RESEARCH ARTICLE
Murine Embryonic Stem Cell Plasticity Is
Regulated through Klf5 and Maintained by
Metalloproteinase MMP1 and Hypoxia
Aya Abou Hammoud1,2,3, Nina Kirstein1,2¤a, Virginie Mournetas1,2¤b, Anais Darracq1,2,
Sabine Broc1,2, Camille Blanchard1,2, Dana Zeineddine3, Mohamad Mortada3,
Helene Boeuf1,2*
1 Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France, 2 CNRS, CIRID, UMR 5164, F-33 000
Bordeaux, France, 3 Lebanese University, Beyrouth, Liban
¤a Current address: DNA Replication and Epigenetics group, Research Unit Gene Vectors, Helmholtz
Zentrum München, German Research Center for Environmental Health, Munich, Germany
¤b Current address: I-STEM, CECS/Inserm UMR861, 91030 Evry, France
* helene.boeuf@u-bordeaux.fr
Abstract
OPEN ACCESS
Citation: Hammoud AA, Kirstein N, Mournetas V,
Darracq A, Broc S, Blanchard C, et al. (2016) Murine
Embryonic Stem Cell Plasticity Is Regulated through
Klf5 and Maintained by Metalloproteinase MMP1 and
Hypoxia. PLoS ONE 11(1): e0146281. doi:10.1371/
journal.pone.0146281
Editor: Johnson Rajasingh, University of Kansas
Medical Center, UNITED STATES
Received: July 20, 2015
Accepted: December 15, 2015
Published: January 5, 2016
Copyright: © 2016 Hammoud 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.
Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in
the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal
leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells
apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible
phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF–mediated reprogramming are poorly understood. By employing
a LIF-dependent ‘plasticity’ test, that we set up, we show that Klf5, but not JunB is a key LIF
effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency,
has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can
replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less
efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O2 despite lower levels of
Pluri and Master gene expression in comparison to 20% O2.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was funded by CNRS, University
of Bordeaux, the SIRIC BRIO program, the Region
Aquitaine, the INCA and the FR TransBiomed. A.A.H.
had a fellowship from the foundation Hariri (Lebanon)
under a Franco-Lebanese co-supervision between
the University of Bordeaux and the Lebanese
University. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Introduction
During the last decades, mouse embryonic stem cells (mESCs) have been intensively studied to
reveal genetic programs essential for control of pluripotency and early cell fate decisions. This
led to the characterization of signaling pathways and transcription effectors essential for the
maintenance of mESCs pluripotency. These include the leukemia inhibitory factor (LIF)/signal
transducer and activator of transcription 3 (STAT3)/suppressor of cytokine signaling 3
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Plasticity Control in mESCs
Competing Interests: The authors have declared
that no competing interests exist.
(SOCS3) pathway, along with the ‘Master genes’ like Octamer 4 (Oct4), Nanog, SRY (sex determining region Y)-box 2 (Sox2) and estrogen-related receptor, beta (Essrb) [1–3]. Subsequently,
cocktails of genes were identified that could drive reprograming of many types of somatic cells
(like fibroblasts, keratinocytes, hepatocytes or bone marrow-derived cells), from various species
including Humans, to induced pluripotent stem cells (iPSCs), with potential applications in
cell therapies and regenerative medicine [4–6].
The mESCs are derived from pre-implantation blastocysts and are maintained pluripotent
in vitro in i) serum-containing medium with LIF, or ii) bone morphogenetic protein 4
(BMP4)/LIF medium, or iii) serum-free medium supplemented with LIF and cocktails of
inhibitors for key signaling pathways [extracellular regulated kinase (ERK), fibroblast growth
factor (FGF) and glycogen synthase kinase 3β (GSK3β) inhibitors, 3i]. All these cell growth
media maintain mESCs in a naive pluripotent state, the most immature in vitro state defined
by the cells being capable of colonizing embryos and contributing to all cell types in the organism [7–10]. Human embryonic stem cells (hESCs), which are maintained pluripotent in the
presence of FGF2 and activin A are closer to primed mouse epiblast stem cells (EpiSCs), a state
more prone to differentiation and less stable than the naive state. However various studies have
reported strategies to revert hESCs to a naive state by treatment with LIF, STAT3 and/or signaling pathway inhibitors [11–14].
The LIF-induced signaling cascade starts with activation of Janus kinase (JAK) phosphorylating phosphatidylinositol 3-kinase (PI3K), which induces the phosphorylation and activation
of AKT serine/threonine kinase. AKT signaling leads to the activation of T-box 3 (Tbx3)
expression, a transcription factor that induces the expression of pluripotency maintaining
genes. Additionally, AKT inactivates GSK3β by phosphorylation preventing the ubiquitindependent degradation of Myc and inhibition of Nanog expression. GSK3β is also inhibited by
the canonical wingless (Wnt) signaling pathway which acts in synergy with LIF to maintain the
expression of pluripotency related genes [15–18].
