L'utilisation des cellules souches est une approche prometteuse pour le traitement des maladies dégénératives neuromusculaires. De nombreuses études portent actuellement sur les cellules souches embryonnaires (ES) et les cellules pluripotentes induites par reprogrammation (IPs) dont l'utilisation en médecine régénérative reste sujette à caution à cause du potentiel de ces cellules à former des tératomes. Des lors, aussi bien les ES que les IPs nécessitent une différenciation vers un type cellulaire précis. Cette différenciation peut mener à des risques supplémentaires tels que la dérive génique ou diverses sources de contamination.Le muscle squelettique adulte, avec sa grande plasticité et capacité régénératrice, contient une population de cellules souches qui est spécifique de ce compartiment tissulaire et qui a été isolée et étudiée au laboratoire. Les cellules souches du muscle squelettique adulte: skeletal Muscle-Derived Stem Cells, MDSC, repeuplent et réparent en quelques jours le muscle squelettique lésé avec une haute efficacité, même en présence des cellules satellites endogènes. (Arsic et al Exp. Cell Res. 2008). Le laboratoire d'accueil a entrepris de caractériser cette population cellulaire, en particulier par son origine histologique, de tester le potentiel de réparation tissulaire de ces cellules transplantées dans des modèles murins, et de déterminer la bio-distribution de ces cellules en vue d'utilisation thérapeutique.Mon travail de thèse s'est intéressé à cette population de cellules souches issues du muscle qui ont une propriété commune : la faible adhérence au substrat. La faible adhérence est une propriété très intéressante car en plus de définir des cellules plus proches de l'état pluripotent, cette propriété leur confère une grande capacité de migration. Ces cellules seraient donc plus facilement utilisables en médecine régénératrice. Dans cette perspective il est intéressant de disposer de cellules souches multipotentes qui pourrait se comporter comme des cellules pluripotentes en terme de capacité régénératrice, mais sans les inconvénients de ces dernières à savoir ; risque tératogène et prolifération incontrôlée, et manipulation des cultures cellulaires longues et couteuses.Au début de ma thèse je me suis donc intéressée aux différentes populations de cellules présentes dans le muscle et je me suis concentrée sur différents marqueurs connus chez les cellules souches, dont la présence a été établie chez différentes cellules souches y compris chez les cellules souches dérivées du muscle squelettique, mais pas clairement identifiés d'un point de vue histologique. Les cellules souches du muscle expriment le facteur de pluripotence Sox2, mais aussi des marqueurs d'immaturité tels que BCRP1/ABCG2, Sca-1 et SSEA1. J'ai examiné leur potentiel de différenciation in vitro en plusieurs lineages tels que des cellules cardiaques spécifiques (dites pacemakers), des cellules productrices d'insuline et des cellules qui présentent des marqueurs neuronaux. Je me suis également concentrée sur les possibles applications thérapeutiques grâce à l'utilisation de modèles génétiques murins et notamment dans les cas de problèmes du rythme cardiaque, et du diabète insulinodépendant. Pour ces études in vivo du potentiel réparateur des MDSC on procède à une simple injection des cellules souches dérivées du muscle squelettique (MDSC). Le fait de retrouver des MDSC injectées dans les organes