Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.

Regulation of self-renewal by extracellular signals

Mouse ES (embryonic stem) cells are derived from the ICM (inner cell mass) of the blastocyst-stage embryo [1]. ES cell derivation can be considered the ‘capture’ of a transient developmental state, the pluripotent epiblast. When ES cells were first described, they were isolated and expanded by plating epiblasts on to a layer of mitotically inactivated fibroblasts [2,3]. These fibroblasts were thought to provide trophic factors that aided the growth of the ES cells and became known as ‘feeders’. Feeders, together with the FBS (fetal bovine serum) in the culture medium, create a complex environment.

One approach to elucidating the requirement of ES cells for extrinsic stimulation has been to refine the culture conditions, replacing undefined multifactorial components. A key advance was the discovery that LIF (leukaemia inhibitory factor) could substitute for feeders [4,5]. Genetic and biochemical experiments have since identified STAT3 (signal transducer and activator of transcription 3) as the critical downstream effector [6]. The ability of LIF-mediated activation of STAT3 to support long-term self-renewal of mouse ES cells in vitro is reflected in vivo by the requirement for this signalling pathway in diapause [7], the arrest of early development if a dam is still suckling. STAT3 has been reported to function through the regulation of c-Myc [8], Klf4 (Krüppel-like factor 4) [9] and Klf5 (Krüppel-like factor 5) [10] among other proposed target genes, but no single target has been identified that is both required for and is completely sufficient to replace the effect of LIF.

Serum-free medium compositions have been developed, initially for the purposes of directed ES cell differentiation [11]. Adding BMP4 (bone morphogenetic protein 4) replaces the need for serum, an effect which is reproduced by forced expression of Id (inhibitor of DNA binding) genes, allowing ES cells to be cultured in defined conditions [12]. Together, these findings suggest that ES cells are reliant on extracellular signals. The behaviour of ES cells in serum-free medium supplemented with LIF or BMP4 alone provides further insight into how they influence self-renewal. In LIF alone, ES cells retain some capacity for self-renewal, especially at high cell densities, but progressively differentiate into neurectoderm, whereas in BMP4 alone, rapid non-neural differentiation occurs [12]. These observations led to the hypothesis that LIF and BMP4 support self-renewal by restricting differentiation along specific lineages. Only in combination are they sufficient to suppress all differentiation and allow ES cell expansion at low density.

FGF4 (fibroblast growth factor 4)–MAPK (mitogen-activated protein kinase) signalling drives exit from the pluripotent state

In recent years it has become clear that the derivation and propagation of ES cells can be enhanced by suppressing the autocrine signal(s) that induce differentiation [13]. ES cells genetically deficient in Fgf4 initiate neural differentiation inefficiently [14,15] and are also defective in mesoderm differentiation [14]. This suggests that the response to FGF4 signalling lies upstream in the induction of differentiation, initiating a common path down which ES cells proceed before becoming susceptible to instructive signals that dictate which cell lineage they will enter. The phenotype of Fgf4-knockout ES cells can be reproduced using FGFR (FGF receptor) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase] 1/2 inhibitors [1315]. This, together with the observation that Erk2-null ES cells differentiate inefficiently [14], identifies the ERK1/2 signalling pathway as the downstream effector of the FGF4 signal.

In vivo, the pre-implantation embryo segregates the hypoblast and pluripotent epiblast at the blastocyst stage [16], defining the cells that will give rise to extra-embryonic tissues and the embryo proper. Exposing morula stage embryos to MEK inhibitors prevents the emergence of the hypoblast and generates an expanded pluripotent epiblast [17]. A similar phenotype is observed in embryos that lack Grb2 (growth-factor-receptor-bound protein 2) [18], which is responsible for coupling the FGFR to Ras–MAPK. These observations indicate that MAPK signalling is required for hypoblast specification. The observation that all ICM cells become epiblast in the absence of MAPK signalling suggests that this is a default ground state. ES cells express FGF4 under the direction of the transcription factors Oct4 (octamer-binding protein 4) and Sox2 [SRY (sex-determining region Y) box 2] [19], whose activities are critical for the maintenance of pluripotency [20,21]. This indicates that the transcription factors required for establishment and maintenance of the pluripotent state also promote signalling that drives progression from this state. Thus blocking or counteracting FGF4 signalling may be critical to capture pluripotency at this pre-implantation stage. Indeed, the addition of MEK inhibitors to serum-free medium compositions containing LIF permits the derivation of ES cells in the absence of feeders from strains normally considered non-permissive [22].

