Current Stem Cell Research & Therapy, 2006, 1, 103-111103
Intracellular Signaling Pathways Regulating Pluripotency of Embryonic Stem
Keisuke Okita and Shinya Yamanaka*
Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507,
Abstract: The cytokine LIF and its downstream effector STAT3 are essential for maintenance of pluripotency in
mouse ES cells. The requirement for the transcription factor Oct3/4 for ES cell pluripotency is also well-
documented. However, LIF is not involved in self-renewal of human ES cells, suggesting that other pathways
must play an important role in this process. The importance of other signal transduction pathways, including
BMP and Wnt signalings, as well as novel transcription factors such as Nanog, is now being recognized. We
will review the rapid progress that has been made in identifying and dissecting the intracellular signaling
pathways that contribute to self-renewal of pluripotent mouse and human ES cells.
Keywords: Growth factor, Cytokine, Regenerative medicine, Transcription factor, Crosstalk.
by means of somatic cell nuclear transfer from patient skin
cells into donated oocytes . They established those
pluripotent cells on feeders from same patients. However
their protocol still had to use calf serum to establish
fibroblasts from patients.
In order to establish a defined serum free medium for
human ES cells, and to generate ES-like cells from patients’
somatic cells, it is essential to understand how ES cells
maintain their pluripotency and ability to proliferate rapidly.
In this review, we discuss recent progress in unraveling the
intracellular signaling pathways that contribute to self-
renewal of pluripotent mouse and human ES cells.
Embryonic stem (ES) cells are derived from the inner cell
mass, a population of cells in the blastocyst stage embryo
that gives rise to all cells of the embryo proper. Once
established in vitro, ES cells can be cultured indefinitely
without losing their pluripotency, that is the ability to
develop into any cell type in the body. These remarkable
characteristics have made ES cells an extremely useful tool.
The establishment of mouse ES cell lines in 1981 led to the
development of the gene targeting technology used to
generate knockout mice [1, 2], a technique that has quickly
become a standard approach for investigating and modeling
gene function. Moreover, since ES cells have unrestricted
developmental capacity, they represent a promising source
for cell transplantation therapies to treat various human
diseases . Since their isolation in 1998 , human ES
cells have been coaxed to differentiate into such varied cell
types as pancreatic β-cells, neurons, and cardiomyocytes,
simply by changing the culture conditions in which the cells
are grown. Future transplantation of these cells into patients
suffering from diabetes, neurodegenerative diseases, and
myocardial infarction holds a therapeutic promise.
However, the use of human ES cells as therapeutic
treatment presents significant ethical and scientific problems.
Since ES cells are derived from blastocyst stage embryos,
the clinical use of human ES cells stirs up ethical objections
against the destruction of human embryos. In addition,
human ES cells must currently be grown and maintained on
a feeder layer of mouse embryonic fibroblasts (MEFs) in a
medium containing fetal bovine serum, which may lead to
unexpected viral infection
contamination. Thus, for therapeutic applications, ES cells
must be grown in a synthetic medium without factors or
cells from animal-derived. Recently, Hwang et al. reported
the establishment of patient-specific human ES cells
I. LIF/gp130/STAT3 (Fig. 1)
Mouse ES cells have historically been derived and
maintained on a feeder layer of MEFs. However, conditioned
media from MEFs can support the self-renewal of mouse ES
cells, eliminating the need for a feeder layer. It was
subsequently demonstrated that MEFs inhibit ES cell
differentiation via production of the IL-6 family cytokine,
leukemia inhibitory factor (LIF) [6, 7]. With the addition of
recombinant LIF protein into the culture medium, mouse ES
cells can be cultured without MEF feeder cells.
