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

O envelhecimento é caracterizado por diversas alterações no organismo, as quais acarretam em fragilidade, maior susceptibilidade a quedas, perda de autonomia e piora da qualidade de vida. O músculo esquelético também é afetado pelo envelhecimento, levando a alterações na locomoção, adaptação metabólica e em sua plasticidade. Alterações na plasticidade - prejudicam a capacidade regenerativa do músculo esquelético, desencadeando modificações em todos os estágios desse processo. Uma estratégia que tem sido bastante utilizada para minimizar/reverter o impacto do envelhecimento na função e plasticidade muscular é o treinamento físico aeróbico (TFA), o qual promove diversos benefícios à musculatura esquelética. Dessa forma, na presente dissertação investigamos a contribuição do TFA de 4 semanas em esteira rolante na capacidade regenerativa do músculo tibial anterior de camundongos jovens e idosos após lesão mecânica. A capacidade regenerativa foi avaliada por métodos histológicos e de imunofluorescência em tecido aos 2, 4 e 15 dias após a indução da lesão mecânica. Os níveis de RNAm de fatores relacionados à resposta regenerativa muscular foram avaliados por PCR em tempo real. Para confirmar a eficácia do TFA e função muscular, avaliamos a capacidade aeróbica, a deambulação e a produção de força ex vivo. Observou-se que o TFA melhorou a função muscular e a capacidade aeróbica dos animais jovens e idosos. No que diz respeito ao processo de regeneração muscular, os resultados obtidos sugerem, aumento da área necrótica, da inflamação, da deposição de colágeno e redução da área de secção transversa das fibras nos animais idosos sedentários ao longo do curso temporal estudado. Adicionalmente, observou-se redução na expressão de genes envolvidos na ativação de células satélites e atraso no processo de diferenciação dessas células nesses animais. OTFA contribuiu para a redução da área necrótica, da inflamação, levando a menor deposição de colágeno e aumento da distribuição das fibras centro nucleadas nos animais idosos. No entanto, não se observou modificações na expressão dos genes com o TFA nesses animais. Portanto, os dados sugerem que o TFA contribui para melhora do processo de regeneração muscular em camundongos idosos / Aging is a biological process characterized by a progressive impairment in physiological systems, which leads to general frailty and reduced exercise tolerance and performance in daily living activities. Skeletal muscle is directly affected by aging, displaying changes in locomotion, metabolic adaptation, and muscle plasticity. Altered muscle plasticity affects muscle regeneration capacity in elderly. Aerobic exercise training (AET) has been used as a strategy to minimize/reverse the impact of aging on muscle function and regenerative function. Thus, we have investigated the contribution of 4-week AET (running on the treadmill) for tibialis anterior muscle regenerative response from mechanical injury in young and old muscle, which were randomly assigned into untrained and trained groups. The regenerative capacity was evaluated by histology and immunofluorescence at 2, 4 and 15 days after the mechanical injury induction. Muscle mRNA levels of regulatory genes involved in muscle regeneration were evaluated by real time PCR. To verify the effectiveness of AET and muscle function, we assessed the aerobic capacity, step length in ambulation test and ex vivo muscle force production. We observed that AE improved muscle function and aerobic capacity of young and old mice. Regarding the muscle regeneration process, our data suggest an increase in necrotic area, inflammation and collagen deposition paralleled by a reduced fiber cross sectional area in sedentary old mice. These responses were associated with changes in gene expression suggesting reduced satellite cells activation and delayed differentiation. AET contributed to reduction in both necrotic area and inflammation, leading to reduced collagen deposition and increased centronucleated fibers, suggesting improved regeneration process. However no changes were observed in mRNA levels of genes studied after AET. Altogether, our data provide evidence for AET improved regeneration process in muscle of old mice

