Rejuvenation-Responsive and Senolytic-Sensitive Muscle Stem Cells Unveiled by CD200 and CD63 in Geriatric Muscle

Abstract Muscle stem cells (MuSCs) are parenchymal cells in skeletal muscle regeneration and maintenance. With aging, MuSCs experience a decline in their regenerative function and reduction in their number. However, recent evidence points to substantial heterogeneity within the aged MuSC population, raising questions about the underlying mechanisms of age-associated dysfunction. Here, we used Pax7 CreERT2 ;Rosa YFP mice (MuSC YFP ) to label Pax7-expressing MuSCs and chronologically traced MuSCs until geriatric age. Genetic labeling and chronological tracing revealed that the number of YFP + MuSC remained comparable between young, middle and geriatric ages. At geriatric age, YFP + MuSCs exhibited reduced expression of traditional MuSC markers such as VCAM1 and PAX7. A previously unrecognized subpopulation emerged, characterized by loss of VCAM1 and low or absent PAX7. Despite their altered marker profile, these cells retained transcriptional signatures of quiescence and myogenic potential, but displayed significantly reduced proliferative and regenerative capacities. They displayed gene expression patterns indicative of senescence(definition)-like state and were selectively ablated by senolytic treatment. DHT restored regenerative function in aged mice and re-induced VCAM1 expression in YFP + /Pax7 -/low /VCAM1 - cells, indicating responsiveness to rejuvenation. Based on their emergence with aging, functional impairment and responsiveness to rejuvenation, we termed this population GERI-MuSCs (Geriatric Emerging Rejuvenation-responsive and Impaired MuSCs). CD63 and CD200 were identified as novel surface markers that together with VCAM1, reliably detect GERI-MuSCs as well as classical Pax7 + /VCAM1 High MuSCs, providing a tool for comprehensive isolation of MuSCs from aged wild-type mice. Together, our findings provide a refined framework for studying MuSC aging and offer new tools for isolating functionally distinct MuSC subsets from aged skeletal muscle.