Amniotic membrane-derived stromal cells release extracellular vesicles that favor regeneration of dystrophic skeletal muscles

Abstract Background Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing satellite cells (MuSCs) and an impairment of their activation and differentiation which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study we focused on an unexplored strategy to improve MuSC function and their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), multipotent cells that have been recognized to have a role in tissue repair in different disease models. Methods The myogenic capacity of the hAMSC secretome (CM-hAMSC) and of their extracellular vesicles (EVs) were tested on human myoblasts, muscle fibers and dystrophic muscle stem cells in terms of ability to favor MuSC activation and differentiation both in vitro and in vivo. Specifically, the regenerative ability of EV-hAMSC was assessed by histological and molecular analyses after intra-muscular injection in the mouse model of DMD –mdx-. Results We demonstrate that CM-hAMSC and EVs isolated thereof are able to directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Indeed, they significantly increased both nuclei numbers (p<0.0001) and fusion index (p<0.0001). Furthermore, we give evidence that EV-hAMSC provide a direct modulation of the muscle stem cell niche in mdx mice. Interestingly, local injection of EV-hAMSCs in mdx muscles correlated with an increase in the number of activated Pax7+/Ki67+ MuSCs (p<0.001) and in new fiber formation (p<0.0001). EV-hAMSCs reduced muscle collagen deposition by almost 50% (p<0.01), thus counteracting fibrosis and MuSCs exhaustion, two hallmarks of DMD. Conclusions In this study, for the first time, we demonstrate that the hAMSC secretome (CM-hAMSC) and mainly EVs derived thereof are able to promote the muscle regeneration potential supporting proliferation and differentiation of resident muscle stem cells. This study paves the way to develop a novel regenerative treatment to counteract DMD progression based on the capacity of EV-hAMSC to reduce fibrosis and enhance myogenesis in dystrophic muscles.