Le muscle strié squelettique possède de grandes capacités de régénération grâce à ses cellules souches, les cellules satellites. Après une lésion, le processus de régénération musculaire qui se met en place est finement régulé dans le temps et l’espace par le microenvironnement, constitué de cellules avoisinantes mais également par des éléments de la matrice extracellulaire (MEC). Cette dernière se compose de molécules structurales comme les collagènes et de composants possédant un rôle trophique comme les glycosaminoglycanes (GAGs). La MEC musculaire est peu étudiée à cause d’une organisation tridimensionnelle complexe rendant son exploration difficile. Lors d’une lésion avec perte de substance musculaire, la régénération est altérée, associée à une fibrose et une inflammation chronique. Ce type de lésion est fréquemment rencontré en traumatologie mais survient également chez le blessé de guerre. Malgré un traitement optimal, une invalidité fonctionnelle persiste chez ces patients. L’utilisation d’un biomatériau décellularisé, constitué de MEC pourrait fournir ce support physique et trophique faisant défaut dans ce type de lésion. Dans ce travail, nous avons entrepris l'établissement d'une MEC d’origine musculaire et nous avons établi un protocole de décellularisation permettant d’obtenir un biomatériau conservant l’architecture spécifique de la MEC musculaire avec une élimination de la majorité des antigènes cellulaires afin d'éviter une réponse immunitaire délétère après implantation. Néanmoins, le protocole retenu ne permet de conserver certaines molécules trophiques d’intérêt comme les GAGs. Les « ReGeneRaTing Agent®» (RGTA®) sont des mimétiques fonctionnels de ces GAGs, utilisés en clinique pour améliorer la cicatrisation cutanée et cornéenne. Ces mimétiques conservent une capacité de liaison aux facteurs de croissance avec une résistance aux dégradations enzymatiques. Nous avons évalué l’utilisation de ces molécules au cours de la réparation musculaire, dans un modèle in vivo chez le rongeur. Nous avons réalisé une analyse histologique précoce (8e jour de régénération) mettant en évidence une augmentation du nombre de noyaux par myofibre en faveur d’une augmentation de la fusion, validée également in vitro sur des progéniteurs musculaires. Nous avons également observé une augmentation du nombre de vaisseaux, suggérant une amélioration de l’angiogenèse. Le nombre de gouttelettes lipidiques, marqueur d’une mauvaise régénération, était en diminution. L’exploration histologique plus tardive (28e jour de régénération) n’a retrouvé que l’augmentation du nombre de vaisseaux en faveur d’un effet durable sur l’angiogenèse. Ces RGTA® peuvent être couplés aux biomatériaux et sont particulièrement résistants dans un environnement inflammatoire pouvant être rencontré dans les lésions avec perte de substance musculaire. Des chimiokines et des facteurs de croissance pourront également être ajoutés au biomatériau matriciel afin de favoriser la migration des différents progéniteurs nécessaires à une néoformation musculaire. L’efficacité thérapeutique de ces biomatériaux optimisés nécessitera d’être évaluée dans un modèle in vivo de perte de substance / Skeletal muscle exhibits high capacity for regeneration after an injury that relies on resident stem cells. Muscle regeneration is tightly regulated by both the immune response and other resident cells, as well as by cues from the local extracellular matrix (ECM), contributing to a coordinated repair process. Muscle ECM is a network of structural macromolecules with a large majority of collagens and trophic molecules such as glycosaminoglycans (GAGs). In the skeletal muscle tissue, ECM was overlooked due to its complex organization making investigations difficult. Muscle regenerative ability can be overtaken in large muscle wasting, such as in volumetric muscle loss (VML), leading to fibrosis formation and chronic inflammation. This type of injury predominantly occurs in traumatology and in war-wounded patients, with functional disability despite an optimal treatment. The use of biomaterials could provide the biochemical and physical cues that are missing in this pathologic repair. In this work we have focused on obtaining a biomaterial composed of skeletal muscle ECM. We have tested several decellularization protocols both to preserve the three-dimensional architecture of the muscle ECM and to completely remove cell components in order to avoid a deleterious immune response after implantation. However, the protocol did not allow the preservation of trophic molecules such as GAGs, in the scaffold.“ReGenerating Agents” (RGTA®) are functionally analogous of GAGs with a crucial property to resist enzymatic degradation. They function to restore a proper microenvironment for tissue healing with already a clinical application in skin and corneal repair. We have explored the effects of RGTA® in muscle regeneration using an in vivo model in mouse. At early time of regeneration (day 8), we performed histologic analysis. We showed that regenerating myofibers contained more nuclei in the treated animals, in favor of an increase of progenitor fusion, which has been validated in vitro in myogenic cultures. The number of capillaries was higher in favor of a better angiogenesis. Lipid droplets, a marker of impaired regeneration, were reduced by RGTA® administration. At later time of regeneration (day 28), capillary number was still improved in favor of a durable effect of RGTA® on angiogenesis. RGTA® could be incorporated into biomaterials and are particularly resistant in an inflammatory environment, such as that occurring after a VML injury. Chemokines and growth factors could also be added in ECM-based scaffolds to promote the migration of progenitors that are essential for myofiber neoformation. Therapeutic efficacy of these optimized biomaterials will require to be evaluated in an in vivo model of VML

