamine
upon depolarization in vitro (13) and have been shown to
survive transplantation and engraft in rodent models of PD
(1,15,18). Differentiated motor neurons exhibit spontaneous
action potentials and make contacts with co-cultured myotubes
in vitro (17), and transplantation experimen ts of motor neuron
progeny into developing chick embryo and adult rat spinal
cord have resulted in robust engraftments (16). In addition
to these two neural subtypes, the ﬁeld will most certainly
broaden its repertoire of differentiation methods to include
the plethora of neural subtypes that exist. Already, advances
have been reported in the differentiation of peripheral
sensory neurons (19) and neural crest fates (16).
The future of hESC or iPSC neuronal differentiation lies in
improving the relative abundance of the desired neuronal
subtype within the total cell population and by removing non-
speciﬁc contaminating cell types and remaining proliferative
cells from postmitotic neuronal cultures, thereby obtaining
near-pure populations for transplantation experiments. These
goals have been successfully pursued by ﬂuorescent-activated
cell sorting of the cell population, using subtype-speciﬁc pro-
moters driving ﬂuorescent reporter proteins and by antibody
staining for subtype-speciﬁc cell surface markers. For
instance, by utilizing an HB9 promoter green ﬂuorescent
protein (GFP) construct, Soundararajan et al. (20) enriched
for putative motor neuron precursors. They subsequently
showed that these HB9-positive cells engrafted into the
chick neural tube and exhibited proper migration to the
medial motor column, projected axons to the appropri ate
target muscles and showed synaptic activity that resembled
that of endogenous motor neurons. A synapsin promoter-
driving GFP has also been used to successfully isolate
neurons from a diverse cell population (2 1). Similarly, the
research community is just beginning to appreciate the
utility of the cell surface marker proﬁle of different NPC
populations and subtype-speciﬁc cells. For example, cell
surface markers such as CD133, 5E12, CD34, CD45 and
CD24/lo have been employed to enrich for neurosphere-
forming cells from fetal brain (22). Similarly, SSEA-4 and
prominin-1/CD133 have been shown to demarcate fetal
NSCs from human brain (23,24).
An important consequence of furthering the purity and
differentiation speciﬁcity of hESCs has been the opportunity
to systematically study in a genome-wide fashion the molecu-
lar pathways governing self-renewal and lineage speciﬁcation
EPIGENETICS OF STEM CELL BIOLOGY:
CHROMATIN AND METHYLATION
Aside from the key transcription factors, such as Oct4, Nanog
and Sox2, which are necessary and sufﬁcient for specifying
self-renewal in hESCs (25), epigenetic changes mediated by
other factors also play important roles in maintaining a pluri-
potent state. For example, Polycomb group (PcG) proteins are
transcriptional repressors that modify chrom atin structure and
have been implicated in hESC pluripotency (26). PcG proteins
can act in two distinct complexes, PRC1 and PRC2, to repress
gene expression. To identify target genes in murine embryonic
stem cells, Boyer et al. performed genome-wide location
analysis using antibodies against core components of
PRC1 (phc1 and Rnf2) and PRC2 (Suz12 and Eed) (27).
From these stud ies, the view emerged that PRC2 initiates
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transcriptional repression by inhibiting initiation, whereas the
PRC1 complex maintains the repressive conditions. Mutations
in the components of PRCs eliminate the pluripotent ability of
mES cells, leading to embryonic lethality (28,29).
Attesting to the importance of PRC1 and PRC2 in control-
ling gene expression in mESCs, 90% of genomic sequences
detected in chromatin immunoprecipitation (ChIP) exper-
iments using antibodies against PRC1 and PRC2 components
were within 1 kb of a transcription start site, making these
complexes likely regulators of these genes. PRC1 and PRC2
components overlap in a subset of these genes, many of
which encode transcription factors with important roles in a
variety of developmental processes. Also, all these
co-occupied genes are highly enriched for histone H3 tri-
methylated at lysine 27 (H3K27me3), a marker strongly
associated with transcriptionally repressed chromatin (30).
Since H3K27 trimethylation is catalyzed by PcG proteins
(26), it is likely that this mark is deposited at these promoters
by recruited PcG proteins. Upon differentiation of mESCs,
93% of PcG target transcripts were preferentially upregulated.
Speciﬁcally, the differentiation of mESCs cells toward NPCs
increased the expression of neuron-speciﬁc genes, whereas
the loss of H3K27me3 was concomitant with an increase in
RNA polymerase II occupancy and H3K4me3, a histone modi-
ﬁcation associated with active transcription that is created by
trithorax-group (trxG) proteins (26).