The receptor for LIF is a heteromeric complex consisting
of gp130 and the LIF receptor (LIFR, also referred as to
LIFRβ) . The tyrosine kinase Janus kinase (JAK) binds
constitutively to the intercellular domain of this receptor
complex in its inactive form. Upon LIF binding, JAK
kinase phosphorylates tyrosine residues of both gp130 and
LIFR. Phosphorylation of Y765/812/904/914 of the
intracellular domain of gp130 and Y976/996/1023 of LIFR
recruits signal transducers and activators of transcription
(STAT) 1 and STAT3 through their SH2 domains .
STAT proteins are then activated by JAK-mediated tyrosine
phosphorylation to form homodimers and/or heterodimers
and translocate into the nucleus, where they function as
transcription factors . LIF stimulation also induces other
signaling pathways (described below).
Several groups have shown that STAT3 is important for
maintenance of pluripotency of ES cells [11-14]. In
*Address correspondence to this author at the Department of Stem Cell
Biology, Institute for Frontier Medical Sciences, Kyoto University, 53
Kawahara-machi, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; Tel: 81-75-
751-3839; Fax: 81-75-751-4632; E-mail: email@example.com
1574-888X/06 $50.00+.00© 2006 Bentham Science Publishers Ltd.
104 Current Stem Cell Research & Therapy, 2006, Vol. 1, No. 1 Okita and Yamanaka
particular, Mastuda et al. generated an inducible form of
STAT3 by fusing it with the ligand-binding domain of the
estrogen receptor, allowing for activation of STAT3 by
tamoxifen administration. They reported that STAT3
activation is sufficient for self-renewal in the presence of
fetal bovine serum . One of important target genes of
STAT3 is c-Myc, a helix-loop-helix/leucine zipper
transcription factor . However, LIF cannot support clonal
expansion of mouse ES cells in the absence of serum .
This finding indicates that other factors required for ES cell
proliferation and maintenance are present in serum or
produced by MEFs.
Unlike mouse ES cells, LIF cannot promote self-renewal
of human or monkey ES cells [17, 18]. Human ES cells
express relatively low level of LIF signaling components
(LIFR, JAK, and STAT3), and high level of suppressor of
cytokine signaling (SOCS), which negatively regulate LIF
signaling . In monkey ES cells, depression of LIF
signaling by dominant negative form of STAT3 did not
affect their undifferentiated state . Human and monkey
ES cells seem to maintain the pluripotency in LIF/STAT3
These secreted ligands bind to heterodimeric complexes of
type I (ALK2, ALK3, ALK6) and type II (BMPRII, ActRII,
ActRIIB) receptor tyrosine kinases. Binding of BMP
triggers complex formation of the receptor components and
facilitates phosphorylation of Smads, intracellular signal
transduction molecules that fall into three categories:
receptor-regulated Smads (R-Smads), cooperating Smad (Co-
Smad) and inhibitory Smads (I-Smads). Upon BMP
binding, R-Smads (Smad1, Smad5, and Smad8) are
phosphorylated at two C-terminal serine residues and form
heteromeric complexes with Smad4, the sole Co-Smad
known in mammals. The Smad complexes then translocate
to the nucleus and function as transcription factors. I-Smads
(Smad6 and Smad7) suppress the Smad signaling pathway
by inhibiting association between the receptors and R-
Smads, competing with Smad1 for binding to the Co-Smad,
and/or promoting ubiquitin-dependent degradation of
receptors and R-Smads .
Ying et al. reported that BMP4 and LIF cooperate in the
maintenance of pluripotency of mouse ES cells . Under
the serum-free culture conditions they used, LIF alone
stimulated neural differentiation of ES cells. However,
addition of BMP4 was able to suppress neural differentiation
and maintain the undifferentiated state of mouse ES cells,
even in the absence of serum. They also showed that BMP4
induced expression of inhibitor of differentiation (Id), an
inhibitor for basic helix-loop-helix transcription factors
II. BMP/Smad (Fig. 2)
Bone morphogenetic proteins (BMP) are members of the
transforming growth factor β (TGF-β) superfamily .
