Rozprawy doktorskie: „Muscle stem cell”

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Woodhouse, Samuel. "The role of Ezh2 in adult muscle stem cell fate." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610201.

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Theret, Marine. "Cell and non-cell autonomous regulations of metabolism on muscle stem cell fate and skeletal muscle homeostasis." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB120/document.

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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

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Wang, Yu Xin. "Molecular Regulation of Muscle Stem Cell Self-Renewal." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35207.

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Muscle stem cells self-renew to maintain the long-term capacity for skeletal muscles to regenerate. However, the homeostatic regulation of muscle stem cell self-renewal is poorly understood. By utilizing high-throughput screening and transcriptomic approaches, we identify the critical function of dystrophin, the epidermal growth factor receptor (EGFR), and fibronectin in the establishment of cell polarity and in determining symmetric and asymmetric modes of muscle stem cell self-renewal. These findings reveal an orchestrated network of paracrine signaling that regulate muscle stem cell homeostasis during regeneration and have profound implications for the pathogenesis and development of therapies for Duchenne muscular dystrophy.

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Victor, Pedro Sousa. "Skeletal muscle aging: stem cell function and tissue homeostasis." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/81933.

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Muscle aging, in particular, is characterized by the reduction of tissue mass and function, which are particularly prominent in geriatric individuals undergoing sarcopenia. The age-associated muscle wasting is also associated with a decline in regenerative ability and a reduction in resident muscle stem cell (satellite cell) number and function. Although sarcopenia is one of the major contributors to the general loss of physiological function, the mechanisms involved in age-related loss of muscle homeostasis and satellite cell activity are yet poorly understood. Using a microarray-based transcriptome analysis of muscle stem cells isolated from young and physiologically aged/geriatric mice, we uncovered specific changes in the gene expression profile that highlighted key biological processes and potential molecular markers associated with satellite cell aging, which included p16INK4a. We used Bmi1-deficient mice to further explore the implications of p16INK4a up-regulation in satellite cell function. We found premature p16INK4a up-regulation in young/adult Bmi1-deficient satellite cells correlating with defects in satellite cell number, proliferation and self-renewal capacity. In addition we have identified a number of overlapping biological processes dysregulated in physiologically aged and Bmi1-deficient satellite cells, suggesting that Bmi1-dependent epigenetic regulation may underlie many of the intrinsic changes taking place in chronologically aged satellite cells. In addition, we show that Bmi1 loss causes defects of late postnatal/adult muscle growth characterized by reduced muscle mass with smaller muscle fibers, typical of atrophying senescent/sarcopenic muscle. Since p16INK4a expression is specifically up-regulated in muscle satellite cells of geriatric, sarcopenic mice and in a mouse model of accelerated senescence/sarcopenia (SAMP8), we propose that the Bmi1/p16INK4a axis might be particularly operative in muscle stem cells from the elderly. Muscle wasting is one of the physiological consequences of sarcopenia and the identification of novel factors regulating muscle growth and atrophy is of potential relevance for therapeutical applications. We have uncovered a new role for Sestrins as skeletal muscle growth promoting factors in the adult. We found Sestrins expression regulated in mouse models of skeletal muscle atrophy and hypertrophy and in human myopathies. Through a gain of function approach we show that Sestrins induce skeletal muscle growth, by activating the IGF1/PI3K/AKT pathway. El envejecimiento del tejido muscular está caracterizado concretamente por una reducción global de la masa muscular y un empeoramiento de la función de tejido, particularmente prominentes en individuos muy viejos (geriátricos) que padecen sarcopenia. La pérdida muscular asociado a la edad, se acompaña de una reducción en la capacidad de regeneración del músculo y en una reducción del número y la función de las células madre residentes en el músculo (células satélite). Aunque la sarcopenia sea una de las causas principales de la pérdida general de función fisiológica del músculo, los mecanismos implicados en la reducción de la homeostasis muscular y de actividad de las células satélite no han sido completamente caracterizados. Mediante el análisis comparativo del transcriptoma de células madre musculares aisladas de ratones jóvenes y de ratones viejos (geriátricos), hemos encontrado cambios específicos en su perfil de expresión génica que apuntan a los procesos biológicos dominantes y a los marcadores moleculares potencialmente asociados con el envejecimiento de las células satélite, entre los que destaca p16INK4a. Por ello, hemos utilizado ratones deficientes en Bmi1 para explorar más profundamente las implicaciones de la sobreexpresión de p16INK4a en la función de las células satélite. Hemos encontrado que células satélite jóvenes del ratón Bmi1-/- presentan sobrexpresión de p16INK4a, que correlacionan con una reducción en el número de la células, y en su capacidad de proliferación y autorenovación. Además hemos identificado un grupo de procesos biológicos comunes entre las células satélite viejas y las deficientes en Bmi1, sugiriendo que la regulación epigenética mediada por Bmi1 puede ser la base de muchos de los cambios intrínsecos que ocurren en células envejecidas fisiológicamente. Además, demostramos que la pérdida Bmi1 causa defectos en el crecimiento postnatal/adulto del músculo, caracterizado por pérdida de masa muscular con fibras más pequeñas, típico del músculo atrofiado senescente o sarcopénico. Puesto que la expresión de p16 está aumentada específicamente en el músculo de ratones viejos, sarcopénicos y en un modelo del ratón con envejecimiento (senescencia) acelerado (SAMP8), proponemos que el eje Bmi1/p16 puede actuar particularmente en las células madre musculares de los ancianos. La pérdida de masa muscular es una de las consecuencias fisiológicas de la sarcopenia y la identificación de nuevos factores que regulen el crecimiento y atrofia del músculo es de gran importancia para aplicaciones terapéuticas. Hemos descubierto un nuevo papel de las Sestrinas como factores promotores del crecimiento del músculo esquelético en el adulto. Hemos encontrado que la expresión de las Sestrinas se regula en modelos del ratón de atrofia y de hipertrofia muscular y en miopatías humanas. Mediante experimentos de ganacia de función hemos demostrado que las Sestrinas inducen el crecimiento del músculo esquelético, activando el ruta de señalización de IGF1/PI3K/AKT

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Richards-Malcolm, Sonia Angela. "THE ROLE OF STEM CELL ANTIGEN-1(Sca-1) IN MUSCLE AGING." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_theses/519.

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Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

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Feige, Peter. "Molecular Regulation of Satellite Cell Fate." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40804.

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Muscle homeostasis and regeneration are complex cellular processes orchestrated by muscle stem cells and their interaction with their stem cell microenvironment. The fate of a muscle stem cell is influenced by different conditions such as muscle injury, cold stress, or disease. During extensive muscle repair and in the context of muscular dystrophy, we identified the critical function of the Epidermal Growth Factor Receptor (EGFR) in establishing cell polarity and in turn the efficient formation of myogenic progeny able to repair muscle. Using a novel drug screen, we identified the p53 protein to regulate muscle stem cell fate decision to repress the formation of brown adipose tissue as a means to regulate whole-body metabolism. To increase the impact of our research we also optimized protocols evaluating mouse satellite cell transplantation to delineate stem cell hierarchy and developed a new paradigm to model human muscle stem cell fate to better translate our findings into the clinical arena. These findings reveal the tunable nature of stem cell fate decisions and highlight the development of research tools to accelerate the translation of research findings to improve human health.

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Pannerec, Alice. "The skeletal muscle stem cell niche : defining hierarchies based upon the stem cell marker PW1 to identify therapeutic target cells." Paris 6, 2012. http://www.theses.fr/2012PA066440.

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Les cellules satellites permettent la réparation des muscles squelettiques, mais chez les patients atteints de myopathies ces cellules ne fonctionnent pas correctement ce qui conduit à l’atrophie musculaire. Nos travaux ont montré qu’une nouvelle population de cellules souches musculaires, les PICs, favorisent la prolifération des cellules satellites par l’intermédiaire de la follistatine qui contrebalance l’effet négatif de la myostatine. Lorsque la myostatine est inactivée chez des souris par injection d’inhibiteur, le nombre de PICs augmente considérablement et les animaux présentent des muscles hypertrophiés. De récentes études ont montré que la régénération musculaire est impossible sans les cellules satellites, mais si nous inactivons la myostatine dans ces animaux la régénération musculaire est restaurée. Nous postulons que les PICs ont permis cette réparation et constituent donc une bonne cible pour des molécules pharmacologiques à visée thérapeutique Satellite cells are considered the major source of muscle progenitors, however, other populations with myogenic popential have been discovered. We have identified a new muscle-resident non-satellite cell population, termed PICs, which can differentiate into three different lineages, skeletal muscle, smooth muscle and fat. PICs rescue satellite cells from myostatin inhibition in vitro through follistatin release. When myostatin is inactivated in vivo, PICs number is markedly increased and mice display hypertrophied muscles. While recent studies have demonstrated that muscle regeneration cannot occur without satellite cells, we show that muscle regeneration is restored when mice have been previously treated with a myostatin inhibitor. We postulate that PICs have participated in muscle repair rescue, and thus constitute an interesting population to be targeted for pharmaceutical strategies aimed at improving skeletal muscle mass and function

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Pannérec, Alice. "The skeletal muscle stem cell niche : defining hierarchies based upon the stem cell marker PW1 to identify therapeutic target cells." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00833422.

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Les cellules satellites permettent la réparation des muscles squelettiques, mais chez les patients atteints de myopathies ces cellules ne fonctionnent pas correctement ce qui conduit à l'atrophie musculaire. Nos travaux ont montré qu'une nouvelle population de cellules souches musculaires, les PICs, favorisent la prolifération des cellules satellites par l'intermédiaire de la follistatine qui contrebalance l'effet négatif de la myostatine. Lorsque la myostatine est inactivée chez des souris par injection d'inhibiteur, le nombre de PICs augmente considérablement et les animaux présentent des muscles hypertrophiés. De récentes études ont montré que la régénération musculaire est impossible sans les cellules satellites, mais si nous inactivons la myostatine dans ces animaux la régénération musculaire est restaurée. Nous postulons que les PICs ont permis cette réparation et constituent donc une bonne cible pour des molécules pharmacologiques à visée thérapeutique

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Cahill, Kevin Scott. "Enhancement of stem-cell transplantation strategies for muscle regeneration." [Gainesville, Fla.] : University of Florida, 2003. http://purl.fcla.edu/fcla/etd/UFE0002319.

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Zhang, Ting [Verfasser]. "Epigenetic regulation of muscle stem cell expansion / Ting Zhang." Gießen : Universitätsbibliothek, 2015. http://d-nb.info/1076980325/34.

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Geiger, Ashley Elizabeth. "Impacts of dietary obesity on muscle stem cell behaviors." Thesis, Virginia Tech, 2019. http://hdl.handle.net/10919/87757.

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Occurrence of obesity has steadily increased in the human population and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury, however, the mechanism underlying these changes in satellite cell (SC) biology have yet to be explored. To test the negative impacts of obesity on SC behaviors, we fed C57BL/6 mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller numbers (P < 0.001) compared to those isolated from NC mice, and this observation was confirmed (P < 0.05) by BrdU incorporation. Moreover, in vitro differentiation assays consisting of equally seeded SCs derived from NC and HFD muscles showed that HFD SCs exhibited compromised (P < 0.001) differentiation capacity compared to NC SCs. Immunocytochemical staining of cultured SCs demonstrated that the percentage of Pax7+/MyoD- (self-renewed) SC subpopulation decreased (P < 0.001) with HFD treatment group compared to the control. In single fiber explants, a higher ratio of SCs experienced apoptotic events as revealed by the expression of cleaved caspase 3 (P < 0.001). To investigate further the impact of obesity on SC quiescence and cycling properties in vivo, we used an inducible H2B-GFP mouse model to trace the turnover rate of GFP and thus cell division under normal and obese conditions. Flow cytometric analysis revealed that SCs from HFD treatment cycled faster (P < 0.001) than their NC counterparts, as reflected by the quicker loss of the GFP intensity. To test for SC muscle regenerative capacity in vivo, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC quiescence, proliferation, differentiation, and self-renewal in vitro, ex vivo and in vivo. MS

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PAVLIDOU, THEODORA. "Perturbation of muscle stem cell differentiation by small molecules." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2014. http://hdl.handle.net/2108/201951.

