Morphofunctional interactions between mouse mesenchymal stromal/stem cells and myoblasts in co-culture: clues for skeletal muscle regeneration.

Introduction and Aims. Mesenchymal stromal cells (MSCs) have been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On these basis, in the present study, we evaluated in a co-culture system the ability of bonemarrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. Material and methods. C2C12 cells were cultured alone or in co-culture with mouse MSCs and their proliferative attitude were assayed by Time lapse videomicroscopy, cyclin A expression and EdU incorporation. Notch-1 expression and activation were also evaluated by RT-PCR, Western Blot and confocal immunofluorescence in the myoblasts cultured alone or immunomagnetically separated from MSCs after co-culture. Paracrine interaction between the two cell types was evaluated by analyzing the involvement of MSC-derived vascular endothelial growth factor (VEGF) in the myoblastic response. Results. Myoblast proliferation was greatly increased in the co-culture; this phenomenon was accompanied by the up-regulation of Notch-1 signalling, a key determinant of myoblast activation and proliferation. The myoblastic response was mainly depended on the paracrine release of VEGF by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. Treatment with the selective pharmacological VEGFR inhibitor, KRN633, attenuated receptor activation and inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signalling. Conclusions. This study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new cell-based skeletal muscle repair strategies.