Inhibition of GSK3 consolidates ES cell self-renewal

We have described previously the use of a GSK3 inhibitor, CHIR (CHIRON99021), in mouse ES cell culture [23]. GSK3 inhibitors are known to have off-target effects against cyclins and other kinases. CHIR was the most potent and selective of seven GSK3 inhibitors tested against a panel of 70–80 kinases [24]. Although inhibitors of the FGF4–MAPK signalling cascade reduce differentiation, they are insufficient to support clonal propagation of ES cells. CHIR restores clonogenicity, and the combination of CHIR and inhibitors of the FGF4–MAPK signalling cascade (known as 3i or 2i) is sufficient for the derivation and propagation of germline-competent ES cells. The effect of CHIR on ES cell culture can be reproduced by alternative small-molecule inhibitors of GSK3 [23], indicating that these findings are unlikely to be due to unique off-target effects of CHIR. Furthermore, ES cells lacking all four alleles of Gsk3a/Gsk3b {known as DKO (double knockout) cells [25]} can be cultured in the presence of the MEK inhibitor alone and show no functional response to CHIR [23].

GSK3 was initially identified as the kinase responsible for phosphorylation and inhibition of glycogen synthase. Many more substrates have since been identified implicating GSK3 in the regulation of many biological processes (reviewed in [26]). Typically, GSK3 is more active in resting cells and functions to inhibit the activity of its substrates, with an overall catabolic effect. Inhibition of GSK3 downstream of growth factor stimulation of PI3K (phosphoinositide 3-kinase) results in activation of GSK3 substrates and an increase in anabolic processes. GSK3 has a further role in the canonical Wnt pathway forming part of a complex that also includes axin and APC (adenomatous polyposis coli). Cytoplasmic β-catenin is sequestered by this complex where it is phosphorylated by GSK3, earmarking it for ubiquitination and proteolysis. Wnt ligands signal through Frizzled and LRP (low-density lipoprotein receptor protein) 5/6 receptors to inhibit the destruction complex, allowing β-catenin to accumulate and translocate to the nucleus where it can interact with co-activators to drive transcription of target genes (reviewed in [27]). Canonical Wnt signalling becomes constitutively active if either Axin or Apc function is lost. Inhibition of GSK3 using small molecules results in activation of canonical Wnt signalling, but will also affect other GSK3 targets [26].

Several studies have examined the effect of canonical Wnt signalling on ES cells but differ in their conclusions. Wnt signalling has been found to specifically inhibit neural differentiation [28,29], but others report that it may be sufficient to inhibit differentiation altogether [30]. Interpretation is made difficult by the use of Wnt-conditioned medium or GSK3 inhibitors that may have off-target effects. For example, the self-renewal promoting effect of Wnt-conditioned medium was found to depend in part upon activation of STAT3 [31]. Employing a stabilized form of β-catenin to mimic the effect of Wnt has led to the conclusion that canonical Wnt signalling is responsible for the observed phenotypes, although these studies carry qualifications of non-physiological levels of β-catenin and altered cell adhesion properties. However, the phenotypes of ES cells carrying loss-of-function mutations in Apc [32] or Gsk3 [25] are consistent with a role for canonical Wnt signalling. Apc-mutant ES cells were found to exhibit differentiation defects both in vitro and in teratomas. A similar phenotype was observed when endogenous β-catenin was stabilized by deleting the region of the protein carrying the phosphorylation sites that normally target it for proteolysis [32]. Doble et al. [25] found that DKO ES cells, which have highly elevated β-catenin, were severely compromised in their ability to differentiate [25]. Notably, both studies report a dose-dependent effect with the severity of the phenotype correlating with the severity of the Apc mutation or the number of functional Gsk3 alleles. However, even those mutants with the highest levels of β-catenin activity were capable of some differentiation, consistent with reports that activation of canonical Wnt signalling alone is not sufficient to support long-term ES cell self-renewal [23,31]. One complication in interpreting those studies is to discriminate between function in undifferentiated ES cells and the requirement during differentiation.

