REVIEW ARTICLE 279

Molecular Mechanisms Involved

in Self-Renewal and Pluripotency
of Embryonic Stem Cells
NA LIU, MIN LU, XUEMEI TIAN, AND ZHONGCHAO HAN*
State Key Laboratory of Experimental Hematology, National Research Center for Stem Cell Engineering and Technology,
Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Embryonic stem cells (ES cells) are derived from inner cell mass (ICM). The self-renewal and pluripotency are the main specificities of ES
cells, which are likely to reveal a deeper understanding of human cellular biology and which are considered to be promising sources for cell
therapy to treat patients with degenerative diseases in clinical. Growth of ES cells as a pluripotent population requires a balance between
survival, proliferation, and self-renewal signals. In fact, the precise mechanism that regulates stem cell self-renewal and pluripotency
remains largely unknown. Recently, in vitro and in vivo studies have identified several genetic regulators that may play important roles in
the self-renewal and pluripotency process of human and mouse ES cells, including extracellular signaling factors, transcription factors, cell-
cycle regulators, microRNA, genes implicated in chromosomal stability, and DNA methylation. In this review, we will summarize the
currently known molecular regulators for ES cells self-renewal, and we will propose some possibilities to explain the ways in which these
distinct pathways might interact.
J. Cell. Physiol. 211: 279–286, 2007. ß 2006 Wiley-Liss, Inc.

Embryonic stem cells (ES cells) were first established from the
inner cell mass (ICM) of mouse blastocysts in 1981 (Evans and
Kaufman, 1981). Human ES cells were first derived in 1998
(Thomson et al., 1998), which possess the remarkable property
of pluripotency, giving rise to all cells of the organism. For this
reason, ES cells are thought to hold great promise for
regenerative medicine (Pera and Trounson, 2004; Liew et al.,
2005). Therefore, a detailed understanding of the mechanisms
that enable propagation of ES cells in a pluripotent state is
essential to realize their therapeutic potential. Now, a great
deal of effort is focused on experiments aimed at improving
culture conditions, generating new ES cell lines suitable for
clinical purpose and founding efficient differentiation method to
produce human cells suitable for transplantation. All of these
need to understand the molecular mechanism involved in
self-renewal and pluripotency in ES cells. Understanding the
molecular mechanism by which pluripotency is maintained in
human ES cells is important for the development of improved
methods to derive and culture human ES cells. It is of equal
importance to understand the changes which take place in such
signaling networks as a cause or consequence of ES cells
differentiation, as this may help us to develop better strategies
to direct differentiation into defined populations of cells for
therapeutic use (Armstrong et al., 2006).
In fact, the precise mechanism that regulates stem cell
self-renewal and pluripotency remains largely unknown. Thus,
investigation into the molecular and cellular mechanisms of
stem cell self-renewal and pluripotency should help meet these
challenges and provide the necessary tools to harness the
regenerative potential of ES cells for therapeutical purposes.
The past few years have seen remarkable progress in our
understanding of ES cells biology. Pluripotency of mouse and
human ES cells is regulated by distinct signaling pathways.
Recently, in vitro and in vivo studies have identified several
genetic regulators that may play important roles in the human
and mouse ES cell self-renewal and pluripotency process. Extra
signaling factors, transcription factors, cell-cycle regulators,
microRNA, genes implicated in chromosomal stability, and
DNA methylation. In this review, we will summarize the
currently known molecular regulators for ES cells self-renewal,
and we will propose some possibilities to explain the ways in
which these distinct pathways might interact.
Signaling Transduction Pathways
LIF-STAT3 pathway
Mouse embryonic stem cells (mES cells) could be expanded
continuously in culture when co-cultured with mitotic
inactivated embryonic fibroblast cells and resulted in stem cells
capable of multi-lineage differentiation (Evans and Kaufman,
1981). The factor capable of promoting self-renewal and
inhibiting differentiation provided by the feeder cells was later
identified as leukemia inhibitory factor (LIF), a member of the
IL-6 cytokine family (Smith et al., 1988; Williams et al., 1988).
LIF stimulates mES cells through the gp130, which works as a
heterodimer together with LIFR. Activation of gp130 leads to
the activation of the Janus-associated tyrosine kinase (JAK) and
of signal transducer and activation of transcription (STAT). The
ability of LIF to maintain the mES cells self-renewal is dependent
upon activation of STAT3 (Niwa et al., 1998), whose activation
is sufficient to prevent mES cells differentiation in the presence
of serum (Matsuda et al., 1999). In addition to the activation of
STAT3, LIF also stimulates the activation of the
mitogen-activated protein kinase (MAPK), whose activation
promotes differentiation. Suppression of ERKs signal can
promote mES cells self-renewal (Burdon et al., 1999). MAPK
signal may be a negative regulation mechanism for STAT3. mES
cells can maintain their property in the presence of LIF due to
the balance of the STAT3 activation and MAPK effect.
