Control of Muscle Stem Cells to Enhance Regeneration

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PROJECT SUMMARY According to the most recent U.S. Census, the elderly population will more than double to 80 million, encompassing 1 in 5 Americans by 2050. Aging is characterized by a decline in tissue function and regenerative capacity. Sarcopenia, also known as age-dependent loss of skeletal muscle mass and strength, is a major public- health problem that affects 15% of the elderly, leading to loss of mobility and diminished quality of life. Age- related muscle loss is paralleled by a loss in the function of muscle stem cells (MuSCs), key players in muscle homeostasis and regeneration. However, the mechanisms responsible for age-associated MuSC dysfunction remain elusive. Two major barriers to gaining mechanistic insights into MuSC aging are (1) the heterogeneity of the aged MuSC population, which renders standard bulk analysis ineffective, and (2) the lack of tools to resolve this heterogeneity, underscoring the need for single-cell studies. We previously demonstrated that aged MuSCs are a heterogeneous population comprised of functional and dysfunctional subsets. This key observation suggests a therapeutic strategy to regenerate muscle - boosting the activity of resilient functional MuSCs. Here we explore this possibility using a specific cell surface marker and a series of innovative single-cell technologies required to resolve MuSC subsets. Our preliminary data iden- tify CD47 as a cell surface marker whose expression level, not presence or absence, distinguishes functional CD47lo and dysfunctional CD47hi MuSC subsets. Known widely as a receptor for SIRPα, CD47 is also a receptor for thrombospondin-1 (THBS1). We found that CD47hi MuSCs accumulate in aged muscle and aberrantly ex- press THBS1. We hypothesize that during aging the accumulation of CD47hi MuSCs impairs the proliferation of CD47lo MuSCs through secretion of THBS1, hindering regeneration and contributing to sarcopenia. Here, our specific aims are to (1) determine how CD47 signaling goes awry in aging, (2) elucidate how post-transcriptional regulation of CD47 is altered during aging leading to the accumulation of dysfunctional CD47hi MuSCs, and (3) determine the effects of aberrant THBS1 secretion in the aged MuSC niche on regeneration. We capitalize on cutting-edge single-cell technologies, including multidimensional single-cell mass cytometry (CyTOF) and multi- plexed tissue imaging (CO-Detection by indexing (CODEX)). These technologies allow us to track simultane- ously 40+ distinct cell and signaling phenotypes in CD47lo and CD47hi MuSCs (CyTOF) and resolve how spatial changes in the architecture of the multicellular niche lead to MuSC dysfunction in aging (CODEX). We combine this knowledge with in vivo investigation of regenerative capacity and strength in aged mice. Finally, we perturb CD47 signaling in vivo using blocking antibodies to surmount the regenerative deficits in aged mice. The pro- posed analyses of newly identified aged MuSC subsets that can be prospectively isolated will provide fresh mechanistic insights into aging and inform therapeutic strategies to augment endogenous muscle repair.