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Skeletal muscle is one of the most important and plastic tissues of our body. Damaged or stressed skeletal muscle undergoes biological repair and formation of new myofibers upon regeneration signals that activate a complex cross talk between heterogeneous populations of muscle mononucleate cells. The result of this dynamic interplay is the activation of a specialized population of myogenic progenitors, the satellite cells (SCs). Satellite cells are mitotically quiescent and upon activation by regenerative signals they can divide asymmetrically and give rise to myogenic cells (myoblasts) that proliferate, differentiate and fuse to pre-existing myofibers or form new myofibers. The cell context plays an important role in maintaining the satellite cells in the quiescent state and in activating them following regeneration cues. These context dependent effects are often referred to as the stem cell niche. This doctoral thesis aims at understanding the molecular mechanisms that control the activation process and the differentiation decisions involved in the muscle regeneration process. To this end, we performed a highthroughput screening of modulators of differentiation by using the technology of automated fluorescence microscopy. We screened the collection of small molecules of the Prestwick library, containing the drugs approved by the U.S Food and Drug Administration (FDA). We developed fluorescence microscopy readouts in order to monitor the differentiation of mesoangioblasts (MABs), a muscle pluripotent cell line, and of a heterogeneous mix of muscle mononucleate cells. The readouts were designed to monitor differentiation in three different directions, skeletal muscle, adipogenic progenitors and osteoblasts. To date we have screened 560 compounds and we generated a list of “hit” molecules that can perturb the differentiation potential of the cells in the three lineages. We have performed secondary experiments to validate the list of putative interfering small molecules and, in parallel, we have built a similarity tree from a collection of transcriptional expression profile data from cultured human cells treated with bioactive small molecules, developed by the Connectivity Map team in the Broad Institute of MIT. In the second part of this thesis, we decided to further investigate the role of metformin, a drug used in the treatment of diabetes type II, in skeletal muscle differentiation. Even though metformin was one of the screened compounds that did not exhibit perturbation of the differentiation readoutswe were interested in its role in skeletal muscle differentiation since it is implicated in perturbations of the metabolism. Previous studies had suggested that muscle regeneration is improved upon AMPK activation. Short-term calorie restriction enhances the number and the myogenic potential of satellite cells in the muscles of young and old mice with an associated increase in mitochondrial abundance and an enhancement of transplant efficiency. Moreover, different metabolic pathways have been described to be involved in the establishment of the quiescent state. In pathological conditions, as in Duchenne muscular dystrophy (DMD), the repeated cycles of muscle degeneration-regeneration use up the satellite cell pool and decrease their regeneration potential rendering return to quiescence a crucial step in the regeneration process. Recently it has been reported that mTORC1 activity is necessary for the transition of satellite cells from a quiescent G0 phase to a quiescent G-alert phase, characterized by elevated propensity to cycle, increased mitochondrial activity and enhanced myogenic differentiation. Building on the above observations, I asked whether metformin, a calorie restriction-mimicking drug, could affect the activation, proliferation and differentiation of myoblasts in vitro (C2C12 cell line) and of satellite cells in vivo. Our results show that metformin reduces C2C12 myoblasts growth and inhibits their myogenic differentiation in the absence of apoptosis. This inhibition is accompanied by a reversible delay of the cell cycle in the G2/M phase, incompatible with terminal differentiation. When added in terminally differentiated myotubes, metformin induces their degradation and atrophy. Moreover, we demonstrated that metformin delays satellite cell differentiation by postponing exit from quiescence and cell cycle entry. The delayed activation of satellite cells was followed by belated regeneration of skeletal muscle after cardiotoxin injury and was associated with mTOR inhibition and reduced RPS6 phosphorylation. Since the activation, the asymmetric division and differentiation of satellite cells are a prerequisite for the regeneration of the skeletal muscle, metformin treatment could be a way to control the balance between the diverse stem cell fates.

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Collins, Charlotte Anne. "An investigation of the stem cell potential of skeletal muscle satellite cells." Thesis, University College London (University of London), 2004. http://discovery.ucl.ac.uk/1446604/.

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Satellite cells are defined by their position beneath the basal lamina of myofibres, and are a source of new myonuclei in adult skeletal muscles. However, other phenotypes also contribute to muscle regeneration, and the relative importance of satellite cells is not known. This work aimed to analyse the stem cell potential of satellite cells by formally investigating their contribution to muscle regeneration. Myofibres isolated from extensor digitorum longus, soleus, and tibialis anterior muscles were found to have respective means of 7,22 and 10 associated satellite cells. When a single myofibre was grafted into an irradiated dystrophic mouse muscle, the associated satellite cells underwent extensive, stem cell-like proliferation, generating progeny which sometimes gave rise to a cluster of more than 100 new myofibres. Cluster size varied according to the muscle group from which the graft was derived, but was not proportional to satellite cell number. Primary myoblasts derived from equivalent muscle groups did not undergo such extensive proliferation, or show inter-muscle variability, suggesting that stem cell activity is critically dependent on a component of the satellite cell niche. Single myofibres isolated from irradiated muscles were non-myogenic after grafting. Satellite cells associated with single myofibres were found to generate new satellite cells in engrafted muscles, demonstrating that satellite cell compartment is maintained by self-renewal. When single myofibre-engrafted muscles were damaged with myotoxin, graft-derived cells underwent rapid clonal expansion to regenerate compact clusters of donor-derived myofibres. The percentage of engrafted muscles containing identifiable donor-derived nuclei was increased after damage, showing that previously inactive cells had been recruited into an active myogenic program. Without experimentally-induced damage, frequency of muscle formation and cluster size were spontaneously augmented over time. These findings demonstrate that satellite cells have several stem cell-like qualities, and thus constitute a self-sufficient and sustainable source of regeneration in adult muscles.

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Kahatapitiya, Prathibha C. "Enrichment of skeletal muscle stem cell transplantation using chemotherapeutic drugs a paradigm for enhanced stem cell transplantation /." Connect to full text, 2008. http://hdl.handle.net/2123/4050.

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Thesis (Ph. D.)--University of Sydney, 2009. Title from title screen (viewed Apr. 24, 2008) Title from title screen (viewed Feb. 18, 2009) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Paediatrics and Child Health, Faculty of Medicine. Degree awarded 2009 ; thesis submitted 2008. Includes bibliographical references. Also available in print form.

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Benavente, Diaz Maria. "Investigation of the molecular diversity defining muscle stem cell heterogeneity." Electronic Thesis or Diss., Sorbonne université, 2020. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2020SORUS072.pdf.

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Le muscle squelettique adulte a une capacité de régénération remarquable, pouvant guérir après des traumatismes répétés. Cette propriété dépend de la présence de cellules souches musculaires (SCMu), qui sont pour la plupart quiescentes dans des conditions homéostatiques mais qui s'activent après une blessure, réintègrent le cycle cellulaire et prolifèrent pour donner naissance à des myoblastes qui fusionneront pour restaurer les fibres endommagées. De nombreuses études ont étudié les états transitoires que les SCMu empruntent de l'entrée du cycle cellulaire à la différenciation. Malgré le fait que plusieurs souris rapportrices génétiquement modifiées aient été générées pour examiner ces événements, l'initiation de la différenciation, qui est généralement définie par l'expression du facteur de régulation myogénique Myogenin, est difficilement appréciable à cause du manque de souris rapportrice fiable. Par conséquent, nous avons développé une nouvelle lignée rapportrice où la différenciation des cellules myogéniques exprimant le facteur de transcription Myogenin peut être marquée par l'expression d'une protéine fluorescente tdTomato. Cette nouvelle lignée de souris knock-in nous a permis d'analyser la cinétique de l'expression de Myogenin lors de la différenciation cellulaire in vitro et d'effectuer des expériences préliminaires in vivo par imagerie intravitale. De plus, bien que toutes les SCMu de souris soient caractérisées par l'expression du facteur de transcription Pax7, plusieurs études ont décrit des différences de prolifération, de capacité de transplantation et de sensibilité à la maladie entre les SCMu des muscles crâniens et des membres. Pour étudier les réseaux de régulation des gènes qui régissent cette hétérogénéité fonctionnelle, nous avons combiné des analyses transcriptomiques sur cellules uniques avec des approches de biologie cellulaire utilisant des lignées de souris rapportrices pour identifier les régulateurs clés qui confèrent des propriétés distinctes aux SCMu à haute-performance (extraoculaires) et à faible-performance (tibialis antérieur) en quiescence et lors de l'activation. Nous avons identifié un retard dans la différenciation des SCMu extraoculaires en culture, accompagné par l'expression de facteurs de remodelage de la matrice extracellulaire et de récepteurs membranaires distincts et nous avons validé l'expression de certains de ces candidats au niveau protéique. Des analyses informatiques avancées ont mis en évidence la dynamique sous-jacente au maintien d'une population de progéniteurs dans les SCMu extraoculaires, contrôlée par un réseau singulier de facteurs de transcription formant un module de molécules co-régulées. En conclusion, ces études apportent de nouvelles informations sur les mécanismes qui octroient des propriétés différentes des cellules souches musculaires venant d'emplacements anatomiques distincts Adult skeletal muscle has a remarkable regenerative capacity, being able to recover after repeated trauma. This property depends on the presence of muscle stem cells (MuSCs), which are mostly quiescent in homeostatic conditions, re-enter the cell cycle after injury and proliferate to give rise to committed myoblasts that will eventually fuse to restore the damaged fibres. Numerous studies have investigated the cell state transitions that MuSCs undergo from cell cycle entry to differentiation. Although several genetically modified reporter mice have been generated to study these events, detailed studies on the initiation of differentiation, which is generally defined by expression of the myogenic regulatory factor Myogenin, have been hampered by the lack of a reliable reporter mouse. Therefore, we developed a fluorescent reporter line where differentiating myogenic cells expressing Myogenin are marked by the expression of a tdTomato fluorescent protein. This novel knock-in mouse line allowed us to monitor the kinetics of Myogenin expression during cell differentiation in vitro, and perform preliminary experiments on the behaviour of myogenic cells in vivo by intravital imaging. Although all mouse MuSCs are characterised by the expression of the transcription factor Pax7 and they share several properties, some studies have reported differences in proliferation, engraftment ability, and sensitivity to disease of MuSCs from cranial and limb muscles. To investigate the gene regulatory networks that govern this functional heterogeneity, we have integrated single-cell transcriptomic analyses with cell biology approaches using mouse reporter lines to identify key regulators that confer distinct properties to high performing (extraocular muscles) and lower performing (limb, Tibialis anterior muscle) MuSCs in quiescence and activated states. We identified a delayed lineage progression of extraocular MuSCs in culture that was accompanied with the expression of distinct extracellular matrix remodelling factors and membrane receptors, and we validated the expression of some of these candidates at the protein level. Advanced computational analyses highlighted the dynamics underlying the maintenance of a stem-like progenitor population in extraocular MuSCs, controlled by a singular network of transcription factors acting as a co-regulating module. Taken together, these studies provide novel insights into the mechanisms underlying the differential properties of muscle stem cells in distinct anatomical locations

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Kahatapitiya, Prathibha Chathurani. "Enrichment of skeletal muscle stem cell transplantation using chemotherapeutic drugs." Thesis, The University of Sydney, 2009. http://hdl.handle.net/2123/4050.