It is generally assumed that the effect of activated β-catenin is mediated by activation of TCF (T-cell factor)/LEF (lymphoid-enhancer binding factor) target genes. Under standard ES cell culture conditions, the TCF/LEF luciferase reporter TOPFlash has only background levels of activity [31]. Addition of Wnt or GSK3 inhibitors results in TOPFlash activation and modest up-regulation of endogenous Wnt target genes. There are four TCF/LEF family members in mammals, TCF1, TCF3, TCF4 and LEF1, with TCF3 being the most abundant in ES cells. It has been found that Tcf3-null ES cells have increased resistance to differentiation [33]. Genome-wide analysis in Tcf3-null ES cells showed up-regulation of targets of the key pluripotency transcription factors Oct4 and Nanog [34]. In a separate study, TCF3 was found to co-occupy the promoters of many Oct4 and Nanog targets [35]. It has been proposed therefore that TCF3 functions to oppose the effects of the core machinery of pluripotency, limiting the expression of key regulators. It has been speculated further that activation of Wnt signalling converts TCF3 into an activator, elevating expression of these same targets and suppressing differentiation [35]. Luciferase assays suggest that TCF3 can suppress the expression of Oct4 [36] and Nanog [33], but it remains to be formally shown that TCF3 behaves as an activator of these targets in conjunction with β-catenin. Indeed, the biochemical evidence is that TCF3 acts as a constitutive repressor. β-Catenin is known to have TCF/LEF-independent effects, including vitamin D receptor-dependent activity in the epidermis [37], and intriguingly β-catenin has been reported to interact with the pluripotency-associated transcription factor Oct4 [38]. Thus β-catenin may have TCF/LEF-independent functions in ES cells.

Furthermore, GSK3 is likely to exert β-catenin-independent effects in ES cells. Notably, MAPK signalling and GSK3 have opposing effects on protein translation through regulation of mTOR (mammalian target of rapamycin) [27,39] and on c-Myc stability (reviewed in [40]). c-Myc has been identified as a key factor in ES cell self-renewal downstream of STAT3 and GSK3 [8]. However, although we have detected decreased c-Myc transcript and protein in the presence of MEK inhibitors, we observe little or no effect of GSK3 inhibition [23]. The poor clonogenicity of ES cells cultured in the presence of MEK inhibitors alone may in part be due to decreased protein translation. GSK3 inhibition may counteract the undesired effects of MEK inhibition by increasing translation, resulting in cloning efficiencies comparable with standard ES cell culture conditions [23].

Improved ES cell derivation and culture provides mechanistic insight

The recent description of post-implantation embryo-derived pluripotent stem cells, known as EpiSCs (epiblast stem cells) [41,42], suggests that human ‘ES’ cells are in fact more similar to this post-implantation developmental stage. Most of what is known about mouse ES cells comes from studies in lines derived from the inbred 129 strain. Derivation from other mouse strains is much less efficient and, until recently, was not possible in serum- and feeder-free conditions. By using the selective MEK inhibitor PD184352 in combination with LIF and BMP4, Batlle-Morera et al. [22] showed that ES cells could be derived in defined serum-free conditions from mouse strains previously considered refractory to ES cell derivation. Combining a GSK3 inhibitor (CHIR), MEK inhibitor [PD03 (PD0325901)] and LIF (we call this 2i+LIF) it has been possible to derive ES cell lines from completely recalcitrant strains including NOD (non-obese diabetic) mice [43]. The same culture conditions have been applied to derive ES cell lines from the rat that are capable of contributing to adult chimaeras and passing through the germ line [44,45]. This shows for the first time that ES cells are not a mouse-specific phenomenon and that the mechanisms governing establishment of and exit from the pluripotent state are conserved in rodents. If this is true for mammals, it raises the possibility that equivalent cell lines can be established from human embryos.

