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 Pax7 High subset in a deeper quiescent state. However, the conventional method of identifying Pax7 High cells involves intracellular staining, which limits their viability for functional studies. This work investigates whether autofluorescence (AF) could be used 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-AF High and MuRC-AF Low populations into immunodeficient mice assessed survival and regenerative potential using bioluminescence imaging and immunohistochemistry. Results Human MuRC exhibited a threefold increase in autofluorescence intensity compared to MB, with a peak at 405 nm excitation, likely linked to a 1.6-fold increase in lipid content, while NADH/NADPH levels remained comparable. Flow cytometry identified MuRC-AF High as a Pax7 High -enriched subpopulation, indicative of a deeper quiescent state. Functionally, MuRC-AF High cells showed delayed cell cycle re-entry and slower proliferation yet maintained full differentiation capacity. In vivo, both MuRC-AF High and MuRC-AF Low survived transplantation, contributed to Pax7 + MuSC formation, and retained regenerative potential upon re-injury. Conclusion Autofluorescence enables the isolation of distinct human MuRC subpopulations. The AF High subset contains a high proportion of Pax7 High cells and shows delayed activation yet retains engraftment efficiency that is comparable to that of the AF Low subpopulation. These findings suggests that AF could be used as a biomarker to identify functionally distinct human muscle progenitor subsets while preserving their regenerative potential for future use.