These data suggest that PcG proteins have specialized roles
in silencing genes at the embryonic stage that correlate with
differentiation and loss of pluripotency but maintain the poten-
tial to become activated upon lineage commitment (31,32).
In this new model, pluripotent cells can keep important
tissue-speciﬁc regulator genes poised for expression by an
opposing histone modiﬁcation. The fact that PcG proteins
can repress genes that are poised for activation indicates that
the dynamic role of PcG complexes seems to be different
from other irreversible epigenetic mechanisms, such as DNA
methylation. Oct4, Sox2 and Nanog, the three key pluripo-
tency transcription factors in hESCs (33), can bind many
genes identiﬁed as PcG targets. Moreover, PcG proteins can
target a similar set of developmental regulators in hESCs
(34). These data suggest that at least for a subset of genes,
PcG proteins can act as transcriptional repressors by collabor-
ating with a speciﬁc set of transcriptional factors.
Recently, Mikkelsen et al. (35) used ChIP for speciﬁc
histone modiﬁcations coupled with high-throughput sequen-
cing technology to generate genome-wide maps of chromatin
state from mESCs and lineage-committed cells. The authors
used H3K27me3 and H3K4me3 antibodies to study 17, 762
promoter regions, discriminating between CpG-rich promoters
(associated with both ubiquitously expressed housekeeping
genes, and genes with more complex expression patterns),
CpG-poor promoters (generally associated with highly tissue-
speciﬁc genes) and promoters with intermediate CpG content.
In mESCs cells, CpG-rich promoters are associated with
active chromatin, as judged by the presence of the
H3K4me3 mark. However, around 22% of these promoters
are bivalent, exhibiting histone markers of both active
(H3K4me3) and repressed (H3K27me3) chromatin. These
genes show low transcriptional levels, suggesting that the
repressive effect of PcG activity can be dominant over the
TrxG activity. Most promoters marked with H3K4me3 alone
retain this mark upon neural differentiation while about half
Figure 1. Derivation and differentiation of human embryonic stem cells to a neuronal lineage. Traditionally, hESCs are derived by culturing the inner cell mass
of a blastocyst, although recent evidence suggests overexpression of subset of key transcription factors can also result in hES-type cells. Neuronal differentiation
of hESCs is achieved through either co-culture with mouse PA6 cells or embryoid body (EB) formation resulting in neural rosettes which are manually dissected.
Neuronal precursor cells derived from rosettes can be enriched through sorting, used for transplantation studies, or further differentiated in vitro.
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of the promoters with bivalent marks resolve to H3K4me3,
concomitant with an increase in gene expression. Conversely,
CpG-poor promoters behave quite differently. In mESCs, only
6.5% have signiﬁcant H3K4me3 marks and virtually none
showed enrichment for H3K27me3. In neuronally derived
cells, most of these promoters lost the H3K4me3 mark,
whereas 1.5% gained the mark. In both situations mESCs
and neuron-derived cells—the expression levels of the associ-
ated genes—strongly correlated with the presence or absence
of H3K4me3. Altogether, these ﬁndings suggest that
CpG-poor promoters may be selectively activated by cell-type
or tissue-speciﬁc factors. In fact, it was shown that in embryo-
nic stem cells, windows of unmethylated CpG dinucleotides
and possible interacting factors mark enhancers for tissue-
speciﬁc genes (36).
While these studies have been performed in mESCs, it is
likely that hESCs are highly dependent on similar modes of
epigenetic regulation. Not only can genome-wide maps of
chromatin-state provide a rich source of information about
different stages of development, they also reveal stem cell-
and lineage-speciﬁc epigenomic signatures that will ultimately
reveal how a single genome produces such a diversity of cell
MICRORNAS MODULATE PROLIFERATION
Small non-coding RNAs may act as an additional layer con-
trolling the gene expression patterns required for maintaining
the pluripotent state of stem cells on the one hand and promot-
ing lineage-speciﬁc differentiation on the other. MicroRNAs
represent the most abundant class of functional small RNAs
in mammalian cells (37) and are also the best understood.