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The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

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Kahatapitiya, Prathibha Chathurani. "Enrichment of skeletal muscle stem cell transplantation using chemotherapeutic drugs." University of Sydney, 2009. http://hdl.handle.net/2123/4050.

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Doctor of Philosophy (PhD) The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

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Piccoli, Martina. "Mouse amniotic fluid stem cells are able to differentiate into satellite cells replenishing the depauperated muscle stem cell niche." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423564.

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Introduction: Stem cell biology has received much interest because of its potential in both therapeutic application and in vitro modeling of diseases. In particular embryonic stem cells have good proliferative and differentiative abilities, but their use is still associated to ethical concerns and problems related to their teratogenic potential. Adult stem cells have also been described to be pluripotent both in vitro and in vivo. However, their use is limited because they are difficult to isolate and expand, particularly in a clinical setting. In this scenario, it would be advantageous to obtain a cell population with high selfrenewal and differentiation capacities, without ethical problems. In 2007 our group described that amniotic fluid stem (AFS) cells could be derived selecting amniocytes using c-Kit antibody. AFS cells have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Aim: This work aiming at characterize the myogenic potential of mouse AFS cells using a mouse model of spinal muscular atrophy and in particular at analyzing their ability to differentiate into satellite cells and colonize the muscle stem cell niche. Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected for the marker c-Kit with immunomagnetic beads. Freshly isolated AFS cells were analyzed for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) by flow cytometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. For the treatment of HSA-Cre, SmnF7/F7 mutant mice, GFP+ AFS cells were injected via the tail vein and animals were sacrificed one and fifteen months after transplantation. Clinical aspects were observed and analyzed after transplantation to evaluate AFS cells’ effects. Several muscles were stained with hematoxylin and eosin, Masson’s trichrome and analyzed by immunofluorescence with anti-GFP and anti-dystrophin antibodies. To demonstrate the ability of AFS cells to replenish the muscle niche, staining for satellite cell markers and secondary transplantation were performed. The myogenic potential of AFS cells was also evaluated with transplantation after in vitro expansion. Results: Mouse AFS cell number changes during the course of gestation. At E12.5 these cells express hematopoietic markers (CD45, CD34, SCA1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. Gene expression analysis showed that mouse AFS cells express at low levels Oct4 and Sox2 and at high levels c-Myc and Klf4, whereas they are negative for the expression of myogenic genes. Mild muscular mutant HSA-Cre, SmnF7/F7 mice die at the age of 10 months and show evident clinical complications such as kyphosis and muscle shrinkage. After transplantation with GFP+ AFS or bone marrow (BM) cells mice survival rate increased by 75% and 50% respectively. Animals treated with AFS cells recovered more than 75% of force compared to the untreated. One month after transplantation, muscles obtained from AFS-treated mice displayed 37% of GFP+ fibers, with very low number of regenerating myofibers (<1%) and normal dystrophin expression. Fifteen months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a normalized phenotype, close to the same age WT mice, and 58% of the myofibers were GFP+. Similar results were obtained with transplantation of mouse AFS cells expanded in culture. Discussion: Mouse AFS cells are a heterogeneous population, and their phenotype changes during the course of gestation. At E12.5 they express mesenchymal, hematopoietic and endothelial markers, but most importantly don not express myogenic factors, indicating that no myogenic progenitor cells are present in this stem cell population. When injected in a muscular mutant mouse model, AFS cells showed a myogenic potential, even after long-term transplantation, suggesting an interesting therapeutic potential. They indeed were able to differentiate into satellite cells localizing in the muscle stem cell niche and expressing Pax7, a7integrin and SM/c-2.6, exclusively markers of satellite cell population. Moreover, AFS cells could contribute to the formation of new myofibers even after in vitro expansion. Introduzione: Negli ultimi anni lo studio delle cellule staminali ha suscitato molto interesse, sia per il grande potenziale di queste cellule nelle terapie e applicazioni cliniche, sia come modello di studio in vitro per diversi tipi di malattie. In particolare, le cellule staminali embrionali hanno una elevata capacità proliferativa e di differenziazione, ma il loro utilizzo è ancora associato a problematiche etiche. Anche le cellule staminali adulte possiedono grandi potenzialità differenziative sia in vitro che in vivo, tuttavia il loro utilizzo è limitato in quanto difficili da isolare ed espandere, soprattutto in ambito clinico. In questo scenario sarebbe vantaggioso poter ottenere una popolazione di cellule con elevata capacità di proliferazione e differenziazione, senza dover affrontare però problemi di tipo etico. Nel 2007 il nostro gruppo ha isolato una popolazione di cellule staminali dal liquido amniotico (cellule AFS), utilizzando come marcatore il recettore c-Kit. Queste cellule hanno capacità clonogenica e possono essere dirette a differenziare in una vasta gamma di tipi cellulari appartenenti a tutti e tre i foglietti germinativi. Obiettivo: Questo lavoro mira a caratterizzare il potenziale miogenico delle cellule staminali del liquido amniotico di topo utilizzando un modello murino di atrofia spinale muscolare. In particolare è volto ad analizzare la capacità delle cellule AFS di dare origine a cellule staminali muscolari e colonizzare la nicchia staminale del muscolo scheletrico. Materiali e Metodi: Le cellule AFS sono state ottenute mediante amniocentesi e selezionate per la positività al marcatore c-kit con metodo immmunomagnetico. Appena isolate le cellule AFS sono state analizzate per l'espressione di diversi marcatori (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) tramite citometria a flusso; inoltre, attraverso qRT-PCR è stata analizzata l'espressione di Oct4, Sox2, c-Myc, Klf4 e Sca-1 delle cellule AFS isolate a diversi stadi embrionali. Per la terapia di topi transgenici HSA-Cre, SmnF7/F7, le cellule AFS GFP+ sono state iniettate per via sistemica attraverso la vena caudale; gli animali sono stati poi sacrificati a uno e a quindici mesi dopo il trapianto. Sono stati osservati e analizzati alcuni parametri clinici per valutare l’effetto del trapianto cellulare. Diversi muscoli sono stati raccolti ed analizzati con ematossilina e eosina, tricromica di Masson e mediante immunofluorescenza con anticorpi anti-GFP e anti-distrofina. Per dimostrare la capacità delle cellule AFS di colonizzare la nicchia staminale del muscolo, sono state eseguite delle immunofluorescenze per i marcatori specifici delle cellule satelliti e sono stati eseguiti dei trapianti secondari. Il potenziale miogenico delle cellule AFS è stato valutato anche con trapianto dopo espansione in vitro. Risultati: Il numero medio di cellule AFS presenti nel liquido amniotico varia nel corso della gestazione murina; all’età di 12.5 giorni queste cellule sono circa l’1% del totale ed esprimono marcatori ematopoietici (CD45, CD34, SCA1), marcatori mesenchimali (CD90, CD105) unitamente a Flk1, CD31 e CD44. L’analisi di espressione genica ha dimostrato che le cellule AFS esprimono a bassi livelli Oct4 e Sox2 e alti livelli di c-Myc e Klf4, mentre, nonostante la composizione mista di questa popolazione, non è stata rilevata espressione di marcatori o fattori di trascrizione tipici dei precursori muscolari. I topi HSA-Cre, SmnF7/F7 mediamente muoiono all'età di 10 mesi e durante il corso della loro vita mostrano evidenti complicazioni cliniche come una pronunciata cifosi e atrofia a livello muscolare. Dopo il trapianto con cellule AFS GFP+ o con cellule del midollo osseo, il tasso di sopravvivenza di questi animali aumenta rispettivamente del 75% e 50%. Gli animali trattati con cellule AFS hanno recuperato più del 75% della forza rispetto agli animali non trattati. Un mese dopo il trapianto, i muscoli di topi trattati con cellule AFS presentano il 37% di fibre GFP+, un numero molto basso di miofibre rigeneranti (< 1%) ed una normale espressione di distrofina. Quindici mesi dopo il trapianto, gli animali trattati con cellule del midollo osseo mostrano un elevato numero di fibre centro nucleate, un’importante infiltrazione di tessuto interstiziale e nessuna miofibra GFP+, mentre i topi trattati con cellule AFS hanno un fenotipo molto simile a quello di topi sani della stessa età, e il 58% delle miofibre è GFP+. Risultati simili sono stati ottenuti trattando lo stesso modello animale con cellule AFS dopo espansione in cultura. Discussione: Le cellule AFS isolate dal liquido amniotico di topo sono una popolazione eterogenea; queste cellule esprimono marcatori mesenchimali, ematopoietici e marcatori endoteliali. Va evidenziato che, nonostante la composizione mista di questa popolazione staminale, non esistono precursori muscolari al suo interno, e quindi qualunque differenziamento in senso muscolare di queste cellule è dovuto ad una differenziazione delle cellule AFS e non ad una maturazione di cellule già pre-commited. Quando vengono iniettate in un modello di atrofia muscolare, le cellule AFS mostrano un grande potenziale miogenico, anche a lungo termine, dimostrandosi una interessante fonte cellulare per scopi terapeutici. Queste cellule infatti sono state in grado di differenziare in cellule satelliti localizzandosi nella nicchia delle cellule staminali muscolari ed esprimendo Pax7, a7integrina e SM/c-2.6, tutti marcatori esclusivi delle cellule satelliti. Inoltre, le cellule AFS possono contribuire alla formazione di nuove miofibre anche dopo espansione in cultura, aumentando così lo spettro di possibili applicazioni terapeutiche.

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Yin, Xiaoke. "Protein changes associated with embryonic stem cell differentiation to vascular smooth muscle cells." Thesis, Queen Mary, University of London, 2006. http://qmro.qmul.ac.uk/xmlui/handle/123456789/1764.

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Embryonic stem (ES) cells can differentiate into many different cell lines, including vascular smooth muscle cells (SMCs). The aim of this project is to characterize protein changes during this differentiation process. Mouse ES cells are pre-differentiated by withdrawal of the leukemia inhibitory factor-1 from the culture medium. Subsequently, stem cell antigen-1 positive (Sca-1) cells are sorted by magnetic labelling cell sorting with anti-Sca-1 microbeads and cultured in differentiation medium with or without platelet-derived growth factor (PDGF). Protein extracts of ES cells and Sca-1+ cells are separated by two-dimensional electrophoresis. About 300 protein species of each cell lines are analyzed by mass spectrometry. Proteome maps are available online (http:/ /vwvw.v ascular-proteomicsc. om). After stimulation with PDGF for 5 passages, Sca-1+ cells differentiate into SMCs (esSMCs) with 95% staining positive for SMC markers such as smooth muscle a-actin, calponin, and smooth muscle myosin heavy chain. Protein profiles of esSMCs and mouse aortic SMCs are compared using the difference gel electrophoresis approach. esSMCs display decreased expression of myofilaments but increased oxidation of redox-sensitive proteins due to higher levels of reactive oxgen species (ROS). While immunoblotting reveals an upregulation of numerous antioxidants in esSMCs, enzymatic assays demonstrate lower glutathione concentrations compared to aortic SMCs despite a 3-fold increase in glutathione reductase activity. Mitochondrial superoxide measurement revealed the mitochondria-derived superoxide is the main source of ROS in esSMCs and inhibition of electron transport chain complex III by antimycin A showed remarkable increase of ROS in esSMCs. Moreover, depletion of glutathione by diethyl maleate or inhibition of glutathione reductase by carmustine (BCNU) results in a remarkable loss of cell viability in esSMCs compared to aortic SMCs while adding 2-mercaptoethanol increased esSMCs survival. These results indicate that esSMCs require additional antioxidant protection for survival and consequently, treatment with anti-oxidants could be beneficial for tissue repair from ES cells.

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Thumiah-Mootoo, Madhavee. "The Role of Mitophagy in Muscle Stem Cell Fate and Function During Muscle Regeneration." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42239.