The stringent assay for the sufficiency of an ES cell culture environment is the ability of isolated single cells to form undifferentiated colonies. In 2i+LIF medium, mouse ES cells form colonies efficiently and the colonies are composed purely of undifferentiated cells. This contrasts with conventional serum and LIF culture conditions where many of the colonies are formed of a mixture of undifferentiated and differentiated cells (Figure 1A). By assessing the colony-forming efficiency in different combinations of the three components of 2i+LIF, we gain some insight into how they influence ES cells. Any two of the three components are sufficient to support the formation of undifferentiated colonies (Figure 1B). In combination CHIR+LIF, PD03+LIF or CHIR+PD03 (2i) support long-term culture and a high efficiency of colony formation. This shows that none of the three components is an absolute requirement for ES cell maintenance and raises the question of whether they exert their effects by distinct mechanisms or converge on common targets. Used alone, PD03 is incapable of supporting clonal colony formation, whereas LIF or CHIR alone does support the formation of partially undifferentiated colonies, but cannot sustain ES cell cultures long-term. Since we know that inhibition of the FGF4–MAPK signalling cascade prevents differentiation, this suggests that the failure to form colonies in PD03 alone may be the result of poor viability. In this assay, both LIF and CHIR are clearly sufficient to support cell viability and to reduce differentiation sufficiently for some undifferentiated cells to persist for the duration of the experiment. These experiments were carried out in 129-background E14 ES cells. From the studies into derivation from other strains, we predicted that other genetic backgrounds might have slightly different responses, particularly with respect to MEK inhibition. In C57Bl6, CBA or FVB ES cell lines, few or no colonies formed in LIF+BMP4 or CHIR alone (Figure 2). Adding PD03 restored efficient colony formation in all cases. This confirms that a major difference between strains is their sensitivity to the differentiation-promoting effects of FGF4–MAPK signalling. This is consistent with the efficacy of MEK inhibition in deriving ES cells from non-129 strains [22]. Interestingly, all of the lines tested in this experiment also formed colonies efficiently in CHIR+LIF, showing little or no increase in colony number in response to PD03. These findings suggest that the combination of LIF and CHIR renders ES cells unsusceptible to FGF4 either by making them insensitive to its inductive effects or by inhibiting the signalling cascade, presumably indirectly since we do not observe suppression of ERK1/2 phosphorylation in response to LIF or CHIR (J. Wray and T. Kalkan, unpublished work).

Effect of culture conditions on ES cell colony formation

Figure 1
Effect of culture conditions on ES cell colony formation

(A) Phase-contrast images showing alkaline-phosphatase-stained ES cell colonies formed from single cells in the presence of LIF and 10% (v/v) FBS (LIF+serum; left-hand panel) or N2B27 plus 2i+LIF (1 μM PD03, 3 μM CHIR and LIF; right-hand panel). Note the ‘skirt’ of differentiated (alkaline-phosphatase-negative) cells surrounding the undifferentiated ‘core’ in the colony formed in LIF+serum. (B) Percentage of isolated single ES cells giving rise to undifferentiated colonies in N2B27 alone, or plus PD03 (1 μM), CHIR (3 μM), LIF, PD03+LIF, 2i (CHIR+PD03) or CHIR+LIF.

Figure 1
Effect of culture conditions on ES cell colony formation

(A) Phase-contrast images showing alkaline-phosphatase-stained ES cell colonies formed from single cells in the presence of LIF and 10% (v/v) FBS (LIF+serum; left-hand panel) or N2B27 plus 2i+LIF (1 μM PD03, 3 μM CHIR and LIF; right-hand panel). Note the ‘skirt’ of differentiated (alkaline-phosphatase-negative) cells surrounding the undifferentiated ‘core’ in the colony formed in LIF+serum. (B) Percentage of isolated single ES cells giving rise to undifferentiated colonies in N2B27 alone, or plus PD03 (1 μM), CHIR (3 μM), LIF, PD03+LIF, 2i (CHIR+PD03) or CHIR+LIF.

Strain differences in sensitivity to MAPK activity

Figure 2
Strain differences in sensitivity to MAPK activity

Number of mixed (partially alkaline-phosphatase-negative) and undifferentiated colonies formed from 600 129-, C57Bl6 (B6)-, CBA- or FVB-ES cells plated in N2B27 plus LIF+BMP4, CHIR (3 μM) or CHIR+LIF in the absence (None) or presence of PD03. Undifferentiated colonies were identified by staining for alkaline phosphatase.