Fig. (1). Intracellular signaling pathways activated by LIF. Association of LIF with its heteromeric receptor, which consists of LIFR
and gp130, results in activation of several intracellular signaling pathways, including the STAT3 pathway, the Ras/ERK pathway, and
the PI3 kinase pathway (see crosstalk section). The STAT3 pathway is crucial for the maintenance of pluripotency in mouse ES cells,
but not in primate ES cells. LIF also activates the Ras/ERK and the PI3 kinase pathways (see Fig. 4).
Intracellular Signaling Pathways Regulating PluripotencyCurrent Stem Cell Research & Therapy, 2006, Vol. 1, No. 1 105
Fig. (2). Intracellular signaling pathway activated by BMP. Receptors of the TGF-β superfamily of ligands consist of a heteromeric
complex of type I and type II receptor serine/threonine kinases. Binding of BMP to the receptor induces phosphorylation of R-Smads
by type I receptors. Phosphorylated R-Smads form complexes with Co-Smad and accumulate in the nucleus, where together they
regulate gene transcription. I-Smads suppress the pathway via several mechanisms. In mouse ES cells, BMP4 can induce expression of
Id and suppress neural differentiation. In human ES cells, in contrast, several groups reported that BMP4 induces differentiation.
known to be involved in many cell fate determinations,
including neural differentiation . When Id protein was
overexpressed in mouse ES cells, LIF was able to maintain
self-renewal of ES cells in the absence of either BMP4 or
serum. Thus BMP4 appears to prevent neural differentiation
of mouse ES cells through the induction of Id expression.
By contrast, BMP4 alone
differentiation of mouse ES cells. These findings suggest
that the self-renewal of mouse ES cells is achieved by a
delicate balance between the two cytokines, LIF and BMP.
In human ES cells, BMP4 induces differentiation into
mesoderm and ectoderm , whereas BMP2 promotes
extraembryonic endoderm differentiation. Repression of
BMP signaling in human ES cells by adding the BMP
antagonist Noggin and high doses of bFGF supports long-
term self-renewal in the absence of serum and feeder cells
It also acts as an intracellular signaling molecule of the
canonical Wnt signaling pathway . In the absence of Wnt
activation, β-catenin is phosphorylated by a complex
consisting of adenomatous polyposis coli gene (APC),
Axin, and glycogen synthase
Phosphorylated β-catenin is degraded by the ubiquitin-
proteasome system, thereby keeping the level of cytoplasmic
β-catenin low. Upon binding of Wnt to its receptors,
Frizzled and LRP5/6, GSK3β is inactivated through a
interaction of Axin with LRP5/6, and/or the action of an
Axin-binding molecule, Dishevelled. As a result, β-catenin
accumulates in the cytoplasm and travels to the nucleus,
where it associates with lymphoid enhancer factor (LEF)/T-
cell factor (TCF) transcription factors.
Aubert et al. showed that neural differentiation of mouse
ES cells was attenuated by the activation of Wnt signaling
by overexpression of Wnt1 or treatment with lithium
chloride, an inhibitor of GSK3β . Moreover, Wnt3a
mutant mice display ectopic neural tube formation in the
primitive streak of gastrulating embryo . In addition, ES
cells with a mutant form of APC show impaired ability to
differentiate into the three germ layers . Together, these
kinase (GSK) 3β.
involving the direct
III. Wnt/ -Catenin/TCF (Fig. 3)
β-catenin is a cytoplasmic protein that functions in cell-
cell adhesion by linking cadherins to the actin cytoskeleton.
106 Current Stem Cell Research & Therapy, 2006, Vol. 1, No. 1 Okita and Yamanaka
Fig. (3). Intracellular signaling pathway activated by Wnt. Wnt binds to its receptor, Frizzled, and coreceptor, LRP5 or LRP6. The
downstream effector Dishevelled is then activated through mechanisms that are poorly understood. Activated Dishevelled inactivates
the APC/Axin/GSK3β complex. Since this complex induces degradation of β-catenin in the absence of Wnt ligand, its inactivation
results in the stabilization and accumulation of β-catenin protein in the nucleus. β-catenin binds to and activates LEF/TCF
transcription factors. In both mouse and human ES cells, the Wnt/β-catenin pathway may promote self-renewal.
data suggest that Wnt signaling may suppress differentiation
in early embryos and in ES cells.
