Differential Coupling of Self-Renewal Signaling

Pathways in Murine Induced Pluripotent Stem Cells

Luca Orlando1,2*, Yolanda Sanchez-Ripoll1, James Foster1, Heather Bone1, Claudia Giachino2, Melanie J.
Welham1
1 Centre for Regenerative Medicine, Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom, 2 Department of Clinical and Biological
Sciences, University of Turin, Orbassano (Torino), Italy

Abstract
The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of
embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation
and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular
signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To
address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line.
We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced
proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this
iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a NanogGFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling
decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line
displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second
feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key
role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the
molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in nave
pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors.
Citation: Orlando L, Sanchez-Ripoll Y, Foster J, Bone H, Giachino C, et al. (2012) Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced
Pluripotent Stem Cells. PLoS ONE 7(1): e30234. doi:10.1371/journal.pone.0030234
Editor: Andreas Androutsellis-Theotokis, Universitatsklinikum Carl Gustav Carus an der Technischen Universitat Dresden, Germany
Received September 29, 2011; Accepted December 12, 2011; Published January 23, 2012
Copyright: 2012 Orlando et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Marie Curie Early Stage Training program MEST-CT-2005-019822 and a VIP Award 087870/Z/08/Z from the Wellcome Trust.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]

ESCs, exemplified by major differences in mRNA and miRNA

expression profiles [1012]. Moreover, the starting conditions of
reprogramming appear to influence the behavior of iPSC lines
generated [1315] and recently iPSC lines have been shown to
retain a transcriptional and epigenetic memory of the differentiated cells from which they were derived [16,17]. This raises the
prospect that iPSC lines may respond to a different repertoire of
signals, compared to pluripotent ESCs, that is, at least in part,
dictated by their cellular origin.
Several extrinsic factors, signaling pathways and transcription
factors are known to play important roles in controlling selfrenewal and pluripotency of mouse ESCs. The transcription
factors include those used to generate iPSCs, the most important
being Oct4, Sox2 and Nanog [2]. Of the extrinsic factors,
leukemia inhibitory factor (LIF) plays a key role through activation
of the Signal transducer and activator of transcription factor 3
(Stat-3) and c-Myc [1820]. Bone morphogenetic proteins 2 and 4
(BMP2/4), present in serum or when added exogenously to serumfree media, cooperate with LIF to promote self-renewal by
inducing expression of Inhibitor of Differentiation, Id2 [21].
Although LIF also activates the extracellular-regulated kinases
Erk1 and Erk2, the activity of these Mitogen-activated protein
(MAP) kinases opposes, rather than promotes pluripotency [22]

Introduction
Induced pluripotent stem cells (iPSCs) are somatic cells
reprogrammed to pluripotency by the over-expression of specific
sets of genes. Mouse iPSCs were first generated by introducing the
combination of Oct3/4, Sox2, Klf4 and c-Myc [1] and they have
now been obtained using many approaches [2]. Since the
discovery of iPSCs, the main goal of researchers has been to
obtain them with increased efficiency and using techniques that
could allow for their use in clinical applications. Many initial
studies focused on the similarities between embryonic stem cells
(ESCs) and iPSCs, including assessment of pluripotency by testing
for their ability to contribute to formation of chimeras (germline
transmission) [37], as well as assessing histone modifications [8]
and methylation patterns [9]. In spite of significant technical
improvements in the ability to achieve reprogramming, as well as
in understanding the biological mechanisms underlying iPSC
generation, an in-depth analysis of the fine molecular regulation of
iPSC fate and the response of iPSCs to different stimuli is still
lacking in literature. In fact, despite the similarities in morphology
and the ability to pass the most stringent test of pluripotency
(germline transmission), it has become apparent more recently that
iPSCs exhibit some important differences when compared to

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Signaling Pathways Controlling iPSC Fate

and in serum-free conditions it has been demonstrated that

inhibition of Erk1 and 2, in addition to inhibition of glycogen
synthase kinase 3 (Gsk-3), is sufficient to maintain mouse ESCs in a
pluripotent ground state [23]. Other studies have also reported
that inhibition of Gsk-3 promotes self-renewal [24,25], via bcatenin-dependent and independent mechanisms [26]. Activation
of the Phosphoinositide-3 kinase (PI3K) pathway has been
associated with controlling proliferation of ESCs [2730], with
mTOR playing a key role [29]. PI3K-dependent signaling has also
been shown to play a role in optimal maintenance of mouse ESC
self-renewal [3134], at least in part through PI3K-mediated
inhibition of Gsk-3 activity [35].
In this study, we investigated whether the fate of mouse iPSCs is
controlled via similar mechanisms to those established in mouse
ESCs. Activation of LIF-Stat-3 and PI3K signaling appear
comparable between iPSCs and ESCs. However, we demonstrate
that the model iPSC line we have studied, which was derived from
fibroblasts [3], exhibits enhanced responsiveness to serumcontaining factors, reduced sensitivity to LIF withdrawal and
increased responsiveness to inhibition of Gsk-3. Furthermore,
activation of Erk1 and 2 is impaired in this iPSC line. We also
report that following extended passage this model iPSC line
exhibits an altered karyotype, indicative of genomic instability,
which correlates with altered behavior. The intriguing possibility
that in addition to transcriptional and epigenetic memory the
signaling pathways controlling the fate of iPSCs may also be
influenced by their cell of origin is discussed on the basis of our
results.

