Influence of mesenchymal stromal cell extracellular vesicles on myofiber repair and regeneration

Mechanical stresses and myofiber fragility caused by physical activity, aging and/or genetic diseases can seriously damage the skeletal muscles. The process of myofiber repair and regeneration requires a highly coordinated mechanisms based on the action of various intercellular factors such as proteins, cytokines, miRNA and membrane lipids mainly released by extracellular vesicles (EVs). Although the cellular and molecular mechanisms in skeletal muscle regeneration are well known, the influence of EVs in the intercellular communications to coordinate the repair and regeneration of injured myofibers is still under investigation. Recently, it has been suggested that mesenchymal stromal cells (MSCs) showed a great potential in treating muscular damage due to their paracrine factors. However, the role of EVs released by MSCs in myogenic repair and regeneration has not been extensively studied. The aim of this study was to investigate the role of EVs isolated from MSCs on muscle repair and regeneration. EVs were isolated from cell culture medium by liquid exchange method based on polyethylene glycol precipitation protocol. The EVs isolated from control, differentiating murine myoblasts and MSCs, were characterized for their size, membrane markers and presence of myokines by electron microscopy, western blot, Elisa assays and proquantum immunoassays. EVs isolated from MSCs were then added to the medium of damaged differentiated myoblasts to investigate their influence on the process of myofiber repair. The ability to produce myotubes was investigated by inverted light microscopy and the expression of the muscle differentiation markers MyoD and myogenin were evaluated by western blot.Results showed the ability of damaged myoblasts to release inflammatory factors enclosed in EVs and the influence of EVs isolated by MSCs in reducing the inflamed microenvironment and promoting the myogenic repair and regeneration. In conclusion, our data demonstrated the powerful role of MSC - EVS in regulating myogenic differentiation. In the future, we aim to investigate treatments, such as photobiomodulation, that could increase the release of specific myokines to accelerate the repair and regeneration of injured muscle. This project has received funding from the Italian Ministry of University and Research, under the PRIN 2022 programme (20222P2NAJ; CUP I53D23003030006) (finanziato dall’Unione europea – Next Generation EU)