Most stem cells are found in complex microenvironments, termed ‘niches’ which reside in
low oxygen concentration ([O2]), [19,20]. mESCs are derived from embryos which also remain
in 1.5–5% [O2]. This low oxygen environment is physiologically normal, not only for ESCs but
also for many other types of stem cells including neural stem cells (NSCs), hematopoietic stem
cells (HSCs) and mesenchymal stem cells (MSCs) [21–23]. The effect of low [O2] on ESCs fates
remains controversial and poorly understood. Several reports have shown that low [O2] inhibits differentiation and maintains pluripotency of hESCs [24–27] and improves clonal survival
of mESCs, particularly when the GSK3β pathway is repressed [7,28,29]. Also the Wnt/ b-Catenin pathway is stimulated, under hypoxia in mESCs which could be differentiated into the
three cell lineages in vitro, indicating that hypoxia did not alter their pluripotency [30,31]. Activation of the Notch signaling pathway by hypoxia stabilizes hypoxia induced factor 1α (Hif1α),
the major effector of early steps of hypoxia, maintains the undifferentiated state and thus
enhances the generation of both human and mouse iPSCs [32]. In addition, hypoxia can also
revert hESCs or iPSC-derived differentiated cells back to the stem cell-like state [33]. In contrast, other studies reported that low [O2] facilitates the differentiation of mESCs by impairing
LIF signaling [34]. In addition, hypoxia enhances differentiation of hESCs towards cardiomyocytes and chondrocytes [35,36] and stimulates vascular differentiation through HIF1α-mediated repression of Oct4 and activation of vascular endothelial growth factor (Vegf) [37].
Recently, the metalloproteinase MMP1, shown to degrade the extracellular matrix (ECM) of
mESCs, came as another microenvironment component essential for maintenance of mESC
pluripotency. Indeed, it was shown that MMP1, either secreted by feeder cells or added to cell
culture medium could release the ECM-trapped ciliary neurotrophic factor (CNTF) cytokine
which then activates the JAK/STAT3 pathway, as does LIF [38,39].
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By various microarray analyses performed on pluripotent versus differentiated cells, the
core pluripotency genes were characterized by specific knockdown strategies [40,41]. In such
analysis, we defined a cluster of Pluri genes [including muscle and microspikes RAS (Mras),
Esrrb and T cell lymphoma breakpoint 1 (Tcl1)] whose expression diminishes within the first
day (d1) of LIF removal. Among these genes a particular subset, involved in cell adhesion and
cell-cell contacts, had also been characterized (including carcinoembryonic antigen-related cell
adhesion molecule 1 (Ceacam1), gap junction protein beta 3 (Gjb3), gap junction protein beta
5 (Gjb5) and junction adhesion molecule 2 (Jam2). Furthermore, a cluster of early differentiation genes, named Diff, is induced starting d1 of LIF withdrawal (including lymphoid enhancer
binding factor 1 (Lef1), carbonic anhydrase 4 (Car4) and protocadherin 1 (Pcdh1), [42]. In
addition, we identified LIF-induced (Lifind) genes, whose expression is stimulated by very
short LIF treatment after a period of LIF withdrawal of 1 or 2 days. There are two categories of
Lifind genes: those expressed after LIF induction specifically in cells depleted of LIF for 24
hours (speLifind) like Kruppel-like factor 5 (Klf5) and those induced in a more general way, in
immature as well as differentiated cells (pleioLifind) like JunB. [42–44]. Klf5, a transcription
factor of the Kruppel gene family, well conserved upon evolution, has been shown to be essential for the derivation of mESCs from Inner Cell Mass (ICM) as well as for the maintenance of
mESCs cell pluripotency in vitro [45–48]. Klf5-/- ES cells differentiates spontaneously at high
frequency and in knock-down experiments an essential effect of Klf5 to block specific mesodermal differentiation has been demonstrated. In reverse experiments it has been shown that
overexpression of Klf5 leads to LIF-independent self-renewal with high level of Tcl1 expression
and increased cell proliferation along with AKT phosphorylation and stimulation
[45,46,49,50].
By studying early steps of differentiation initiated by LIF withdrawal, we have previously
shown that cells enter commitment phases during which LIF plays differential roles. While at
d1 of LIF depletion re-addition of LIF blocks differentiation and/or reverts cells towards a
pluripotency state, at d2 and later, re-addition of LIF is less efficient in altering the differentiation process and blocking apoptosis [43,51]. To understand the molecular mechanisms
involved in the regulation of this early LIF-dependent mESC plasticity we set up an in vitro
test for studying the impact of genes and signaling pathways which could modulate these
properties in mESCs. In addition, we analysed the effects of MMP1, a new stemness player,
and of hypoxia in mESC plasticity. We discovered that Klf5 but not JunB is acting on cell
plasticity. We also show that MMP1 can replace LIF to a certain extent in the plasticity test
and that mESCs remain plastic under hypoxic conditions, despite alterations in gene expression patterns.
Materials and Methods
Cell culture
The mESC line CGR8 (from A. Smith laboratory) was grown in 6 well plates coated with 0.2%
gelatin under 7% CO2 humidified atmosphere at 37°C in Dulbecco’s Modified Eagle’s Medium,
Sigma Aldrich (DMEM-glutamax) supplemented with 10% fetal calf serum (FCS, Dutscher) or
10% KnockOut™ Serum Replacement (SR), (Gibco), 0.1 mM β-mercaptoethanol (SigmaAldrich), 400 μg/mL gentamicin (Gibco Invitrogen) in the presence of 10 ng/mL of human
LIF. Cells were passaged every 2 or 3 days by trypsinization and resuspended in complete
medium.
For experiments performed under hypoxia (3% O2), cultures were performed in cell chamber with O2 and CO2 regulators (BioSpherix) under water saturated atmosphere.