To test whether iPSCs and ESCs cultured under these different

conditions retained the capacity to self-renew, we conducted clonal
assays, based on alkaline phosphatase staining, which have been
widely used to assess the capacity of ESC populations to retain
their pluritpotency. Alkaline phosphatase is expressed only by
undifferentiated ESCs and its expression is rapidly lost upon
differentiation, hence these assays provide a convenient way to
measure self-renewal. iPSCs cultured in GMEM plus serum and
LIF exhibited a greatly reduced ability to self-renew, compared
with the same cells cultured in KO SR plus LIF (Fig. 1B). This
effect seems not to be directly correlated to the presence of serum,
but rather the combination of GMEM and serum, as when 10%
serum was added to KO SR medium iPSCs retained a
morphology similar to ESCs (Fig. 1A) and their capacity to selfrenew was similar to that observed when cultured in KO SR plus
LIF without serum (Fig. 1B).
We also investigated the growth of iPSCs and ESCs in different
media. iPSCs clearly grew more slowly in KO SR plus LIF than
ESCs, with a doubling time of approximately 24 hours against
17 hours for ESCs (Fig. 1C). We did not observe any increase in
the number of non-viable cells in iPSC cultures compared to ESC
cultures, so we consider it unlikely that this decrease in growth is
due to increased apoptosis. The data shown in Fig. 1B also suggest
that differentiation is not increased in iPSCs under these
conditions, so this also seems unlikely to contribute to the
decreased growth observed. Interestingly, when serum was
included, proliferation of iPSCs increased considerably, such that
their growth was more rapid than that of ESCs, with a doubling
time of about 13 hours against 15 hours for ESCs (Fig. 1D). This
effect was not influenced by the gelatin-coated plate system of
culture, since we observed similar growth rate results when cells
were cultured on MEF feeders (data not shown). These data
suggest that our model iPSC line exhibits a strong proliferative
response to serum and, depending on the media used, the presence
of serum in iPSC cultures increases their propensity to
differentiate, demonstrated by a reduction in self-renewal in
GMEM plus serum conditions (Fig. 1B).
mTor has been reported to be a key effector regulating somatic
cell proliferation [37], while ESCs from which mTor has been
conditionally deleted fail to expand [29]. Given the clear
difference in the proliferative capability of iPSCs compared to
ESCs we investigated what effect treatment with the mTor
inhibitor, rapamycin, would have. Rapamycin inhibited proliferation of both iPSCs and ESCs (Fig. 1C and D). Intriguingly, the
proliferation rate of ESCs in KO SR medium after treatment with
rapamycin was almost the same as that of untreated iPSCs
(Fig. 1C) suggesting a putative impairment in mTor signaling in
iPSCs. Immunoblotting to assess inhibition of mTor was carried
out and as shown in Figure 1E, phosphorylation of S6 ribosomal
protein, a downstream effector of the mTor-S6K1 pathway, was
reduced upon addition of rapamycin, while phosphorylation of
mTor itself was not. Interestingly, addition of serum to iPSCs led
to an enhancement in S6 phosphorylation, which correlates with
the enhanced proliferation of iPSCs in the presence of serum
(Fig. 1C and D).
To further investigate the influence of culture environment on
the fate of iPSCs, we made use of the fact that both the iPSC and
ESC lines express GFP under the control of the endogenous
Nanog promoter [3,36]. Expression of Nanog has been reported to
be heterogeneous in ESC cultures, with a low Nanog state making
cells more susceptible to differentiation inducing cues and a high
Nanog state being associated with maintenance of pluripotency
[36]. The profiles of GFP expression, a read-out for Nanog
expression, were analyzed by flow cytometry following culture of

Results
Dependency of murine iPSC fate on culture conditions
There have been few studies examining the influence of
extracellular factors on and the molecular signaling pathways
involved in the regulation of cell fate of iPSC lines. Therefore, we
have undertaken a study, using one of the earliest murine iPSC
lines generated as our model [3], to characterize both the influence
of different stimuli on the fate of this line and compare these
responses to those of a widely used murine ESC line, namely
E14tg2a [36]. Importantly, the iPSC line selected has been
demonstrated to be pluripotent and able to give rise to chimeras
[3]. Both lines express GFP under the control of the endogenous
Nanog promoter, providing a facile means of assessing cell fate.
The extracellular environment can influence the ability of ESCs
to self-renew and retain pluripotency and so we first examined the
effect of different culture conditions on the growth and fate of our
model iPSC line in comparison to murine ESCs. As the iPSC line
was originally cultured on a feeder layer of mouse embryo
fibroblasts (MEFs), we investigated the effect of culturing this iPSC
line on gelatin-coated plates, instead of MEFs, in different media.
As shown in Figure 1A, when cultured in KnockOut-DMEM
(KO) supplemented with KO Serum Replacement (SR) and LIF,
the iPSC line exhibited a morphology typical of that observed with
ESCs, with compact, phase-bright colonies predominating. When
KO medium was supplemented with serum (we retained SR
supplementation in this medium so that we could assess the effect
of different treatments using the same basal conditions) the
morphology of both ESCs and iPSCs were similar, with a mixture
of compact and flattened colonies typically observed. However,
when cultured in GMEM plus 10% Hyclone serum (HY) the
morphology of iPSCs quickly changed such that they had a more
flattened appearance, resembling cells with a more differentiated
phenotype. ESCs cultured in the same conditions exhibited a more
flattened morphology, but still grew tightly packed within colonies.
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Figure 1. Culture conditions influence iPSC self-renewal and proliferation. A ESCs or iPSCs (passage 21) were plated onto gelatin-coated
dishes in KnockOut-DMEM (KO) supplemented with KO Serum Replacement (SR) and LIF in the presence or absence of 10%(v/v) Hyclone serum (HY)
or GMEM supplemented with LIF and 10% (v/v) HY, as indicated. 48 hours after plating cultures were observed by light microscopy and photographs
taken. Representative images are shown, scale bar = 200 mm. B The self-renewal capacity of ESCs and iPSCs were evaluated by alkaline phosphatase
(AP) expression in clonal assays following 4 days culture in different conditions described in A. The average percentage of AP positive colonies 6 SEM
are shown from three independent experiments. Two-tailed paired t-tests indicate the following significance *** = p,0.005. C and D Cells where
plated at day 0 in KO SR+LIF (C) or the same media supplemented with 10% (v/v) HY (D). Rapamycin (5 nM) or DMSO (1:10000, as control) were
added 24 hours after seeding, and kept in the medium for the duration of the experiment. Cells were harvested after 48 (D2) 72 (D3) and 96 (D4)
hours and counted in triplicate. The average values 6 SEM are shown from three independent experiments. E ESCs or iPSCs were seeded in KO
SR+LIF at 8000 cells/cm2. After 24 h DMSO 1:10000 (ctrl), 5 nM Rapamycin (Rap), 10% (v/v) Hyclone serum (HY) or serum and rapamycin together (HY
Rap) were added to the cultures. After 24 h treatment protein extracts were prepared, separated through 10% acrylamide gels using SDS PAGE and
immunoblotted using the antibodies indicated.
doi:10.1371/journal.pone.0030234.g001