Figure 2
Strain differences in sensitivity to MAPK activity

Number of mixed (partially alkaline-phosphatase-negative) and undifferentiated colonies formed from 600 129-, C57Bl6 (B6)-, CBA- or FVB-ES cells plated in N2B27 plus LIF+BMP4, CHIR (3 μM) or CHIR+LIF in the absence (None) or presence of PD03. Undifferentiated colonies were identified by staining for alkaline phosphatase.

Heterogeneity in ES cell populations

In recent years, reports have emerged of heterogeneous expression of transcription factors in ES cells. These include several known to have roles in the establishment and/or maintenance of pluripotency. Nanog [46], Rex1 [47] and Stella [48] reporters all show heterogeneous expression. Sorted fractions can re-establish the parental distribution, showing that the expression is dynamic. Functionally, Nanog-low cells were shown to be more prone to differentiate [46], Stella-low cells to be closer to a post-implantation epiblast phenotype [48], and Rex1-low cells to be unable to contribute to the developing embryo upon blastocyst injection [47]. Thus their heterogeneous expression reflects different cell states that coexist within the population despite their genetic homogeneity. It is not yet clear what causes this heterogeneity, but the sensitivity of ES cells to medium composition (Figures 1 and 2) and the functional differences between ES cell sub-populations suggest that it is a consequence of the culture environment. We examined the profile of GFP (green fluorescent protein) knockin reporters for Nanog [46] and Rex1 by flow cytometry. Nanog–GFP and Rex1–GFP expression is heterogeneous in serum + LIF (Figure 3A). In contrast, a single peak of Nanog–GFP or Rex1–GFP expression is observed in 2i. Immunofluorescence reveals that, in serum + LIF, Nanog protein expression also varies significantly between individual ES cells within the undifferentiated Oct4-positive population (Figure 3B). In 2i, Nanog protein levels are homogeneously high. The absence of Nanog- or Rex1-low cells from 2i suggests that the ground state is stabilized in 2i and is not intrinsically fluctuating. As discussed above, this is likely to result from the elimination of the differentiation-promoting effects of FGF4–MAPK signalling, together with the consolidating effect of GSK3 inhibition. We propose that the pluripotency network, headed by Oct4/Sox2, Nanog and KLF2/KLF4 is inherently stable, but extremely sensitive to destabilization by exogenous signals. Eliminating or neutralizing those signals appears to be the key to capturing a naïve state that may then self-maintain.

ES cells are homogeneous in 2i

Figure 3
ES cells are homogeneous in 2i

(A) Flow cytometry analysis showing Nanog–GFP or Rex1–GFP expression profiles in reporter cell lines cultured in the presence of LIF and 10% (v/v) FBS (LIF+serum) or in N2B27 plus 2i (1 μM PD03 and 3 μM CHIR). (B) Fluorescent images showing immunostaining for Oct4 and Nanog expression in ES cells cultured LIF+serum or 2i.

Figure 3
ES cells are homogeneous in 2i

(A) Flow cytometry analysis showing Nanog–GFP or Rex1–GFP expression profiles in reporter cell lines cultured in the presence of LIF and 10% (v/v) FBS (LIF+serum) or in N2B27 plus 2i (1 μM PD03 and 3 μM CHIR). (B) Fluorescent images showing immunostaining for Oct4 and Nanog expression in ES cells cultured LIF+serum or 2i.

Revolutionizing Drug Discovery with Stem Cell Technology: A Biochemical Society Focused Meeting held at GlaxoSmithKline, Stevenage, U.K., 18–19 January 2010. Organized and Edited by Aaron Chuang (GlaxoSmithKline, U.K.), Katy Gearing (GlaxoSmithKline, U.K.) and Melanie Welham (Bath, U.K.).

Abbreviations

     
  • APC

    adenomatous polyposis coli

  •  
  • BMP

    bone morphogenetic protein

  •  
  • CHIR

    CHIRON99021

  •  
  • DKO

    double knockout

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • ES

    embryonic stem

  •  
  • FBS

    fetal bovine serum

  •  
  • FGF

    fibroblast growth factor

  •  
  • FGFR

    FGF receptor

  •  
  • GFP

    green fluorescent protein

  •  
  • GSK

    glycogen synthase kinase

  •  
  • ICM

    inner cell mass

  •  
  • KLF

    Krüppel-like factor

  •  
  • LEF

    lymphoid-enhancer binding factor

  •  
  • LIF

    leukaemia inhibitory factor

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEK

    MAPK/ERK kinase

  •  
  • PD03

    PD0325901

  •  
  • Oct4

    octomer-binding protein 4

  •  
  • 2i

    combination of two inhibitors (i.e. CHIRON99021 and PD0325901)

  •  
  • STAT3

    signal transducer and activator of transcription 3

  •  
  • TCF

    T-cell factor

We thank Ge Guo for Rex1–GFP reporter ES cells, and Jenny Nichols and Laura Batlle-Morera for C57Bl6, CBA and FVB cell lines. We also thank Jenny Nichols for critical reading of the manuscript prior to submission.

