SummarySkeletal muscle maintenance and repair is dependent on the resident adult muscle stem cell (MuSC). During injury, and in diseased muscle, stem cells are engaged to replace or repair damaged muscle, which requires the stem cells to exit quiescence and expand, followed by differentiation to regenerate myofibers and self-renewal to replenish the stem cell population. Following an injury, little is known regarding the timing of MuSC (skeletal muscle stem cell) self-renewal, myoblast expansion or myoblast differentiation. To determine the timing and kinetics of these cell fate decisions, we employed DNA-based lineage tracing to label newly replicated cells and followed cell fates during skeletal muscle regeneration. MuSCs activate and expand as myoblasts rapidly following injury, where the majority differentiate into myonuclei, establishing the centrally located myonuclear pool. Re-establishing the majority MuSC pool by self-renewal occurs after 5 days post-muscle injury, accompanied by low levels of myonuclear accretion that generate only peripheral myonuclei. In aged mice, possessing ∼1/2 the number of MuSCs present in young adult mice, the timing of post injury MuSC self-renewal is delayed, and although MuSCs expansion as myoblasts in aged muscle is impaired, the number of MuSC unexpectedly recovers to young adult levels during regeneration. Following an induced muscle injury, we found that myonuclei are generated within the first four days post injury derived from myoblasts expanding from activated MuSCs. Only later during regeneration, from 5 d to 14 d post injury, is the MuSC pool replenished by self-renewal, accompanied by generation of peripheral myonuclei.
