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

## TL;DR

This study shows that autofluorescence can identify distinct human muscle reserve cell populations with different quiescence levels and regenerative potential.

## Contribution

The novel use of autofluorescence as a biomarker to isolate viable, functional human muscle reserve cells with distinct Pax7 levels and stem cell states.

## Key findings

- MuRC-AFHigh cells are enriched in Pax7High cells and show delayed activation but retain regenerative potential.
- Autofluorescence intensity correlates with lipid content and quiescence in human muscle reserve cells.
- Both MuRC-AFHigh and MuRC-AFLow subpopulations survive transplantation and contribute to muscle regeneration.

## Abstract

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, the conventional method of identifying Pax7High 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.

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.

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-AFHigh as a Pax7High-enriched subpopulation, indicative of a deeper quiescent state. Functionally, MuRC-AFHigh cells showed delayed cell cycle re-entry and slower proliferation yet maintained full differentiation capacity. In vivo, both MuRC-AFHigh and MuRC-AFLow survived transplantation, contributed to Pax7 + MuSC formation, and retained regenerative potential upon re-injury.

Autofluorescence enables the isolation of distinct human MuRC subpopulations. The AFHigh subset contains a high proportion of Pax7High cells and shows delayed activation yet retains engraftment efficiency that is comparable to that of the AFLow 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.