These 19- to 23-nucleotide RNAs are transcribed embedded
in long primary transcripts, from which they are released in
a multi-step processing pathway (38). Upon incorporation of
the mature microRNAs into the effector complex, they nega-
tively regulate gene expression by recognizing mRNAs
through partial sequence complimentarity within mRNA 3
untranslated regions, thereby targeting these transcripts for
degradation, destabilization and/or repression of productive
Experimental evidence indicates that a single microRNA
can directly target at least 100 distinct mRNA species
(40,41). Progress in determining the functional targets of
microRNAs has largely been driven by improvements in the
design and delivery of microRNA mimics and antagonists to
cultured cells and in live animals (42–44). For instance, the
microRNA paralogs miR-125a and miR-125b are dramatically
upregulated during RA-induced neuronal differentiation of
P19 mouse embryonic carcinoma cells, and they have been
shown to directly target and downregulate the product of the
lin-28 gene, a regulator of developmental timing (45). The
brain-enriched microRNA miR-124a targets the laminin
gamma 1 and integrin beta 1 mRNAs, and ectopic expression
of miR-124a in chick embryo causes defects in the basal
lamina of the developing neural tube (46). Similarly, another
brain-enriched microRNA, miR-134, has been shown to be
involved in control of spine development via downregulating
expression of the protein kinase Limk1 (47). These examples
illustrate the profound yet speciﬁc effects that microRNAs
exert on central biological processes.
Several lines of evidence from mouse embryonic stem cell
studies indicate that the microRNA pathway is critical both for
stem cell self-renew al and differentiation in general and for
neurogenesis in particular. Although mESCs deleted fo r
either of the two RNase enzymes essential for microRNA
maturation are viable and do not lose their capacity to generate
embryonic stem cell colonies, the knock-out cells display pro-
liferation defects (48 – 50). Despite the differences in embryo-
nic stem cell maintenance regulation between mouse and
human (51), it is likely that stem cell maintenance in
humans is similarly dependen t on microRNAs. This view is
supported by the fact that many stem cell enriched microRN As
that have been found by cloning in mouse (52) and human (53)
embryonic stem cells are highly related to each other in
sequence, suggesting that they have similar mRNA targets.
In humans, this hESC-enriched microRNA family consists of
miR-93, miR-302a-d, miR-371, miR-372, miR-373 and
miR-520a-h. Since miR-302a-d are also found expressed
highly in embryonic carcinoma cells (53), only miR-371,
miR-372 and miR-373 appear speciﬁc to normal hESCs
cells. While no functional studies of these microRNAs have
been conducted in hESCs cells, the results from primary
human somatic and cancer cells point to a role for miR-3 72
and miR-373 in allowing cells to proliferate by promoting
the bypass of the G1/S cell cycle checkpoint (54). A similar
role for microRNA-dependent regulation has been observed
for stem cell division in Drosophila (55), attesting to the evol-
utionary conservation of their role in stem cell maintenance.
In mESCs, several individual microRNAs have recently
been shown to be able to promote and direct neurogenesis at
all stages. For example, over-expression of mir-134, which
is highly expressed in the adult CNS, boosts mESC differen-
tiation toward the ectodermal lineage, even in the presence
of the stem cell maintenance factor LIF (56). Interestingly,
miR-134 may exert this effect in part by directly downregulat-
ing the Oct4 activators Nanog and LRH1 (56). Modulation of
the relative levels of functional miR-124a and miR-9—both
brain-enriched microRNAs—during differentiation of mESCs-
derived neural progenitors alters the ratio of cells of the glial
versus neuronal lineages, perhaps by acting via the STAT3
pathway (57). Lastly, miR-133b, a midbrain-enriched micro-
RNA, negatively regulates the maturation and function of
mES-derived DAs (42). It appears that miR-133b directly
targets the transcription factor Pitx3, an activator of tyrosine
hydroxylase, the enzyme catalyzing the rate-limiting step in
dopamine (42). In agreement with the notion that microRNAs
may regulate neuronal subtype speciﬁcation, recent micro-
RNA proﬁling of hESC lines revealed cell line-speciﬁc intrin-
sic biases toward particular neuronal lineages, which were
accompanied by distinct microRNA expression patterns
Thus, although we are still awaiting experimental proof, it
seems reasonable to hypothesize that the manipulation of cel-
lular levels of speciﬁc microRNAs may, in the near future,
become a viable option to improve upon—and perhaps even
substitute for—current differentiation protocols for hESCs.
Such an approach would likely entail the dosed delivery of
R70 Human Molecular Genetics, 2008, Vol. 17, Review Issue 1
agonists and inhibitors of sets of microRNAs at several stages
during the differentiation procedure.