cibles des souris modèles pose aussi la question de la biodistribution de ces cellules dans l'organisme. J'ai donc consacré plus d'un an de mon financement doctoral pour examiner cette biodistribution et montré un recrutement ciblé dès 48h après injection, vers les organes ou tissus lésés. / The use of stem cells is a promising approach for the treatment of neuromuscular degenerative diseases. Many studies currently focus on embryonic stem cells (ES) and induced pluripotent stem cells (IPs) for use in regenerative medicine. But some problems remain for their use in cell therapy in particular the potential of these cells to form teratomas. This problem requires both ES and IPs to be differentiated towards a specific cell type. Such induction of differentiation can lead to additional risks such as genetic drift or various sources of contamination.The adult skeletal muscle, has a high plasticity and regenerative capacity, it contains a stem cell population that is specific for muscle, and has been isolated and studied in the laboratory. Adult skeletal Muscle-Derived Stem Cells, MDSC repopulate and repair damaged skeletal muscle with high efficiency in a few days, even in the presence of endogenous satellite cells. (Arsic et al Exp. Cell Res. 2008). The host laboratory is characterizing this cell population and its histological identity and testing the tissue repair potential of transplanted MDSC in mouse models, as well as their bio-distribution for therapeutic use.My thesis work addressed the study of this stem cells population isolated from skeletal muscle showing low adhesion to substrate. Poor/low adherence is an interesting property because in addition to be defined as closer to the pluripotent state, this property is associated with a higher migration capability. This population of muscle stem cells should be easier to use than pre-differentiated stem cells in regenerative medicine. In this perspective it is interesting to use multipotent stem cells that are close to pluripotent cells in terms of differentiation and regenerative capacity, but without the inconveniencies like teratogenic risk and uncontrolled proliferation, as well as expensive and time-consuming cell culture.At the beginning of my thesis I was interested by the different populations of cells present in muscle and I focused my work on known markers of stem cells, whose presence has been established in skeletal muscle, but not clearly identified histologically. Muscle stem cells expressed the pluripotency factor Sox2, but also markers, such as BCRP1/ABCG2, Sca-1 and SSEA1. I have examined the potential of MDSC to differentiate in vitro into several cell types such as cardiac pacemaker-like cells, insulin-producing cells and cells that exhibit neuronal markers. I also focused on the possible therapeutic applications of MDSC, particularly in the case of heart rhythm problems and in the case of insulin-dependent diabetes. For these in vivo studies of the repair potential of MDSC, a single systemic injection is carried out in mouse models of the diseases. The histological recovery of injected MDSC into target organs also raises the question of the biodistribution of MDSC in the body. Therefore I spent more than a year of my doctoral thesis to address this issue and showed a targeted recruitment of MDSC to injured tissue or organs within 48h of their systemic injection.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