cells in different conditions. When cultured in KO SR and LIF

(Fig. 2A +LIF panel), the profile of Nanog-GFP expression was
similar for both iPSCs and ESCs, with the majority of cells having
high GFP expression and a smaller proportion of cells exhibiting
low levels of GFP expression, consistent with previous reports [36].
When serum was added (Fig. 2B +LIF panel), there was a shift
towards more heterogeneous Nanog-GFP expression in ESCs such

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that the majority of cells in the population exhibited intermediate

levels of GFP. In contrast, iPSCs maintained a larger proportion of
cells expressing high levels of GFP. When cultured in GMEM
supplemented with LIF and Hyclone serum (Fig. 2C +LIF panel),
expression of the GFP reporter was again heterogeneous. The
effect of removing LIF from the cultures on Nanog-GFP
expression was also examined. Surprisingly, even after 4 days in

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Figure 2. iPSCs are less sensitive to LIF withdrawal than ESCs. ESCs or iPSCs were plated at a concentration of 8000 cells/cm2, harvested after
4 days and analyzed for Nanog-GFP expression by flow cytometry. Cells were grown in KO SR (A), KO SR supplemented with 10% (v/v) Hyclone serum
(HY) (B) or GMEM supplemented with HY (C) in presence or absence of LIF, as indicated. Numbers shown on the histograms are the mean of FL1
fluorescence intensity (MFI). One representative experiment out of four is shown. D ESCs or iPSCs were seeded at 8500 cells/cm2 in KO SR in the
presence or absence of LIF. Protein extracts were prepared after 4 days incubation and immunoblotting performed with the antibodies indicated.
Following detection of pY705 Stat-3, blots were stripped and reprobed to detect total levels of Stat-3. E and F Cells were seeded at 8500 cells/cm2 in
KO SR plus 10% (v/v) HY (E) or GMEM supplemented with 10% (v/v) HY (F), in the presence or absence of LIF. After 48 h protein extracts were
prepared and immunoblotting performed as indicated.
doi:10.1371/journal.pone.0030234.g002

we decided to examine the status of Stat-3 phosphorylation, since

it is a direct target of LIF-induced signaling. In iPSCs cultured in
KO SR Stat-3 phosphorylation at Y705 was retained even after 4
days of LIF withdrawal, whereas phosphorylated Y705 Stat-3 was
undetectable in ESCs in the same conditions (Fig. 2D). In KO SR
plus serum (Fig. 2E) or GMEM plus serum (Fig. 2F), pY705 Stat-3
decreased following removal of LIF in both ESCs and iPSCs.
Taken together with earlier results, these data suggest that the
iPSC line is less sensitive to LIF withdrawal than is typical for
mouse pluripotent ESC lines and retains characteristics of self-

KO SR medium in the absence of LIF, the majority of iPSCs

retained high levels of GFP expression (Fig. 2A, -LIF panel), which
was in stark contrast to the lower levels of GFP detected in ESCs
under the same conditions. This effect was less pronounced when
serum was also present in the KO medium (Fig. 2B -LIF panel)
and GFP expression appeared to be more heterogeneous, although
again expression was higher in iPSCs than ESCs. In GMEM plus
serum both ESCs and iPSCs showed similar patterns of lowered
GFP expression (Fig. 2C LIF panel). These results suggested that
the iPSCs are less sensitive to withdrawal of LIF than ESCs and so
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previous studies [24,25,31]) and expression of GFP, as a read-out

of Nanog expression, was assessed by flow cytometry.
To investigate the effect of PI3K signaling perturbation, we used
LY294002 [39], a broad selectivity PI3K inhibitor which has been
shown to lead to loss of ESC self-renewal [31]. As shown in
Fig. 4A, in KO SR in the presence of LIF, inhibition of PI3K
signaling with LY294002 led to a reduction in Nanog-GFP
expression in ESCs, but not in iPSCs. In the absence of LIF,
Nanog-GFP expression decreased in both ESCs and iPSCs,
although the reduction was less for iPSCs, consistent with the data
shown in Fig. 2B and the inclusion of LY294002 had no additional
effect on either cell type. To analyze the effect of Gsk-3 inhibition,
we used BIO, a commercially available Gsk-3 inhibitor that has
been reported to enhance ESC self-renewal and Nanog expression
[25] and 1 m, a Gsk-3 inhibitor that demonstrates greater
selectivity than BIO and that also enhances self-renewal of mouse
ESCs [24]. Figure 4A shows that inhibition of Gsk-3 with either
BIO or 1 m led to a pronounced increase in GFP expression in
both cell types, consistent with previous results [24,25]. BIO had
modest effects, only significant in ESCs, while 1 m prevented the
decrease in GFP expression apparent in the control LIF-deprived
ESCs (-LIF, Fig. 4A) and significantly elevated GFP expression in
iPSCs.
To complement these analyses, we also assessed the effects of
small molecule inhibitors on cell fate control of iPSCs using
alkaline phosphatase staining as an alternate measure of selfrenewal. Based on our earlier observations showing that selfrenewal of iPSCs is best maintained in KO SR medium (Fig. 1),
we carried out these studies in KO SR plus LIF in the presence or
absence of serum. In the presence of LIF and serum, inhibition of
PI3K signaling with LY294002 decreased the proportion of
alkaline phosphatase positive, self-renewing iPSC and ESC
colonies (Fig. 4B). In the absence of serum inhibition of PI3Ks
had no significant effect on iPSC self-renewal (+LIF HY, Fig. 4B).
These results, along with those shown in Fig. 4A, suggest that
whereas inhibition of PI3K signaling in ESCs leads to loss of selfrenewal, as reported previously [28,31], under the different
conditions tested, self-renewal of iPSCs is only perturbed by
LY294002 treatment in the presence of serum. This may be
related to the requirement for factors contained in serum to
maintain cells as they differentiate. In contrast, incubation with
Gsk-3 inhibitors in the presence of LIF and serum increased the
proportion of alkaline phosphatase positive colonies (Fig. 4B). In
this case the absence of serum didnt change the effect in a
significant way (+LIF HY, Fig. 4B), as addition of 1 m enhanced
the proportion of alkaline phosphatase positive colonies present
over the levels in DMSO-treated controls by approximately 40%
in each case.
Based on our finding that 1 m treatment maintains Nanog-GFP
expression in iPSCs following LIF removal, we also tested whether
iPSCs could generate alkaline phosphatase positive colonies in the
absence of LIF. While ESCs formed very few colonies without LIF
in KO SR plus serum (data not shown), iPSCs did generate
colonies, albeit in reduced numbers compared to in the presence of
LIF (Fig. 4C). Importantly, the inclusion of 1 m to iPSCs cultured
in KO SR plus serum without LIF led to the formation of
comparable proportions of alkaline phosphatase positive colonies
(Fig. 4D) to those formed in the presence of LIF (Fig. 1B), as well
as similar numbers of colonies (Fig. 4C). These results are
consistent with our earlier data demonstrating decreased sensitivity
of iPSCs to LIF withdrawal (Fig. 2A and B, -LIF panel). Thus, in
the absence of LIF, inhibition of Gsk-3 in iPSCs not only
maintains Nanog-GFP expression (Fig. 4A), but also enhances self-

