Myoblast and fibroblast derived small extracellular vesicles differentially affect myoblast migration dynamics.

Muscle injury activates satellite cells and fibroblasts, with extracellular vesicles (EVs) mediating the related intercellular communication. The influence of EVs released by either cell type on recipient cell behaviour is still unclear. This study investigated the uptake and effects of EVs derived from C2C12 myoblasts (myo-EVs) and L929 fibroblasts (fibro-EVs) on proliferating myoblasts. Both cell lines were cultured in media largely depleted of FBS-derived EVs. Myo-EVs and fibro-EVs isolated from conditioned media were characterised using conventional methods. Effects of these EVs on myoblast function were assessed via PKH67-labelled EV uptake, proliferation, scratch closure, leading front migration rate and individual cell trajectories and western blot analysis for MyoD and Myogenin. Myoblasts preferentially internalised myo-EVs at 5 h (myo-EVs: 3.41 ± 1.34 vs fibro-EVs: 1.25 ± 1.13 puncta per cell) and 48 h (myo-EVs 16.55 ± 12.60 vs fibro-EVs 9.67 ± 4.88) (both p < 0.05). Under proliferative EV-depleted conditions, added EVs did not change myoblast proliferation. However, the elevated expression of Myogenin indicating a subtle shift toward differentiation. Myo-EVs increased myoblast migration rate into a scratch, compared to controls (13.77 ± 1.7 vs 11.08 ± 2.23 µm/h, p < 0.01), but had no effect under conditions of FBS EV depletion. On the other hand, fibro-EVs increased the speed of individual cells, but negatively affected leading front migration due to impaired myoblast persistence. These findings highlight the importance of cell-specific EV-mediated communication in muscle regeneration. Further, tissue explants used to generate cell-specific EVs for treatment should be free of contaminating cell types.