Abstract
Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (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. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, 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 and more efficient cell-based skeletal muscle repair strategies.
Highlights
Skeletal muscle has a robust capacity of repair/regeneration in response to injury or disease, relying in large part upon the presence of a population of skeletal myoblast precursors, the satellite cells, whose activation, re-entry the cell cycle and differentiation require signals emanated by damaged fibers and infiltrating inflammatory cells [1,2]
mesenchymal stromal cells (MSCs) Stimulate C2C12 Cell Proliferation To examine whether myoblast proliferation was modulated by interactions with MSCs, we used a model of direct coculture of native C2C12 cells and MSCs marked with Dil (DilMSCs), in order to distinguish the behavior of the two cell types
In this line of thought, it is worth of noting that a close relationship between stromal and satellite cells has been shown within the skeletal muscle niche and a role for the stromal cells as a natural scaffold on which stem cells interact and proliferate, has been proposed [31], supporting the instructive potential of MSCs for the recruitment of the intrinsic muscle stem cell pool
Summary
Skeletal muscle has a robust capacity of repair/regeneration in response to injury or disease, relying in large part upon the presence of a population of skeletal myoblast precursors, the satellite cells, whose activation, re-entry the cell cycle and differentiation require signals emanated by damaged fibers and infiltrating inflammatory cells [1,2]. The full potential of satellite-cell therapy is affected by several limitations, including the high heterogeneity of this cell population [7], the loss of their myogenic potential upon in vitro expansion [6] and the predetermination dependent from the source of muscle fibers [8,9] This has shifted the attention to other cell sources of non-myogenic origin as additional candidates for skeletal muscle repair/regeneration. MSCs are a rare population of cells that can be isolated from the bone marrow, adipose tissue and many other regions of the body, rapidly expanded ex vivo and utilized for experimental and clinical studies They have the potential to acquire lineage of any-mesenchymal-derived tissue in vitro. We have recently reported in a co-culture system that MSCs support proliferation of neonatal cardiomyocyte precursors through combined paracrine/juxtacrine mechanisms [22], suggesting the potential ability of these cells to determine the fate of local stem cells and augment the endogenous repair of the damaged tissues
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