renewing cells, i.e., Nanog expression and Stat-3 Y705 phosphorylation, for extended periods in the absence of LIF.

iPSCs exhibit reduced levels of Erk MAP kinase activation

Having established the basic growth characteristics of iPSCs, we
next investigated whether the molecular signaling pathways known
to be involved in regulating pluripotency of mouse ESCs are also
involved in regulating the cell fate decisions of iPSCs. Initially we
assessed the basal levels of activation of key pathways after cells
had been cultured for 48 h in KO SR supplemented with LIF in
the absence or presence of serum (Fig. 3A) or cultured in KO SR
in the presence or absence of LIF (Fig. 3B). As shown in Figures 3A
and 3B, the levels of protein expression for key signaling
components, including Stat-3, Erks, Gsk-3 and Akt were similar
in ESCs and iPSCs. In addition, the levels of phosphorylation of
key activation motifs on Stat-3 (Y705) and Akt (S473) were similar
in ESCs and iPSCs. However, we consistently observed that Erk1
and 2 exhibited reduced levels of phosphorylation in iPSCs, which
was even more dramatic when cells were cultured in GMEM plus
serum (Figure S1). In relation to phosphorylation of the S6
ribosomal protein at S235 and 236, in some experiments we
observed lower levels of phosphorylation in iPSCs compared to
ESCs in basal conditions (Fig. 3A and see below), but in other
experiments no difference was observed (Fig. 3B(i); also see Fig. 1E
and below). It is known that S6 phosphorylation is sensitive to the
rate of cell proliferation, so this variability may be more a
reflection of growth rates, with the slower rate of proliferation of
iPSCs versus ESCs resulting in reduced S6 phosphorylation in
some cases. However, we consistently observed enhanced levels of
S6 phosphorylation when serum was included in iPSC culture
medium (Fig. 1E and Fig. 3A). Interestingly, levels of S9/20
phosphorylation on Gsk-3 a/b isoforms were found to be
consistently increased in iPSCs, compared to ESCs and declined
in the presence of serum or upon LIF removal (Fig. 3A and B(ii)).
As we observed a reduction in levels of phosphorylated and
active Erk1 and 2 in iPSCs, we examined the ability of acute
stimulation with LIF (in the absence of serum and following 4 h
LIF withdrawal) to induce phosphorylation and activation of Stat3 and Erk1 and 2. As shown in Fig. 3C, short-term stimulation
with LIF (5 and 30 mins) induced phosphorylation of Y705 of
Stat-3 in both ESCs and iPSCs. A 30 min treatment with serum
also induced Stat-3 phosphorylation at Y705. Interestingly, while a
5 min treatment with LIF induced a robust increase in Erk1 and 2
phosphorylation in ESCs, LIF appeared to be unable to activate
Erk1 and 2 in iPSCs, despite similar overall levels of Erk
expression in both cell types. Serum treatment served as a control
and induced similar levels of Erk1 and 2 phosphorylation in ESCs
and iPSCs. These data indicate that in the iPSC line used as our
model, LIF receptor signaling is not as efficiently coupled to MAP
kinase signaling as it is in pluripotent ESCs. However, this
pathway is intact and presumably the basal phosphorylation
measured in iPSCs arises due to the actions of other factors
present, either produced by the iPSC cells themselves (ESCs
produce Fgf4 which strongly induces Erk1 and 2 activation [38])
or present in the culture media.

