A l’état basal, les cellules souches musculaires sont quiescentes. Après blessure, ces cellules s’activent, s’amplifient et se différencient afin de réparer les myofibres lésées. Cependant, une petite population de ces cellules myogéniques activées ne va pas entrer dans la voie de la myogenèse, mais va retourner en quiescence par un phénomène appelé auto-renouvellement. Cette étape est cruciale afin de maintenir une réserve de cellules souches musculaires tout au long de la vie. Mais, les mécanismes cellulaires et moléculaires régulant ce phénomène sont peu décrits dans la littérature. La régénération musculaire est composée d’une série d’évènements complexes et bien orchestrés selon une cinétique précise. Le challenge de son étude est donc de pouvoir distinguer les évènements les uns des autres, tout en sachant qu’ils sont interconnectés. Bien que les cellules souches musculaires aient un fort potentiel de régénération, elles ont besoin d’interagir avec d’autres cellules au cours de la régénération, notamment avec les macrophages qui ont un rôle prépondérant dans ce processus. Après une blessure, les monocytes circulants sont recrutés sur le site de lésion et se différencient en macrophages inflammatoires. Ensuite, ces macrophages changent leur statut inflammatoire et acquièrent un profil anti-inflammatoire. Plusieurs études in vitro ont suggéré un rôle pour le métabolisme et son régulateur principal, la kinase activée par l’AMP (AMPK), dans la résolution de l’inflammation et dans le devenir des cellules souches adultes. Ainsi, j’ai étudié l’influence extrinsèque (via les macrophages) et intrinsèque du métabolisme sur le devenir des cellules souches musculaires au cours de la régénération. Pour cela, j’ai utilisé divers modèles déficients pour l’AMPK1 dans le macrophage, dans la cellule souche musculaire et dans la myofibre qui m’ont permis d’établir des cultures primaires de macrophages et de cellules musculaires. Dans un premier temps, grâce à ces outils, nous avons pu démontrer le rôle primordial de l’AMPK dans la résolution de l’inflammation au cours de la régénération musculaire et dans l’acquisition des fonctions anti-inflammatoires des macrophages. Dans ce contexte, l’activation de l’AMPK est dépendante de la kinase CAMKK et régule la phagocytose, principal phénomène cellulaire permettant le changement de statut inflammatoire des macrophages. Ce travail a été publié en 2013 dans le journal Cell Metabolism. Ensuite, j’ai mis en évidence un lien entre le métabolisme et le devenir des cellules souches musculaires. La suppression de l’AMPK dans les cellules souches musculaires augmente leur auto-renouvellement. Cette modification du devenir des cellules souches est due à un changement de métabolisme similaire à l’effet Warburg observé dans les cellules souches cancéreuses, qui s’effectue via la modulation de l’activité de l’enzyme Lactate Déshydrogénase, enzyme clé de la glycolyse. En conclusion, j’ai pu mettre en évidence deux nouveaux rôles de l’AMPK dans le devenir des cellules souches musculaires, établissant un lien de causalité entre métabolisme, inflammation et devenir des cellules souches. / During skeletal muscle regeneration, muscle stem cells activate and recapitulate the myogenic program to repair the damaged myofibers. A subset of these cells does not enter into the myogenesis program but self-renews to return into quiescence for further needs. Control of muscle stem cell fate choice is crucial to maintain homeostasis but molecular and cellular mechanisms controlling this step are poorly understood. A difficulty of understanding muscle stem cell self-renewal is that skeletal muscle regeneration is a coordinated and non-synchronized process. Various and dissociated molecular and cellular mechanisms regulate muscle stem cell fate. Indeed, skeletal muscle regeneration requires the interaction between myogenic cells and other cell types, among which the macrophages. Macrophages infiltrate the muscle and adopt distinct and sequential phenotypes. They act on the sequential phases of muscle regeneration and resolving the inflammation by skewing their inflammatory profile to an anti-inflammatory state. Some in vitro studies suggested a role for the metabolism and the AMP-activated protein Kinase (AMPK), the master metabolic regulator of cells, in both inflammation and stem cell fate. Thus, I investigated the role of metabolism on muscle stem cell fate within the muscle stem cells (cell autonomous regulations) and through the action of macrophages (non-cell autonomous regulations) during skeletal muscle regeneration. To analyze muscle stem cell fate, I used in vitro (macrophages and muscle stem cell primary cultures), ex vivo (isolated myofibers) and in vivo (using specific mice model deleted specifically for AMPK1 in the myeloid lineage, in muscle stem cells or in myofibers) experiments. First, I highlighted that macrophagic AMPK1is required for the resolution of inflammation during skeletal muscle regeneration and for the trophic functions of macrophages on muscle stem cell fate. Moreover, CAMKK-AMPK1 activation regulates phagocytosis, which is the main cellular mechanism inducing macrophage skewing. This work was published in 2013 in Cell Metabolism. Second, I demonstrated that depletion of myogenic AMPK1 tailors muscle stem cell metabolism in a LKB1 independent manner, orients their fate to the self-renewal by promoting metabolic switch from an oxidative to a glycolytic metabolism pathway, through the over activation of a new molecular target, which is a key enzyme for glycolysis: the Lactate Dehydrogenase. To conclude, during my thesis, I established two new crucial roles of AMPK1 in muscle stem cell fate choice, linking for the first time metabolism, inflammation and fate choice.

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

During aging there is a decline in (MuSCs) and muscle regeneration, though the underlying reason is unknown. Interestingly, mitochondrial fragmentation is a common feature in aging, however, how this impacts MuSC function and maintenance has not been investigated. To address the effect of mitochondrial fragmentation in MuSCs, we generated a knockout mouse model using the Pax7CreERT2 inducible system to target deletion of the mitochondrial fusion protein Opa1 specifically within MuSCs (Opa1-KO). Analysis of MuSC function following muscle injury revealed a defect in the regenerative potential of Opa1-KO MuSCs. Moreover, following injury there was a substantial decrease in the number of MuSC in Opa1-KO animals with a concomitant increase in the number of committing cells, illustrating that loss of Opa1 drives MuSC towards commitment at the expense of self-renewal. Furthermore, loss of Opa1 in MuSCs alters the quiescence state, priming MuSCs for activation, as indicated by a reduction in quiescence-related genes, increased EdU incorporation, and enhanced cell cycle kinetics. To address the impact of mitochondrial dysfunction on muscle stem cell capacity, we generated a model of chronic Opa1 loss. Analysis of muscle stem cell function 3 months after Opa1 ablation revealed mitochondrial dysfunction and a defect in proliferation upon activation, leading to failed muscle regeneration. These data are the first to demonstrate a novel role for mitochondrial structure in the regulation of MuSC maintenance and regenerative capacity.

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.