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Embryoid bodies and differentiation procedures
mESCs were grown in bacterial Petri-dishes in DMEM glutamax supplemented with 10% SR
for 8 days under normoxia or hypoxia (BioSpherix cell chamber). At d8, embryoid bodies
(EBs) were plated in gelatin-coated flasks for 7 days under normoxia or hypoxia. Beating cardiomyocytes were detected the day after plating and the neuronal cells with dense networks were
observed from 3 days after plating.
Drug treatment (LY294002)
The analysis of the effect of the PI3K inhibitor LY294002 (Cell signaling) was done by pretreating the cells with 50 μM LY294002 during 1h before treatment with LIF. Then LIF induction was performed for 1 to 4 days in the presence of the drug added for the first 24h. The efficiency and specificity of the inhibitor was verified by Western blot analysis on Phospho-AKT
(anti-Phospho-ser473 AKT from Cell signaling, data not shown).
MMP1 treatment
Cells grown in complete medium were washed with PBS and incubated in medium without
LIF in the presence of 100 ng/mL of recombinant human metalloproteinase 1 (MMP1; cat.
420–01, PeproTech). Medium was changed every day.
Small interfering RNA transfection
Cells were depleted from antibiotics 24h before transfection. 20 nM of siRNAs targeting Klf5 or
JunB (ON-TARGETplus SmartPOOL (Dharmacon), and 5 μL Lipofectamine 2000 (Invitrogen)
were incubated separately in 250 μL DMEM glutamax for 5 min at room temperature. Then,
both solutions were gently mixed and incubated for another 20 min before being added to cell
culture. As a control, the same concentration of siRNA [ON-TARGETplus Non-targeting Pool
(Dharmacon)] was used. SiRNA transfection was done on about 5.105 cells in suspension in
cell medium without antibiotics. The medium was changed 24h after transfection and medium
with LIF was added for 3 days. To determine siRNA efficiency at the beginning of the induction
process, the expression of each target gene was verified by RT-qPCR analysis after 1h of LIF
induction. The knock-down was considered to be efficient when gene expression during LIF
induction equals the level of expression in non-induced cells, which was the case for both studied genes (data not shown).
RNA isolation and quantitative real-time PCR
For RNA isolation, 800 μL of TRIzol reagent (Invitrogen) were added to the cells and RNAs
were prepared according to the manufacturer’s instructions. The reverse transcriptase (RT)/
DNase step was performed with the QuantiTect Rev Transcription kit (Qiagen). Quantitative
real time (q)-PCR was performed using the Applied Biosystems StepOneTM Real-Time PCR
System in a 25 μL reaction volume containing 5 μL of cDNA (1:20 dilution of the reverse transcribed sample), 12.5 μL of B-R SYBR1 Green SuperMix for iQ (Quanta, BioSciences) and
primers pair at a final concentration of 0.5 μM. The PCR program included a denaturation step
at 95°C for 10 min, an amplification step for 40 cycles (15 s at 95°C, 1 min at 60°C) and a final
dissociation curve step in order to determine the specificity of the product. Samples were duplicated for each run. Quantification was done by calculating the 2ΔΔCt value. Data were normalized using the Hprt mRNA, known to remain constant in the experimental condition.
Statistical significance of differential gene expression was calculated from four or more independent experiments by using IBM SPSS Statistics 21 software.
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Protein cell lysates and Western blot
Cells were lysed in mild RIPA buffer [PBS 1x, 1% Triton-X-100 (Sigma), 1% NP-40 (Sigma),
0.05% SDS] supplemented with protease inhibitor cocktail (Sigma), 1 mM Pefabloc (Sigma),
40μM NAF, 1mM Na3VO4 and centrifuged for 20 min at 15 000 g. Antibodies were diluted
1:1000 as follows: rabbit polyclonal anti-gp190 (Novus, NBP2-32070); rabbit monoclonal antiPhospho-tyr705 STAT3 (D3A7, Cell Signaling); rabbit monoclonal Anti-STAT3 (ABCAM,
Ab68153); rabbit polyclonal Anti-Oct4 (ABCAM, Ab18976); rabbit polyclonal anti-Nanog
(ABCAM, Ab80892); goat polyclonal anti-Sox2 (Santa Cruz, sc-17320); Rabbit polyclonal
Anti-Erk2 (Santa Cruz, sc-154). Primary antibody incubation was done overnight in a sealed
plastic bag at 4°C with slight agitation in the Odyssey blocking buffer (LI-COR company).
After washing with TBS 0,1% Tween (twice for 10 min), the membrane was incubated for 1 h
with the fluorescent far-red coupled secondary antibody, in accordance with the primary antibody: IRDye 680RD goat (polyclonal) anti-rabbit IgG (H+L), (LI-COR) or IRDye 800RD goat
(polyclonal) anti-mouse IgG (H+L), (LI-COR) or HRP-labeled anti-Goat (IgG (H+L) (Vector
Laboratories), diluted 1:20 000 in Odyssey blocking buffer. After washing twice for 10 min with
TBS 0.1% Tween and with TBS1x, membranes were revealed with the OdysseyFC (LI-COR)
apparatus with the Image studio software as recommended by manufacturer. Quantification of
the correct size band for each antibody was performed with the OdysseyFC (LI-COR) quantification image studio software.