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Skeletal muscles have a remarkable capacity to repair and regenerate in response to injury by virtue of their unique population of resident muscle stem cells (MuSCs). Recently, several studies have reported that mitochondria are important regulators of fate and function in various types of stem cells including MuSCs. Furthermore, emerging evidence has shown that accumulation of dysfunctional mitochondria leads to stem cell aging, premature commitment and impaired self-renewal. Preliminary evidence from publicly available transcriptomics datasets processed by our lab showed that Phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) and Parkin/PARK2 genes, two key regulators of mitophagy are expressed in quiescent MuSCs and are transiently down-regulated as MuSCs activate. This led us to hypothesize that maintenance of an optimally functioning population of mitochondria through mitophagy would be important for self-renewal and muscle repair. In vitro single myofiber cultures isolated from mitophagy reporter mice (mito-QC mice), show that mitophagy is active in quiescent MuSCs and is transiently decreased upon MuSCs activation. We also show that mitophagy is re-activated in differentiating and self-renewing MuSCs. To further study muscle regeneration, we used a cardiotoxin (CTX) injury model of the Tibialis anterior (TA) muscle in mouse models harboring a knockout (KO) of PINK1 and Parkin. We show that loss of PINK1 in vivo promotes commitment of MuSCs in response to acute injury and ultimately leads to depletion of the MuSC pool and impaired muscle regeneration compared to wild type (WT) mice following repetitive injuries. Similarly, loss of Parkin in MuSCs in vivo impaired their self-renewal capacity. Consistent with these results, in vitro single myofiber cultures isolated from PINK1-deficient mice showed increased MuSCs commitment and impaired self-renewal. In vitro preliminary results from MuSCs-specific KO of Parkin revealed altered lineage progression, differentiation and self-renewal of MuSCs. Together, these findings suggest that PINK1/Parkin-dependent mitophagy acts as an important mitochondrial quality control mechanism which could be required for regulating MuSCs fate and function during muscle regeneration.

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Goel, Aviva J. "Niche Regulation of Muscle Stem Cell Quiescence by Classical Cadherins." Thesis, Icahn School of Medicine at Mount Sinai, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10743988.

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Many adult stem cells are characterized by prolonged quiescence, promoted by cues from their niche. Upon tissue damage, a coordinated transition to the activated state is necessary for successful repair. Non-physiological breaks in quiescence often lead to stem cell depletion and impaired tissue restoration. Here, I identify cadherin-mediated adhesion and signaling between muscle stem cells (satellite cells; SCs) and their myofiber niche as a mechanism that orchestrates the quiescence-to-activation transition. Conditional removal of N-cadherin and M-cadherin in mice leads to a break in SC quiescence with long-term expansion of a regeneration-proficient SC pool. These SCs have an incomplete disruption of the myofiber-SC adhesive junction, and maintain niche residence and cell polarity, yet show properties of SCs in a state of transition from quiescence towards full activation. Among these properties is nuclear localization of b- catenin, which is necessary for this phenotype. These findings are consistent with the conclusion that injury-induced perturbation of niche adhesive junctions is a first step in the quiescence-to-activation transition.

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Berti, Federica. "Protection of the muscle stem cell state from premature differentiation." Thesis, University of Portsmouth, 2016. https://researchportal.port.ac.uk/portal/en/theses/protection-of-the-muscle-stem-cell-state-from-premature-differentiation(6886509a-35fa-43b1-8971-7cb2dcaa7da3).html.

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The extraordinary capacity of regeneration exists in just certain species and tissue types. Invertebrates, the cells that regenerate tissues are called stem cells. During adulthood, damaged muscle tissue regenerates through Satellite stem cells that re-enter the cell cycle from a quiescent state, giving rise to new muscle tissue. During development, newly forming organisms build tissues through proliferating stem cells that renew the stem cell pool whilst generating myogenic stem cells which will eventually differentiate into muscle cells. For many years the Myogenic Regulatory Factors (Mrf) genes have been considered to be the main genes driving this proliferation in adult and fetal stages. Mrf genes have been shown to be capable of inducing non-myogenic cells to enter the myogenic lineage in vitro, but their role and capabilities in vivo have been less well characterised. Here we show, using the developing chicken model, that Mrf genes and related genes are not capable of prematurely upregulating terminal muscle differentiation before HH20. MyoD and other combinations of gene misexpression were however shown to be capable of inducing Myosin upregulation between HH20 and HH24 indicating the existence of a time-frame dependent protection of premature development. These results indicate that the Mrf genes have a reduced proficiency for inducing differentiation in vivo compared with in vitro likely due to the presence of currently unidentified additional factors. Our results demonstrate that the current understanding of the signals and cues of muscle stem cell differentiation are still insufficient to exploit the regenerative capabilities of muscle tissues towards regenerative therapies. The possible additional factors required for muscle stem cell differentiation are also discussed.

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Yu, Xiaotian. "Functional impact of microRNA-34a on stem cell differentiation towards smooth muscle cell." Thesis, Queen Mary, University of London, 2014. http://qmro.qmul.ac.uk/xmlui/handle/123456789/9121.

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MicroRNAs play an important role in biological regulation. Recently miR-34a has been reported to regulate tumour cell cycle progression and apoptosis. However, the functional role of miR-34a in smooth muscle cell (SMC) differentiation from stem cells is yet unclear. Main objectives of this PhD project are to determine the functional role of miR-34a and its target genes in SMC differentiation and underlying mechanisms. Mouse embryonic stem (ES) cells were seeded on collagen coated flasks in differentiation medium to allow SMC differentiation. Upon analysis, miR-34a was significantly up-regulated during SMC differentiation. Results demonstrated that overexpression of miR-34a significantly promoted SMC-specific gene expression, while knockdown of miR-34a inhibited expression of SMC specific gene. Enforced expression and knockdown of miR-34a in differentiating ES cells up-regulated and down-regulated, respectively, several SMC transcription factors in a similar manner. It was also found that miR-34a overexpression in stem cells promoted SMC differentiation in vivo. Furthermore, deacetylase sirtuin 1 (Sirt1) was identified as one of the top targets of miR-34a. Surprisingly, Sirt1 was demonstrated to be positively regulated by miR-34a during SMC differentiation in a cellular context and RNA sequence dependent manner. VIII Mechanistically, the data suggested that miR-34a promoted differentiating stem cells arrest at G0/G1 phase, and a significant decreased incorporation of miR-34a and SirT1 RNA into Ago2-RISC complex was observed upon SMC differentiation. The results demonstrated that Sirt1 acted as a transcriptional activator in the regulation of SMC gene during ES cell differentiation. Finally, H3K9 tri-methylation around the promoter regions of the SMαA and SM22α genes was also found to be significantly inhibited by SirT1 overexpression. These findings suggest that miR-34a plays an important role in SMC differentiation from ES cells. Meanwhile, Sirt1 can be regulated by miR-34a through an unexpected pathway and it was identified as a functional modulating target in miR-34a mediated SMC differentiation.

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Yeo, Wendy Wai Yeng. "Differentiation of skeletal muscle-derived stem cells into beta pancreatic lineage." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS091.

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Le diabète de type 1 (DT1) est caractérisé par des niveaux élevés de glucose en raison de la destruction des cellules ß pancréatiques sécrétrices d'insuline. Cependant, les thérapies actuelles de remplacement des cellules bêta du pancréas impliquant la transplantation d'îlots pancréatiques sont techniquement difficiles et limitées par la disponibilité de don d'organes. Bien que les cellules souches embryonnaires et les cellules souches pluripotentes induites soient intensément étudiées, aucune de ces deux sources de cellules souches ne peut être utilisée directement sans le risque de développement de tumeurs. Les cellules souches dérivées du muscle squelettique (MDSC) sont une source de cellules alternative intéressante car elles sont multi-potentes et peuvent donc se différencier vers plusieurs lignages cellulaires tels que des cellules cardiaques à battement autonome “pacemaker-like” et des cellules neuronales. Par conséquent, nous avons émis l'hypothèse qu'elles pourraient se différencier en lignées de type pancréatique. Les objectifs de cette étude étaient donc d'étudier le potentiel des MDSC (1) à se différencier in vitro en cellules beta pancréatiques exprimant l'insuline et (2) à se différentier in vivo dans le pancréas et ainsi réduire l'hyperglycémie chez la souris modèle d'un diabète de type 1. Dans cette étude, les MDSC de muscle de souris ont été isolées via une série de passages des cellules les moins adhérentes en culture. Les cellules souches ainsi isolées peuvent adhérer sur une couche de cellules de types fibroblastes ou sur une matrice extra-cellulaire de type laminine pour ensuite se différentier in vitro ou bien être utilisées comme cellules souches MDSC non-adhérentes et non différentiées pour les études in vivo. In vitro, les MDSC peuvent se différencier spontanément en agrégats de cellules formant des îlots et exprimant des marqueurs de cellules bêta identifiés par immunofluorescence et analyse “PCR transcription inverse”. Ceci a été confirmé par immuno-analyse montrant l'expression des protéines nécessaires à la fonction des cellules ß, comme Nkx6.1, MafA et Glut2. Les MDSC différenciées en aggrégats cellulaires de type îlots pancréatiques montrent une sécrétion d'insuline en réponse au glucose in vitro. Cependant, dans des modèles murins de DT1 induit par la streptozotocine, l'injection intra-péritonéale des MDSC n'a pas permis de rétablir chez les souris diabétiques une normoglycémie du glucose sanguin en dépit d'un engreffement des MDSC dans les tissus pancréatiques. Ces données montrent que les MDSC peuvent constituer une source de cellules souches alternative intéressante pour le traitement du diabète Type 1 Diabetes (T1D) is characterized by high and poorly controlled glucose levels due to the destruction of insulin-secreting pancreatic ß-cells. However, current ß-cell replacement therapies, involving pancreas and pancreatic islet transplantation are technically demanding and limited by donor availability. While embryonic stem cells and induced pluripotent stem cells are intensely investigated, neither can be used due to safety issues. Skeletal muscle-derived stem cells (MDSC) are an attractive alternative cell source as they have the potential to undergo multilineage differentiation into beating pacemaker-like cells and neuronal cells. Hence, it is hypothesised that they can differentiate into pancreatic lineages. This led to the goals of this study, which were (1) to investigate the potential of MDSC to differentiate into mature insulin expressing cells in vitro and (2) to reduce hyperglycemia in mouse model type 1 diabetes. In this study, MDSC were isolated from mouse via a serial pre-plating based on the adhesive characteristics of cultured cells, in which the cells of interest adhered to plates at a later time for in vitro differentiation, while the non-adherence undifferentiated MDSC were used for in vivo study. The MDSC were found to spontaneously differentiate into islet-like aggregates and expressed ß-cell markers in vitro, as determined by immunofluorescence and reverse transcription PCR analyses. This was further confirmed by immunoblotting analysis showing expression of proteins required for ß-cell function, such as Nkx6.1, MafA and Glut2. The differentiation of MDSC into islet-like clusters demonstrated glucose responsiveness in vitro. In streptozotocin-induced T1D mouse models, intraperitoneal injection of the undifferentiated MDSC did not restore the blood glucose levels of the diabetic mice to normoglycemia despite successful engraftment of MDSC into the pancreatic tissues. Taken together, these data show that MDSC may serve as an alternative source of stem cells for the treatment of diabetes

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25

Swaminathan, Ganesh. "Evaluation Of Adult Stem Cell Derived Smooth Muscle Cells For Elastic Matrix Regenerative Repair." University of Akron / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=akron1462209321.

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26

Ruan, Travis. "Identification of Terminal Differentiation Enhancers in Human Embryonic Stem Cell Derived Skeletal Muscle Cells." Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/27257.

