Year 2

Muscle stem cells are a rare population of specialized cells dedicated to the regeneration of functional muscle. They play a critical role in maintaining muscle tissue, and mount a remarkable response to injury, whereupon they rapidly repair damaged muscle fibers. Thus, transplantation of muscle stem cells has the potential to treat numerous muscle conditions caused by disease, injury and aging, which constitute major unmet medical needs. Although well characterized in mice, remarkably little is known about the molecular and cellular characteristics of this important stem cell population in humans, or the mechanisms controlling their contribution to muscle regeneration. Our proposal aims to characterize the stem cell population in human muscles with the goal of defining the optimal cells for transplantation and repair of muscle tissue. Additionally, we are developing strategies for propagating human muscle stem cells in culture and expanding their number and functional properties, with the overarching goal of improving their therapeutic potential in clinical settings. During the second year of this grant period, we developed a novel method for enriching muscle stem cells present in human biopsies, using a newly discovered stem cell biomarker. This population appears to be highly enriched for stem cells with potent regenerative potential. We used the method to isolate human muscle stem cells from muscle biopsies obtained from patients representing a range of ages. Strikingly, the muscle stem cell content of the biopsy from the oldest patient (aged 77 years) showed a marked reduction in muscle stem cell content, compared with the biopsies from younger donors (aged 41-51) which were remarkably consistent. When transplanted, cells were visible as early as five days after transplant, and a robust signal persisted for the subsequent 20-week duration of the experiment, indicative of rapid and long-term engraftment. Remarkably, the transplanted human cells responded to injury induced by injection of a toxin into the leg by increasing in number to a striking degree, a hallmark of their ability to regenerate damaged tissue. Microscopic examination revealed that, importantly, the human muscle stem cells also homed to their correct cellular “niche” within the mouse leg with exquisite precision, indicating their capacity to reconstitute the stem cell reservoir. Moreover, the cells contributed to muscle fibers with the morphological characteristics and gene expression patterns typical of human muscle. No tumors or abnormal cellular growths were observed, providing evidence for the safe use of these cells for transplantation therapies. Thus, we have demonstrated our improved strategies for isolation, characterization and transplantation of human MuSCs in a mouse model will be effective in testing whether they can restore muscle strength in atrophied limbs.