Les cellules souches embryonnaires (ESCs) sont dérivées d'embryons au stade blastocyste. Elles sont caractérisées par la capacité de se diviser et de maintenir un phénotype indifférencié et en présence de stimuli, de se différencier en cellules spécialisées dérivées des trois feuillets embryonnaires, c'est la pluripotence. Elles sont un outil puissant pour modéliser des maladies génétiques à des fins de découvertes en recherche fondamentale et aussi dans un but d’applications cliniques. Les mESCs sont maintenues pluripotentes in vitro en présence de LIF (Leukemia Inhibitor Factor), une cytokine de la famille des Interleukines 6 (IL6) présentant des effets pléiotropes en fonction du type et de la maturité cellulaire. Le retrait de LIF conduit à la différenciation hétérogène des mESCs dont une partie meurt par apoptose. Lors du retrait de LIF, les cellules entrent séquentiellement dans des phases d'engagement réversible (jusqu'à 36h après retrait du LIF) et irréversible, au cours desquelles la re-stimulation par le LIF induit des effets différents. Afin de mieux caractériser cet effet de LIF, nous avons mis au point un « test de plasticité » in vitro et avons étudié l'impact de paramètres environnementaux qui pourraient moduler cette plasticité dans les mESCs. Nous avons montré que la MMP1 (Matrix Metalloproteinase 1), qui peut remplacer le LIF dans le maintien de la pluripotence, est moins efficace pour le maintien de la plasticité cellulaire des mESCs. Nous avons aussi montré que les mESCs restent pluripotentes et plastiques à 3% d'O2, in vitro, et qu’elles se caractérisent par un nouvel équilibre d'expression des gènes et des protéines en comparaison à 20% d'O2. / Embryonic Stem Cells (ESCs) are derived from embryo at the blastocyst stage. These cells are characterized by their properties of self-renewal and pluripotency: ability to divide and maintain an undifferentiated phenotype and to differentiate into specialized cells of the three primary germ layers in the presence of stimuli. ESCs are a powerful tool to modelize genetic diseases for fundamental research and clinical applications. Mouse Embryonic Stem Cells (mESCs) are maintained pluripotent in vitro in the presence of Leukemia Inhibitory Factor (LIF), an Interleukin 6 (IL6) cytokine family member which displays pleiotropic functions, depending on both maturity and type of cells. LIF withdrawal leads to heterogeneous differentiation of mESCs and part of the differentiated cells die by apoptosis. During the kinetics of LIF withdrawal, we show that cells enter a LIF-dependent reversible (up to 36h of LIF withdrawal) and irreversible phase of differentiation in which LIF-restimulation induces differential effects. To better characterize this period and LIF-dependent processes, we settled up an in vitro « plasticity test » and investigated the impact of environmental parameters that could regulate cell plasticity in mESCs. Our results reveal that the Matrix Metalloproteinase 1 (MMP1), which can replace LIF cytokine for maintenance of mESCs pluripotency, mimics its effects in the plasticity window, but with less efficiency. In addition, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro, under 3% O2 (physioxic condition) with a new equilibrium of gene and protein expression levels compared to 20% O2.

Mouse embryonic stem cells (mESCs) can be maintained in a proliferative and undifferentiated state over many passages (self-renewal) while retaining the potential to give rise to every cell type of the organism (pluripotency). Autocrine FGF4/Erk signalling has been identified as a major stimulus for fate decisions and lineage commitment in these cells. Recent findings on serum-free culture conditions with specific inhibitors (known as 2i) demonstrate that the inhibition of this pathway reduces transcription factor heterogeneity and is vital to maintain ground state pluripotency of mESCs. We suggest a novel mathematical model to explicitly integrate FGF4/Erk signalling into an interaction network of key pluripotency factors (namely Oct4, Sox2, Nanog and Rex1). The envisaged model allows to explore whether and how proposed mechanisms and feedback regulations can account for different expression patterns in mESC cultures. We demonstrate that an FGF4/Erk-mediated negative feedback is sufficient to induce molecular heterogeneity with respect to Nanog and Rex1 expression and thus critically regulates the propensity for differentiation and the loss of pluripotency. Furthermore, we compare simulation results on the transcription factor dynamics in different self-renewing states and during differentiation with experimental data on a Rex1GFPd2 reporter cell line using flow cytometry and qRT-PCR measurements. Concluding from our results we argue that interaction between FGF4/Erk signalling and Nanog expression qualifies as a key mechanism to manipulate mESC pluripotency. In particular, we infer that ground state pluripotency under 2i is achieved by shifting stable expression pattern of Nanog from a bistable into a monostable regulation impeding stochastic state transitions. Furthermore, we derive testable predictions on altering the degree of Nanog heterogeneity and on the frequency of state transitions in LIF/serum conditions to challenge our model assumptions.

Mouse embryonic stem cells (mESCs) can be maintained in a proliferative and undifferentiated state over many passages (self-renewal) while retaining the potential to give rise to every cell type of the organism (pluripotency). Autocrine FGF4/Erk signalling has been identified as a major stimulus for fate decisions and lineage commitment in these cells. Recent findings on serum-free culture conditions with specific inhibitors (known as 2i) demonstrate that the inhibition of this pathway reduces transcription factor heterogeneity and is vital to maintain ground state pluripotency of mESCs. We suggest a novel mathematical model to explicitly integrate FGF4/Erk signalling into an interaction network of key pluripotency factors (namely Oct4, Sox2, Nanog and Rex1). The envisaged model allows to explore whether and how proposed mechanisms and feedback regulations can account for different expression patterns in mESC cultures. We demonstrate that an FGF4/Erk-mediated negative feedback is sufficient to induce molecular heterogeneity with respect to Nanog and Rex1 expression and thus critically regulates the propensity for differentiation and the loss of pluripotency. Furthermore, we compare simulation results on the transcription factor dynamics in different self-renewing states and during differentiation with experimental data on a Rex1GFPd2 reporter cell line using flow cytometry and qRT-PCR measurements. Concluding from our results we argue that interaction between FGF4/Erk signalling and Nanog expression qualifies as a key mechanism to manipulate mESC pluripotency. In particular, we infer that ground state pluripotency under 2i is achieved by shifting stable expression pattern of Nanog from a bistable into a monostable regulation impeding stochastic state transitions. Furthermore, we derive testable predictions on altering the degree of Nanog heterogeneity and on the frequency of state transitions in LIF/serum conditions to challenge our model assumptions.