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Skeletal myogenesis is a tightly coordinated process resulting from the temporal expression of signalling cascades that specify myogenic cell fate. Identification of signalling pathways and small molecules that can modulate this developmental process, continues to be an active area of research. Utilising the pluripotent nature of human embryonic stem cells (hESC) and combined with next generation sequencing, we demonstrate our in vitro skeletal muscle differentiation system accurately recapitulate major skeletal muscle developmental process. We show myotubes formation can be further enhanced using a combination of anabolic factors and myokines. Multi-omics analysis revealed oxidative phosphorylation as the major up-regulated pathway, suggesting energy metabolism is coupled to enhanced skeletal muscle differentiation. Finally, to identify novel drug candidates that could reinforce muscle strength, we performed a high throughput drug screening of over 1000 drugs in hESC derived skeletal muscle cells (hESC-SkMC) and identified several candidate compounds that significantly increased the muscle marker Myosin Heavy Chain (MyHC) expression level. We further demonstrate this enhanced muscle differentiation is also closely associated with an up-regulation of energy metabolism. Together, this work presents a genetic dissection of hESC-SkMC development in vitro, which may assist in the identification and development of more effective drug therapies targeting skeletal muscle development or diseases.

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27

Pasut, Alessandra. "Regulation of Muscle Stem Cell Function by the Transcription Factor Pax7." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32448.

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Pax7 is a paired box transcription factor expressed by all satellite cells which are critically required for muscle regeneration and growth. The absolute requirements of Pax7 in the maintenance of the satellite cell pool are widely acknowledged. However the mechanisms by which Pax7 executes muscle regeneration or contributes to satellite cell homeostasis remain elusive. We performed cell and molecular analysis of Pax7 null satellite cells to investigate muscle stem cell function. Through genome wide studies, we found that genes involved in cell cell interactions, regulation of migration, control of lipid metabolism and inhibition of myogenic differentiation were significantly perturbed in Pax7 null satellite cells. Analysis of satellite cells in vitro showed that Pax7 null satellite cells undergo precocious myogenic differentiation and have perturbed expression of genes involved in the Notch signaling pathway. We showed that Notch 1 is a novel Pax7 target gene and by using a genetic approach we demonstrated that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) in Pax7 null satellite cells is sufficient to maintain the satellite cell pool as well as to restore their proliferation. Instead of differentiating into myogenic cells and in the absence of a myogenic cue, NICD1 Pax7 null satellite cells become a source of ectopic brown fat within muscles and give rise to brown adipocytes both in vivo and in vitro. In conclusion we showed that Notch 1 partially rescues Pax7 deficient satellite cells loss and proliferation. Additionally we provide the first evidence that Notch signalling contributes to satellite cell fate by inhibiting terminal myogenic differentiation and inducing brown adipogenesis.

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28

Baker, Nicole. "Muscle Stem Cell Fate is Directed by the Mitochondrial Fusion Protein OPA1." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/41974.

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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.

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29

Schabort, Elske Jeanne. "The effect of TGF-[beta] isoforms on progenitor cell recruitment and differentiation into cardiac and skeletal muscle /." Link to the online version, 2007. http://hdl.handle.net/10019.1/1295.

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30

Tan, Kah Yong. "Stem Cell-Based Strategies to Enhance Muscle Regeneration through Extrinsic and Intrinsic Regulators." Thesis, Harvard University, 2011. http://dissertations.umi.com/gsas.harvard:10009.

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Skeletal muscle has a remarkable capacity for regeneration, mediated by muscle stem cells that can self-renew or differentiate to form the mature myofibers of the tissue. Several human diseases are characterized by a loss of function and strength in skeletal muscle, with impairments in the ability to regenerate and consequent decreases in quality of life and increases in mortality. The work in this dissertation has focused on developing methods for combating muscle disease. This goal has been approached through attempts at cell replacement therapy - by generating muscle cells that can be engrafted in vivo. I also investigated the influence on regeneration of the skeletal muscle microenvironment (skeletal muscle-resident fibroblasts), and systemic environment (inflammation in myogenic and non-myogenic tissues), both of which were found to affect skeletal muscle stem cell behavior and the efficiency of myogenic regeneration. Ultimately, these studies identified novel factors that impair or improve skeletal muscle differentiation, and that offer the potential to modulate the process of muscle regeneration. In the process of investigating if induced pluripotent stem cells from skeletal muscle retain an epigenetic memory conducive to myogenic differentiation, I discovered that precursor cells in skeletal muscle reprogram to a pluripotent state more efficiently. However, these induced pluripotent stem cells, like embryonic stem cells, retain strong barriers to skeletal muscle differentiation. Together, these findings offer insights into the process of muscle regeneration, and suggest new potential pathways towards treatment of muscle disease.

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31

Steele-Stallard, Heather. "Induced pluripotent stem cell platforms for disease modelling of skeletal muscle laminopathies." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10058072/.

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Laminopathies are a clinically and genetically heterogeneous group of 16 disorders caused by mutations in LMNA. This gene codes for lamin A and lamin C, which together with lamin B1 and B2 form the nuclear lamina, a mesh-like structure located underneath the inner nuclear membrane. Laminopathy disorders show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and others causing multisystem disease with accelerated aging. The exact mechanisms underlying the pathology of laminopathies, and the cause of the tissue specific phenotypes are unknown, although several mechanisms have been proposed. Understanding the pathology of these disorders is limited by the rarity of cases, and lack of easily accessible cell types. Induced pluripotent stem cells (iPSCs) can be derived from easily accessible cell types, have unlimited proliferation potential, and can be differentiated into cell types that would otherwise be difficult and invasive to obtain. This PhD project aimed to use iPSCs from patients with skeletal muscle laminopathies to model disease phenotypes in vitro. In this thesis, fibroblasts from a patient with a skeletal muscle laminopathy were reprogrammed into iPSCs. This line, along with three already reprogrammed iPSC lines from skeletal muscle laminopathy patients were differentiated into mesodermal/mesenchymal progenitors, myogenic precursor cells and myotubes. Disease-associated phenotypes were observed in these cells, namely abnormal nuclear shape and mislocalisation of nuclear lamina proteins. Furthermore, work towards developing a therapy based on lamin A/C exon skipping was conducted. These results demonstrate that iPSCs from skeletal muscle laminopathy patients can be used to model disease-associated phenotypes in vitro. This lays the foundation for future therapy testing and disease modelling in skeletal muscle laminopathies using patient specific iPSCs.

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32

Fittipaldi, R. "ROLE OF SMYD3 IN SKELETAL MUSCLE ATROPHY AND MOUSE EMBRYONIC STEM CELL." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/238009.

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Epigenetic regulation of gene expression plays a pivotal role in the establishment of developmental programs and the maintenance of the differentiated state. Among the different actors involved in this scenery, the modifications of histones tail are implicated with the propagation of gene expression patterns. Our group is focused on SMYD3, a histone-methyltransferase which is reported to be is highly expressed in normal conditions only in the embryo, in adult skeletal muscle and in few other tissues. In light of SMYD3 restricted expression, we asked whether it might play a role during myogenesis and/or muscle maintenance but also when it plays its role during development. We firstly have clarified the role of the histone-methyltransferase SMYD3 as a modulator of two factors involved in muscle-growth regulation and muscle atrophy, myostatin and c-Met transcription. Our results uncover a novel role for SMYD3 in recruiting the bromodomain protein BRD4. SMYD3 engages BRD4 and the positive transcription elongation factor complex (p-TEFb), triggering the phosphorylation on Serine 2 of the RNA Polymerase II, which favors the transcription elongation. Our data show that treatment with the BRD4 inhibitor JQ1 protects myotubes size reduction induced by dexamethasone administration and hinders pro-atrophic factors upregulation. These results suggest that JQ1 may represent a novel pharmacological avenue to alleviate muscle loss associated with muscle atrophy. We then clarify the role played by SMYD3 in embryonic development. Recent study in zebrafish model suggests that SMYD3 plays an important role in the development of heart. Therefore we decided to investigate the role of SMYD3 by employing mouse embryonic stem cells (mESC) as a model of developmental differentiation toward cardiomyocytes. We observed that the expression levels of cardiac markers as well as cardiovascuolar progenitor markers were increased in SMYD3 depleted embryoid bodies. To further disclose the role of SMYD3 in embryoid bodies differentiation we also analyzed markers of the primitive streak and transcripts involved in EMT. We report the SMYD3 played a role during early stages of embryonic stem cells differentiation.

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33

Harutiun, Minas Nalbandian Geymonat. "Characterization of hiPSC-Derived Muscle Progenitors Reveals Distinctive Markers for Myogenic Cell Purification Toward Cell Therapy." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/265184.

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京都大学 新制・課程博士 博士(医学) 甲第23412号 医博第4757号 新制||医||1052(附属図書館) 京都大学大学院医学研究科医学専攻 (主査)特定拠点教授妻木範行, 教授戸口田淳也, 教授松田秀一 学位規則第4条第1項該当 Doctor of Medical Science Kyoto University DFAM

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34

Kocharyan, Avetik. "Derivation and Characterization of Pax7 Positive Skeletal Muscle Precursor Cells from Control and HGPS-derived induced Pluripotent Stem Cells." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37517.

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Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder associated with premature aging in various tissues and organs of the afflicted individuals, including accelerated skeletal muscle atrophy. Classical HGPS manifests due to single-base substitution in the LAMNA gene which encodes Lamin A/C proteins. As a result of the mutation, a truncated form of Lamin (known as Progerin) is produced which undergoes persistent farnesylation during post-translational modification. Accumulation of Progerin in the nucleus has been linked to various cellular abnormalities including abnormal nuclear morphologies and altered chromatin organization, among others. However, the exact molecular mechanisms leading to skeletal muscle atrophy have not yet been elucidated. In this study, the iPSC approach was implemented in order to study the skeletal muscle phenotype of HGPS by generating and characterizing a population of Pax7 positive skeletal muscle precursor cells (SMPs). During the course of this project, we have demonstrated the need for excessive optimization of the previously developed directed differentiation protocol for successful application on induced Pluripotent Stem Cells. Furthermore, we have successfully modified the protocol to allow for a more rapid expansion of the SMPs through regular passaging of the myogenic cells starting on day 20 of differentiation. Additionally, this new method produced more uniform distribution of the myogenic cells and allowed for successful freezing/thawing of the myogenic cells. When compared to the controls, the HGPS-derived SMPs did not appear to be defective in formation, proliferation or differentiation. Abnormal nuclear morphology and DNA damage, documented in HGPS fibroblasts and vascular smooth muscle cells, were not detected the in myogenic cells. Furthermore, we were not able to detect Progerin protein accumulation in the generated myogenic cultures, offering an explanation for the absence of these phenotypes in the skeletal muscle system.

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35

Ding, Can. "The influence of Notch over-stimulation on muscle stem cell quiescence versus proliferation, and on muscle regeneration." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066399/document.