Flow cytometry
mESCs were grown in the presence or absence of LIF with or without LIF re-addition as indicated, as described in the Fig 1, on a kinetic of 4 or 5 days. At the end point of the experiment,
cells were trypsinized and the centrifugated pellets were washed in PBS 1X. 100 000 cells of
each point were distributed in 96 round well plates. Cells were incubated in 50 μL of primary
anti-Ceacam1-Phycoerythrin antibody (Biolegend, BLE 134506, diluted 1/10), in PBS 1X, 0.5%
BSA, 1mM EDTA for 30 min. at 4°C in dark. Cells were then washed twice with 250 μL of PBS
1X and the resuspended pellets in PBS 1X were transferred in specific flow cytometry tubes
and processed on the Fortessa cytometer (Becton Dickinson). Non labelled and isotype labelled
cells were used as negative control. The percentage of positive cells and the MFI were obtained
with the DIVA software BD Biosciences. The overlay “half off set graph” was displayed with
the gated P1 population, with the Flow Jo software.
Results
Set up of a ‘Plasticity test’ in the mESC model
To characterize the behavior of mESCs during the first days of LIF withdrawal and to precisely
determine how cells respond to LIF during the commitment phases, we set up an in vitro assay,
hereafter referred to the ‘plasticity test’. In this test, cells were stimulated to differentiate by
simple LIF withdrawal for 1 to 4 or 5 days and then LIF was reintroduced at different time
points as depicted in Fig 1A. Analysis of gene expression by RT-qPCR was performed at each
time point of the experiment. This assay enabled us to study the effects of key genes by siRNA
depletion as depicted in Fig 1B, or signaling pathways by the use of chemical inhibitors, as
depicted in Fig 1C.
In this assay, we analyzed the expression profiles of the Master genes (Oct4, Sox2 and
Nanog) and of the newly identified Pluri (Ceacam1, Mras and Esrrb) and Diff (Lef1, Car4 and
Pcdh1) genes (Fig 2). The expression level of Master genes was not strongly regulated during
the first days of LIF withdrawal as expected [42,52]. These genes were also highly expressed
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Fig 1. General scheme of the ‘mESC Plasticity Test’. (A) mESCs maintained in pluripotency (+LIF) or
cultivated without LIF for the indicated period of time in days (d1 to d5) are induced with LIF at different time
points. (B) The effect of specific genes was tested by a siRNA strategy in which addition of siRNA was
performed the first day of LIF withdrawal. (C) PI3K inhibitor LY was added for 24 hours to the committed or
differentiated cells as indicated.
doi:10.1371/journal.pone.0146281.g001
after LIF reintroduction, up to d3 (Fig 2A). In contrast, the expression level of Pluri genes
(known to decrease after one day of LIF withdrawal [42]), was re-established by LIF treatment
at d1 but not restored or less efficiently re-established at d2 and d3 (Fig 2B). In addition, the
expression level of the examined Diff genes was also regulated: their expression was stimulated
upon LIF withdrawal and repressed upon LIF treatment at d1 but less efficiently at d2 and d3
(Fig 2C). Interestingly, the LIF effect observed after 4 days [(-LIFd1+LIFd3), Fig 2] started
shortly after LIF addition [within the first 24h, (-LIFd1+LIFd1), S1 Fig], ruling out a selection
process of LIF-resistant cells, enriched after 4 days of culture.
In addition to gene expression analysis by RT-qPCR we also analyzed endogenous protein
expression by flow cytometry with an antibody raised against the membrane-associated protein
Ceacam1 (encoded by the Ceacam1 Pluri gene). Mean of Fluorescence Intensity (MFI) analysis
of the bulk population (gated P1 cell population) revealed a global decrease of Ceacam1 expression, starting at d1 of LIF withdrawal with a continuous loss until the end of LIF kinetic starvation. Expression is regained at the -LIFd1+LIFd3 condition but not at–LIFd2+LIFd2, which
follows the RNA profile (Fig 2D). This experiment indicates that, upon LIF withdrawal, there
are no detectable resistant cells expressing higher level of Ceacam1 protein which could have
remained and should be observed as a double peak in such analysis.
The Klf5 gene is involved in the regulation of the plasticity window
We previously identified two groups of genes induced by LIF (speLifind and pleioLifind genes)
and postulated that some of the speLifind genes, induced by LIF preferentially at d1 but less at
d2 after LIF withdrawal, could be involved in such reversion process [43]. As a proof of principle, by using siRNA strategy, we analyzed the impact of Klf5 (speLifind) and JunB (pleioLifind)
knockdown on cell plasticity as described in Fig 1B. The down-regulation of Klf5 during LIF
induction led to specific repression of the Pluri genes and induction of early Diff markers like
Lef1, Nestin and Brachyury (Brach) (Fig 3A). In contrast, no effect on Pluri or Diff gene
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Fig 2. LIF has differential effects on mESC-derived cells, after d1, d2 or d3 of LIF starvation. Cells were depleted from LIF or induced with LIF after a
period of LIF depletion as indicated. After 4 days, expression of the selected genes (A) Master genes, (B) Pluri genes and (C) Diff genes was analyzed by RT-
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qPCR with Hprt used for normalization. Graphs represent the average level of expression and standard error of the mean (SEM) bars, calculated from 4
independent experiments. +LIF condition was arbitrarily set as 1. One sample t-test was performed for each condition versus the +LIF sample: *pvalue<0.05; **p-value<0.01, ***p-value<0.001; a: p-value = 0.051; if not stated: not significant. (D) A representative experiment of flow cytometry,
performed in the indicated conditions with the Ceacam-PE antibody, is presented: "off set graph" of the MFI of the total P1 gated cell population of each cell
growth condition, with the Flo Jo software.
doi:10.1371/journal.pone.0146281.g002
expression was detected with JunB siRNA, although it efficiently repressed JunB expression
(repression was 100% in the first hours of LIF induction and was at least of 50% after 4 days, at
the end point of the experiment, (data not shown and Fig 3B). These results indicate that Klf5
is involved in the LIF response at d1 while JunB does not regulate this process.