Although the essential functions of LIF and STAT3 in self-
renewal of mES cells have been thoroughly documented, the
downstream target genes of activated STAT3 in mES cells have
remained elusive. Recently, in order to isolate these genes,
researchers performed a microarray-based kinetic comparison
of LIF-stimulated (undifferentiated) ES cells versus ES cells
*Correspondence to: Zhongchao Han, Institute of Hematology,
Chinese Academy of Medical Sciences and Peking Union Medical
College, Tianjin 300020, China. E-mail: tihzchan@public.tpt.tj.cn
Received 2 November 2006; Accepted 3 November 2006
DOI: 10.1002/jcp.20978

ß 2 0 0 6 W I L E Y - L I S S , I N C .
280 LIU ET AL.
induced to differentiate by shutting down STAT3 activity
through either LIF deprivation or, more specifically, expression
of a STAT3 dominant-negative mutant. In this experiment,
some growth factors such as Lefty1 or transcriptional
regulators such as Id1 and Id2 and the groucho-like protein
Aes1 were found to be related to STAT3. Aes1 may be the
direct target of STAT3 (Sekkai et al., 2005). There may be some
links between LIF-STAT3 and signaling pathways involved in
the maintenance of stem cell properties such as BMP and Wnt.
Furthermore, Esg-1 (ES-cell-specific protein) may be another
direct or indirect target gene of STAT3, although the actual
binding and transcriptional activation of Esg-1 by STAT3
remains to be tested (Tanaka et al., 2002). These identified
genes are required to better understand the molecular
mechanisms involved in ES cells self-renewal.
Cartwright indicated a role for the transcription factor c-Myc in
self-renewal by functioning as a key target of LIF-STAT3
signaling. The expression of c-Myc is rapidly downregulated
within the first 36 h following LIF withdrawal. Expression of
c-Myc renders self-renewal independent of LIF, while
expression of a dominant negative form of c-Myc antagonizes
self-renewal and promotes differentiation (Cartwright et al.,
2005). The induction of c-Myc by STAT3 is in agreement with
infinite proliferation of ES cells through c-Myc induction of the
regulatory subunit of telomerase (Wang et al., 1998).
It is known that BCR-ABL also can stimulate STAT3 activity.
Further studies reveal that BCR-ABL expressing mESC
persistently exhibited a primitive morphology, despite LIF
withdrawal (Coppo et al., 2003). In this progress, MEKK1
plays a key role in self-renewal and STAT3 activation in
BCR-ABL-transformed mES cells (Nakamura et al., 2005).
LIFR and gp130 are also expressed in human embryonic stem
cells (hES cells) and functional activation of the STAT3 by human
LIF (Daheron et al., 2004). However, this is not sufficient to
maintain hES cell pluripotency in vitro. Furthermore studies
found that hES cells can maintain their property in vitro, which is
independent of LIF-STAT3 (Humphrey et al., 2004). The
different effect of LIF-STAT3 in the hES cells and mES cells may
be due to the different stage in their development.
MAPK-ERK pathway
mES cells show high ERK activity when they are stimulated to
undergo differentiation. Suppression of ERK signaling pathway
promotes mES cells self-renewal. And, BMP activation inhibits
mES cells differentiation cooperated with LIF due to Id
expression and ERK inactivation (Burdon et al., 1999; Qi et al.,
2004; Lodge et al., 2005). However, hES cells display high-level
basal activity of ERK in undifferentiation status. Its activation
relates to the bFGF signaling which can maintain human ES cells
self-renewal for a long time (Dvorak et al., 2005a; Kang et al.,
2005; Levenstein et al., 2006). ERK activation may be at
downstream of PI3K, which is also important in human ES cells
self-renewal (Armstrong et al., 2006).
PI3K pathway
Phosphoinositide 3-kinase (PI3K) pathway is important for
proliferation, survival, and maintenance of pluripotency in ES
cells. Eras is specifically expressed in ES cells, which stimulates
PI3K. PI3K activation promotes ES cell proliferation (Takahashi
et al., 2003). In addition to proliferation for ES cells, PI3K activity
might be also crucial for self-renewal of ES cells (Takahashi et al.,
2005). It has been reported that the inhibition of PI3K and Akt
induces differentiation of mouse and human ES cells in the
presence of LIF and feeder cells, respectively (Paling et al., 2004;
Kim et al., 2005), suggesting that P13K/Akt signaling is necessary
for the maintenance of ES cell pluripotency. Watanabe et al.
(2006) also demonstrate that activation of Akt signaling can
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
maintain the pluripotency of mouse ES cells independent of
Wnt/b-catenin signaling. PI3K/Akt signaling can be activated by
the growth factors, which participate in ES cell pluripotency,
such as LIF and bFGF (Jirmanova et al., 2002; Xu et al., 2005b).
Akt controls various cellular processes through a number of
target proteins. The mTOR is a critical downstream effector for
PI3K/Akt signaling. But the addition of rapamycin, an inhibitor of
mTOR, to the Akt-expressing mouse ES cells inhibited
proliferation, but did not induce differentiation. Additional
activation of mTOR is not sufficient for the LIF-independent
maintenance of pluripotency, suggesting that mTOR is not a
major downstream effector in ES cells pluripotency
maintenance (Watanabe et al., 2006). Short G1 phase
contributes to the ES cells undifferentiation status, and Akt can
promote a G1 phase to S phase transition by facilitating the
formation of cyclin/CDK complexes (Brazil et al., 2004). This
mechanism may be the key role of Akt for ES cells self-renewal.
