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. 1 111
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Received: June 15, 2005Revised: June 30, 2005Accepted: July 13, 2005