PI3K does not regulate early commitment but it acts later for cell fate
choices
LIF induces the PI3K pathway allowing the maintenance of mESCs pluripotency [15,16,52].
We investigated the effect of a specific PI3K inhibitor (LY294002 abbreviated LY) in the ‘plasticity test’ as described in Fig 1C. We analyzed gene expression at the end point of the kinetics
(5 days) in the presence or absence of LY, added for 24 hours in cell medium at each point of
the kinetics. PI3K inhibition had faint effects on the Sox2 master gene while displaying no
effects neither on Oct4 nor on Nanog, Pluri and early Diff genes indicating that PI3K is not
strongly involved in LIF-dependent cell plasticity (Fig 4A–4C). However, addition of the inhibitor at later time points (at d3 and d4 upon LIF withdrawal) led to changes in the expression of
Fig 3. Klf5 but not JunB disturbs the LIF-dependent plasticity in early committed cells. Cells were
depleted from LIF for 24 hours and either transfected with siControl (siCtrl) (A) siKlf5, or (B) siJunB and then
induced with LIF for three days (d3). Graphs represent the average level of expression and SEM from 3 to 6
independent experiments. Paired sample t-test was performed to determine the significance of the difference
in the expression levels observed with the non specific (siCtrl) versus specific siRNA: *p-value<0.05; **pvalue<0.01, ***p-value<0.001; if not stated: not significant.
doi:10.1371/journal.pone.0146281.g003
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Fig 4. PI3K does not regulate the LIF-dependent plasticity window but stimulates differentiation towards early mesoderm lineage while inhibiting
ectoderm and endoderm differentiation processes. Cells were depleted from LIF or induced with LIF in the presence of LY, for the first 24 hours as
described in Fig 1C. After 5 days, expression of the selected genes (A) Master genes, (B) Pluri genes, (C) early Diff genes and (D) specific germ layer genes,
was analyzed by RT-qPCR. Graphs represent the average level of expression and SEM from 4 independent experiments. Paired sample t-test was
performed to evaluate the significance of the difference in the expression levels observed with or without LY: *p-value<0.05; **p-value<0.01, ***pvalue<0.001; a: pvalue = 0.051; if not stated: not significant.
doi:10.1371/journal.pone.0146281.g004
some early lineage markers: while expression of Nestin and to less extend of Ncam1 (ectoderm
marker) and of Gata4 (endoderm marker) was induced, the expression of Brachyury (early
mesoderm marker) but not of Kdr1 (late mesoderm marker) was strongly repressed. Thus,
active PI3K seems to repress ectoderm and endoderm while inducing mesoderm during early
onset of differentiation (Fig 4D).
Slight variations in cell responses in Serum versus SR-containing
medium
Since the behavior of mESCs can fluctuate depending on serum batches which have various
effects on cell proliferation and pluripotency ([53] and our unpublished results), we also performed the ‘plasticity test’ in medium supplemented with the serum replacement (SR) compound instead of foetal calf serum. While similar results were found for Pluri gene expression
(like Ceacam1, Mras and Esrrb), we observed that the plasticity window was slightly delayed
with an effect of LIF extended up to d2 for Diff genes (S2 Fig). To reduce serum-side effects in
our experiments, we performed all the following assays in medium supplemented with 10% SR
rather than in medium containing serum.
MMP1 is a novel stemness factor that regulates the plasticity window
We analyzed the impact of the MMP1 metalloproteinase, recently shown to be secreted by
feeder cells and to degrade the extracellular matrix (ECM) of mESCs leading to the release of
the ECM-trapped ciliary neurotrophic factor (CNTF) cytokine [39]. This LIF family member
displays similar effects to LIF on mESCs, and maintains mESC pluripotency by inducing the
JAK1/ STAT3 pathway [39]. In our assay, we replaced LIF by MMP1 and observed cells
remaining pluripotent for up to 11 days, as previously shown ([39] and our data not shown).
In the ‘plasticity test’, MMP1 mimicked the LIF effect, however, with a less potent effect on the
expression of Diff genes (Fig 5). Nevertheless, this experiment showed that MMP1-dependent
remodeling of the extracellular matrix is involved in cell plasticity.
Hypoxia maintains pluripotency and plasticity in vitro despite gene
expression changes versus normoxia
We also analyzed the potential plasticity of mESCs grown under low O2 (3%), which corresponds to physioxic (physiologic in vivo) conditions of pre-implantation embryos [7,54–57].