However, overexpression of cyclin D, regulated by Akt, does
not maintain the undifferentiated status of ES cells. Thus, the
critical target molecules remain to be identified (Watanabe
et al., 2006). Beside these, ERK pathway may be another
mechanism for PI3K effect in maintaining ES cells pluripotency
(Armstrong et al., 2006).
In ES cells, p53 promotes differentiation by suppression of
Nanog expression (Lin et al., 2005). This effect depended on the
phosphorylation status of Ser315 of p53, which is a substrate of
GSK3b (Watcharasit et al., 2002; Qu et al., 2004). GSK3b is
negatively regulated by PI3K and Akt (Hay and Sonenberg,
2004). Taken together, PI3K may contribute to self-renewal of
ES cells through GSK3b, p53, and Nanog (Takahashi et al.,
2003).
Furthermore, PI3K/Akt signaling is required to suppress
apoptosis in ES cells. The density of ES cells is very important for
ES cells self-renewal. Thus, this may be another mechanism of
PI3K in ES cell self-renewal (Gross et al., 2005).
Wnt
Recently, evidence has been presented that the Wnt pathway
could be involved in the maintenance of pluripotency of both
human and mouse ES cells. Activation of Wnt pathway by
6-bromoindirubin-3-oxime (BIO), a specific pharmacological
inhibitor of glycogen synthase kinase-3 (GSK-3), can maintain
the undifferentiated phenotype in ES cells and sustain
expression of the ES cells specific markers. Wnt signaling is
endogenously activated in ES cells and is downregulated upon
differentiation (Sato et al., 2004). In contrast to LIF-STAT3 and
BMP signaling, the effect of Wnt/b-catenin signaling in ES
self-renewal has no difference between mouse and human ES
cells. Wnt signaling activation can upregulate STAT3
expression, suggesting that it has synergistic effect with
LIF-STAT3 (Hao et al., 2006). Furthermore, the Wnt signaling
pathway has been shown to elevate the level of c-Myc, which is
also the target gene of STAT3 that was described above. Thus,
Wnt and LIF-STAT3 may converge on c-Myc (Cartwright et al.,
2005; Kristensen et al., 2005).
TGFb pathways
TGFb is a prototypic member of a large superfamily of related
growth and differentiation factors. The family has more than
40 members, including TGFb, Activin, Nodal, and bone
morphogenetic proteins (BMPs). These ligands are all
associated with ES cells. The TGFb transduces signal from the
membrane to the nucleus by binding to heteromeric complex of
serine/threonine kinase receptors known as TGFb type I and
type II receptors. TGFb and activin have high affinity for the type
II receptors but do not bind to the type I receptors in the
absence of type II receptor, whereas BMPs have higher affinity
 MECHANISMS IN ES CELLS SELF-RENEWAL
for their type I receptors than for type II (Valdimarsdottir and
Mummery, 2005).
BMP4. BMP4 is a member of the TGFb superfamily but it
functions via a different signaling pathway with TGFb.
Activation of STAT3 is sufficient for mES cells self-renewal. But
4 years later, researchers found that LIF-STAT3 cannot
maintain mES cells in serum-free medium. That is to say that
there are many unknown factors present in serum which are
required to co-affect with LIF-STAT3. This serum factor is likely
to be BMP4 acting via activation of Smad1/5/8. Addition of BMP4
to the media enables LIF to maintain mES cells in serum-free
culture (Ying et al., 2003). BMP4 have been shown to
phosphorylate Smad1/5 in both human and mouse ES cells.
Smad1/5 activation results in the expression of inhibitor of
differentiation (Id) protein, which functionally antagonizes
neurogenic bHLH transcription factors and blocks the neural
differentiation (Ying et al., 2003; Gerrard et al., 2005).
Exogenous expression of Id mimics the effect of BMP4 in mouse
ES cells (Ying et al., 2003). Furthermore, BMP4 can maintain
mouse ES cells in the presence of LIF, possibly by blocking the
MAPK signaling cascade (Qi et al., 2004). In a word, BMP4
contributes to mESC self-renewal; this effect needs LIF
presence.
In contrast to mES cells, BMP4 does not maintain the hES cells
self-renewal and in fact induces hES cells trophoblast or
primitive endoderm differentiation (Xu et al., 2002). Noggin, an
inhibitor of BMP4 signaling, sustains undifferentiated states of
human ES cells in the presence of basic FGF (Xu et al., 2005a).
GDF3 is a secreted factor of the TGFb family. It is expressed at
high levels during pluripotency and declines dramatically when
ES cells undergo differentiation (Sato et al., 2003). GDF3 may
play a role in the ES cells pluripotency, but recent study found
that GDF3 effect is different in human ES cells and mouse ES
cells. In human ES cells, GDF3 overexpression contributes to
the undifferentiation status. By contrast, in mouse ES cells,
reduced GDF3 levels helped to support pluripotency (Levine
and Brivanlou, 2006). GDF3 may be an inhibitor of BMP4; that is
why GDF3 function in ES cells opposes to BMP4.