Funding

Research in our laboratory is funded by the Wellcome Trust, the Medical Research Coucil, the Biotechnology and Biological Sciences Research Council and the European Commission Framework 7 project Euro System.

References

References
1
Brook
F.A.
Gardner
R.L.
The origin and efficient derivation of embryonic stem cells in the mouse
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
5709
-
5712
)
2
Evans
M.J.
Kaufman
M.H.
Establishment in culture of pluripotential cells from mouse embryos
Nature
1981
, vol. 
292
 (pg. 
154
-
156
)
3
Martin
G.R.
Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells
Proc. Natl. Acad. Sci. U.S.A.
1981
, vol. 
78
 (pg. 
7634
-
7638
)
4
Smith
A.G.
Heath
J.K.
Donaldson
D.D.
Wong
G.G.
Moreau
J.
Stahl
M.
Rogers
D.
Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides
Nature
1988
, vol. 
336
 (pg. 
688
-
690
)
5
Williams
R.L.
Hilton
D.J.
Pease
S.
Willson
T.A.
Stewart
C.L.
Gearing
D.P.
Wagner
E.F.
Metcalf
D.
Nicola
N.A.
Gough
N.M.
Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells
Nature
1988
, vol. 
336
 (pg. 
684
-
687
)
6
Niwa
H.
Burdon
T.
Chambers
I.
Smith
A.
Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3
Genes Dev.
1998
, vol. 
12
 (pg. 
2048
-
2060
)
7
Nichols
J.
Chambers
I.
Taga
T.
Smith
A.
Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines
Development
2001
, vol. 
128
 (pg. 
2333
-
2339
)
8
Cartwright
P.
McLean
C.
Sheppard
A.
Rivett
D.
Jones
K.
Dalton
S.
LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism
Development
2005
, vol. 
132
 (pg. 
885
-
896
)
9
Li
Y.
McClintick
J.
Zhong
L.
Edenberg
H.J.
Yoder
M.C.
Chan
R.J.
Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4
Blood
2005
, vol. 
105
 (pg. 
635
-
637
)
10
Bourillot
P.Y.
Aksoy
I.
Schreiber
V.
Wianny
F.
Schulz
H.
Hummel
O.
Hubner
N.
Savatier
P.
Novel STAT3 target genes exert distinct roles in the inhibition of mesoderm and endoderm differentiation in cooperation with Nanog
Stem Cells
2009
, vol. 
27
 (pg. 
1760
-
1771
)
11
Ying
Q.L.
Smith
A.G.
Defined conditions for neural commitment and differentiation
Methods Enzymol.
2003
, vol. 
365
 (pg. 
327
-
341
)
12
Ying
Q.L.
Nichols
J.
Chambers
I.
Smith
A.
BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3
Cell
2003
, vol. 
115
 (pg. 
281
-
292
)
13
Burdon
T.
Stracey
C.
Chambers
I.
Nichols
J.
Smith
A.
Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells
Dev. Biol.
1999
, vol. 
210
 (pg. 
30
-
43
)
14
Kunath
T.
Saba-El-Leil
M.K.
Almousailleakh
M.
Wray
J.
Meloche
S.
Smith
A.
FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment
Development
2007
, vol. 
134
 (pg. 
2895
-
2902
)
15
Stavridis
M.P.
Lunn
J.S.
Collins
B.J.
Storey
K.G.
A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification
Development
2007
, vol. 
134
 (pg. 
2889
-
2894
)
16
Gardner
R.L.
Beddington
R.S.
Multi-lineage ‘stem’ cells in the mammalian embryo
J. Cell Sci. Suppl.
1988
, vol. 
10
 (pg. 
11
-
27
)
17
Nichols
J.
Silva
J.
Roode
M.
Smith
A.
Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo
Development
2009
, vol. 
136
 (pg. 
3215
-
3222
)
18
Chazaud
C.
Yamanaka
Y.
Pawson
T.
Rossant
J.
Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway
Dev. Cell
2006
, vol. 
10
 (pg. 
615
-
624
)
19
Yuan
H.
Corbi
N.
Basilico
C.
Dailey
L.
Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3
Genes Dev.
1995
, vol. 
9
 (pg. 
2635
-
2645
)
20
Niwa
