As humans age, the ability to regenerate skeletal muscle tissue is impaired. Injuries to the musculoskeletal system that require extended periods of immobilization lead to muscle atrophy and are particularly devastating to the elderly population. Loss of skeletal muscle mass and function reduces mobility, which negatively affects quality of life, and increases the risk of mobility-related accidents. Currently, strategies to ameliorate injury-related or heritable muscle atrophy are limited and consist primarily of exercise-based regimens to increase strength. Here we propose to develop a human stem cell therapy to prevent and/or reverse localized skeletal muscle atrophy in muscles of the aged. Specifically, we aim to (1) refine a strategy we have developed that enables us to isolate muscle stem cells (MuSCs) from human muscle biopsies, (2) apply a strategy which we developed for murine MuSCs to human MuSCs that rejuvenates and expands the stem cells in culture to clinically useful numbers using a combined bioengineering and small molecule treatment, and (3) demonstrate that transplantation of human MuSCs results in increased in vivo force generation and strength in atrophied muscles of aged recipients. Together, these studies will culminate in the validation of a novel stem cell-based "development candidate" to the treat skeletal muscle atrophy afflicting the ever increasing aged community and will advance the use of stem cells for therapy in the clinic.
Statement of Benefit to California:
Skeletal muscle is critical to our day-to-day movement and loss of muscle mass and function impairs quality of life and increases the risk of mobility related accidents particularly in the elderly. Currently there are no clinical approaches to prevent or reverse the muscle atrophy that occurs following musculoskeletal injury and subsequent immobilization other than physical activity. Since California is projected to be the fastest growing state in the U.S. in terms of population, with an elderly population that is projected to grow twice as fast as the total population of the state, it is more important than ever to develop strategies that positively impact the health of this demographic. The research in this CIRM application aims to provide a muscle stem cell transplantation therapy to overcome muscle atrophy. Initially, the application of this strategy to the function of small muscles of the eye critical to vision, to the pharynx critical to swallowing, or to the muscles of the hand are envisioned. A major effort will also entail scaling up muscle stem cell production for the treatment muscle atrophy occurring in ~60% of the aged knee or hip revision rehabilitation patients. This work will not only advance the use of stem cells in the clinic, but will also provide a novel therapy to ameliorate the devastating effects of muscle atrophy in our ever growing aged community.
Year 1Maintenance and repair of skeletal muscle throughout adulthood relies on the essential contributions of resident muscle stem cells. Our lab has pioneered the prospective isolation of mouse skeletal muscle stem cells by FACS and has shown that these cells contribute extensively to the regeneration of damaged muscles and also replenish the endogenous stem cell pool. Although much insight has been gained in mice, in humans there is a scarcity of knowledge of the molecular identities of the resident stem and progenitor cells and the fundamental cellular and molecular mechanisms regulating their contributions to muscle repair. This unmet need is particularly underscored in dystrophic and aged individuals in which muscle function is compromised. Our project aims to elucidate the stem cell populations in young and aged human muscles to better understand the poor ability to repair damage to aged muscles. Furthermore, we are developing in vitro strategies to propagate human muscle stem cells with the goal of enhancing their numbers and improving their therapeutic function in clinical settings.
During the first year of this grant period, we have made substantial progress on Milestones 1, 2 and 3. Human muscle stem cells have been isolated by flow cytometry in our laboratory based on the expression of positive and negative cell surface selection markers. We have shown that the purified cells can be transplanted in vivo into damaged muscles of immunodeficient recipient mice and generate muscle fibers, demonstrating the potential of isolated human cells to make muscle in vivo. We observed using non-invasive bioluminescence imaging that the purified human muscle stem cells expand in vivo more than 100-fold during the repair process, in contrast to human myoblast progenitors, which do not expand in vivo. Notably, we now have data demonstrating that using our culture conditions, these cells can be successfully maintained as stem cells as shown by their ability to engraft and generate muscle in vivo. We are actively investigating factors that expand skeletal muscle stem cells (MuSCs) purified from human muscle tissues obtained from aged individuals ex vivo. Thus, we have been able to translate our prior findings with murine MuSCs to human MuSCs.
Year 2Muscle 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.