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.

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.

La remarquable capacité de croissance musculaire des poissons est liée notamment à leur capacité à produire durablement de nouvelles fibres (hyperplasie). Nous avons cherché à caractériser au niveau cellulaire et moléculaire les propriétés intrinsèques des précurseurs myogéniques de muscle hyperplasique de la truite. Ainsi, nous avons comparé la prolifération in situ, le comportement in vitro, et les transcriptomes (ARN messagers et microARNs) de précurseurs myogéniques de muscle hyperplasique de truites juvéniles (JT) et de muscles non-hyperplasiques de truites juvéniles soumises à un jeûne prolongé (FJT) ou de truites adultes (AT). Nous avons observé un fort taux de prolifération des cellules satellites au sein du muscle hyperplasique comparé au muscle non-hyperplasique.L’analyse du transcriptome montre que les gènes surexprimés dans les précurseurs du muscle hyperplasique (JT) sont principalement impliqués dans l'activité mitochondriale, le cycle cellulaire et la différenciation myogénique. L’analyse du miRnome a mis en évidence des miRNA surexprimés chez les JT dont les cibles potentielles régulent la prolifération ou la différenciation. En accord avec ces résultats, les précurseurs myogéniques JT présentent in vitro des capacités de prolifération et de différenciation plus élevées que les précurseurs myogéniques FJT et AT. L’ensemble de nos résultats montrent que les précurseurs myogéniques extraits de muscle en croissance hyperplasique présentent, de par les expressions géniques dont ils sont le siège et leur comportement in vitro, de plus grandes capacités intrins / The dramatic increase in myotomal muscle mass in fish is related to their unique ability to lastingly produce new muscle fibres, a process termed hyperplasia. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from hyperplasic fish muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomes (RNAs and miRNAs) of myogenic precursors from hyperplasic muscle of juvenile trout (JT) and from non-hyperplastic muscle of fasted juvenile trout (FJT) and adult trout (AT). We showed an higher proliferation rate of trout satellite cells in vivo in hyperplastic muscle compared to non-hyperplastic muscle. Functional analysis of transcriptomics data showed, among the over-expressed genes in JT myogenic cells compared to AT and FJT, an enrichment in genes related to mitochondrial activity, cell cycle and myogenic differentiation.We found also an enrichment in genes involved in Notch signaling pathway, indicating a repression of quiescency markers in JT myogenic cells compared to AT and FJT. Moreover, the miRnome analysis also revealed overexpressed miRNAs in JT whose targets may regulate the proliferation and the differentiation of myogenic precursors. In line with our results, JT myogenic precursors displayed in vitro higher proliferation and differentiation capacities than FJT and AT myogenic precursors. On the whole, our results converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracte

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

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.

Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.

Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.