UC Irvine

Skeletal muscle is a highly regenerative tissue that is responsible for voluntary, contractile movement of the body. Endogenous muscle stem cells are responsible for regenerating skeletal muscle upon injury, although in diseased contexts this regeneration becomes impaired to the deficit of the patient. There remains a pressing need for the discovery of viable, long-lasting therapeutic treatments for patients with genetic congenital neuromuscular diseases as current standard of care fails to improve patient quality of life. Thus, the introduction of induced pluripotent stem cell (iPSC) derived muscle stem cell therapies offers an exciting avenue to introducing an autologous healthy donor stem cell into a diseased patient. While this platform is promising, current research has found that iPSC derivations generate immature skeletal muscle stem cells, transcriptomically on par with embryonic human muscle stem cells. One major limitation of these stem cells is the ineffective retention of the stem cell population upon engraftment, an objective highlighted in this dissertation.We describe the heterogenous myogenic populations generated upon engraftment in vivo, importantly finding distinctly different myofibers that resemble either an immature, regenerative state (marked by fetal cardiac actin, ACTC1) or a mature, developed state that arises through fusion with host mouse myofibers. We found that human PAX7+ muscle stem cells are better retained in regions of regenerating ACTC1+ myofibers rather than the larger, chimeric myofibers. We then harnessed spatial transcriptomic approaches to study the early stages of human myogenic regeneration upon engraftment, discovering niche factors expressed by regenerating myofibers that likely support stem cell retention and self-renewal. In line with these findings, after rapid cell loss upon transplantation, MYOG+ and PAX7+ myogenic populations stabilized upon the formation of myofibers. With an understanding of the importance of regenerative myofiber population in the retention of muscle stem cells upon engraftment, we then applied this workflow to a dystrophic microenvironment. We identified that the dystrophic microenvironment induces a massive change in the interactome of myogenic populations, and these changes are detrimental towards the regenerative program after subsequent injuries.Multiple epidermal growth factor like domains 10 (MEGF10) is a poorly understood stem cell niche receptor that appeared as both a candidate niche factor on regenerative myofibers and downregulated in dystrophic microenvironments. Therefore, we postulated that overexpression of MEGF10 may be sufficient to improve stem cell retention in transplantation models. We leveraged CRISPR genomic engineering to generate a TET-On inducible MEGF10 expression iPSC line which can robustly be differentiated to skeletal muscle and engrafted in vivo. We found that overexpression of MEGF10 during the first ten days following engraftments significantly supported more PAX7+ muscle stem cells and myofiber regeneration. Further, spatial transcriptomic analysis reveals that MEGF10 accelerates myofiber regeneration but stalls further maturation, arresting myofibers in a regenerative ACTC1+ state, marked by potential downstream MEGF10 signaling candidates LRRN1, CDH15, DLK1, and JAM2. Our findings are the first that describe advantageous heterogeneity of iPSC muscle stem cells upon engraftment, how they regenerate in real time and in a dystrophic host environment, and the temporal significance of stem cell niche receptors in their roles of transplanted niche formation. These breakthroughs may shed light on how to effectively generate novel stem cell therapy treatments for patients with neuromuscular disorders.