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La transplantation de cellules souches de muscle possède un grand potentiel pour la réparation à long terme du muscle dystrophique. Cependant, la croissance ex vivo des cellules souches musculaires réduit de manière significative l'efficacité de leur greffe puisque le potentiel myogénique est considérablement réduit lors de la mise en culture. La voie de signalisation Notch a émergé comme un régulateur majeur des cellules souches musculaires (MuSCs) et il a également été décrit que la sur-activation de Notch est crucial pour le maintien du caractère souche des MuSC. Cette découverte pourrait être traduite comme un bénéfice thérapeutique potentiel. Des MuSCs murines ont été fraîchement isolées et ensemencées sur des boîtes de culture recouverte de Dll1-Fc, le domaine extracellulaire de Delta-like-1 est fusionné au fragment Fc humain, afin d'activer la voie de signalisation Notch et avec un IgG hu-main comme contrôle. Nous avons utilisé le rAAV afin d’exprimer le Dll1 spécifique-ment dans les muscles de souris. Les souris P3 ont été traitées avec de l’AAV pendant 3 semaines et 6 semaines afin d’étudier l'effet de Dll1 au cours du développement postnatal. Afin d’étudier le processus de régénération, l'AAV a également été injecté dans les muscles de souris mdx alors que les souris de type sauvage ont été utilisées comme contrôle. Un potentiel caractère souche supérieur (marquée avec le Pax7) est observé dans les cultures des MuSCs qui sont recouverte de Dll1-Fc par rapport à leurs homologues contrôles, par contre le taux de proliférer est réduit. Au cours du développement postnatal, la sur-activation de la voie de signalisation Notch par Dll1 sur les fibres musculaires a été en mesure d'élargir le pool des cellules Pax7+, cependant elle entraîne une diminution de la masse musculaire avec réduction de la taille des fibres et ceci sans affecter l'accumulation des myonuclei. Dans les MuSCs quiescentes (de type sauvage), la sur-activation de la voie de signalisation Notch ne présente pas de réel effet. La surexpression de Dll1 dans le muscle mdx a diminué la masse musculaire et agrandit le pool de cellules souches musculaires, ce-pendant le taux de régénération n'a pas été affecté. L’augmentation des MuSCs est attribuée à une différenciation entravée des cellules souches musculaires. En étudiant la stimulation de la voie de signalisation Notch dans les MuSCs à la fois in vitro et in vivo, nous démontrons que sur-activation de Notch préserve le caractère souche des cellules via l’inhibition de la prolifération et de la différenciation myogénique des MuSCs Muscle stem cell transplantation possesses great potential for long-term repair of dys-trophic muscle. However expansion of muscle stem cells ex vivo significantly reduces their engraftment efficiency since the myogenic potential is dramatically lost in culture. The Notch signaling pathway has emerged as a major regulator of muscle stem cells (MuSCs) and it has recently been discovered that high Notch activity is crucial for maintaining stemness in MuSCs. This feature might be exploited and developed into a novel therapeutic approach.Murine MuSCs were freshly isolated and seeded on culture vessels coated with Dll1-Fc, which fused Delta-like-1 extracellular domain with human Fc, to activate Notch sig-naling and with human IgG as a control. The rAAV gene delivery system was em-ployed to express Dll1 in murine muscles. P3 mice were treated with AAV for 3 weeks and 6 weeks to investigate the effect of Dll1 during postnatal development. To investi-gate the regeneration process, AAV were injected into mdx muscles whereas wild-type mice were used as control.Higher potential stemness (marked by Pax7 positivity) was observed in MuSCs grow-ing on a Dll1-Fc surface as compared to their counterparts on the control surface, while their proliferation rate was reduced. During postnatal development, overstimulation of Notch signaling by Dll1 on the mus-cle fibers was able to enlarge the Pax7+ cell pool, while also resulting in decreased muscle mass and smaller muscle fibers without affecting the accretion of myonuclei into the fiber. In quiescent (wild-type) MuSCs, overstimulation of Notch signaling did not have any discernible effect. Overexpression of Dll1 in mdx muscle decreased the muscle mass and enlarged the muscle stem cell pool, while muscle regeneration re-mained unaffected. By investigating Notch stimulation in MuSCs both in vitro and in vivo, we demonstrate that high Notch activity preserves stemness via inhibition of MuSCs proliferation and myogenic differentiation. Our findings point out that the Dll1 molecule, as a canonical Notch ligand, might have a therapeutic potential in cell-based therapies against muscu-lar dystrophies

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36

Ding, Can. "The influence of Notch over-stimulation on muscle stem cell quiescence versus proliferation, and on muscle regeneration." Electronic Thesis or Diss., Paris 6, 2015. http://www.theses.fr/2015PA066399.

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La transplantation de cellules souches de muscle possède un grand potentiel pour la réparation à long terme du muscle dystrophique. Cependant, la croissance ex vivo des cellules souches musculaires réduit de manière significative l'efficacité de leur greffe puisque le potentiel myogénique est considérablement réduit lors de la mise en culture. La voie de signalisation Notch a émergé comme un régulateur majeur des cellules souches musculaires (MuSCs) et il a également été décrit que la sur-activation de Notch est crucial pour le maintien du caractère souche des MuSC. Cette découverte pourrait être traduite comme un bénéfice thérapeutique potentiel. Des MuSCs murines ont été fraîchement isolées et ensemencées sur des boîtes de culture recouverte de Dll1-Fc, le domaine extracellulaire de Delta-like-1 est fusionné au fragment Fc humain, afin d'activer la voie de signalisation Notch et avec un IgG hu-main comme contrôle. Nous avons utilisé le rAAV afin d’exprimer le Dll1 spécifique-ment dans les muscles de souris. Les souris P3 ont été traitées avec de l’AAV pendant 3 semaines et 6 semaines afin d’étudier l'effet de Dll1 au cours du développement postnatal. Afin d’étudier le processus de régénération, l'AAV a également été injecté dans les muscles de souris mdx alors que les souris de type sauvage ont été utilisées comme contrôle. Un potentiel caractère souche supérieur (marquée avec le Pax7) est observé dans les cultures des MuSCs qui sont recouverte de Dll1-Fc par rapport à leurs homologues contrôles, par contre le taux de proliférer est réduit. Au cours du développement postnatal, la sur-activation de la voie de signalisation Notch par Dll1 sur les fibres musculaires a été en mesure d'élargir le pool des cellules Pax7+, cependant elle entraîne une diminution de la masse musculaire avec réduction de la taille des fibres et ceci sans affecter l'accumulation des myonuclei. Dans les MuSCs quiescentes (de type sauvage), la sur-activation de la voie de signalisation Notch ne présente pas de réel effet. La surexpression de Dll1 dans le muscle mdx a diminué la masse musculaire et agrandit le pool de cellules souches musculaires, ce-pendant le taux de régénération n'a pas été affecté. L’augmentation des MuSCs est attribuée à une différenciation entravée des cellules souches musculaires. En étudiant la stimulation de la voie de signalisation Notch dans les MuSCs à la fois in vitro et in vivo, nous démontrons que sur-activation de Notch préserve le caractère souche des cellules via l’inhibition de la prolifération et de la différenciation myogénique des MuSCs Muscle stem cell transplantation possesses great potential for long-term repair of dys-trophic muscle. However expansion of muscle stem cells ex vivo significantly reduces their engraftment efficiency since the myogenic potential is dramatically lost in culture. The Notch signaling pathway has emerged as a major regulator of muscle stem cells (MuSCs) and it has recently been discovered that high Notch activity is crucial for maintaining stemness in MuSCs. This feature might be exploited and developed into a novel therapeutic approach.Murine MuSCs were freshly isolated and seeded on culture vessels coated with Dll1-Fc, which fused Delta-like-1 extracellular domain with human Fc, to activate Notch sig-naling and with human IgG as a control. The rAAV gene delivery system was em-ployed to express Dll1 in murine muscles. P3 mice were treated with AAV for 3 weeks and 6 weeks to investigate the effect of Dll1 during postnatal development. To investi-gate the regeneration process, AAV were injected into mdx muscles whereas wild-type mice were used as control.Higher potential stemness (marked by Pax7 positivity) was observed in MuSCs grow-ing on a Dll1-Fc surface as compared to their counterparts on the control surface, while their proliferation rate was reduced. During postnatal development, overstimulation of Notch signaling by Dll1 on the mus-cle fibers was able to enlarge the Pax7+ cell pool, while also resulting in decreased muscle mass and smaller muscle fibers without affecting the accretion of myonuclei into the fiber. In quiescent (wild-type) MuSCs, overstimulation of Notch signaling did not have any discernible effect. Overexpression of Dll1 in mdx muscle decreased the muscle mass and enlarged the muscle stem cell pool, while muscle regeneration re-mained unaffected. By investigating Notch stimulation in MuSCs both in vitro and in vivo, we demonstrate that high Notch activity preserves stemness via inhibition of MuSCs proliferation and myogenic differentiation. Our findings point out that the Dll1 molecule, as a canonical Notch ligand, might have a therapeutic potential in cell-based therapies against muscu-lar dystrophies

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37

Li, Xiang. "Mitochondrial transfer from induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial and smooth muscle cells attenuates oxidative stress-induced injury." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/58260.

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Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airflow limitation that is not fully reversible and is usually caused by cigarette smoke (CS). The disease is predicted to be the fourth leading cause of death by 2030, but none of the currently available treatments can alleviate the progressive decline in lung function. Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can be isolated from various tissues such as bone marrow (BM-MSCs). Despite numerous reports of their efficacy in COPD-related pre-clinical models, BM-MSCs have not demonstrated efficacy in a clinical trial of COPD, highlighting the need for improved MSC-based therapy. The in vitro derivation of MSCs from induced pluripotent stem cells (iPSCs) has provided a new source of MSCs. Compared to BM-MSCs, iPSC-derived MSCs (iPSC-MSCs) are a more abundant source, have a higher expanding capacity and are possibly not subject to the ageing-associated dysfunction seen in BM-MSCs. In this study I determined the effects of human iPSC-MSCs in a rat COPD model using BM-MSCs as comparison. Rats were exposed to CS for 1 hr/day for 56 days. iPSC-MSCs or BM-MSCs were administrated at days 29 and 43. iPSC-MSCs demonstrated superior effects over BM-MSCs in attenuating CS-induced lung airspace enlargement, fibrosis, inflammation and apoptosis. In a mouse model of ozone-induced lung damage, intravenous administration of iPSC-MSCs 24 hours before ozone exposure for 3 hours alleviated airway hyper-responsiveness, inflammation and apoptosis in the lung. There is increasing evidence demonstrating that mitochondrial dysfunction may play an important role in COPD pathogenesis, indicating mitochondria as a potential therapeutic target. Meanwhile, mitochondrial transfer from MSCs to injured airway cells has been reported as a novel mechanism of action for MSCs. In this study mitochondrial transfer from iPSC-MSCs to the airway epithelium of CS-exposed rats was detected. iPSC-MSCs also transferred mitochondria to bronchial epithelial BEAS-2B cells and primary airway smooth muscle cell (ASMCs) in vitro in a direct co-culture system, an effect that was enhanced by CS medium (CSM). Direct co-culture with iPSC-MSCs alleviated CSM-induced ATP deprivation in BEAS-2B cells, as well as CSM-induced mitochondrial reactive oxygen species (ROS), apoptosis and reduction of mitochondrial membrane potential (ΔΨm) in ASMCs. Administration of iPSC-MSCs also prevented ozone-induced mitochondrial ROS and ΔΨm reduction in mouse lungs. The paracrine effects of iPSC-MSCs were also investigated. iPSC-MSC-derived conditioned medium (iPSC-MSCs-CdM) protected BEAS2-B cells from CSM-induced apoptosis. The effect was reduced by depleting stem cell factor (SCF) from iPSC-MSCs-CdM. However, both iPSC-MSCs-CdM and trans-well inserts containing iPSC-MSCs were only able to alleviate CSM-induced mitochondrial ROS, but not ΔΨm reduction and apoptosis, in ASMCs. I demonstrated the capacity of iPSC-MSCs to alleviate oxidative stress-induced COPD phenotype in vivo. Mitochondrial transfer from iPSC-MSCs was able to alleviate oxidative stress-induced mitochondrial dysfunction and apoptosis in target cells. The full capacity of iPSC-MSCs to achieve these effects may rely on a combination of cell-cell contact and release of paracrine factors. These findings define iPSC-MSCs as a promising candidate for the development of MSCs-based therapy of COPD.

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38

Zhou, Lili. "The role of Lasp in the «Drosophila» male stem cell niche and in muscle development." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95064.

