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

Abstract

BackgroundHuman 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. MethodsHuman 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. ResultsHuman 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. ConclusionAF 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.