H.
Miyazaki
J.
Smith
A.G.
Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells
Nat. Genet.
2000
, vol. 
24
 (pg. 
372
-
376
)
21
Masui
S.
Nakatake
Y.
Toyooka
Y.
Shimosato
D.
Yagi
R.
Takahashi
K.
Okochi
H.
Okuda
A.
Matoba
R.
Sharov
A.A.
, et al. 
Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells
Nat. Cell Biol.
2007
, vol. 
9
 (pg. 
625
-
635
)
22
Batlle-Morera
L.
Smith
A.
Nichols
J.
Parameters influencing derivation of embryonic stem cells from murine embryos
Genesis
2008
, vol. 
46
 (pg. 
758
-
767
)
23
Ying
Q.L.
Wray
J.
Nichols
J.
Batlle-Morera
L.
Doble
B.
Woodgett
J.
Cohen
P.
Smith
A.
The ground state of embryonic stem cell self-renewal
Nature
2008
, vol. 
453
 (pg. 
519
-
523
)
24
Bain
J.
Plater
L.
Elliott
M.
Shpiro
N.
Hastie
C.J.
McLauchlan
H.
Klevernic
I.
Arthur
J.S.
Alessi
D.R.
Cohen
P.
The selectivity of protein kinase inhibitors: a further update
Biochem. J.
2007
, vol. 
408
 (pg. 
297
-
315
)
25
Doble
B.W.
Patel
S.
Wood
G.A.
Kockeritz
L.K.
Woodgett
J.R.
Functional redundancy of GSK-3α and GSK-3β in Wnt/β-catenin signaling shown by using an allelic series of embryonic stem cell lines
Dev. Cell
2007
, vol. 
12
 (pg. 
957
-
971
)
26
Doble
B.W.
Woodgett
J.R.
GSK-3: tricks of the trade for a multi-tasking kinase
J. Cell Sci.
2003
, vol. 
116
 (pg. 
1175
-
1186
)
27
Wu
D.
Pan
W.
GSK3: a multifaceted kinase in Wnt signaling
Trends Biochem. Sci.
2009
, vol. 
35
 (pg. 
161
-
168
)
28
Aubert
J.
Dunstan
H.
Chambers
I.
Smith
A.
Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation
Nat. Biotechnol.
2002
, vol. 
20
 (pg. 
1240
-
1245
)
29
Haegele
L.
Ingold
B.
Naumann
H.
Tabatabai
G.
Ledermann
B.
Brandner
S.
Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression
Mol. Cell. Neurosci.
2003
, vol. 
24
 (pg. 
696
-
708
)
30
Sato
N.
Meijer
L.
Skaltsounis
L.
Greengard
P.
Brivanlou
A.H.
Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor
Nat. Med.
2004
, vol. 
10
 (pg. 
55
-
63
)
31
Ogawa
K.
Nishinakamura
R.
Iwamatsu
Y.
Shimosato
D.
Niwa
H.
Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells
Biochem. Biophys. Res. Commun.
2006
, vol. 
343
 (pg. 
159
-
166
)
32
Kielman
M.F.
Rindapaa
M.
Gaspar
C.
van Poppel
N.
Breukel
C.
van Leeuwen
S.
Taketo
M.M.
Roberts
S.
Smits
R.
Fodde
R.
Apc modulates embryonic stem-cell differentiation by controlling the dosage of β-catenin signaling
Nat. Genet.
2002
, vol. 
32
 (pg. 
594
-
605
)
33
Pereira
L.
Yi
F.
Merrill
B.J.
Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal
Mol. Cell. Biol.
2006
, vol. 
26
 (pg. 
7479
-
7491
)
34
Yi
F.
Pereira
L.
Merrill
B.J.
Tcf3 functions as a steady-state limiter of transcriptional programs of mouse embryonic stem cell self-renewal
Stem Cells
2008
, vol. 
26
 (pg. 
1951
-
1960
)
35
Cole
M.F.
Johnstone
S.E.
Newman
J.J.
Kagey
M.H.
Young
R.A.
Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells
Genes Dev.
2008
, vol. 
22
 (pg. 
746
-
755
)
36
Tam
W.L.
Lim
C.Y.
Han
J.
Zhang
J.
Ang
Y.S.
Ng
H.H.
Yang
H.
Lim
B.
T-cell factor 3 regulates embryonic stem cell pluripotency and self-renewal by the transcriptional control of multiple lineage pathways
Stem Cells
2008
, vol. 
26
 (pg. 
2019
-
2031
)
37
Palmer
H.G.
Anjos-Afonso
F.
Carmeliet
G.
Takeda
H.
Watt
F.M.
The vitamin D receptor is a Wnt effector that controls hair follicle differentiation and specifies tumor type in adult epidermis
PLoS ONE
2008
, vol. 
3
 pg. 
e1483
 