Sato et al. compared the gene expression profile of
undifferentiated and differentiated human ES cells in detail,
and revealed the enrichment of Frizzled 5 in undifferentiated
ES cells . They also reported that 6-bromoindirubin-3’-
oxime (BIO), a newly identified pharmacological inhibitor
of GSK3β, could maintain mouse ES cell pluripotency in
the absence of LIF . Noteworthy, BIO also sustained
human ES cells at the undifferentiated state, and maintained
the expression of Oct3/4, Rex1, and Nanog. Considering
LIF has little effect on human ES cells, BIO would useful to
prove the culture techniques of human ES cells.
Wnt also accelerates the proliferation of stem cells in the
intestinal, epidermal and hematopoietic systems; it may
represent a common factor controlling stem cell proliferation
. However, it has been reported that Wnt facilitates
neural differentiation of ES cells and induces the expression
of mesoderm marker, Brachyury
experiments are required to clarify the precise function of
Wnt/β-catenin signaling in ES cells and other stem cell
Wnt can signal independently of β-catenin through so-
called non-canonical pathways . These include calcium
flux, JNK and both small and heterotrimeric G proteins. In
F9 mouse embryonic carcinoma cells, Wnt5A can activate
cGMP-specific phosphodiesterase through heterotrimeric G
proteins . The roles of these non-canonical Wnt
pathways in pluripotency remain elusive.
IV. Phosphatidyl Inositol 3 (PI3) Kinase (Fig. 4)
PI3 kinases are lipid kinases that catalyze the
phosphorylation of inositol phospholipids at the third
carbon position of the inositol ring . They are divided in
three major classes according to substrate specificity, amino
acid sequence, and homology of lipid kinase domains. Class
1A PI3 kinases are heterodimers consisting of an
Intracellular Signaling Pathways Regulating PluripotencyCurrent Stem Cell Research & Therapy, 2006, Vol. 1, No. 1 107
Fig. (4). Activation of the Ras/ERK pathway and PI3 kinase pathway by growth factors. Binding of growth factors to their receptors
induces autophosphorylation of receptors and/or phosphorylation of receptor-associated proteins. The adaptor protein Grb2 binds to
the phosphorylated tyrosines through its SH2 domains and activates the Ras/ERK pathway through the GTP-GDP exchange factor
SOS. Activation of the Ras/ERK pathway induces differentiation in mouse ES cells. The PI3 kinase pathway can be activated via three
routes. First, Gab1 can bind to Grb2, resulting in tyrosine phosphorylation and activation of the PI3 kinase pathway. Second, the PI3
kinase-regulatory subunit p85 can bind to a phosphorylated tyrosine residue of the receptor. Alternatively, activated Ras can induce
membrane localization and activation of the p110 catalytic subunit of PI3 kinase. In addition, the PI3 kinase pathway is
constitutively activated by ERas in mouse ES cells. PTEN is a negative regulator of the PI3 kinase pathway. The PI3 kinase pathway
can promote self-renewal of mouse and human ES cells, possibly by suppression of the ERK pathway.
adaptor/regulatory and a catalytic subunit. The regulatory
subunits come in seven isoforms generated by alternative
splicing from three genes (p85α, p85β, and p55γ), while
there are three isoforms of the catalytic subunit (p110α,
p110β, and p110γ). Activation of the class 1A PI3 kinases
is induced by many different receptor tyrosine kinases for
growth factors, such as FGF, EGF, and PDGF, and leads to
generation of the second messenger phosphatidylinositol-
3,4,5-tris-phosphate (PIP3). A serine/threonine kinase, Akt1,
binds to PIP3 through its pleckstrin homology (PH) domain
and is translocated to the inner cell membrane, where it is
phosphorylated and activated by another serine/threonine
kinase PDK1. Activated Akt1 modulates the function of
numerous substrates, such as Mdm2, IKK, and mammalian
target of rapamycin (mTOR), and elicits various cellular
responses, including proliferation and suppression of cell
death. PI3 kinase is also known to act as a downstream
effector of Ras .