GENE EXPRESSION AND STEM CELL
Genome-wide expression proﬁling provides a basis to examine
changes of genes and pathways in response to hESC differen-
tiation. Several groups have utilized high-throughput proﬁling
methods such as microarrays, expressed sequence tags (ESTs),
serial analysis of gene expression, massively parallel signature
sequencing proﬁling (MPSS) and RT-PCR methods to
compare transcriptional proﬁles in different hESC lines and
to study expression changes during the differentiation of
hESCs to various lineages (60–64). By studying ESTs,
Brandenberger et al. (63) identiﬁed 32 000 unique transcripts
as being expressed in undifferentiated hESCs, among them
500 were signiﬁcantly upregulated, and 150 were downre-
gulated. Wright et al. (65) identiﬁed ‘expressed’ and ‘not
expressed’ genes in NPCs isolated from the human embryonic
cortex; Cai et al. (66) used the MPSS technique to analyze
expression of fetal NPCs in comparison to hESCs and astro-
cyte precursors; Maisel et al. (67) used Affymetrix Gene
Chip arrays to compare adult and fetal NPCs propagated as
neurospheres. Using SAGE and MPSS, Richards et al. (68)
and Miura et al. (62) identiﬁed 190 or 50 genes that were
upregulated in hESCs compared with differentiated cells.
Some genes were often found in different expression datasets,
which included Oct4, Sox2 and Nanog, and were considered to
be molecular signatures of ESCs.
Apart from transcriptional changes, co- or posttranscrip-
tional mRNA regulation also occurs through alternative spli-
cing (AS), whereby more than one transcript isoform of a
gene is produced. In the case of stem cells, AS generates
different transcripts at different stages of differentiation and
frequently contributes to the regulation of gene expression
by generating tissue-speciﬁc mRNA and protein isoforms
(69–72). Underscoring the importance of AS in gene regu-
lation, recent studies using splicing-sensitive microarrays
suggested that up to 75% of human genes undergo AS (73).
Hence, it is not surprising that AS plays important roles in reg-
ulating neuronal gene expression and function (74,75).
A number of regulatory proteins are likely involved in regu-
lating AS. These proteins are in the spliceosome particles, the
SR protein family, the heterogeneous nuclear ribonucleopro-
teins, as well as auxiliary splicing factors, typically RNA-
binding proteins (76). These splicing factors bind to short,
degenerate cis-regulatory elements located in the exonic and
intronic regions of AS exons regulate splicing choices, and dis-
ruptions in these elements lead to genetic diseases (77). While
almost nothing is known about which splicing factors are
important for AS regulation in human or mouse ESCs,
several factors have been implicated in being important in reg-
ulating neuronal differentiation and maturation. Recently, three
publications demonstrate that the polypyrimidine tract-binding
protein (PTB/PTBP1) is critical in keeping non-neuronal cells
from differentiating into neurons (78–80). PTB is known as a
splicing repressor of neuron-speciﬁc exon usage in a myriad
of pre-mRNAs (81). The three studies showed that knocking
down PTB protein is sufﬁcient to trigger neuronal-speciﬁc AS
in non-neuronal cells. Interestingly, neurons express a paralog
of PTB, nPTB (PTBP2), which acts as a weaker splicing repres-
sor compared with PTB; and PTB and nPTB are expressed in a
mutually exclusive fashion. PTB directly represses nPTB
expression in non-neuronal cells by preventing exon 10
inclusion in nPTB, which introduces a premature translation ter-
mination codon, thereby degrading nPTB mRNA via the
nonsense-mediated pathway. To answer why and how PTB is
excluded from neurons, Makeyev et al.(79)showthata
neuronal-enriched microRNA (mir-124) directly targets the 3
untranslated region of PTB, silences PTB expression, and
does not induce but promotes neuronal differentiation of
mouse P19 cells. It is an open question whether these same
regulatory networks pertain to hESCs, a non-neuronal cell
and differentiated NSCs.
The splicing ﬁeld is currently in a cataloguing phase of
attempting to identify all isoforms in various tissues and cell
lines. To our knowledge, AS has not been implicated in stem
cell biology, until recently—a newly identiﬁed isoform of
FGF4, FGF4si counters the growth-promoting effects of
FGF4 (82). Interestingly, while FGF4 ceases to be expressed
in late differentiated cells, FGF4si does not. It is still unclear
which splicing factor regulates the generation of the FGFsi
isoform. To systematically identify AS isoforms, two
approaches have been taken, namely ESTs and microarrays.