One of the major drawbacks of using stem cell therapy to treat muscular dystrophies is the challenge of isolating sufficient numbers of suitable precursor cells for transplantation. As such, a deeper understanding of the molecular mechanisms involved during muscle development, which would increase the proportion of embryonic stem cells that can differentiate into skeletal myocytes, is essential. In conditional SOX7-/- mice, we observed that the loss of SOX7 in satellite cells resulted in poor differentiation and fusion. In vivo, we observed fewer Pax7+ satellite cells in the mice lacking SOX7 as well as smaller muscle fibers. RT-qPCR data also revealed that Pax7, MRF and MHC3 transcript levels were down-regulated in SOX7 knockdown mice. Surprisingly, when SOX7 was over-expressed in embryonic stem cells, we found that there was a defect in making muscle precursor cells, specifically a failure to activate Pax7 expression. Taken together, these results suggest that SOX7 expression is required for the proper regulation of skeletal myogenesis.

Adult skeletal muscles have remarkable regenerative capacity. Muscle regeneration occurs when muscle tissue experiences injury, causing a population of normally quiescent muscle-resident stem cells, called satellite cells, to become activated. The CCAAT/enhancer binding proteins known as C/EBPs are transcription factors belonging to the bZIP family. Previous work from our lab has identified C/EBPβ as an important negative regulator of myogenesis. C/EBPβ expression is localized to muscle satellite cells and is downregulated upon induction to differentiate, mirroring the loss of Pax7 expression in early myogenesis. C/EBPβ expression also negatively regulates MyoD protein expression. Leaky ribosomal scanning of the Cebpb mRNA produces three C/EBPβ isoforms: LAP*, LAP and LIP, though the individual role of each of these isoforms has not been investigated in myoblasts. This thesis focuses on determining the role of each of the C/EBPβ isoforms during skeletal muscle differentiation. Forced expression of the C/EBPβ-LIP isoform in myoblasts led to a decrease in Myf5, MyoD, and myogenin expression under differentiation conditions when compared to empty vector controls. Further, the fusion of cells was greatly reduced following differentiation. C/EBPβ-LIP expressing cells also demonstrated a growth defect, with pronounced G1 arrest and features of senescence. In contrast, myoblasts expressing the C/EBPβ-LAP isoform has impaired differentiation, though this was not as pronounced as in C/EBPβ-LIP expressing cells and proliferated normally. While LIP is not normally expressed in primary myoblasts from healthy muscle, the ratio of LIP:LAP was increased in primary myoblasts isolated from mdx mice, an animal model for Duchenne muscular dystrophy. These findings suggest that the regulation of C/EBPβ isoform expression could regulate stem cell stamina and may contribute to defects in muscle regeneration in disease.

Comment les signalisations dynamiques et les mouvements morphogénétiques régionalisent et permettent la formation de tissus complexes durant l'embryogenèse est très peu compris. J’ai caractérise au cours de ma thèse, les évènements signalisants qui sont mis en place au cours de la myogenèse précoce chez l'embryon de poulet. J'ai montre que les progénitures musculaires présents dans les somites requièrent l'activation dynamique des voies de signalisation Wnt et Notch. L’activation transitoire de la signalisation Notch est requise pour adopter un destin myogénique. Le ligand de Notch Dll1 est exprime de manière mosaïque dans les cellules migrantes des crêtes neurales qui passent près du somite. Gain et perte de fonction de Dll1 dans les crêtes neurales modifient la signalisation Notch dans les somites, résultant en un délai ou une prématuré myogenèse. Nos résultats indiquent que les crêtes neural régulent la formation précoce du muscle par un mécanisme unique mené par la migration des cellules des crêtes neurales exprimant Dll1 qui déclenche l'activation transitoire de la signalisation Notch dans certains progénitures musculaires sélectionnes. Cette dynamique signalisation garantie une différentiation progressive du pool de progénitures musculaires. / How dynamic signalling and extensive tissue rearrangements interplay to generate complex patterns and shapes during embryogenesis is poorly understood. During my PhD, I have characterized the signalling events taking place during early morphogenesis of chick skeletal muscles. I observed that muscle progenitors present in somites require dynamic activation of Wnt and Notch signalling. I showed that a transient activation of NOTCH signalling is required to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. These results suggest that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.

Activin B, a member of the transforming growth factor-β superfamily, is ubiquitously expressed in diverse tissues and is a regulator of reproduction, embryonic development, and adult tissue homeostasis. We aimed to determine whether activin B is involved in skeletal muscle injury and if selective inhibition of activin B would provide a regenerative benefit. The local introduction of activin B into normal skeletal muscle increased the expression of inflammatory and muscle atrophy genes TWEAK, TNFα, GDF3 and TRIM63, by 2-, 10-, 10-, and 4-fold, respectively. The data indicate a sensitive response of skeletal muscle to activin B. Six hours after cardiotoxin-induced skeletal muscle damage, circulating activin B protein expression in serum increased by 9-fold and InhβB gene expression increased by 30-fold in muscle. After cardiotoxin-induced skeletal muscle damage, activin B protein expression in muscle was significantly increased at 48- and 120-hours by 1.5 and 2-fold, respectively. Muscle histopathological features showed that activin B antibody–treated mice displayed a reduction in necrotic debris, with a concomitant reduction in intramyocellular space at 9-days after injury. Activin B treated C2C12 myoblasts also displayed a dose-dependent reduction in active myogenesis. Furthermore, the increased presence of activin B early in muscle injury impedes muscle repair and remodeling. In summary, acute muscle injury leads to increases in activin B levels and when selectively neutralized with a monoclonal antibody, there is augmented skeletal muscle repair.

No description available.