Perturbation of PI3K and Gsk-3-dependent signaling

have opposing effects on the fate of iPSCs
Having established the signaling pathways that appear active in
iPSCs, we then examined which of these played a role in
controlling their fate using a series of small molecule inhibitors.
ESCs and iPSCs were cultured for 4 days in the presence or
absence of inhibitors (the doses of which had been optimized in
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Figure 3. Expression and phosphorylation status of key signaling intermediates in ESCs and iPSCs. A and B ESCs or iPSCs were seeded
8000 cells/cm2 in KO SR plus LIF. After 24 hours, 10% (v/v) Hyclone serum (HY) was added where indicated (A), or cells were washed 3 times with PBS
before LIF-containing or LIF-free KO SR was added to the cultures as indicated (B). After a further 24 hours incubation, protein extracts were
prepared, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using the antibodies indicated. Blots in A and B(i) were all
generated using cell extracts from one replicate and those in B(ii) from an independent experimental replicate. C ESCs or iPSCs were seeded at 8500
cells/cm2 in GMEM 10% (v/v) Hyclone serum (HY) plus LIF, then 24 h later washed and deprived of LIF and serum for 4 h. 5000 U/ml of LIF or 10% (v/
v) HY were added and proteins extracted following 0, 5, 30 min treatment with LIF or 30 min treatment with serum. The phospho-proteins indicated
were detected by immunoblotting of the same membrane, which had been cut referring to the size of the protein marker, then stripped and reprobed with the corresponding pan antibody. Results generated from the same blots are grouped, with each series terminating with the respective
Gapdh as loading control.
doi:10.1371/journal.pone.0030234.g003

the presence and more particularly the absence of serum, we were

initially most interested to examine the consequences of Gsk-3
inhibition on signaling in iPSCs. In the presence of LIF or LIF and
serum, inhibition of Gsk-3 with 1 m for 24 hours resulted in
significant elevation of b-catenin levels in both ESCs and iPSCs,
consistent with stabilization of b-catenin protein (Fig. 5A).
Interestingly, basal levels of S6 phosphorylation declined upon
LIF withdrawal in both ESCs and iPSCs. However, inclusion of
1 m maintained levels of pS6 phosphorylation in ESCs and iPSCs

renewal (Fig. 4D) and survival (Fig. 4C) suggesting modulation of

Gsk-3 activity is key to determining the fate of iPSCs.
In light of the results of our functional analyses, we next assessed
the effects that treatment of ESCs and iPSCs with these small
molecule inhibitors had on the activity of key signaling pathways.
As the Gsk-3 inhibitor BIO was shown to have similar effects to
the 1 m molecule, but is known to be less selective [24], only 1 m
was used for the following experiments. Given the ability of Gsk-3
inhibition to promote maintenance of iPSC self-renewal in both

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Figure 4. iPSCs demonstrate enhanced responsiveness to Gsk-3 inhibition. A ESCs and iPSCs were plated at a density of 8000cells/cm2 in
KO SR plus or minus LIF as indicated. After 24 hours, inhibitors were added to the medium at the following concentrations: LY294002: 5 mM; BIO:
2 mM; 1 m: 2 mM; DMSO 1:10000 as control, and cultured for 4 days. Following treatment, Nanog-GFP expression was assessed by flow cytometry and
the mean of fluorescence intensity (MFI) of Nanog-GFP was calculated for each condition. The histogram bars represent the variation in Nanog-GFP
MFI in treated samples, compared to the KO SR plus LIF (DMSO treated) control condition, expressed as a percentage. The mean of three

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independent experiments 6 SEM are shown. Two-tailed paired t-tests indicate the following significance values: *** = p,0.005, * = p,0.05 are
referred to the DMSO plus LIF condition; {{{ = p,0.005 { = p,0.01 are referred to the DMSO minus LIF condition; # = p,0.01 is referred to the plus
LIF 1 m-treated condition. B The percentage of alkaline phosphatase positive ESC or iPSC colonies generated in KO SR plus LIF in the presence or
absence of 10% (v/v) Hyclone serum (HY) were measured. The data represent the percentage variation in alkaline phosphatase (AP) positive colonies
compared with the DMSO treated control. The average values 6 SEM are shown from three independent experiments. Two-tailed paired t-test:
*** = p,0.005, * = p,0.05 are referred to the DMSO plus LIF. C iPSCs were cultured in KO SR without LIF and supplemented with 10% (v/v) HY. The
total numbers of colonies generated are shown and are compared with the total number of colonies generated by ESCs and iPSCs in KO SR plus LIF
plus serum. The percentage of alkaline phosphatase positive colonies are shown in histogram D.
doi:10.1371/journal.pone.0030234.g004

cultured without LIF (Fig. 5B), which could contribute to

enhanced survival. In ESCs, when LY294002 was used to inhibit
PI3K signaling, a decline in phosphorylation of Ser 473 of Akt, a
key downstream target of PI3K signaling, was only readily
detectable in the presence of serum (Fig. 5C), although
phosphorylation of S6 was clearly reduced by LY294002
treatment in both the absence and presence of serum. Interestingly, in iPSCs, LY294002 had less effect on S6 phosphorylation in
the presence of serum, compared to a dramatic reduction in the
absence of serum, consistent with increased responsiveness of this
iPSC line to serum. Taken together, these data suggest that Gsk-3
plays a predominant role in regulating the fate of iPSCs and that
S6 phosphorylation is particularly sensitive to presence of serum.

maintained in KO SR supplemented with LIF (Fig. 6A panel (iii)).

The change of morphology was just one of the several differences
apparent between iPSCs at low passage (iPSC LP, considered as
passages ,25) compared with the high passage iPSCs (iPSC HP,
considered as .40 passages). First of all, iPSC HP showed genetic
instability, with an abnormal karyotype and chromosomal
aberrations (Fig. 6B and Table S1). This observation was
reinforced by the observation that phosphorylation of Ser 15 of
p53 was dramatically increased in iPSC HP (Fig. 6C), which is
linked with genetic instability. Moreover, iPSC HP were found to
proliferate at a rate comparable with that of ESCs (Fig. 6D),
raising the possibility that these cells had undergone some form of
adaptation. In spite of these changes, some characteristics
remained stem cell-like, such as a high level of expression of the
Nanog-GFP, even though the GFP negative population (indicated
by the arrow in Fig. 6E panel (iii)) is lost in iPSCs HP. When the
ability of iPSC HP to self-renew was tested in clonal assays, a lower
proportion of alkaline phosphatase positive colonies were generated in KO SR plus LIF, in both the presence (Fig. 6F) and
absence of serum (Fig. 6G). However, in the presence of 1 m, the
percentage of alkaline phosphatase positive colonies increased
dramatically (Fig. 6F and G). This effect, coupled with a
reinstatement of an ESC-like morphology in the presence of 1 m
(Fig. 6A panel (iv)), suggests that high passage iPSC HP, like their
low passage predecessors, are highly sensitive to Gsk-3-dependent
signaling. Finally, iPSC HP were not able to grow in self-renewal
assays without LIF (data not shown), suggesting a recovery in LIF
dependence.