Cells were incubated at 3% O2 in a dedicated hypoxic cell chamber and incubator with cell
medium changed in the hypoxia chamber without normoxic shock during the entire experiment. Under continuous 3% O2, protein expression levels of the LIF receptor subunit (gp190)
and of Phospho-tyr705-STAT3 were lower than under normoxia indicating an impairment of
LIF signaling in these conditions as previously shown (Fig 6A, S3 Fig and [34]). In addition, we
showed that the expression levels of Oct4, Sox2 and Nanog proteins were decreased upon hypoxia (Fig 6A and S3 Fig). In addition, protein expression levels and STAT3 phosphorylation
decrease upon LIF withdrawal under normoxia (unless for Oct4), as expected, but also under
hypoxia. This attests that LIF signalling is activated, even if weaker under hypoxia. Hypoxia
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Fig 5. MMP1 metalloproteinase partly mimics the LIF effect. Cells were depleted from LIF for the indicated time and induced with 100 ng/mL of purified
MMP1 as indicated. After 4 days, expression of the selected genes (A) Master genes, (B) Pluri genes and (C) Diff genes was analyzed by RT-qPCR. Graphs
represent the average level of expression and SEM as depicted in Fig 2. One sample t-test was performed for each condition versus the +LIF sample: *pvalue<0.05; **p-value<0.01, ***p-value<0.001; if not stated: not significant.
doi:10.1371/journal.pone.0146281.g005
also induced the expression of Hif1α protein targets, like the BCL2/adenovirus E1B interacting
protein 3 (Bnip3) and glucose transporter 1 (Glut1) genes, attesting that the Hif1α protein was
stabilized and active (Fig 6B). However, despite delayed cell growth compared to 20% O2, as
previously observed [34], cells in 3% O2 remained pluripotent as shown by morphological criteria (alkaline phosphatase staining, S4 Fig) and by their capacity to form embryoid bodies
(EBs) and to differentiate into neurons and beating cardiomyocytes (data not shown). Analysis
of expression levels of the three germ layer markers in our differentiation procedure (eight
day-formed EBs followed by seven days of plated EBs) showed their efficient expression in
both normoxia and hypoxia (Fig 6C). In addition, specific markers [neuronal like tubulin β3
(Tubb3/Tuj1) and microtubule associated protein 2 (Map2), glial like glial fibrillary acidic
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Plasticity Control in mESCs
Fig 6. mESCs grown under 3% O2 have an impaired LIF signaling, express less ‘pluripotency-involved’ proteins but maintain pluripotency. Cells
were grown with (+LIF) or without LIF for 1 to 3 or 4 days under normoxia or hypoxia as indicated. (A) Protein cell lysates (60 μg) from cells grown under the
indicated conditions were analyzed by Western blot. ERK2 was used as a loading control. A representative experiment is shown. Quantification of signals
obtained with the different antibodies in the + LIF condition (n = 4) is provided in S3 Fig (B) Gene expression of the indicated genes was analyzed by RTqPCR. Graphs represent the average level of expression and SEM as depicted in Fig 2 (n = 4). Paired sample t-test was performed to evaluate the
significance of the difference in the expression levels observed in normoxia versus hypoxia: *p-value<0.05; **p-value<0.01, ***p-value<0.001; if not stated:
not significant. (C) early lineage markers, (D) specific lineage markers in mESCs induced for in vitro differentiation under normoxia (20% O2, N) or hypoxia
(3% O2, H). Gene expression was analyzed by RT-qPCR in cells grown for two days under N or H conditions (+LIFd2), in EBs at day 8 of their formation (EBs
d8) and seven days after EBs plating. Y axis is in log scale.
doi:10.1371/journal.pone.0146281.g006
protein (Gfap) and cardiac like myocyte enhancer factor 2C (Mef2c)] were highly expressed
under hypoxia with comparable levels to the expression under normoxia highlighting the pluripotent state of the starting cells. Interestingly, we show that cells kept their plasticity potential
under 3% O2 conditions (Fig 7), despite lower Oct4, Nanog and Sox2 protein expression (Fig
6A and S3 Fig), lower expression of the Pluri genes (Fig 7B) and higher expression of Lef1 and
Fgf5 in 3% O2 compared to 20% O2 (Fig 7D). Collectively, these results indicate that a novel
equilibrium of gene expression and of post-translational regulation (as observed for Oct4,
Nanog and Sox2) are established under low O2 in pluripotent cells. However, the expression
levels of lineage markers (SRY (sex determining region Y)-box 17 (Sox17), Nestin, Kdr1,
Ncam1 and Afp (Fig 7D) and also Brachuyry, (data not shown) remained low in + LIF condition under 3% as well as 20% O2 concentration, emphasizing the point that cells do not
PLOS ONE | DOI:10.1371/journal.pone.0146281 January 5, 2016
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Plasticity Control in mESCs
Fig 7. Hypoxia maintains cell plasticity with a novel equilibrium of Pluri and early Diff genes. Cells grown under normoxia (N) or hypoxia (H) were
depleted from LIF or induced with LIF after a period of LIF depletion as indicated. After 4 days, expression of the selected genes, (A) Master, (B) Pluri, (C)
Pluri Adhesion and (D) Diff was analysed by RT-qPCR. Graphs represent the average level of expression and SEM as depicted in Fig 2 (n = 4). One sample
t-test was performed for the +LIF condition and paired sample t-test was performed for the other conditions to evaluate the significance of the difference in the
expression levels observed in normoxia versus hypoxia: *p-value<0.05; **p-value<0.01, ***p-value<0.001; if not stated: not significant.
doi:10.1371/journal.pone.0146281.g007
differentiate in the presence of LIF, under low O2, at least for the time in culture used here, up
to four days.