Treatment of mouse ES cells with LY294002, a potent
PI3 kinase inhibitor, suppressed progression of cells from
the G1 to S phase and decreased cell proliferation .
Targeted disruption of phosphatase and tensin homologue
deleted on chromosome ten/PTEN/a negative regulator of the
PI3 kinase pathway through PIP3 dephosphorylation,
promotes ES cell proliferation and tumorigenicity .
Thus, the PI3 kinase pathway is likely to be a crucial
regulator of ES cell proliferation. In addition, the PI3 kinase
pathway may be involved in the maintenance of pluripotency
in both mouse and human ES cells [39, 40].
V. Ras/Raf/ERK (Fig. 5)
Ras proteins belong to a superfamily of low molecular
weight GTP-binding proteins, and control cell proliferation
and differentiation in variety of cells [41, 42]. Like other
GTP-binding proteins, Ras exists in two states – an active
GTP-bound state and an inactive GDP-bound state. The
conformational changes from the inactive to the active state
are mediated by Ras-GTP exchange factors.
Many different growth factors are able to activate Ras
signaling by binding to receptor tyrosine kinases, resulting
in autophosphorylation of tyrosine residues on the receptors.
SH2 domain-containing tyrosine phosphatase (SHP) 2 and
growth factor receptor binding protein (Grb) 2 then bind to
the phosphotyrosines on the receptors and activate Ras
through Ras-GTP exchange factors, son of sevenless (SOS),
which is constitutively associated with Grb2. Activated Ras
binds to many downstream effector proteins, including Raf
108 Current Stem Cell Research & Therapy, 2006, Vol. 1, No. 1 Okita and Yamanaka
Fig. (5). Potential crosstalk between intracellular signaling pathways in mouse ES cells. STAT3 induces c-Myc expression and then c-
Myc activated several target gene for self-renewal. GSK3β inactivates c-Myc by phosphorylation and proteasome-mediated
degradation. However, it is not clear whether c-Myc is the only downstream effecter of LIF/STAT pathway accounted for pluripotency.
BMP/Smad pathway prevents differentiation by inducing target genes like Id, and their signal also suppress the ERK pathway
through indirect mechanisms. The PI3 kinase pathway inhibits the ERK signaling probably thorough the phosphorylation of Raf by
Akt. Wnt stimulation and inhibition of GSK3β was reported to enhance self-renewal of mouse and human ES cells. Maintenance of
pluripotency by Wnt signaling may be achieved by accumulation of c-Myc and/or β-catenin. c-Myc has been identified as one of the
target genes of Ras/ERK pathway in fibroblasts [75, 76]. Whether this is also the case in ES cells is uncertain.
serine/threonine kinases, which activate extracellular signal-
regulated kinase (ERK).
phosphorylation and activation of transcription factors, such
as c-Jun, c-Fos, Ets, and Elk .
Although the Ras/ERK pathway promotes proliferation
and survival of many different types of cells, multiple lines
of evidence indicate that
differentiation and suppresses proliferation of mouse ES
cells. For example, the tyrosine residue in gp130 required
for activation of Ras/ERK pathway is dispensable for
maintaining self-renewal of mouse ES cells . In
addition, the ERK inhibitor PD98059 promotes efficient
derivation of ES cells from blastocysts . Grb2-null ES
cells fail to differentiate into endoderm lineages , while
ectopic expression of the active form of HRas in ES cells
results in massive differentiation into primitive endoderm
lineages . Taken together, these results suggest that the
This pathway leads to
Ras/ERK pathway promotes differentiation of and
suppresses self-renewal of mouse ES cells.