iPSCs change in morphology and behavior following

long-term culture
During the course of culturing iPSCs we observed that between
passage 25 and 40 their morphology began to alter, even when

Discussion
In this study we undertook a comparison of the molecular
signaling pathways that contribute to maintenance of the stem cell
state of mouse iPSCs and ESCs. Our studies demonstrate that the
iPSC line we used as our model exhibits a significant response to
factors contained within serum, accompanied by reduced
sensitivity to the withdrawal of LIF. While basal levels of
expression and phosphorylation of components of a number of
signaling pathways known to regulate self-renewal of mouse ESCs
were similar in iPSCs and ESCs, our studies revealed a key role for
Gsk-3 in controlling iPSC fate. Upon LIF withdrawal, inhibition of
Gsk-3 was able to maintain iPSCs in a self-renewing state for short
periods, a response not observed with ESCs. Furthermore,
inhibition of Gsk-3 improved survival of iPSCs in the absence of
LIF and also increased the ability of high passage iPSCs to selfrenew. High passage iPSCs exhibited evidence of genomic
instability and altered responsiveness. Together our findings
highlight the importance of characterizing not only the molecular
signatures of reprogrammed iPSCs, but also their responses to the
external environment and signals controlling their behavior.
The equivalency between iPSC lines and ESC lines is still
somewhat controversial. Many reports indicate that there is a high
degree of similarity between the cell types [3,7,9,40,41], but more
recently important differences have also been reported [16,17,42
44]. Although we have only examined the responses of one iPSC

Figure 5. Effect of perturbation of key self-renewal signaling

pathways in ESCs and iPSCs. ESCs and iPSCs were seeded in KO SR
plus or minus LIF as indicated. After 24 hours 2 mM 1 m and 10% (v/v)
Hyclone serum (HY) alone or together (A), 2 mM 1 m (B), 5 mM
LY294002 and 10% (v/v) HY (C) or DMSO 1:10000 (in all controls), were
added. After a further 24 hours proteins were extracted and
immunoblotting performed as indicated. Blots were stripped and reprobed with Gapdh, pan Akt or Shp2 antibodies to assess loading.
doi:10.1371/journal.pone.0030234.g005

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Signaling Pathways Controlling iPSC Fate

Figure 6. High passage iPSCs exhibit altered morphology and response to stimuli. Cells were plated at a density of 8500 cells/cm2 in KO SR
plus LIF. A iPSCs changed morphology after several passages (iii), compared with ESC (i) or iPSC low passage (ii), but reinstate an ESC-like morphology
after 24 h treatment with 2 mM 1 m (iv). iPSCs were considered as low passage (iPSC LP) with passages ,25 and high passage (iPSC HP) with
passages .40. B Chromosome spreads of ESCs and low/high passage iPSCs. The arrow indicates chromosome aberrations in iPSC HP. One
representative karyotype out of 20 from each cell line is shown. C 4 days after seeding, ESC or iPSC proteins were extracted, separated through 10%
acrylamide gels using SDS-PAGE and immunoblotted using phospho-S15 p53 antibody. b-actin antibody was used as loading control. As a positive
control, protein extracted from irradiated T lymphocytes was used. D Cells were plated at day 0 and then harvested after 48 (D2), 72 (D3) and 96 (D4)
hours. Cells were counted in triplicate. The average doubling times 6 SEM are shown from two independent experiments. Doubling times were
calculated using free software available from www.doubling-time.com. Two-tailed paired t test: ** = p,0.01 * = p,0.05. E iPSCs low/high passage or
ESCs were harvested 4 days after seeding and Nanog-GFP expression analyzed by flow cytometry. Contour plot graphs are shown and the missing
Nanog-GFP low population in iPSC HP indicated by an arrow. F and G Alkaline phosphatase positive colonies were counted after growing in KO SR
plus LIF supplemented with (F) or without 10% (v/v) Hyclone serum (HY, G). After 24 hours, 2 mM 1 m was added and kept in the medium until the
cells were fixed and stained. The average values 6SEM are shown from three independent experiments.
doi:10.1371/journal.pone.0030234.g006
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Signaling Pathways Controlling iPSC Fate