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Drosophila Lasp is the only member of the nebulin family in Drosophila. Lasp has an amino-terminal LIM domain, two actin-binding nebulin repeats and a carboxyl-terminal SH3 domain and exhibits very high homology to human Lasp. To assess Lasp function in vivo, we generated a null mutant in Drosophila Lasp, named Lasp1. Lasp1 mutants are homozygous viable, but male sterile. Lasp localizes to cyst cells, early germ cells, hub cells and actin cones. In Lasp1 mutants, the stem cell niche is no longer anchored to the apical tip of the testis, and actin cone migration is perturbed resulting in improper spermatid individualization. Lasp colocalizes with βPS integrin and genetically interact with βPS integrin resulting in complete hub cell mislocalization, which indicates that Lasp modulates integrin adhesion in this context. Lasp1 mutant larvae and flies also have impaired crawling, climbing and flying ability. Lasp localizes to Z lines of third instar larval body wall muscles. In Lasp1 mutant indirect flight muscle, thin filament and sarcomere length is reduced while sarcomere ultrastructure is not significantly affected. The same applies to larval body wall muscles, where we observe a misregulation of sarcomere length in both absence and overexpression of Lasp. This phenotype is very similar to nebulin mutant knock-out mice indicating that Lasp plays a role in regulating thin filament lengths, but with only two nebulin repeats. Chez la drosophile, Lasp est la seule protéine représentante de la famille des Nébuline. Lasp contient un domaine LIM, deux répétitions de type Nébuline et un domaine SH3, et présente une forte homologie avec la famille Lasp des mammifères. Afin identifier le rôle de Lasp, nous avons généré une mutation nulle, nommée Lasp1. Les mutants Lasp1 sont homozygotes viables, mais les mâles stériles. Lasp se localise dans cellules kyste, dans les cellules germinales, les cellules hub et au niveau des cônes d'actine. Chez les mutants Lasp1, les cellules souches ne sont plus ancré à l'extrémité apicale du testicule, et la migration des cônes d'actine est perturbée, conduisant à une individualisation irrégulière des spermatides. Lasp est colocalisée avec l'intégrine βPS et interagit génétiquement avec l'intégrine βPS, amenant une délocalization des cellules hub, indiquant que Lasp module adhésion intégrine dans ce contexte. Les larves mutantes pour Lasp se déplacent avec difficulté et les adultes ont avec une capacité d'escalade et de vols réduite. Lasp se localise aux lignes Z dans les muscles des larves du troisième stade. Chez les adultes Lasp1, les muscles des ailes présentent une longueur réduite des filaments minces ainsi que des sarcomères, alors que l'ultrastructure du sarcomère ne semble pas être significativement affectée. Les muscles larvaires présentent le phenotype. De plus, on observe un dérèglement de la longueur du sarcomère en surexprimant Lasp dans un contexte sauvage. Ce phénotype est très similaire à celui des souris mutantes pour la nébuline, indiquant que Lasp joue un rôle dans la régulation de la longueur du filament mince, mais avec seulement deux répétitions nébuline.

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39

Luk, Hui Ying. "Effect of the Resistance Exercise-Induced Hormonal Changes on Satellite Cell Myogenic State." Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1157528/.

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Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

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40

Neal, A. "Satellite cell subpopulations and environmental mediators of their function : implications for stem cell therapy in skeletal muscle." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1383594/.

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Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre (myofibre). Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. There is a need to investigate factors that enable satellite cell survival and/or proliferation post engraftment in order to obtain the optimal donor cell and host environment for efficient satellite cell transplantation. I have investigated sex differences in mouse satellite cell populations across the lifespan in vitro and in vivo. I show that satellite cell number and myogenic regulator factor expressions differ according to sex and developmental stage. Despite this, I show that engraftment efficiency is not mediated by the age or sex of the host or the donor. I hypothesise that there are two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. I have used high doses of ionising radiation to separate radio-resistant from radio-sensitive satellite cells. I demonstrate that radio resistant satellite cells do not contribute to growth, but are able to contribute to host muscle regeneration post transplantation and have compared their expression pro files using microarray. I hypothesise that satellite cells able to survive high dose ionizing radiation are the same population of satellite cells that are able to survive transplantation. Engraftment efficiency is greatly improved if host muscle is exposed to ionizing radiation prior to engraftment. I demonstrate that elimination of the host satellite cell pool is not sufficient to account for the improved engraftment efficiency with radiation and I have therefore investigated the role of the vasculature as a mediator of radiation induced improvement in engraftment efficiency.

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41

Mademtzoglou, Despoina. "Coordinating growth arrest and myogenesis in muscle stem cells : a molecular and cellular analysis." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066231/document.

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Ce travail de thèse a porté sur l'étude de l'équilibre entre la prolifération et la différenciation dans le cadre de la myogenèse embryonaire et postnatale. Chez l'embryon, le sortie du cycle cellulaire est contrôlé par p57 et p21 pendant la myogenèse. Nous avons montré que la voie de signalisation Notch ainsi que les facteurs de régulation myogéniques (MRFs) régulent l'expression de p57 dans les cellules progénitrices et les myoblastes en différentiation. Chez l'adulte, p21 et p57 ne sont pas exriés dans la population quiescente de cellules souches du muscle (cellules satellites - SCs). p21 et p57 sont rapidement induits après activation et durant la différentiation des SCs. Ex vivo, les myoblastes déficients pour le gène p21 présentent des défauts de prolifération et de différenciation. In vivo, l'étude de la régénération musculaire chez les mutants p21 a montré une réduction précoce des SCs, avec un retard de reconstitution du tissu musculaire. Afin de pouvoir étudier le rôle de p57 après la naissance (les mutants p57 meurent à la naissance) nous avons généré un modèle murin permettant de muter le gène p57 de manière spatio-temporelle avec le système de recombinaison Cre/LoxP. Nous avons combiner notre allèle p57 conditionnel avec une Cre exprimée de manière ubiquitaire, et observé des phénotypes identiques aux phénotypes décrits précédemment chez les souris présentant une perte du p57. L'ablation conditionnelle de p57 dans les SCs adultes, a conduit à une diminution de la différenciation myogénique in vitro. Notre travail suggère que p21 et p57 jouent un rôle important dans la régulation de la différenciation et le cycle cellulaire dans le muscle adulte This thesis focuses on the coordination of proliferation and differentiation in embryonic and adult myogenesis. During development, we demonstrated that skeletal muscle progenitors interact with the differentiating myoblasts via the Notch pathway to maintain their pool. It has previously been established that p57 and p21 redundantly promote cell cycle exit in developing muscle and we showed that Notch and Myogenic Regulatory Factors act through muscle-specific regulation of p57. We then examined p21 and p57 in adult skeletal muscle stem cells, called satellite cells (SCs). Although absent from quiescent SCs, p21 and p57 are expressed upon activation (including proliferating myoblasts) and differentiation. p21-null myoblasts exhibited proliferation and differentiation defects in myofiber cultures, implicating p21 at the early activation phase. In vivo muscle regeneration studies with p21 mutants showed an early impact on the SC pool, while SCs and muscle structure were re-established by the end of regeneration. Since p57-deficient mice die at birth, we generated a conditional knock-out (KO) allele for postnatal studies using the loxP/Cre recombination system. With a ubiquitous Cre we observed developmental and perinatal phenotypes similar to previously described KO embryos. The new p57 allele also includes a β-galactosidase reporter and we showed that it recapitulates p57 expression profile in embryonic and adult tissues. Conditional ablation of p57 in adult SCs reduced myogenic differentiation in primary myoblast culture. Our work suggests that p21 and p57 are involved in adult myogenesis and cell cycle exit, working at the early steps of satellite cell activation

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42

Lý, Hà My. "Characterization of a novel molecular pathway linking metabolic regulation and muscle stem cell fate." Electronic Thesis or Diss., Lyon 1, 2024. http://www.theses.fr/2024LYO10193.

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Le muscle strié squelettique possède une capacité de régénération grâce à une population de cellules souches musculaires (CSM ou cellules satellites) qui gardent une capacité à exprimer un programme myogénique lors d'une lésion musculaire. Une perte de la capacité myogénique des CSM est associée à la perte musculaire avec l'âge, mais aussi à des maladies invalidantes telles que les myopathies. Une des caractéristiques de la fonction des CSMs est leur plasticité métabolique qui supporte le programme myogénique. Sur la base de travaux antérieurs du laboratoire mettant en évidence le rôle central du régulateur métabolique AMPK dans la myogenèse adulte, j'ai identifié que NUAK1, une kinase liée à l'AMPK dont la fonction est associée à la régulation métabolique dans les neurones en développement, est un nouvel acteur dans la régulation du programme des CSMs et de la myogenèse adulte. L'activité de NUAK1 est nécessaire à la capacité de régénération musculaire et NUAK1 contrôle les aspects séquentiels du programme myogénique, c'est-à-dire l’engagement, la différenciation et la fusion des CSMs pour produire des myofibres matures et le renouvellement des CSMs. A l’échelle moléculaire, mes données préliminaires suggèrent que NUAK1 agit par la régulation du métabolisme des acides gras et de la biogenèse du cholestérol. Pendant ma doctorat, je confirme ce lien et décrypterai comment le facteur de transcription SREBP1, qui contrôle les voies métaboliques des lipides, intervient dans les fonctions de NUAK1 lors de la myogenèse adulte. Cette approche-candidat complétes par une approche non biaisée visant à identifier les changements du protéome et du fonctionnement mitochondrial. Par conséquent, mon travail de doctorat démontres un lien original entre le métabolisme des lipides et le devenir des CSMs, ce qui permettra d’identifier des cibles thérapeutiques potentielles dans les maladies du muscle Muscle stem cells (MuSCs) provide a constant regenerative capacity to the skeletal muscle owing to their capacity to recapitulate a myogenic program upon muscle lesion. Alterations of MuSC myogenic capacity are associated to muscle loss with age, but also debilitating diseases such as myopathies. Metabolic plasticity is a hallmark of MuSC fate program. Based on previous work from the lab highlighting a central role for the metabolic regulator kinase AMPK in adult myogenesis, I adopted a candidate-based study that identified NUAK1, an AMPK-related kinase whose function is associated to metabolic regulation in developing neurons, as a novel player in the regulation of MuSC fate and adult myogenesis. NUAK1 expression is required for muscle regeneration capacity and NUAK1 controls sequential aspects of the myogenic program, ie. MuSC commitment and differentiation into myoblast, myoblast fusion to produce mature myofibers, and MuSC renewal. On a molecular level, I obtained exciting preliminary data suggesting that NUAK1 acts through the regulation of fatty acids and cholesterol biogenesis. During my PhD, I confirm this link and decipher how the transcription factor SREBP1, which controls lipid metabolic pathways, mediates the functions of NUAK1 during adult myogenesis. This candidate-based study completes by an unbiased approach aiming at identifying changes in mitochondrial metabolism. Overall, my PhD work demonstrate an original link between lipids metabolism and MuSC fate, which will give hints toward putative targets of therapeutic value

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43

Pala, Francesca. "Metabolic characterisation of skeletal muscle stem cells in distinct physiological states." Electronic Thesis or Diss., Paris 6, 2017. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2017PA066607.pdf.