Discussion
Plasticity, which is a hallmark of stem cells, is defined in this study as the potential of committed cells to revert to a more immature state when grown in a particular environment. The characterization of regulators of this process is of major interest. For example, understanding the
mechanism of the reversion of primed ESCs to the more immature naive state, is an essential
step for future development of cellular therapies. Indeed, human ESCs or iPSCs are closer to
primed than to naive states. Primed mouse EpiSCs can be reverted to the naive state of mESCs
by exposure to LIF–STAT3 signaling, especially when cultured in the presence of signaling
pathway inhibitors (e.g. 2i) or during transient expression of kruppel-like factor 4 (Klf4) or the
myelocytomatosis oncogene (Myc) [58,59]. In Humans, the reversion of the naturally primed
hESCs to naive state also involves the LIF-/STAT3 signaling pathway [11,12,14].
Here, we developed an in vitro assay showing that mESCs are equipped with a time-dependent window of plasticity during their early differentiation steps in which LIF or MMP1 metalloproteinase are essential. We characterized a differential effect of LIF on mESC-derived cells
(either at a committed or differentiated state) and identified Klf5, a known pluripotency gene
which blocks differentiation towards mesoderm lineage [50,60], as one of the factors controlling this effect. This new property of Klf5 on modulating mESCs plasticity could be in link with
its potentiality to reprogram somatic cells, a mechanism favoured by specific miRNAs [61,62]
and to its role to govern homeostasis and oncogenesis in stem and transit-amplifying cells of
the intestinal epithelia [63].
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Plasticity Control in mESCs
Other speLifind genes, mainly induced by LIF in the reversible window of cell differentiation, could also be essential effectors of plasticity rather than the PleioLifind genes and will
deserve further investigation.
We observed serum batch-dependent variations in the plasticity window. Indeed serum
composition fluctuates from batch to batch, probably including varying amounts of BMP2/
BMP4, respectively involved in cardiomyocyte differentiation (BMP2) or maintenance of cell
pluripotency in synergy with LIF (BMP4) [52,64]. To avoid the control of foetal calf serum
(FCS) parameter for each serum batch change, we performed the plasticity test in the serum
replacement medium in which cell differentiation occurs upon LIF withdrawal with less morphological differences than in serum medium (data not shown), but in which differentiated
cells undergo apoptosis as previously shown under classical cell growth condition in FCS [65–
67]. We observed that the plasticity window was slightly delayed with an effect of LIF extended
up to d2 for some genes in the presence of SR containing medium. This is in agreement with a
previous study demonstrating that cells in G1 were more prone to differentiate than cells in
G2/M. Indeed, in this report it was shown, in a ‘LIF rescue assay’, that LIF regulates the extent
of cell cycle phases up to d2 [68].
MMP1 was recently shown to maintain cell pluripotency by releasing trapped cytokine in
the ECM. Our results demonstrated that MMP1 maintained also cell plasticity in committed
cells up to d2, probably by CNTF delivery, a process which remains to be demonstrated in our
assay. Whether ECM remodeling upon MMP1 activity could synergize with LIF during this
process, remains also to be determined.
Since the starting population of mESCs is heterogeneous [69–71], there was a possibility
that after one day of starvation, LIF addition could favor the clonal expansion of a sub-population of cells which are resistant to LIF withdrawal and remain pluripotent. However, when following protein expression encoded by one of the Pluri gene (Ceacam1) by flow cytometry
analysis, we did not detect any residual resistant population which could have been observed
by additional peak of fluorescence in LIF withdrawal kinetic, in such analysis (Fig 2D). Also,
the fact that we observed an induction of Pluri genes and a repression of Diff genes after one
day of treatment (in the -LIFd1+LIFd1 condition, as shown in S1 Fig) argues against a clonal
expansion of resistant cells. In addition, in a ‘LIF rescue assay’, it has been shown that LIF
reverts the differentiation-dependent increase of G1 phase up to d2 [57]. Moreover, the LIF
effect is lost on cells differentiated for two days or more, stressing the point that LIF displays
differential effects depending upon cell maturity and that there is a decisive step for commitment between two and three days of LIF withdrawal. Furthermore, EBs grown for more than
20 days in vitro can re-express Oct4 and recover pluripotent properties, presenting another
window of potential plasticity in the mESC model [72]. However, it has not been described
whether this late effect is LIF-dependent, which could be a possibility since LIF is expressed in
differentiated cells from day 6 and later ([42] and our unpublished results).
PI3K regulates developmental and physiologic processes during myogenesis and promotes
growth and osteogenic differentiation of rat MSCs upon mechano-growth factor (MGF) activation [73,74]. We showed that the PI3K pathway is involved in mESC-derived cell choices and
particularly in the expression of the key Brachyury early mesodermal marker, but not at early
times of LIF-dependent plasticity. Since PI3K is known to maintain mESCs pluripotency
[16,75], this result indicates that the mechanisms of pluripotency and plasticity, despite having
common regulators like Klf5, are not similar. Further characterization of this LIF-dependent
window of plasticity, by chemicals or siRNA library screenings, will require new tools as tagged
cells sorted by gain or loss of a short life fluorescent protein expressed under the control of a
LIF-dependent promoter (the Pluri genes Ceacam 1, Esrrb or early Diff gene like Lef1).
