Pierre Savatier

Signalling, cell cycle and pluripotency in embryonic stem cells

Pluripotent mouse embryonic stem (ES) cells can be expanded in large numbers in vitro owing to a process of symmetrical self-renewal. Self-renewal entails proliferation with a concomitant suppression of differentiation. Here we describe how the cytokine leukaemia inhibitory factor (LIF) sustains self-renewal through activation of the transcription factor STAT3, and how two other signals - extracellular-signal-related kinase (ERK) and phosphatidylinositol-3-OH kinase (PI3K) - can influence differentiation and propagation, respectively. We relate these observations to the unusual cell-cycle properties of ES cells and speculate on the role of the cell cycle in maintaining pluripotency.

Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro.

Multipotential stem cells can be selected from the bone marrow by plastic adhesion, expanded, and cultured. They are able to differentiate not only into multiple cell types, including cartilage, bone, adipose and fibrous tissues, and myelosupportive stroma, but also into mesodermal (endothelium), neuroectodermal, or endodermal (hepatocytes) lineages. Our goal was to characterize the multipotential capacities of human mesenchymal stem cells (hMSCs) and to evaluate their ability to differentiate into insulin‐secreting cells in vitro. hMSCs were obtained from healthy donors, selected by plastic adhesion, and phenotyped by fluorescence‐activated cell sorter and reverse transcription–polymerase chain reaction analysis before and after infection with adenoviruses coding for mouse IPF1, HLXB9, and FOXA2 transcription factors involved early in the endocrine developmental pathway. We found that native hMSCs have a pluripotent phenotype (OCT4 expression and high telomere length) and constitutively express NKX6‐1 at a low level but lack all other transcription factors implicated in beta‐cell differentiation. In all hMSCs, we detected mRNA of cytokeratin 18 and 19, epithelial markers present in pancreatic ductal cells, whereas proconvertase 1/3 mRNA expression was detected only in some hMSCs. Ectopic expression of IPF1, HLXB9, and FOXA2 with or without islet coculture or islet‐conditioned medium results in insulin gene expression. In conclusion, our results demonstrated that in vitro human bone marrow stem cells are able to differentiate into insulin‐expressing cells by a mechanism involving several transcription factors of the beta‐cell developmental pathway when cultured in an appropriate microenvironment.

Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells

Mouse embryonic stem (ES) cells are known to express D-type cyclins at very low levels and these levels increase dramatically during in vitro and in vivo differentiation. Here, we investigate some of the signalling pathways regulating expression of cyclin D1 and progression to S phase, the Ras/Extracellular signal-regulated protein kinase (ERK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. We demonstrate that ERK phosphorylation is fully dispensable for the regulation of cyclin D1 level and for the progression from G1 to S phase in ES cells. By contrast, PI3-kinase activity is required for both. Differentiation induced by retinoic acid results in the gain of ERK-dependent control of cyclin D1 expression and of S phase progression. Differentiation is also paralleled by an increase in PI3-kinase activity. This leads (a) to an increase in the p70 S6 kinase-dependent regulation of the steady-state level of cyclin D1, and (b) to a concomitant decrease in the GSK3β-dependent rate of cyclin D1 degradation. Altogether, these multiple pathways account for the dramatic increase in the level of cyclin D1 protein which parallels ES cell differentiation. Our studies suggest that PI3-kinase is an important regulator of the ES cell cycle and that its activity is not regulated by mitogen stimulation.

Cell cycle features of primate embryonic stem cells.

Using flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non‐phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth‐arrest in G1 after γ‐irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen‐activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent.

Novel STAT3 Target Genes Exert Distinct Roles in the Inhibition of Mesoderm and Endoderm Differentiation in Cooperation with Nanog

Leukemia inhibitory factor (LIF) activates the transcription factor signal transducer and activator of transcription 3 (STAT3), which results in the maintenance of mouse embryonic stem cells in the pluripotent state by inhibiting both mesodermal and endodermal differentiation. How the LIF/STAT3 pathway inhibits commitment to both mesoderm and endoderm lineages is presently unknown. Using a hormone‐dependent STAT3 and with microarray analysis, we identified 58 targets of STAT3 including 20 unknown genes. Functional analysis showed that 22 among the 23 STAT3 target genes analyzed contribute to the maintenance of the undifferentiated state, as evidenced by an increase in the frequency of differentiated colonies in a self‐renewal assay and a concomitant elevation of early differentiation markers upon knockdown. Fourteen of them, including Dact1, Klf4, Klf5, Rgs16, Smad7, Ccrn4l, Cnnm1, Ocln, Ier3, Pim1, Cyr61, and Sgk, were also regulated by Nanog. Analysis of lineage‐specific markers showed that the STAT3 target genes fell into three distinct categories, depending on their capacity to inhibit either mesoderm or endoderm differentiation or both. The identification of genes that harness self‐renewal and are downstream targets of both STAT3 and Nanog shed light on the mechanisms underlying functional redundancy between STAT3 and Nanog in mouse embryonic stem cells. STEM CELLS 2009;27:1760–1771

Regulation of Nanog Expression by Phosphoinositide 3-Kinase-dependent Signaling in Murine Embryonic Stem Cells

Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process.

Contrasting Effects of Basic Fibroblast Growth Factor and Neurotrophin 3 on Cell Cycle Kinetics of Mouse Cortical Stem Cells

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27kip1 and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27kip1 and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency

A short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling.

Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.

Self-renewal of murine embryonic stem cells is supported by the serine/threonine kinases Pim-1 and Pim-3.

pim‐1 and pim‐3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim‐1 and pim‐3 by the leukemia inhibitory factor (LIF)/gp130/signal transducer and activator of transcription‐3 (STAT3) pathway and the role of Pim‐1 and Pim‐3 kinases in mouse embryonic stem (ES) cell self‐renewal. Making use of ES cells expressing a granulocyte colony‐stimulating factor:gp130 chimeric receptor and a hormone‐dependent signal transducer and activator of transcription‐3 estrogen receptor (STAT3‐ERT2), we showed that expression of pim‐1 and pim‐3 was upregulated by LIF/gp130‐dependent signaling and the STAT3 transcription factor. ES cells overexpressing pim‐1 and pim‐3 had a greater capacity to self‐renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim‐1 and pim‐3 increased the rate of spontaneous differentiation in a self‐renewal assay. Knockdown of pim‐1 and pim‐3 was also detrimental to the growth of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim‐1 and Pim‐3 kinases in the control of self‐renewal of ES cells.