VI. Crosstalk Between Intracellular Signaling Pathways
the pathway promotes
Some of the intracellular signaling pathways mentioned
above are known to engage in crosstalk with one other. In
addition to STAT, LIF stimulation induces other signaling
pathways through effector protein, SHP2, which can bind to
Y757 of the intracellular domain of gp130 (Y118 in the
cytoplasmic domain) and Y969 of LIFR (Y115 in the
cytoplasmic domain) [9, 47]. When phosphorylated by JAK,
SHP2 binds the adaptor protein Grb2 and activate the Ras/
ERK signaling pathway. Phosphorylated SHP2 also binds
Grb2-associated binder (Gab), activating the PI3 kinase
pathway. The activation of SHP2 is thought to be
dispensable for ES cell self-renewal, but is required for
proper differentiation [44, 48].
Intracellular Signaling Pathways Regulating PluripotencyCurrent Stem Cell Research & Therapy, 2006, Vol. 1, No. 1 109
Myc is reported to be one of the STAT3 target genes
participating in self-renewal and maintenance of pluripotency
in mouse ES cells . Forced expression of stable c-Myc
rendered self-renewal without LIF, whereas dominant
negative form of c-Myc induced differentiation even in the
LIF existence. GSK3β negatively regulates c-Myc activity
by phosphorylation following degradation by the proteasome
system . Thus c-Myc is a common target both LIF and
Wnt signaling pathways.
The PI3 kinase pathway is activated by exogenous factors
such as insulin, but also activated endogenously by ERas
(ES cell-expressed Ras), a novel small GTP binding protein
specifically expressed in mouse ES cells . Although its
identity with HRas is less than 40%, the five domains
essential for Ras function, as well as the CAAX motif
required for plasma membrane localization, are highly
conserved. However, ERas lacks several amino acids that are
conserved among other Ras family proteins and essential for
GTPase activity. As a consequence, ERas shows high GTP
affinity and is constitutively active. Among known
downstream effectors of Ras, including Raf1, BRas,
RalGDS, and PI3 kinase, ERas specifically binds to and
activates PI3 kinase.
The PI3 kinase pathway  and BMP4  appear to
maintain the undifferentiated state of mouse ES cells via
inhibition of ERK and both ERK and p38, respectively.
Suppression of the ERK activity by the PI3 kinase pathway
was also observed in ES cells deficient in the catalytic
subunit p85α  or PDK1, which activates Akt . PI3
kinase pathway inhibits ERK signaling probably thorough
the phosphorylation of Raf by Akt . The PI3 kinase
pathway can activate the Wnt/β-catenin pathway via
inhibition of GSK3β by Akt, but its role in ES cells is not
Another crosstalk of each signal components also
reported. For instance, Wnt was reported to inhibit neuronal
differentiation in ES cells by inducing BMP expression
. LEF/TCF1, a transcription factor downstream of Wnt
signaling, complexes with Smad4, Co-Smad in the BMP
pathway, to cooperatively control gene expression in mouse
ES cells .
Crosstalk between signaling pathways has also been
described in neural progenitor and stem cells. For example,
cooperation between LIF and BMP2 signaling is mediated
by interaction between STAT3 and Smad1, which form a
complex together with p300, resulting in astrocyte induction
. BMP4 induces smooth muscle differentiation via
Smads in a low cell density culture, whereas it induces glial
cell differentiation via mTOR and STAT3 at a higher cell
density . Whether such crosstalk between pathways also
functions in ES cells or not is currently unknown.
In human ES cells, the best known factor promoting self-
renewal is fibroblast growth factor (Fgf) 2 . Exogenous
Fgf2 is capable of maintaining human ES cells in the
absence of serum and feeder cells. Binding of Fgf to its
receptor and heparin leads to receptor autophosphorylation
and activation of intracellular signaling cascades, including
the Ras/ERK pathway, the PLCγ/Ca2+ pathway, and the
PI3 kinase pathway . Fgf2 may promote self-renewal of
human ES cells by activating the PI3 kinase pathway .