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Plasticity Control in mESCs
Classical cell growth cultures are far from being ideal, in particular because O2 is much
lower in vivo than in cell culture incubators (20%). However, studies of mESCs aiming at taking
this parameter into consideration remain controversial and the outcome of hypoxia is poorly
understood. The various O2 levels (1 to 5% O2) and mESC lines, grown with or without feeders
(D3 versus CGR8 for example) could explain differences in published reports. Also, avoiding
normoxic shock during medium changes, by using a hypoxia cell chamber, is an important
parameter to diminish technical variations in the effect of hypoxia (our unpublished observation). We showed in this study that under hypoxia, mESCs respond poorly to LIF, probably
because of low expression of the specific LIF receptor subunit, gp190, as seen both at the RNA
and protein levels. We showed that the level of STAT3 repressor, Socs3, is not changed between
normoxia and hypoxia at the RNA level (data not shown), but we cannot rule out that a stabilized Socs3 protein could be responsible for the low level of phospho-Tyr 705-STAT3 protein
detected under hypoxia. We also observed that the level of Oct4, Nanog and Sox2 proteins was
reduced under hypoxia compared to normoxia, despite similar RNA expression levels. This
indicates hypoxia-dependent translational regulation in agreement with a recent report showing variation in mRNA translation under hypoxia in the adrenal gland phaerochromocytoma
PC12 model [76]. However, we showed that cells under hypoxia maintain in vitro pluripotent
criteria. Indeed, they are able to form EBs that differentiate into the three mature germ layers
and to neurons and beating cardiomyocytes. Also, we demonstrated that LIF-dependent cell
plasticity is maintained under hypoxic conditions at d1. Whether this reversible state of cells
could be maintained under hypoxia at later times after LIF withdrawal remains to be investigated. Though, we speculate that mESCs, which have been derived under 20% O2, adapted
their potential to remain pluripotent and plastic by expressing Master and Pluri genes at higher
levels than needed under physioxic conditions (3% O2). It will be of great interest to perform
exhaustive transcriptomic and proteomic analyses in fresh mESCs directly derived under low
O2 and to compare them with data so far obtained under 20% O2. This might lead to characterization of new stemness factors that could operate in hypoxic niches in embryos.
In conclusion, we set up an in vitro test allowing to characterize the impact of cytokines
(like LIF in this study), of genes (by siRNA strategy), of signalling pathways (by use of chemicals) and of various environmental parameters on mESCs plasticity. We unveiled a new property of Klf5 to regulate this LIF-dependent effect which is not under strict control of the PI3K
pathway. We also show that MMP1 can replace LIF to a certain extent in the plasticity test and
that mESCs remain plastic under hypoxic conditions, despite alterations in gene expression
patterns.
Supporting Information
S1 Fig. Gene modulation starts within the first 24h of LIF addition. Cells were depleted
from LIF or induced with LIF after a period of LIF depletion as indicated. After two days, RNA
was extracted and expression of the selected genes was analyzed by RT-qPCR with Hprt used
for normalization. Mean and SEM (Standard Error of Mean) bars were calculated from 4 independent experiments. +LIF condition was arbitrarily set as 1.
(TIF)
S2 Fig. Experiments in serum replacement versus FCS-containing medium slightly modulates the plasticity window. Cells were depleted from LIF or induced with LIF after a period of
LIF depletion as indicated. After 4 days, expression of the selected genes (A) Master genes, (B)
Pluri genes and (C) Diff genes was analyzed. Graphs represent the average level of expression
and SEM as depicted in Fig 2. One sample t-test was performed for each condition versus the
PLOS ONE | DOI:10.1371/journal.pone.0146281 January 5, 2016
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Plasticity Control in mESCs
+LIF sample: p-value<0.05; p-value<0.01, p-value<0.001; if not stated: not significant.
(TIF)
S3 Fig. Quantification of Western blot experiments. Graph represents the mean of ratio of
normoxia versus hypoxia signals obtained in the +LIF condition for each antibody, as indicated, with normalization performed with the ERK2 protein as a loading control. n = 4. Quantification was performed with the Odyssey FC (LI-COR) quantification Image Studio software.
(TIF)
S4 Fig. mESCs maintain alkaline phosphatase activity and mESC-like morphology under
hypoxia. Pictures of mESCs grown with LIF under normoxia or hypoxia for four days and
stained with the Alkaline phosphatase kit (Sigma-Aldrich, 86R-1KT). Scale bar is 100 μM.
(TIF)
S5 Fig. List of primers used for RT-qPCR.
(DOCX)
Acknowledgments
We thank all members of the “Pluripotency and early steps of differentiation” team at CNRSUMR-5164-CIRID for their helpful insights and discussions and in particular A. Villacreces, V.
Praloran and Z. Ivanovic for good advices and help with the experiments performed under
hypoxia conditions. We thank X. Gauthereau for good advice and contribution concerning cell
cultures and RT-qPCR experiments, V.Pitard and S. Gonzales for their good help concerning
flow cytometry experiments and S. Daburon for providing supernatant of CHO cells overexpressing human LIF. We thank Austin Smith for the gift of the CGR8 cell line and JF Moreau
and C Goding for careful reading of the manuscript.
Author Contributions
Conceived and designed the experiments: AAH HB. Performed the experiments: AAH NK
VM AD SB DZ CB. Analyzed the data: AAH NK VM AD SB DZ MM HB. Contributed
reagents/materials/analysis tools: HB. Wrote the paper: AAH NK VM HB. Aministrative and
financial support: DZ MM HB.
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