38
Takao
Y.
Yokota
T.
Koide
H.
β-Catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells
Biochem. Biophys. Res. Commun.
2007
, vol. 
353
 (pg. 
699
-
705
)
39
Meloche
S.
Pouyssegur
J.
The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition
Oncogene
2007
, vol. 
26
 (pg. 
3227
-
3239
)
40
Sears
R.C.
The life cycle of c-myc: from synthesis to degradation
Cell Cycle
2004
, vol. 
3
 (pg. 
1133
-
1137
)
41
Brons
I.G.
Smithers
L.E.
Trotter
M.W.
Rugg-Gunn
P.
Sun
B.
Chuva de Sousa Lopes
S.M.
Howlett
S.K.
Clarkson
A.
Ahrlund-Richter
L.
Pedersen
R.A.
Vallier
L.
Derivation of pluripotent epiblast stem cells from mammalian embryos
Nature
2007
, vol. 
448
 (pg. 
191
-
195
)
42
Tesar
P.J.
Chenoweth
J.G.
Brook
F.A.
Davies
T.J.
Evans
E.P.
Mack
D.L.
Gardner
R.L.
McKay
R.D.
New cell lines from mouse epiblast share defining features with human embryonic stem cells
Nature
2007
, vol. 
448
 (pg. 
196
-
199
)
43
Nichols
J.
Jones
K.
Phillips
J.M.
Newland
S.A.
Roode
M.
Mansfield
W.
Smith
A.
Cooke
A.
Validated germline-competent embryonic stem cell lines from nonobese diabetic mice
Nat. Med.
2009
, vol. 
15
 (pg. 
814
-
818
)
44
Buehr
M.
Meek
S.
Blair
K.
Yang
J.
Ure
J.
Silva
J.
McLay
R.
Hall
J.
Ying
Q.L.
Smith
A.
Capture of authentic embryonic stem cells from rat blastocysts
Cell
2008
, vol. 
135
 (pg. 
1287
-
1298
)
45
Li
P.
Tong
C.
Mehrian-Shai
R.
Jia
L.
Wu
N.
Yan
Y.
Maxson
R.E.
Schulze
E.N.
Song
H.
Hsieh
C.L.
, et al. 
Germline competent embryonic stem cells derived from rat blastocysts
Cell
2008
, vol. 
135
 (pg. 
1299
-
1310
)
46
Chambers
I.
Silva
J.
Colby
D.
Nichols
J.
Nijmeijer
B.
Robertson
M.
Vrana
J.
Jones
K.
Grotewold
L.
Smith
A.
Nanog safeguards pluripotency and mediates germline development
Nature
2007
, vol. 
450
 (pg. 
1230
-
1234
)
47
Toyooka
Y.
Shimosato
D.
Murakami
K.
Takahashi
K.
Niwa
H.
Identification and characterization of subpopulations in undifferentiated ES cell culture
Development
2008
, vol. 
135
 (pg. 
909
-
918
)
48
Hayashi
K.
Lopes
S. M.
Tang
F.
Surani
M. A.
Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states
Cell Stem Cell
2008
, vol. 
3
 (pg. 
391
-
401
)