Skeletal muscle associated disorders are correlated with significant morbidity, including frailty, fatigue, reduced mobility and poor resistance to treatments as well as mental health repercussions resulting from a loss of independence. Thus, conditions affecting skeletal muscle put considerable pressure on the health care system. In response to injury, skeletal muscle can regenerate and the molecular mechanisms underlying this unique process has been the subject of intense research with the goal of developing better treatment modalities for muscle-related diseases. Our laboratory has previously demonstrated that C/EBPβ is a negative regulator of postnatal myogenic differentiation. Expressed in muscle satellite cells (MuSCs), the primary source of regenerative potential in skeletal muscle, C/EBPβ inhibits entry into the myogenic differentiation program and is required for MuSC self-renewal after injury. Despite the important role of C/EBPβ in muscle homeostasis, little is known about the genes it regulates. To better understand how C/EBPβ regulates these processes, I used both a candidate-based approach to identify the inhibitor of DNA binding and differentiation protein ID3 as a C/EBPβ target gene that mediates inhibition of myogenic differentiation, and an unbiased approach using RNA-seq. I compared gene expression profiles from C2C12 myoblasts overexpressing C/EBPβ to control cells under growth and differentiation conditions. I observed that more than 20% of the molecular signature found in quiescent MuSCs is regulated by C/EBPβ. Caveolin- 1 was implicated as a direct target of C/EBPβ and part of the molecular mechanism by which C/EBPβ maintains MuSCs quiescence. Interestingly, the RNA-seq data identified numerous C/EBPβ-regulated secreted proteins including growth factors and cytokines. Co-culture experiments indicate that secreted proteins mediate the inhibition of cell differentiation and fusion, suggesting that C/EBPβ functions in an autocrine and paracrine fashion to influence activation of myoblasts in the absence of cell-to-cell contact. Given the role of C/EBPβ in regulating secretory proteins that inhibit myogenic differentiation, I examined the requirement of C/EBPβ in the expression of anti myogenic proteins secreted by cancer cells that affect MuSCs function and drive the development of cancer cachexia. Indeed, the expression of C/EBPβ in cancer cells was found to be required for the production of a cachexia-inducing secretome by tumours in vitro and in vivo. Furthermore, C/EBPβ was found to be sufficient to convert non-cachectic tumours into cachexia-inducing ones. In comparing differentially expressed C/EBPβ-regulated secreted protein transcripts from our RNA-seq data to that from 27 different types of human cancers revealed an ~18% similarity between C/EBPβ-regulated secreted proteins and those enriched in cachectic tumours including pancreatic, gastric and brain cancers. Three of these C/EBPβ-regulated secreted proteins (SERPINF1, TNFRSF11B and CD93) were tested further and found to be inducers of muscle atrophy. This work provides molecular insight into the role of C/EBPβ in the regulation of MuSC function and the regulation of cachexia-inducing factors by tumours, placing C/EBPβ as a novel therapeutic target for the treatment of cancer cachexia and other muscle-related diseases.

No description available.

Miniswine underwent procedures to evaluate treating chronic ischemia with the implantation of autologous satellite cells and laser transmyocardial revascularization (TMR). The objective was to combine two therapies to restore cardiac function. This experiment involved three surgical procedures: (1) placing a constrictor on the coronary artery; (2) producing channels and implanting cells; (3) obtaining samples. The swine were divided into groups: Group 1, Ischemia; Group 2, Ischemia + Laser TMR; Group 3, Ischemia + Laser TMR+ Cells; Group 4, Ischemia + Cells. Sonomicrometry and Millar pressure transducers were used to determine contractility, left ventricular pressure, and pressure-volume loops. There were no significant differences (p<0.05)among the hemodynamic data except for Group 4, which produced significantly lower output values. Morphological evaluation revealed a significantly reduced scar area in Group 3. Although there was a significant difference in scar area, the phenomena behind this improvement as compared to the unimproved hemodynamic function is not understood.