VII. Transcription Factors Controlling Self-Renewal of
Oct3/4 and Nanog are the two well-known homeobox
transcription factors that are specifically expressed in mouse
ES cells and early embryos, and are essential for maintaining
pluripotency . Knockdown experiments using siRNA
indicated that the two homeoproteins are also indispensable
in human ES cells . The intracellular signaling pathways
described above are likely to regulate these transcription
factors to determine the fate of ES cells. However, the
precise mechanisms by which these factors are regulated
Oct3/4 (also known as POU5F1) is specifically expressed
in ES cells, early embryos, and germ cells [60, 61]. Oct3/4-
deficient embryos die at peri-implantation stages of
development . Although Oct3/4-null embryos reach
blastocyst stage, the inner cell mass of these mutants only
produces differentiated cells of trophoblast lineages when
cultured in vitro. Inactivation of Oct3/4 in mouse ES cells
also results in trophoblast differentiation . In contrast,
overexpression of Oct3/4 in ES cells induces differentiation
into primitive endoderm and mesoderm lineages .
Hence, the expression level of Oct3/4 is an important
determinant of cell fates in mouse ES cells.
Nanog is also specifically expressed in pluripotent cells
[64, 65]. Nanog null embryos at E5.5 show disorganization
of extraembryonic tissues with no discernible epiblast or
extraembryonic ectoderm . Nanog-deficient blastocysts
appear to be normal, but the inner cell mass fails to generate
epiblast and only produces parietal endoderm-like cells when
cultured in vitro. Similarly, ES cells lacking Nanog
preferentially differentiate into extraembryonic endoderm
lineages even in the presence of LIF. Importantly,
overexpression of Nanog allows mouse ES cells to self-
renew without LIF. Thus, Nanog blocks primitive endoderm
differentiation and actively maintains pluripotency.
VIII. Epigenetic Modification
Epigenetic modifications, including CpG methylation
and histone modifications, regulate gene transcription and
are also important in the maintenance of pluripotency. ES
cells having mutation in DNA methyltransferase (Dnmt) can
differentiate but show abnormal gene expression . CpG
binding protein (CGBP) has a unique DNA-binding
specificity for unmethylated CpG dinucleotides. Mouse ES
cells lacking the CGBP show reduced levels of genomic
methylation and maintenance DNA methyltransferase
activity. The cells remain undifferentiated even in LIF-
withdrawal . CpG dinucleotides of Oct3/4 and Nanog
gene are hypomethylated in undifferentiated human ES cells,
whereas methylation progresses during neural differentiation
. In mouse development, the upstream regulatory region
of Oct3/4 gene is unmethylated in blastocysts and in
epiblast at E6.25 and methylation initiates at E6.5 . The
methylation pattern of Oct3/4 and Nanog is highly
comparable with their expression pattern, and the
methylation would suppress
differentiated cells. In vitro fusion with ES cells or treatment
with demethylating agent, 5-azacytidine, could erase, at least
in part, epigenetic status of differentiated cells [70, 71].
their transcription in
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Our understanding of the molecular mechanisms
underlying pluripotency of ES cells has progressed
remarkably in the last few years . In addition to the
factors we discussed in this review, it is highly likely that
other factors and pathways are also involved, including
miRNA  and Bcl2 . However, some data are still
controversial. As described above, Wnt was reported as a
self-renewal factor by some groups, but as a differentiation
initiation factor by others. This discrepancy may at least in
part be due to the different ES cell lines used in the different
Many unsolved questions remain. For instance, what is
the relationship between the intracellular signaling pathways
and the transcription factors described in this review? Do
mouse and human ES cells share common pathways to
maintain pluripotency? To answer these questions and to
further the possibility of medical applications of stem cell
technology, further studies into the mechanisms underlying
the maintenance of pluripotency are essential.
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Received: June 15, 2005Revised: June 30, 2005Accepted: July 13, 2005