Background: The capacity of skeletal muscles to regenerate relies on Pax7⁺muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.

Methods: In the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.

Results: We observed that Notch signaling is active and essential for the generation of quiescent human Pax7⁺MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7^Highsubpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7^HighMuRC were less primed for myogenic differentiation as compared to Pax7^LowMuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.

Conclusions: Overall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7^Highsubpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7^HighMuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.