TGFb/activin/nodal. Gene expression profile indicates
TGFb and its correlate factors, including Nodal Cripto Lefty1
and Lefty2, all expressed at high levels in undifferentiated human
ES cells (Sato et al., 2003). Activin A, a member of the TGFb
superfamily, is secreted by mouse embryonic feeders, and that
cultured medium enriched with activin A maintains human ES
cells undifferentiated without condition medium (CM) or
STAT3 activation (Beattie et al., 2005). These data are
supported by James and colleagues who reported that the
TGFb/Activin/Nodal pathway is activated through the
transcription factors Smad2/3 in undifferentiated cells. Smad2/3
activation suppresses Smad1/5 activity, thus TGFb/Activin/
Noda can negatively regulate BMP4 in human ES cells (Beattie
et al., 2005; James et al., 2005). But in mouse ES cells, upon
withdrawal of LIF, levels of Smad2/3 phosphorylation are
reduced, but the SB431542-mediated inhibition of Smad2/3
signaling in the context of LIF resulted in no significant change in
Oct4 levels, suggesting that the Smad2/3 activity is not
necessary for maintenance of undifferentiated state in mouse ES
cells (James et al., 2005). BIO can maintain undifferentiation of
human ES cells by activation of Wnt signaling. In this condition,
the level of Smad2/3 phosphorylation is also elevated (James
et al., 2005). This signaling may be correlated with Wnt. But the
role of the TGFb superfamily in ES self-renewal is not well
understood.
bFGF pathway
ESC can maintain their pluripotency with feeder cells. And
mouse ES cells can also maintain their undifferentiated status in
the presence of LIF without feeder cells. However, LIF cannot
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
281
maintain human ES cells. Human ES cells have most commonly
been cultured in the presence of basic fibroblast growth factor
(bFGF) either on fibroblast feeder layers or in
fibroblast-conditioned medium. bFGF signaling appears to be of
central importance to human ES cells self-renewal (Amit et al.,
2000; Xu et al., 2001).
Investigations that examined the expression of different
elements of signaling pathways in human ES cells have shown the
presence of elements of FGF signaling, including all four FGF
receptors and certain components of their downstream
cascade, which are enriched in undifferentiated human ES cells
in comparison to their differentiated derivatives (Bhattacharya
et al., 2004; Brandenberger et al., 2004; Dvash et al., 2004; Ginis
et al., 2004). Among the four FGFRs, the expression of FGFR1 is
most abundant in undifferentiated human ES cells. SU5402
(inhibitor of FGFR) causes rapid differentiation (Dvorak and
Hampl, 2005).
Human ES cells are routinely cultured on feeder layers of
fibroblasts in medium that is supplemented with bFGF at the
concentration of 4 ng/ml (Thomson et al., 1998). Due to
expected clinical requirements, many laboratories have begun
culturing human ES cells in a feeder-free system. In this case,
they increased the concentration of bFGF to 8 ng/ml
(Carpenter et al., 2004). Recently, researchers have shown that
bFGF (40 ng/ml) combined with noggin (inhibitor of BMP4)
supports the undifferentiated proliferation of human ES cells in
the absence of feeder cells (Dvorak et al., 2005a; Wang et al.,
2005; Xu et al., 2005a). Furthermore, high concentration bFGF
(100 ng/ml) alone is sufficient to maintain human ES cells (Xu
et al., 2005b). Although it is likely that exogenous bFGF
supports growth of undifferentiated human ES cells, the
mechanism of bFGF action is not yet clearly established. Some
researchers found that bFGF can stimulate MAPK signaling
cascade and induces the expression of c-fos in human ES cells
(Kang et al., 2005; Dvorak et al., 2005a). bFGF also can inhibit
BMP activity by preventing the nuclear translocation of
phosphorylated Smad1 (Xu et al., 2005a).
In serum-free media not substituted with bFGF, the cells
differentiate, while its addition makes the cells morphology
more compact and enables prolonged undifferentiated culture
under these conditions. In addition, bFGF enhances the cloning
efficiency of cells (Amit et al., 2000). But the detailed mechanism
of bFGF in ES cells with undifferentiated maintenance requires
to be declared.
Transcription Factors
Recent experiments have also elucidated transcriptional
regulatory circuitry responsible for ES cells self-renewal and
differentiation, which involves the transcription factors Oct4,
Sox2, Nanog, and et al. Some of these factors are expressed
specifically in pluripotent cells, such as Oct4 and Nanog. These
transcriptional factors may be essential for ES cells self-renewal
and differentiation. They are switched ON/OFF by input
environmental signals, such as the signals we described above.
And they are also regulated by themselves. When these genes
are expressed, the self-renewal genes are activated, and the
differentiated genes are repressed. So the ES cells can maintain
their pluripotency.
Oct4/Sox2
Oct4 (also known as Oct3 and encoded by Pou5f1) is a POU
domain-containing transcription factor that binds to an
octamer sequence, ATGCAAAT. During mouse
preimplantation development, Oct4 expression is activated at
the four-cell stage and is later restricted to the pluripotent cells,
such as ES cells, ICM, and germ cells. Oct4 is highly expressed in
human and mouse ES cells, and its expression diminishes when
282 LIU ET AL.
these cells differentiate and lose pluripotency. The precise
levels of this gene are required to maintain the ES cells state.