line in our studies, meaning we cannot exclude the possibility that

our data reflect features exclusive to this particular line, our data
are consistent with the idea proposed that iPSC lines retain a
memory of their cell of origin. Several recent high profile
publications have demonstrated that iPSC lines can retain both
genetic and epigenetic memories of the differentiated cell from
which they were derived [16,17,42,43]. In addition, comparison of
human ESC and iPSC line proteomes and phosphoproteomes has
shown there are reproducible differences which are consistent with
iPSC lines retaining residual regulatory properties characteristic of
their somatic cell of origin [45]. Our study suggests that this may
also translate into the response of iPSCs to external signals. The
iPSC line we used as our model was derived from primary mouse
fibroblasts [3], which require serum to support their proliferation.
It was interesting to find, therefore, that this iPSC line displayed a
dramatic response to serum. In the absence of serum, iPSCs grew
significantly slower than ESCs. However, when supplemented
with serum, iPSC proliferation exceeded that of ESCs and this was
accompanied by an increase in phosphorylation of the S6
ribosomal protein, consistent with enhanced cell metabolism/
proliferation. We propose that a useful additional measure to
assess the completeness of reprogramming may be to investigate if
iPSC lines retain any response to factors that normally act upon
the differentiated cells from which they were derived. We consider
this important as residual responsiveness to such stimuli could also
be retained in differentiated progeny derived from iPSCs, which
could lead to aberrant cellular behaviors, highly undesirable in a
cell-transplantation setting.
In addition to exhibiting responsiveness to serum-containing
factors, our analyses reveal that our model iPSC line shows
diminished sensitivity to withdrawal of LIF. Normally, mouse
iPSCs require LIF for their self-renewal, although at least two
examples of LIF-independent IPSC lines have been reported.
Whether the reprogramming conditions used, epiblast stem cell
conditions [14] or presence of a human fibroblast feeder layer
[15], influenced the generation of these LIF-independent iPSCs is
currently unclear. In KO SR in the presence or absence of serum,
iPSCs retained higher levels of Nanog-GFP reporter expression
when LIF was removed compared to ESCs, which rapidly lost
GFP expression (Fig. 2A&B). This characteristic of iPSCs could be
linked to the fact that they proliferate more slowly, meaning that
they may also differentiate more slowly. However, we do not think
this is the case because in self-renewal assays, iPSCs formed
colonies in the absence of LIF (Fig. 4C), whereas very few ESC
colonies survived under the same conditions. It also important to
note that although Stat-3 phosphorylation in basal conditions and
in short-term LIF stimulations followed similar kinetics to those of
ESCs, in extended cultures, Stat-3 phosphorylation could be
detected in iPSCs after several days of culture without LIF. Under
the same conditions, Nanog-GFP was also retained in a high
proportion of cells, consistent with iPSCs maintaining features of a
pluripotent cell state for a period of time following withdrawal of
LIF.
Several of the signaling pathway components we examined as
part of this study were expressed and phosphorylated at similar
levels in iPSCs and ESCs under basal conditions (KO SR plus or
minus serum in the presence of LIF) including Stat-3, Akt and
mTor. Therefore, it was interesting that consistent differences
were observed in phosphorylation of Erk1, Erk2 and Gsk-3. The
MAPK pathway is involved in cell proliferation, survival and
differentiation [46]. Unexpectedly, in the iPSCs we used for the
majority of these studies (at between 19 and 24 passages) acute
stimulation with LIF was unable to induce detectable phosphorylation of Erk1 and Erk2. The reason for this un-responsiveness to
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LIF is currently unclear, however, the pathway appears intact as

serum was shown to induce Erk1 and Erk2 phosphorylation in
short-term treatments. Gsk-3 has been implicated in the regulation
of mouse ESC self-renewal by a number of groups, including our
own [2325]. It has also been reported that inhibition of Gsk-3 can
accelerate the full reprogramming of pre iPSC [47]. Therefore, it
was of considerable interest to discover that our model iPSC line
demonstrated elevated levels of Gsk-3 phosphorylation compared
to ESCs and the fate of this iPSC line was particularly sensitivity to
inhibition of Gsk-3 activity. For example, treatment with the Gsk-3
inhibitor 1 m was able to promote maintenance of Nanog-GFP
expression in both ESCs and iPSCs in the presence of LIF, but
also, most notably for iPSCs, in the absence of LIF. Treatment of
iPSCs with 1 m was also able to promote cell survival and support
formation of alkaline phosphatase positive colonies in the absence
of LIF. It has been demonstrated that when activation of Erk MAP
kinases is diminished, down-modulation of Gsk-3 becomes crucial
to maintenance of pluripotency [23], which reflects the situation
we observe in this iPSC line.
Another key aspect of our study was the finding that the
characteristics of our iPSC model line changed with increasing
passage. There are a number of possible explanations that could
account for these observations; this line may be genetically
unstable, it may have adapted to the tissue culture conditions used
or the iPSC line could have regressed to a more differentiated
state. In support of the first possibility are our data demonstrating
that at higher passage numbers (greater than 40) this iPSC line
exhibited karyotypic and chromosomal abnormalities. Furthermore, a change of morphology of iPSCs was consistently observed
when they reached passage 40 or more and this was not stochastic.
The high phosphorylation state observed for p53 in HP iPSC is
also consistent with genetic instability, although p53 is known to be
involved in many biological responses, not just in response to DNA
damage. In previous studies iPSC lines have been demonstrated to
be genetically stable [1,7,48,49], although it is worth noting that
cells at lower passage numbers were investigated in these cases,
compared with our present work. Regarding regression to a
more differentiated state, it is quite common during iPSC
generation to observe colonies that are not completely undifferentiated, but share some markers and characteristics with
pluripotent stem cells, although they fail the common pluripotency
test [50]. Such cells have been referred to as pre iPSC cells. So,
a partial but not complete differentiation could occur in iPSCs
during long-term passage and in support of this is the fact that the
proportion of self-renewing cells was considerably lower in iPSC
HP compared to iPSC LP. This was somewhat surprising in view
of the fact that iPSC HP have still very high Nanog GFP
expression. Interestingly, we demonstrate that inhibition of Gsk-3
in iPSC HP restores their ability to generate self-renewing colonies
and cells treated in this way revert to a morphology that more
closely resembles low passage iPSCs and ESCs. This response is
very reminiscent of the effects of Gsk-3 inhibition reported by Silva
et al., [47], where they demonstrated that inhibition of Gsk-3
accelerated the reprogramming of pre iPSC to a fully reprogrammed state.
Taken together our data show an iPSC cell line, even if
considered stable and respecting all the stringent parameters of
pluripotency, can have important differences in response to
external stimuli and regulation of self-renewal compared with
ESCs. It has been shown that iPSCs are very sensitive to the
environmental conditions during the reprogramming [1315] and
they can also adapt to their culture conditions [17]. Here we
confirm and extend these findings. Although our results contrast
with some previous reports, and we have used only one line for our
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Signaling Pathways Controlling iPSC Fate

pan b-catenin (CST 9562), anti-pan Akt (CST 9272); 1:2000 antipan Erk (panErk, Santa Cruz Biotechnology, sc-93), anti-pan
STAT-3 (panStat-3, sc-482), anti-pan SHP-2 (sc 293), anti-pan
mTOR (CST 2983) anti-pan p70S6 kinase (CST 9202) anti bActin (SIGMA A5316) and anti GAPDH (CST 2118). An antirabbit secondary antibody conjugated to horseradish peroxidase
(DAKO) was used for detection and blots were developed using
ECL according to the manufacturers directions (GE Healthcare).
Protein relative quantification was carried out using an ImageQuantTM RT-ECL imager and analyzed using ImageQuantTM
TL software (GE Healthcare). Blots were stripped and reprobed as
previously described [53].

studies, our findings do raise an important general issue for iPSC

biology, especially for those who want to use iPSCs for applications
such as drug discovery/screening and cell-based therapies. Further
more extensive analyses are required to determine if the
characteristics described here are commonly observed with other
iPSC lines. Should such differences be more generally observed it
will be very interesting to link them with different tissues of origin
and possibly also the reprogramming technique. As the field of
iPSC biology progresses it is becoming increasingly clear that
establishing strict parameters for evaluating full reprogramming
and stability will be vital to ensuring safety and efficacy.