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Les cellules souches musculaires, ou cellules satellites, adoptent différents états en transitant de quiescence à prolifération et différentiation. Ces transitions s'accompagnent de variations des demandes énergétiques. Il demeure cependant incertain comment la modulation du métabolisme énergétique peut dicter la spécification d'un état cellulaire donné. Mon projet de thèse a eu pour objectif principal la caractérisation des voies du métabolisme énergétique à l’œuvre dans les différents états cellulaires, et comment leur modulation peut influencer ces états. Nous montrons ainsi que les cellules satellites quiescentes ont de faibles besoins énergétiques et que la phosphorylation oxydative est altérée au cours du vieillissement ainsi que dans les cellules survivant après la mort de l'animal. Au cours de la formation du tissu en croissance ou en régénération chez l'adulte, nos résultats indiquent de larges différences dans leurs demandes énergétiques. Les cellules fœtales ont une faible demande respiratoire et reposent essentiellement sur la glycolyse par rapport aux cellules adultes en cours de régénération. L'altération de la b-oxidation peroxisomale et non mitochondriale induit une différentiation précoce des cellules satellites. L'inhibition pharmacologique des b-oxidations peroxisomale et mitochondriale après blessure aiguë montre différentes contributions de ces organelles à la régénération musculaire. Les transitions entre différents états des cellules satellites s'accompagnent de modifications drastiques de leurs besoins énergétiques et l'altération de vois métaboliques spécifiques peut altérer le destin des cellules myogéniques et la régénération musculaire Muscle stem (satellite, MuSC) cells acquire different cell states as they need to pass from quiescence to proliferation and differentiation to support muscle homeostasis. Some of these changes are accompanied by changes in energy demands. However, it is currently unclear whether modulation in the energy metabolism pathways can in turn influence the commitment to a specific cell state. A central focus of my thesis project is to characterise the energy metabolism pathways that act in the different phases of lineage progression and how their modulation can influence the state of the cell. We show that quiescent cells have low energetic demands and OxPhos is perturbed during aging, as well as in cells that survive after death. We also compared different proliferative states, both during muscle growth and regeneration, and our results indicate a surprising difference in their metabolic requirements. Gene expression profiling and bioenergetics analysis showed that foetal cells have a low respiration demand and rely mostly on glycolysis when compared to regenerating MuSCs. Furthermore, we show distinct requirements for peroxisomal and mitochondrial mediated fatty acid oxidation (FAO) in myogenic cells. Altering peroxisomal but not mitochondrial FAO promotes early differentiation of satellite cells. Experiments using acute muscle injury and pharmacological block show differential requirements for these organelles during regeneration. These observations indicate that changes in the cell state of muscle stem cells lead to significant changes in metabolic requirements and altering specific metabolic pathways can have an impact on myogenic cell fate and the regeneration process

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44

Pala, Francesca. "Metabolic characterisation of skeletal muscle stem cells in distinct physiological states." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066607/document.

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Les cellules souches musculaires, ou cellules satellites, adoptent différents états en transitant de quiescence à prolifération et différentiation. Ces transitions s'accompagnent de variations des demandes énergétiques. Il demeure cependant incertain comment la modulation du métabolisme énergétique peut dicter la spécification d'un état cellulaire donné. Mon projet de thèse a eu pour objectif principal la caractérisation des voies du métabolisme énergétique à l’œuvre dans les différents états cellulaires, et comment leur modulation peut influencer ces états. Nous montrons ainsi que les cellules satellites quiescentes ont de faibles besoins énergétiques et que la phosphorylation oxydative est altérée au cours du vieillissement ainsi que dans les cellules survivant après la mort de l'animal. Au cours de la formation du tissu en croissance ou en régénération chez l'adulte, nos résultats indiquent de larges différences dans leurs demandes énergétiques. Les cellules fœtales ont une faible demande respiratoire et reposent essentiellement sur la glycolyse par rapport aux cellules adultes en cours de régénération. L'altération de la b-oxidation peroxisomale et non mitochondriale induit une différentiation précoce des cellules satellites. L'inhibition pharmacologique des b-oxidations peroxisomale et mitochondriale après blessure aiguë montre différentes contributions de ces organelles à la régénération musculaire. Les transitions entre différents états des cellules satellites s'accompagnent de modifications drastiques de leurs besoins énergétiques et l'altération de vois métaboliques spécifiques peut altérer le destin des cellules myogéniques et la régénération musculaire Muscle stem (satellite, MuSC) cells acquire different cell states as they need to pass from quiescence to proliferation and differentiation to support muscle homeostasis. Some of these changes are accompanied by changes in energy demands. However, it is currently unclear whether modulation in the energy metabolism pathways can in turn influence the commitment to a specific cell state. A central focus of my thesis project is to characterise the energy metabolism pathways that act in the different phases of lineage progression and how their modulation can influence the state of the cell. We show that quiescent cells have low energetic demands and OxPhos is perturbed during aging, as well as in cells that survive after death. We also compared different proliferative states, both during muscle growth and regeneration, and our results indicate a surprising difference in their metabolic requirements. Gene expression profiling and bioenergetics analysis showed that foetal cells have a low respiration demand and rely mostly on glycolysis when compared to regenerating MuSCs. Furthermore, we show distinct requirements for peroxisomal and mitochondrial mediated fatty acid oxidation (FAO) in myogenic cells. Altering peroxisomal but not mitochondrial FAO promotes early differentiation of satellite cells. Experiments using acute muscle injury and pharmacological block show differential requirements for these organelles during regeneration. These observations indicate that changes in the cell state of muscle stem cells lead to significant changes in metabolic requirements and altering specific metabolic pathways can have an impact on myogenic cell fate and the regeneration process

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45

Mademtzoglou, Despoina. "Coordinating growth arrest and myogenesis in muscle stem cells : a molecular and cellular analysis." Electronic Thesis or Diss., Paris 6, 2016. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2016PA066231.pdf.

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Ce travail de thèse a porté sur l'étude de l'équilibre entre la prolifération et la différenciation dans le cadre de la myogenèse embryonaire et postnatale. Chez l'embryon, le sortie du cycle cellulaire est contrôlé par p57 et p21 pendant la myogenèse. Nous avons montré que la voie de signalisation Notch ainsi que les facteurs de régulation myogéniques (MRFs) régulent l'expression de p57 dans les cellules progénitrices et les myoblastes en différentiation. Chez l'adulte, p21 et p57 ne sont pas exriés dans la population quiescente de cellules souches du muscle (cellules satellites - SCs). p21 et p57 sont rapidement induits après activation et durant la différentiation des SCs. Ex vivo, les myoblastes déficients pour le gène p21 présentent des défauts de prolifération et de différenciation. In vivo, l'étude de la régénération musculaire chez les mutants p21 a montré une réduction précoce des SCs, avec un retard de reconstitution du tissu musculaire. Afin de pouvoir étudier le rôle de p57 après la naissance (les mutants p57 meurent à la naissance) nous avons généré un modèle murin permettant de muter le gène p57 de manière spatio-temporelle avec le système de recombinaison Cre/LoxP. Nous avons combiner notre allèle p57 conditionnel avec une Cre exprimée de manière ubiquitaire, et observé des phénotypes identiques aux phénotypes décrits précédemment chez les souris présentant une perte du p57. L'ablation conditionnelle de p57 dans les SCs adultes, a conduit à une diminution de la différenciation myogénique in vitro. Notre travail suggère que p21 et p57 jouent un rôle important dans la régulation de la différenciation et le cycle cellulaire dans le muscle adulte This thesis focuses on the coordination of proliferation and differentiation in embryonic and adult myogenesis. During development, we demonstrated that skeletal muscle progenitors interact with the differentiating myoblasts via the Notch pathway to maintain their pool. It has previously been established that p57 and p21 redundantly promote cell cycle exit in developing muscle and we showed that Notch and Myogenic Regulatory Factors act through muscle-specific regulation of p57. We then examined p21 and p57 in adult skeletal muscle stem cells, called satellite cells (SCs). Although absent from quiescent SCs, p21 and p57 are expressed upon activation (including proliferating myoblasts) and differentiation. p21-null myoblasts exhibited proliferation and differentiation defects in myofiber cultures, implicating p21 at the early activation phase. In vivo muscle regeneration studies with p21 mutants showed an early impact on the SC pool, while SCs and muscle structure were re-established by the end of regeneration. Since p57-deficient mice die at birth, we generated a conditional knock-out (KO) allele for postnatal studies using the loxP/Cre recombination system. With a ubiquitous Cre we observed developmental and perinatal phenotypes similar to previously described KO embryos. The new p57 allele also includes a β-galactosidase reporter and we showed that it recapitulates p57 expression profile in embryonic and adult tissues. Conditional ablation of p57 in adult SCs reduced myogenic differentiation in primary myoblast culture. Our work suggests that p21 and p57 are involved in adult myogenesis and cell cycle exit, working at the early steps of satellite cell activation

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46

Rigon, Matteo [Verfasser], and Rüdiger [Akademischer Betreuer] Rudolf. "Stem cell-induced regeneration of skeletal muscle tissue: characterization of a glycerol-induced muscle damage model / Matteo Rigon ; Betreuer: Rüdiger Rudolf." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1227585527/34.

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47

Girardi, Francesco. "TGFbeta signalling pathway in muscle regeneration : an important regulator of muscle cell fusion." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS114.

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La régénération musculaire s’appuie sur une réserve de cellules souches résidant dans le muscle appelées cellules satellites (MuSCs). Les MuSCs sont quiescentes et peuvent s’activer à la suite d’une blessure du muscle afin de former des progéniteurs amplificateurs (myoblastes) qui se différencieront et fusionneront pour former de nouvelles myofibres. Durant ce processus, un réseau complexe de voies de signalisation est impliqué, parmi lequel la signalisation du facteur de croissance transformant bêta (TGFβ) joue un rôle fondamental. Précédents rapports ont proposé de nombreuses fonctions pour la signalisation TGFβ dans les cellules musculaires, comme leur quiescence, activation et différenciation, mais l’impact de TGFβ sur la fusion de myoblastes n’a jamais été étudié. Dans cette étude, nous avons montré que cette signalisation réduit la fusion des cellules musculaires indépendamment de leur différenciation. Au contraire, l’inhibition de la signalisation TGFβ accroît la fusion cellulaire et favorise les ramifications entre myotubes. Une pharmaco-modulation de la voie in vivo perturbe la régénération musculaire après blessure. Une addition exogène de la protéine TGFβ conduit à une perte de fonction du muscle, tandis que l’inhibition de la voie induit la formation de myotubes géants. Les analyses transcriptomiques et fonctionnelles ont montré que TGFβ agit sur la dynamique de l’actine afin de réduire la diffusion cellulaire à travers une modulation des protrusions à base d’actine. Nos résultats ont donc révélé une voie de signalisation qui limite la fusion de myoblastes et ajoutent un nouveau niveau de compréhension sur la régulation moléculaire de la myogenèse Muscle regeneration relies on a pool of muscle-resident stem cells called satellite cells (MuSCs). MuSCs are quiescent and can activate following muscle injury to give rise to transient amplifying progenitors (myoblasts) that will differentiate and finally fuse together to form new myofibers. During this process, a complex network of signalling pathways is involved, among which, Transforming Growth Factor beta (TGFβ) signalling cascade plays a fundamental role. Previous reports proposed several functions for TGFβ signalling in muscle cells including quiescence, activation and differentiation. However, the impact of TGFβ on myoblast fusion has never been investigated. In this study, we show that TGFβ signalling reduces muscle cell fusion independently of the differentiation step. In contrast, inhibition of TGFβ signalling enhances cell fusion and promotes branching between myotubes. Pharmacological modulation of the pathway in vivo perturbs muscle regeneration after injury. Exogenous addition of TGFβ protein results in a loss of muscle function while inhibition of the TGFβ pathway induces the formation of giant myofibres. Transcriptome analyses and functional assays revealed that TGFβ acts on actin dynamics to reduce cell spreading through modulation of actin-based protrusions. Together our results reveal a signalling pathway that limits mammalian myoblast fusion and add a new level of understanding to the molecular regulation of myogenesis

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48

Libergoli, Michela. "CD90 marks satellite cells into two subpopulations with distinct dynamics of activation and proliferation." Doctoral thesis, Università degli studi di Trento, 2021. http://hdl.handle.net/11572/323156.

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Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

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49

Hori, Shimpei. "PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice." Kyoto University, 2019. http://hdl.handle.net/2433/245311.

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50

Awaya, Tomonari. "Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells." Kyoto University, 2013. http://hdl.handle.net/2433/180602.

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