Loss of Oct4 causes inappropriate differentiation of ES cells into
trophectoderm, whereas overexpression of Oct4 results in
differentiation into primitive endoderm and mesoderm (Yeom
et al., 1996; Niwa, 2001). Several target genes of Oct4 in ES cells
have been identified, and these include Fgf4, Utf1, Opn, Rex1/
Zfp42, Fbx15, and Sox2 (Nishimoto et al., 1999; Tomioka et al.,
2002; Zeng et al., 2004). Sox2 is an HMG-family protein that
occupies many gene targets with Oct4 and is also required in ES
cells with pluripotent sustenance.
LIF does not appear to regulate Oct4, and Oct4 does not appear
to regulate Jak-STAT signaling, suggesting that the Oct4
pathway is a parallel pathway for maintaining ES self-renewal.
Many of Oct4 target genes also contain STAT-binding sites,
suggesting that the two transcription factors may cooperate in
ES cells. ESG1 (embryo-specific gene 1, originally called Dppa5)
is highly expressed in human ES cells, and is downregulated as ES
cells differentiate as much as in mouse ES cells. ESG1 appears to
be downstream of both STAT3 and Oct4 pathways. But, how
ESG1 integrates Oct4 and STAT3 remains to be determined
(Tanaka et al., 2002; Ginis et al., 2004).
In contrast with its target genes, little is known about upstream
regulators. Oct4 promoter contains conserved distal and
proximal enhancers that can either repress or activate its
expression depending on the binding factors occupying these
sites (Pan et al., 2002). The quantitative level of Oct4 is
important for the fate determination of primitive cells. To
maintain the pluripotent state, it is required that the cells
maintain the Oct4 protein level within the narrow window of
abundance. Its expression can be regulated by itself (Chew et al.,
2005; Okumura-Nakanishi et al., 2005). This circuit regulation
may contribute to a robust maintenance of the steady levels of
Oct4 in ES cells. Furthermore, Nanog and FoxD3 also can
activate Oct4 expression (Pan et al., 2006).
Nanog
Nanog is another homeobox-containing transcription factor
with an essential role in maintaining the pluripotent cells of the
inner cells mass and ES cells. It is expressed in pluripotent cells,
and is absent from differentiated cells. Nanog disruption in ES
cells results in differentiation to endoderm lineages.
Overexpression of Nanog in mouse ES cells renders ES cells
self-renewal independent LIF, although the cells self-renewal
capability is reduced, suggesting that Nanog is a major regulator
of the pluripotent state (Chambers et al., 2003; Mitsui et al.,
2003). The Nanog overexpression effect is neither dependent
on STAT3 activation nor needs BMP4 presence. But the
overexpression of Nanog can maintain Id expression (Ying
et al., 2003).
The mechanism through which Nanog regulates stem cell
pluripotency remains entirely unknown. Based on the
differences in gene expression between wild-type and Nanog
null cells, it has been proposed that Nanog regulates
pluripotency mainly as a transcription repressor for
downstream genes that are important for cell differentiation
such as Gata4 and Gata6 (Chambers et al., 2003; Mitsui et al.,
2003). However, Nanog can also activate the genes necessary
for self-renewal such as Rex1. Its C-terminus may contribute to
its transcriptional activity (Pan and Pei, 2005; Shi et al., 2006). As
we described above, Rex1 is a target gene of Oct4/Sox2
complex. Rex1 is also directly regulated by Nanog, suggesting
that Rex1 may be an intersection of Nanog and Oct4/Sox2
signaling (Shi et al., 2006). And Nanog can also activate Oct4
promoter (Pan et al., 2006).
In a recent experiment, Nanog may be regulated by STAT3 and
Brachyury (Brachyury may be a target of Wnt/b-catenin); then
it directly binds to Smad1 and blocks the transcriptional
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
activation by interfering with the recruitment of coactivators
(Suzuki et al., 2006), suggesting that Nanog may interact with
Wnt and BMP4 signaling pathways. These can explain the
phenomenon that mouse ES cells can maintain self-renewal
when overexpressing Nanog, but the capability is reduced
(Chambers et al., 2003; Mitsui et al., 2003). Furthermore,
Nanog promoter has been suggested as a direct target of the
Oct4/Sox2 complex through Chip analysis, in vitro-binding
experiments and RNAi-mediated knockdowns (Boyer et al.,
2005; Kuroda et al., 2005; Rodda et al., 2005).
Network between these factors
Oct4, Sox2, and Nanog may be the main transcriptional factors
in regulating ES cells pluripotency. Recent studies have enabled
the construction of transcriptional regulatory networks in ES
cells that provide a foundation for understanding how these
factors control pluripotency and influence subsequent
differentiation events. These key transcription factors have also
been identified that form an intrinsic core-regulatory circuit
that maintains ES cells in the pluripotent state in vitro. Using
RNA interference method, microarray analysis, and genome
wide chromatin immunoprecipitation experiments, numerous
target genes bound by Nanog, Oct4, and Sox2 have been
identified. These factors appear to form a tight transcriptional
regulatory circuit that maintains ES cells in a pluripotent state
(Boyer et al., 2005, 2006a; Ivanova et al., 2006; Loh et al., 2006;
Rao and Orkin, 2006).