Materials and Methods

Flow cytometry

ESC and iPSC cell culture

ESCs and iPSCs were trypsinised, washed and resuspended in

PBS containing 5% (v/v) FCS, 0.1% (w/v) sodium azide. Flow
cytometry was carried out using a FACSCanto instrument (Becton
Dickinson). Dead cells were excluded from the analysis based on
forward and side scatter parameters. Mean fluorescence intensity
(MFI) of the GFP reporter in the viable cell population of each cell
sample was determined using FACSDiva software. The variation
between MFI values of treated samples compared to control
samples were calculated and expressed in percentage terms as
either increases or decreases. The average difference in MFI values
between samples from three independent experiments were
determined and data presented on histograms.

The culture of mouse ESCs and iPSCs were carried out as

described previously [31,51,52]. The E14tg2a ESC line [36] and
the iPSC line [3] (less than 25 passages from derivation) were used
throughout this study. Culture media used were KnockOutDMEM (GIBCO) supplemented with 15% (v/v) KO Serum
Replacement (GIBCO) and 1000 units\ml of LIF; GMEM
(GIBCO) supplemented with 10% (v/v) Hyclone FBS and 1000
units/ml LIF; KnockOut-DMEM supplemented with 15% (v/v)
KO Serum Replacement, 10% (v/v) Hyclone serum and 1000
units/ml LIF.

Cell proliferation
Cells were cultured on 24-well trays coated with gelatin at a
starting concentration of 24000 cells/cm2 for the KO SR
condition and 8000 cells/cm2 for the KO SR HY condition. At
each time point cells were harvested, stained with trypan-blue and
viable versus non-viable cells counted in triplicate using a
Neubauer chamber. Doubling times were calculated by the freesoftware available from the web site www.doubling-time.com.

Karyotyping
Cells were seeded 8000/cm2 24 h before treatment, then
0.1 mg/ml Colcemid (Sigma) was added into the medium and cells
were incubated for 4 h at 37uC. Cells were then trypsinized,
incubated for 10 min in 0.075 M KCl solution and then fixed with
3:1 methanol: glacial acetic acid. Fixed cells were then dropped on
to a cooled glass slide from a height approximately 1 meter, air
dried and DAPI stained. The karyotypes of 20 independent cell
spreads from each cell line were counted (Table S1).

Alkaline phosphatase staining

To assess self-renewal, ESCs or iPSCs were plated at 1500 cells
per gelatin-coated well of a six well tray and cultured either
without LIF or with the addition of 1000 units/ml of LIF. 6 hours
after plating (to permit adherence) inhibitors (5 mM LY294002,
2 mM BIO, 2 mM 1 m or 1:10000 DMSO as control) were added
without medium change. To detect colonies expressing the
alkaline phosphatase enzyme, a marker of pluripotency, after 5
days of culture cells were washed in PBS, fixed in 100% methanol
and stained with Fast Violet salt (Sigma) dissolved in 0.1 M Tris
pH 9.2, containing 200 mg/ml Naphtol AS-MX phosphate.
Alkaline phosphatase-positive colonies were counted in triplicate
for each experiment using an Olympus XI51 inverted microscope.

Supporting Information
Figure S1 ESCs and iPSCs were plated at a density of
8500 cells/cm2 in GMEM plus 10% (v/v) Hyclone serum
plus or minus LIF as indicated. After 48 h proteins were
extracted and immunoblotting performed with the indicated
antibodies.
(TIF)
Table S1 After chromosome spreading and DAPI staining, 20 single nuclei fields were counted for each cell
line. The different chromosome counts were expressed as a
percentage and shown in the table.
(TIF)

Protein extraction and immunoblotting

Cell extracts were prepared, protein concentrations determined
and 20 mg of protein separated by SDS-PAGE and transferred to
nitrocellulose as previously described [53]. Immunoblotting was
carried out with the following primary rabbit polyclonal
antibodies: 1:1000 for rabbit polyclonal antibodies recognizing
dual phosphorylation of Erk1 and 2 at Thr202/Tyr204 (anti-pErk,
Cell Signaling Technology 9101), phospho-tyrosine 705 of Stat-3
(anti-pSTAT-3, CST 9131), phospho-serine 473 of Akt (anti-pAkt,
CST 9271), phospho-serines 21 or 9 of Gsk-3 a/b (anti-pGsk-3 a/
b, CST 9331), phospho-serines 235/236 of S6 (anti-pS6 CST
4856), phospho-serines 411/418 of p70 S6 kinase (anti- p70S6
kinase CST 9204), phospho-serine 2448 of mTOR (anti-pmTOR
CST 2976), phospho-serine 15 of p53 (anti p53 CST 9284), anti-

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Acknowledgments
We are grateful to Ian Chambers for the gift of Nanog-GFP ESC cells and
RIKEN cell bank for provision of the iPSC line. We wish to thank Adrian
Rogers in the University of Baths Bioimaging Suite for assistance with flow
cytometry.

Author Contributions
Conceived and designed the experiments: LO YS-R HB MJW. Performed
the experiments: LO YS-R JF HB. Analyzed the data: LO CG MJW.
Wrote the paper: LO CG MJW.

11

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Signaling Pathways Controlling iPSC Fate

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January 2012 | Volume 7 | Issue 1 | e30234