Evidence for cell density affecting C2C12 myogenesis: Possible regulation of myogenesis by cell-cell communication

Introduction: Community effect is a phenomenon caused by cell-cell communication during myogenesis. In myogenic C2C12 cells in vitro, the confluent phase is needed for myogenesis induction. Methods: To examine the cell-density effect, growth kinetics and myogenic differentiation were investigated in cells plated at four different cell densities. Results: We found that expression of a myogenic differentiation marker was high in a density-dependent manner. At high density, where cell-cell contact was obvious, contact inhibition after the proliferation stage was accompanied by microarray findings demonstrating upregulation of negative regulating cell-cycle markers, including CDKI p21 and the muscle differentiation markers MyoD and myogenin. Interestingly, developmentally regulated protein expression (drebrin) protein expression was also upregulated in a density-dependent manner. Conclusions: These results suggest that contact inhibition after the proliferation stage may induce growth arrest via cell-cell communication through the expression of CDKI p21 and may be responsible for progressing cell fusion.