Oct4 activity is modulated by interactions with other
transcription factors that include Sox2, FoxD3, and ESG1 which
are highly expressed in ES cells. Sox2 (SRY-related HMG box2),
a member of the HMG-domain DNA-binding protein family,
forms a ternary complex with Oct4 protein in the enhancer
element of target gene and regulates its expression as a binary
complex. Sox2 has an expression pattern similar to Oct4. All of
the Oct4 target genes have octamer and sox heptamer
elements separated by either 0 or 3 bp. Oct4 interacts with
Sox2 to regulate downstream genes. As we described above,
Oct4/Sox2 complex can regulate the Oct4 and Sox2 expression.
Beside these target genes, recently, researchers found that
Nanog is another target of Oct4/Sox2 complex (Kuroda et al.,
2005; Rodda et al., 2005). There is Oct4/Sox2-binding site in the
Nanog promoter. That is to say that Oct4/Sox2 complex plays a
key role in maintaining the expression of essential transcription
factors in ES cells through autoregulatory and multicomponent
loop network motifs. Conserved Oct3/4 and Sox2 co-binding
domains were identified in most ES expressed genes,
highlighting the importance of this transcriptional pathway.
In addition to Oct-sox cooperative binding, interaction
between FoxD3 (forkhead family member D3) and Oct has also
been reported. FoxD3 appears to be nonessential, and differing
expression has been reported in various human ES cell lines
(Ginis et al., 2004; Richards et al., 2004). As we described above,
Oct4 can regulate Nanog expression. Oct4 maintains Nanog
activity by directly activating its promoter when expressed
below steady state, yet represses it at par with or above steady
state concentration in ES cells. This can explain the evidence
that overexpression of Oct4 induces differentiation. FoxD3,
isolated as a transcription factor with restrictive expression in
ES cells, is also required for the maintenance of embryonic cells
in early mouse embryos, suggesting that it may play a role in
regulating stem cells self-renewal and pluripotency alongside
Oct4 and Nanog. FoxD3, a novel activator of Nanog,
counteracts the repressive activity of Oct4 on Nanog promoter
at the steady state (Pan et al., 2006). These transcription factors,
playing important roles in ES cells, form regulatory circuit. This
regulatory circuit maintains ES cells self-renewal and
pluripotency by sustaining transcription factors expression at
precise level.
 MECHANISMS IN ES CELLS SELF-RENEWAL
Factors involved in somatic cells’ reprogramming
Fusion experiments with mouse ES cells have shown the
dominance of the ES cell phenotype over that of somatic cells,
implying that proteins in the nucleus of ES cells are able to
reprogram more differentiated cells to an embryo-like state
(Cowan et al., 2005). In ES cell–NS cell (neural stem cell) fusion,
the frequency of reprogramming was markedly enhanced by
modestly increasing Nanog expression in ES cells. This is
consistent with the view that Nanog is a major driver of
pluripotency (Rao and Orkin, 2006; Silva et al., 2006). Recently,
further studies found that forcing the expression of ES
cells-specific genes, particularly transcription factors (Oct4,
Sox2, c-Myc, Klf4), in somatic cells might induce them to take on
a more embryonic character (Takahashi and Yamanaka, 2006).
Unexpectedly, Nanog was dispensable. However, c-Myc and
Klf4 is essential in this experiment. The c-Myc may be associated
with histone acetyltransferase (HAT) complex, and induces
global histone acetylation, thus allowing Oct4 and Sox2 to bind
to their specific target loci. Klf4 might contribute to activation
of Nanog and other ES cells-specific genes through p53
repression (Takahashi and Yamanaka, 2006). These factors
induce somatic cell de-differentiation, always following the
chromatin modification. That is to say, chromatin modification,
as a consequence of these transcription factors, may play an
important role in the reprogramming progress.
Cell Cycle
Abbreviated cell cycle
In contrast to somatic cells cycle, ES cells cell cycle has some
specific features, the most striking feature of ES cells cell cycle is
that ES cells have an abbreviated cell cycle (human ES cells:
15–16 h; mouse ES cells: 10 h) (Stead et al., 2002; Becker et al.,
2006). The abbreviated cell cycle of ES cells appears to be due to
the short G1 phase as compared to somatic cells. The short G1
phase in ES cells cell cycle may contribute to the self-renewal. ES
cells in G1 phase are more vulnerable to differentiation by
retinoic acid (RA) (Lukaszewicz et al., 2005). ES cells lack the
cyclin-dependent kinase (CDK) 4-associated kinase activity that
characterized all somatic cells. ES cells are known to express
D-type cyclins at very low levels, and these levels increase
dramatically during in vitro and in vivo differentiation.
Expression of D1 type cyclin is regulated by PI3K-dependent
pathways. And PI3K signaling pathway plays a critical role in the
G1/S transition in ES cells. However, PI3K activity is not
dependent on persistent serum stimulation but rather largely
rely both on stimulation of the LIF receptor and expression of
the ES cells-specific Eras factor. ERK activity also regulates
cyclin D1 expression in somatic cells, which is dispensable in ES
cells cycle (Schratt et al., 2001; Burdon et al., 2002; Jirmanova
et al., 2002; Takahashi et al., 2003).
