Revealing muscle stem cell heterogeneity in mice and humans through deep single-cell analysis

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PROJECT SUMMARY/ABSTRACT Muscle stem cells (MuSCs), also known as satellite cells, are essential to skeletal muscle regeneration throughout life. In aged individuals, muscle mass and regenerative capacity after injury progressively decline, leading to diminished quality of life. We have recently demonstrated that MuSCs prospectively isolated from aged mice are highly heterogeneous and, as a population, have a marked reduction in regenerative capacity relative to young adult MuSCs, revealing a previously undetected intrinsic stem cell defect in aged MuSCs. The dysfunction of aged MuSCs is characterized by a shift from reversible quiescence to cellular senescence, driven by elevation of the p16Ink4a cell-cycle inhibitor, and inefficient self-renewal, caused by aberrant cell-autonomous activation of the p38 mitogen-activated protein kinase and STAT3 pathways. What causes these inherent alterations and how to prospectively identify and treat dysfunctional MuSCs in aged mice and humans remain unanswered questions. We propose to combine high-throughput deep single-cell RNA-sequencing across varied adult mouse and human muscle samples and stem-cell population reconstruction algorithms to identify cell- surface antigen profiles that unambiguously distinguish between health and diseased MuSCs in mouse and human aging. We hypothesize that this approach will enable discovery of heterogeneously expressed MuSC cell-surface antigens that demark differing stem-cell capacities within a new-found functional hierarchy. We will prospectively isolate and transplant mouse and human MuSCs sub-populations and perform limiting dilution transplantation assays and self-renewal assays using bioengineered culture microenvironments. We will perform conditional deletion and transient knockdown studies to investigate if the identified antigens have mechanistic roles in self-renewal dysfunction in vivo and in vitro. These proposed studies will provide a deeply-profiled organized cellular atlas of muscle stem and progenitor cells in mouse and human aging that should enable rational therapeutic development targeting dysregulated stem cells for enhancing muscle repair in the elderly following trauma.