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

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24637号 / 工博第5143号 / 新制||工||1982(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

<p>Obesity is associated with an increase in low grade systemic inflammation. Skeletal muscle of individuals with obesity undergo numerous biochemical and morphological alterations including an increase in ectopic lipid accumulation in skeletal muscle and increased macrophage infiltration. Increased intermuscular adipose tissue and macrophages contribute to skeletal muscle inflammation and insulin resistance by secreting elevated proinflammatory cytokines and lipids. This also contributes to reduction in skeletal muscle quality, increasing the susceptibility of muscle to damage and impairing the regenerative response to muscle. Exercise training can reduce inflammation and improve skeletal muscle quality. Importantly reductions in inflammation occur without change in adiposity. Peroxisome proliferator activated receptor g coactivator 1-a (PGC-1a) exerts protective effects on skeletal muscle against damaging insults and may improve muscle regeneration.</p><p> The primary aim of my dissertation was to determine the mechanisms that lead to deficits in skeletal muscle integrity and regeneration in persons with obesity. In Chapter 1, an introduction to the various physiological, pathological, and clinical topics is provided. In Chapter 2, we investigated how exercise training and obesity independently alter skeletal muscle extracellular vesicle (EV) miRNA (miR) content. We found that obesity alters EV miR content indicative of altered anabolic signaling, while exercise training altered EV miR content in a manner indicative of reduced inflammation. In Chapter 3, we report that overexpression of PGC-1a reduces cardiotoxin induced damage of primary human myotubes but limits the ability of undifferentiated cells to reenter the cell cycle and produce progeny that could aid in the restoration of myotubes. In Chapter 4, we demonstrate that exposure to an obesogenic environment increases cardiotoxin induced damage of primary human myotubes from obese donors. In this study we also found that the restoration of myotube fusion index was reduced in lean and obese subjects when incubated with obesogenic media. In Chapter 5 is a review and summary of the outcomes described in Chapters 2-4, a discussion of the limitations of these experiments, and a discussion of future directions.</p>

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.

No description available.

Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene resulting in the loss of the dystrophin protein from the plasma membrane of muscle fibers. In the absence of dystrophin, muscles undergo massive muscle degeneration and inflammation. Inflammation is believed to contribute substantially to dystrophic muscle pathology. The transcription factor NF-κB regulates inflammatory gene expression and provides a logical target for therapeutic treatments. Green tea extract and its primary polyphenol, epigallocatechin gallate, have been shown to have anti-inflammatory properties and to improve dystrophic muscle pathology. The purpose of these studies was to determine if dietary treatment with green tea extract or epigallocatechin gallate administered prior to disease onset could reduce dystrophic muscle pathology during the early disease time course and identify potential mechanisms through which NF-κB may be involved. Green tea extract has been shown to decrease muscle pathology and increase muscle function in mdx mice, a dystrophic mouse model. These changes have been attributed to the antioxidant potential of epigallocatechin gallate; however, other mechanisms such as suppression of the inflammatory response have not been evaluated. In the studies reported herein, both green tea extract and epigallocatechin gallate significantly decreased muscle pathology in mdx mice when provided in their diets prior to disease onset. In green tea extract (0.25% and 0.5%) treated mdx mice, serum creatine kinase, a systemic marker of muscle damage, was decreased by 85% at age 42 days. Normal fiber morphology in the tibialis anterior muscle was increased by 32% at this age (P≤0.05). The primary histopathological change was a 21% decrease in regenerating fibers (P≤0.05). NF-κB staining in central nuclei of regenerating fibers was decreased by 34% (P≤0.05). In epigallocatechin gallate (0.1%) treated mdx mice, serum creatine kinase was unchanged; however, normal fiber morphology in the tibialis anterior was increased by 20% at ages 28 and 42 days (P≤0.05). At age 42 days, the primary histopathological change was a 21% decrease in regenerating fibers (P≤0.05). NF-κB staining in central nuclei of regenerating muscle fibers was decreased by 21% at this age (P≤0.05). Epigallocatechin gallate appears to be the primary polyphenol of green tea extract responsible for many of the beneficial changes in dystrophic muscle. These data suggest that both green tea extract and epigallocatechin gallate decrease NF-κB activity in regenerating fibers resulting in reduced muscle pathology. Complimentary and alternative medicine approaches, including the use of green tea, provide important therapeutic options for ameliorating Duchenne muscular dystrophy. Green tea extract and epigallocatechin gallate are effective at decreasing muscle pathology potentially by reducing NF-κB activity in regenerating fibers in mdx mice. Use of these botanicals appears to elicit a beneficial response in dystrophic muscle that may ultimately lead to effective therapies for patients with this incurable disease. / Ph. D.

O objetivo deste trabalho foi contribuir para o melhor entendimento dos mecanismos intracelulares envolvidos na regeneração muscular esquelética, através do estudo da influência das proteínas de choque térmico (HSPs) e do mTORC1 (mammalian target of rapamycin complex 1) no processo regenerativo muscular. O tratamento com radicicol (indutor de HSPs) em músculos lesados induziu aumento da área de secção transversal das fibras musculares em 10 e 21 dias após lesão e aumento do número de células satélites e de fibras musculares em diferenciação em 1 e 10 dias após lesão, respectivamente, quando comparado aos seus respectivos controles apenas lesados. O tratamento com rapamicina (inibidor de mTORC1) em músculos lesados induziu uma diminuição maior da área de secção transversal das fibras musculares em 10 e 21 dias após lesão e menor síntese protéica muscular em 10 dias após lesão quando comparado aos músculos somente lesados. Nossos resultados sugerem que as HSPs e o mTORC1 são importantes para o processo de regeneração muscular esquelética. / The goal of this work was to contribute to a better understanding about the intracellular mechanisms involved in skeletal muscle regeneration by studying the influence of heat shock proteins (HSPs) and mTORC1 (mammalian target of rapamycin complex 1) in the muscle regeneration process. The treatment with radicicol (a HSP inductor) in injured muscles induced increase of myofiber cross section area at 10 and 21 days post lesion and increased number of satellite cells and differentiating myofibers at 1 and 10 days post lesion, respectively, when compared to their respective injured controls. The treatment with rapamycin (a mTORC1 inhibitor) in injured muscles induced a more accentuated decrease in myofiber cross section area at 10 and 21 days post lesion and decreased muscle protein synthesis at 10 days post lesion when compared to only-injured muscles. Our results suggest that HSPs and mTORC1 are important to the process of skeletal muscle regeneration.