Study of myogenic potential of extra cellular vesicles in murine models for muscular dystrophies

Muscular dystrophies are a heterogeneous group of genetic diseases characterized by progressive and irreversible degeneration of skeletal muscles. Cell-based therapies have shown some beneficial effects on muscle repair, but there are still a number of concerns in its use. Some recent studies have shown the potential of extracellular vesicles (EVs) in muscular regeneration, and here, we are testing which cell type would contain the best information of this regenerative potential. Firstly, we are using EVs isolated from C2C12 cells, because of their good muscle proliferation and differentiation capacity. EVs were isolated by ultracentrifugation and characterized through Electron Microscopy and Flow cytometry. The EVs were marked with a green dye and cocultured with C2C12 myoblasts as well as with murine fibroblasts. EVs incorporation in the receptor cells was evaluated in different times of coculture, with an increasing presence after 2 days in both cell types. We concluded that the incorporation of EVs in the cells can occurs quickly but gradually. For the observation in vivo of EVs effect on muscle regeneration, EVs were injected directly into the gastrocnemius muscle of normal (C57black) and dystrophic mice (Dmdmdx and Largemyd), and the muscles were analyzed after 2 and 10 days. Green dye detection by FACS was done on manually dissociated muscles, and the incorporation of EVs was observed in all muscles, independently of the degree of muscle degeneration. In order to verify if EVs could contribute with functional benefit, other animals were monthly injected with EVs, and functional analyzes are being performed in blind test. In the next step, the same experiments will be performed with EVs isolated from diverse lineages of mesenchymal stem cells and muscle progenitor cells. Better understanding the mechanisms of generation and operation of these vesicles would be crucial in order to verify their myogenic potential.