Pritsker et al. (83) identiﬁed alternatively spliced isoforms of
thousands of genes, using ESTs derived from embryonic and
hematopoietic stem cells. Genes identiﬁed as producing splice
variants showed signiﬁcant enrichment for those encoding com-
ponents of signaling pathways, as well those involved in stem
cell self-renewal and differentiation. Using splicing-sensitive
microarrays, Yeo et al. identiﬁed and characterized AS events
that distinguish pluripotent hESCs from NPCs, in order to
pave the way for future candidate gene approaches to study
the impact of AS in hESCs and NPCs. REAP, a regression-
based method for analyzing exon array data, was introduced
and was applied to discover AS events in hESCs, compared
with NPCs derived from hESC (12). REAP detected more
than 1000 AS events. Interestingly, the study showed that
only a minority of AS events was common between various
hESC-to-NPC comparisons. A possible explanation is that the
cell lines were not only genetically different but were also
exposed to different isolation and culture conditions. It is also
possible that posttranscriptional changes such as AS may be
more variable despite the cells being at acknowledged ‘end-
points’ deﬁned by a limited set of immunohistochemical
markers. However, this suggests that utilizing differences in
AS as a molecular signature of various degrees of differen-
tiation may be far more sensitive and accurate than simply
gene expression. These insights clearly highlight the previously
underappreciated complexity in gene regulation by AS in stem
cell and neuronal differentiation.
POSTTRANSLATIONAL CHANGES IN STEM
Although patterns of gene expression and protein activity
are cru cial for biological processes, many of the interesting
Human Molecular Genetics, 2008, Vol. 17, Review Issue 1 R71
regulatory steps, particularly involved in cell proliferation,
migration and differentiation, are likely to depend on proteins’
posttranslational modiﬁcations rather than expression.
However, there is a dearth of literature on the importance of
posttranslational in stem cell biology. A recent example,
Zhang et al. (84) showed that Oct-4, which plays an important
role in maintaining self-renewal in embryonic stem cells, can
be modulated posttranslationally by SUMO. This underscores
Figure 2. Molecular regulation of stem cells. Regulation of biological processes in hESCs occurs at multiple levels such as epigenetic changes (methylation and
acetylation), regulation of alternative splicing and through post-translational modiﬁcations such as phosphorylation, through the action of microRNAs which can
lead to the translational repression and/or degradation of target mRNAs.
R72 Human Molecular Genetics, 2008, Vol. 17, Review Issue 1
the need for proteomics in regulating the posttranslational
modiﬁcations regulating embryonic stem cell biology. For
example, protein phosphorylation can alter the interactions
between the phosphorylated protein and the rest of the pro-
teome. Phosphoproteome analysis perfo rmed on mESCs (85)
found that many chromatin remodeling proteins are differen-
tially regulated by phosphorylation in embryonic stem cells
compared with differentiated cells. Because the activity of
kinases and phophatases can be regulated by small molecules,
identifying the phosphoprot eome will present an excellent
opportunity to identify small molecule chemical regulators
of pluripotency or directed differentiation of hESCs.
The diversity of regulatory processing at multiple layers
during embryonic stem cell differentiation suggests many
opportunities for interventi on to promote self-renewal or
directed neural differentiation. Figure 2 presents a summary
of the layers discussed in this review. For example, small mol-
ecules have been shown to affect the epigenetic regulation of
gene expression (DNA methylation and histone modiﬁcations)
which affects stem cell proliferation and differentiation
(86–88). RNA interference (RNAi) can also be utilized to
silence genes and reveal their potential roles in proliferation
and differentiation (89–91). Endogenous microRNAs may
also be utilized to control proliferation and differentiation
into speciﬁc lineages. For example, mir-133b has been
shown to regulate the maturation and function of midbrain
DAs in mammals (42). However, our overview of the recent
advances in these aspects of molecular control of stem cell
biology reveals more questions than answers, and illuminates
signiﬁcant gaps in our understanding, and much room for
future work. We end with two broad future goals for the
ﬁelds: (i) determining how epigenetic signatures relate to tran-
scriptional changes in protein-cod ing and miRNA-coding
genes will be important for understanding stem cell biology
(92); (ii) with the diversity of neural subtypes that can be gen-
erated from hESCs, it is a matter of time before we see more
crossings between AS, microRN As and posttranslational
medications as modes of biological richness.
The authors would like to thank Jamie Simon for ﬁgure
Conﬂict of Interest statement. None declared.
G.W.Y. is a Junior Fellow at the Crick-Jacobs Center of Com-
putational and Theoretical Biology. B.W. is a Feodor-Lynnen
fellow of the Alexander von Humboldt Foundation. S.A. is a
Damon Runyon Fellow supported by the Damon Runyon
Cancer Research Foundation (DRG-1859-05). A.R.M. is
supported by the Rett Synd rome Research Foundation.
M.C.N.M. is supported by the George E. Hewitt Foundation
for Medical Research. J.A.S. is supported by the California
Institute of Regenerative Medicine. N.C. is supported by the
Lookout Fund. F.H.G. is supported by the Lookout Fund and
the National Institutes of Health; National Institute on Aging
and National Institute of Neurological Disease and Stroke.
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