Sphingolipids in extracellular vesicles released from the skeletal muscle plasma membrane control muscle stem cell fate during muscle regeneration

Extracellular vesicles (EVs) represents a cytokine-independent pathway by which skeletal muscle (SkM) cells modulate the fate of neighbouring cells to control SkM homeostasis. Here we show that SkM release a bulk of small (sEVs) and large (lEVs), both generated from plasma membrane (PM). Conversely to lEVs, sEVs are synthesized from membrane folds enriched in Alix and TSG101 proteins and lipid-rafts, which have never been described for other cell types. During muscle regeneration, sEVs promoted M1 macrophage polarization and migration and MuSC differentiation, contributing to accelerated muscle regeneration, whereas lEVs influenced the shift from a pro-inflammatory to an anti-inflammatory response, promoting MuSC proliferation. The lipid composition of sEVs and lEVs, particularly the ratios of sphingosine 1-phosphate (d18:1 and d16:1), explained their differential effects, with TNF-α altering these ratios and modifying SkM-EV actions. Our work highlight the role of sub-populations of SkM-EVs in muscle regeneration, and their potential for therapeutic applications.