Control in ES cells and somatic cells differs in that the ES cells
cell cycle is not dependent on a functional p16ink4a/
cyclinD:Cdk4/pRB:E2F pathway (Savatier et al., 1996; Burdon
et al., 2002; White et al., 2005). Rather, the cyclinE:Cdk2 and
cyclinA:Cdk2 complexes in ES cells are constitutively active
throughout the cell cycle, suggesting that the mitotic cycle is
constitutively primed for DNA replication (Stead et al., 2002).
p53
After DNA damage, somatic cells undergo cell-cycle arrest in
the G1 phase to get rid of the damaged DNA, but for ES cells, it
chooses apoptosis and differentiation instead of going through
the checkpoint between the G1 phase and S phase in response
to DNA damage. Mechanisms involved in G1/S checkpoint in
somatic cells cycle (chk1/chk2/Cdc25A and p53-p21) are not
functional in ES cells (Aladjem et al., 1998; Mailand et al., 2000;
Bartek and Lukas, 2001; Hong and Stambrook, 2004). ES cells
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
283
undergo apoptosis when relatively high levels of DNA damage
have been introduced. p53 plays an important role in ES cells’
response to low levels of DNA damage. p53 presents in ES cells,
and its activation can induce ES cells differentiation by
repressing Nanog expression (Lin et al., 2005). Furthermore,
the function of p53 in ES cells may be to trigger apoptosis to
eliminate the cells with damaged DNA (Fluckiger et al., 2006).
p53 is mostly cytoplasmic in ES cells but translocated to the
nucleus during differentiation. p53 is another mechanism for
repressing Nanog expression when ES cells differentiate (Xu,
2005).
Telomerase
Eukaryotic chromosomes are capped by special structures
called telomeres, which act to guarantee chromosome
integrity. Synthesis and maintenance of telomeric repeats are
accomplished by telomerase. Analysis of telomerase enzyme
levels or activity has shown that high levels are expressed in ES
cells (Thomson et al., 1998). Telomerase pathway is active and
indeed is required for prolonged self-renewal in human and
mouse ES cells (Kim et al., 1994; Thomson et al., 1998). During
ES cells differentiation, telomerase activity is downregulated
(Miura et al., 2004). In contrast to most somatic cells, stem,
germ, and tumor cells have high telomerase activity. The
catalytic subunit of telomerase (TERT) promoter contains two
E-boxes surrounding several Sp1-binding sites. E-box sites bind
the Myc/Mad/Max family of transcription factors. Suggesting
that c-Myc may play a role in ES cells self-renewal (Miura et al.,
2004). Overexpression of c-Myc is sufficient to maintain mouse
ES cells in an undifferentiated state, and blocking c-Myc
expression promotes differentiation (Cartwright et al., 2005).
Telomerase activity can be regulated by two growth factors:
bFGF and TGFb. bFGF can upregulate telomerase activity in
human endothelial cells (Kurz et al., 2003). Furthermore, bFGF
plays an important role in human ES cells self-renewal (Xu et al.,
2001). But it remains unclear whether bFGF activates
telomerase in ES cells.
Chromatin and Epigenetic Modification
Differentiation of ES cells from pluripotent to developmentally
more restricted states is accompanied by global changes in
genome expression patterns. Genes active in earlier
progenitors are gradually silenced at developmentally later
stages, and subsets of cell type-specific genes are turned on.
This progression is the result of selectivity active expression of
transcription factors in concern with chromatin remodeling and
modification, which includes covalent histone modification,
DNA methylation of CpG dinucleotides, and localization of
chromatin to specialized nuclear domains.
Chromatin and chromatin modification
Gene activity is critically determined by chromatin structure
and interactions of chromatin-binding proteins. To search for
the elusive keys for ES cells pluripotency, many researchers
have turned to the study of ES cells chromatin. Some features of
chromatin, including nuclear architecture, chromatin structure,
chromatin dynamics, and histone modifications, in ES cells are
different from the somatic cells. For example, ES cells
chromatin displays characteristics of loosely euchromatin, such
as an abundance of acetylated histone modifications and
increased accessibility to nucleases (Boyer et al., 2006b;
Meshorer and Misteli, 2006). ES cells undergo functionally
important global and gene-specific remodeling of chromatin
structure during in vitro differentiation. Increase in the
heterochromatin marker tri-methylated residue K9 of histone
H3 (H3-triMeK9) and a decrease in the global levels of
acetylated histones H3 and H4 (a euchromatin marker) were
284 LIU ET AL.
observed during RA-induced differentiation (Lee et al., 2004;
Meshorer et al., 2006). Furthermore, histone deacetylase
(HDACs) and methyl-CpG-binding protein (MECPs) are
expressed in ES and their levels are dynamically regulated as
cells undergo differentiation (Rao, 2004). Consistent with the
fact that the chromatins of pluripotent nuclei is in an open
conformation, recent studies have shown that lineage-specific
genes replicated earlier in pluripotent cells than differentiated
cells, and had unexpectedly high levels of acetylated H3K9 and
methylated H3K4. But this modification is also combined with
H3K27 trimethylation. H3K27 methylation is functionally
important for preventing these genes expression in ES cells
(Azuara et al., 2006; Bernstein et al., 2006; Boyer et al., 2006b).
