Multiomics Characterization Identifies S1P-secreting USSHigh Skeletal Muscle Stem Cells as Essential Drivers of Niche Remodeling and Muscle Regeneration

Abstract

Acutely induced cellular senescence is increasingly recognized as vital for tissue repair but the precise roles of senescent cells in skeletal muscle regeneration remain paradoxical. Here, using single-cell RNA sequencing and a Unified Senescence Score (USS) methodology, we identify a distinct USSHigh (USSHi) muscle stem cell (MuSC) subpopulation emerging in early phase of acute injury induced muscle regeneration. Isolation via the surface marker MHCII enables characterization of these cells as exhibiting canonical senescent-like features. Further characterization through integrated transcriptomics, proteomics, and secretomics reveals their highly pro-inflammatory secretory capacity. Moreover, lipidomic profiling uncovers that the USSHi MuSCs undergo profound sphingolipid metabolic remodeling to generate upregulated bioactive lipid Sphingosine-1-phosphate (S1P). In vivo specific ablation of this subpopulation using a Pax7-CreERT; Ccl2-LSL-DTA genetic strategy significantly impaired muscle regeneration. Furthermore, spatial transcriptomics and cell-cell communication analyses revealed that USSHi MuSCs act as critical niche modulators, directing crosstalk with multiple cell types. Specifically, S1P is essential for mediating direct USSHi interaction with endothelial cells and USSLo cells to promote endothelial angiogenesis and MuSC expansion in the regenerating muscle. Crucially, exogenous S1P rescues the regenerative deficits caused by USSHi MuSC ablation. Altogether our findings define a senescent-like MuSC population emerging in acute injury induced muscle regeneration and provide definitive evidence to demonstrate the positive role of these cells in enhancing muscle repair via their robust secretory phenotypes and inter-cellular communicative capacity.