Intracellular autofluorescence enables the isolation of viable, functional human muscle reserve cells with distinct Pax7 levels and stem cell states.

Background: Human muscle reserve cells (MuRC) represent a quiescent MuSC population generated in vitro that exhibit heterogeneous Pax7 expression, with a Pax7High subset in a deeper quiescent state. However, conventional identification of Pax7High cells requires intracellular staining, limiting their viability for functional studies. This study investigates autofluorescence (AF) as a potential biomarker to identify functionally distinct human MuRC subpopulations. Methods: Human myoblasts (MB) and MuRC were analysed for AF by fluorescence microscopy and flow cytometry. Cellular metabolic composition was assessed by NADH/NADPH quantification and lipid staining. Human MuRC subpopulations were sorted by AF intensity and analysed for Pax7 expression, cell cycle re-entry, proliferation, clonal expansion, and myogenic differentiation. In vivo transplantation of MuRC-AFHigh and MuRC-AFLow populations into immunodeficient mice assessed survival and regenerative potential using bioluminescence imaging and immunohistochemistry. Results: Human MuRC showed a 3-fold increase in mean fluorescence intensity compared to MB, with AF peak at 405 nm excitation. Lipid staining revealed a 1.6-fold increase in lipid content in MuRC, while NADH/NADPH levels were similar between MB and MuRC. Flow cytometry identified MuRC-AFHigh as a Pax7High-enriched subpopulation. Functionally, MuRC-AFHigh cells exhibited delayed cell cycle re-entry and slower proliferation but retained differentiation potential. In vivo, both MuRC-AFHigh and MuRC-AFLow survived transplantation with no significant differences in engraftment efficiency. They contributed to the generation of human Pax7 positives MuSC and both subpopulations retain their regenerative capacity upon re-injury. Conclusion: AF allows the identification of human MuRC subsets, with the AFHigh subpopulation associated with increased lipid content. MuRC-AFHigh cells are enriched in Pax7High cells and show delayed activation, slower proliferation and comparable engraftment efficiency to the AFLow subpopulation. These findings provide a novel perspective on AF as a potential biomarker to identify functionally distinct muscle progenitor subsets and highlight its relevance in muscle regeneration research.