Polycomb group proteins
Polycomb group proteins (PcG) components are required for
early developmental gene expression (Pasini et al., 2004). In
addition, recent studies found that PcG also play an important
role in ES cells self-renewal and pluripotency. They are
chromatin modification factors. They can facilitate
oligomerization, condensation of chromatin structure, and
inhibition of chromatin remodeling activity. PcG proteins are
comprised of at least two distinct repressor complexes (PRC1
and PRC2-PRC3). Studies in ES cells found that PRC1 and PRC2
bind to a large set of genes composed of transcriptional
regulators and signaling factors, which promote ES cells
differentiation. Many of the target genes were de-repressed in
the absence of the PcG components. Thus, PcG proteins make
an essential contribution to the ES cell state by repressing the
premature expression of differentiation genes. But when ES
cells differentiate, these target genes were preferentially
activated. That is to say PcG repress differentiated genes
expression in a flexible manner, which can be reversed later by
gene-specific and lineage-specific signals (Bernstein et al., 2006;
Boyer et al., 2006b; Buszczak and Spradling, 2006; Lee et al.,
2006).
DNA methylation
DNA methylation at CpG islands is another gene silence
mechanism. Silencing of certain genes by DNA methylation is
required for induction of differentiation of ES cells. This was
shown in experiments with ES cells deficient either in the DNA
methyltransferases Dnmt1, both Dnmt3a and Dnmt3b, or the
CpG island-binding protein CGBP that binds to non-methylated
DNA. These cells showed severe DNA hypomethylation and a
complete differentiation block (Jackson et al., 2004; Carlone
et al., 2005). Hypermethylation of Oct4 promoter/enhancer
region in differentiated cells correlates with gene silencing,
whereas hypomethylation in ES cells allows cells to maintain
high level of Oct4 expression, thus keeping them in pluripotent
state. Reprogramming phenomenon can be observed during
somatic cells fusion with ES cells or in the culture containing
extracts of ES cells. Oct4 DNA demethylation in thymocyte
nuclei has been reported after fusion with ES cells (Taranger
et al., 2005).
From Pluripotency to Differentiation
ES cells are capable of unlimited symmetrical self-renewal and
have the potential to differentiate into any cell type in the body.
Differentiation of ES cells is accompanied by downregulation of
the whole series of transcription factors and signaling molecules
that maintain the pluripotent phenotype, such as Oct4 and
Nanog, and upregulation of transcription factors involved in
differentiation. Little is known about how determinants of ES
cell pluripotency are regulated during differentiation of ES cells.
Recently, studies have shown that orphan nuclear receptor
Germ Cell Nuclear Receptor (GCNF) is required for the
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
repression of pluripotency genes during ES cells differentiation.
It can directly repress Oct4 and Nanog expression when ES was
induced by RA. The repression mechanism may be that GCNF
directly binds to the promoter of Oct4 and Nanog (Gu et al.,
2005). Another mechanism is that GCNF recruits DNA
methyltransferase to the Oct-3/4 promoter and facilitates its
methylation (Sato et al., 2006).
Nucleosome remodeling and histone deacetylation (NuRD)
complex also play important role when ES cells differentiate. ES
cells lacking Mbd3 (a component of NuRD) display a profound
defect in differentiation that results in persistent self-renewal. It
is possible that NuRD is important for silencing Nanog during
ES cells differentiation. Thus, NuRD-mediated gene silencing
has a crucial function in the lineage commitment of ES cells as
this process allows cells to exhibit pluripotency (Kaji et al.,
2006).
microRNA
microRNA are a large family of small non-coding RNAs that
consist of more than 200 known members in the mammalian
genomes. They are important in a wide variety of biological
processes including cell-cycle regulation, apoptosis, cell
differentiation, and maintenance of stemness (Ambros, 2004;
Griffiths-Jones, 2004). Distinct sets of microRNA are
specifically expressed in pluripotent ES cells but not in
differentiated embryonic bodies or in adult tissues, suggesting a
role in ES cells self-renewal. As ES cells differentiate, they
downregulate stem cells maintenance genes and activate
lineage-specific genes (Houbaviy et al., 2003; Suh et al., 2004).
Loss of mature microRNA in Dicer1 null mouse ES cells results
in their failure to differentiate into the three germ layers,
suggesting that microRNA are important for ES cells
pluripotency, although the regulatory mechanism is still largely
unknown (Cheng et al., 2005; Kanellopoulou et al., 2005).
Conclusions
This review has made important contributions toward
elucidating the complexity molecular mechanism in ES cells
self-renewal and pluripotency. Mouse ES cells and human ES
cells show similar markers of pluripotent stem cell lines such as
Oct4, Nanog, ALP, and high levels of telomerase activity. But,
there are notable differences between mouse and human ES
cells. These differences between mouse and human ES cells
suggest that although the same pluripotency genes are
expressed in both mouse ES and human ES cells, their function
and downstream signaling pathways may differ between the two
species. An important goal is to understand the mechanism
involved in pluripotency used by human and mouse ES cells. So,
we can reprogram differentiated cells to an embryo-like state,
and then recreate these cells in somatic cells which can be used
in clinic. Despite our limited current knowledge, there is little
doubt that we will eventually uncover the molecular
mechanisms that give rise to the unique properties of ES cells
and that we will then be able to exploit them as powerful tools in
basic discovery and clinical applications.
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