Synergistic effect of laminin and epidermal growth factor (EGF) on co-cultured myoblasts and fibroblasts

Abstract

Myoblast migration is vital in ensuring the regeneration of muscle and the efficiency of transplantation process. However, long-standing problems of myoblasts based therapy are low growth rate of myoblasts and the contamination of the fibroblasts during isolation of cells and culture. Given the role of laminin and EGF synergy on human skeletal muscle cell line has been confirmed previously, we now investigate the effect on primary human skeletal muscle cells which contains myoblasts and fibroblasts. We investigated the attachment, proliferation and migration in describing the biological behaviour of the myoblasts and fibroblast in laminin and EGF synergy. Human skeletal muscle cells containing myoblasts and fibroblasts were isolated and cultured until passage 4. Desmin was used as a marker to distinguish myoblasts from fibroblasts. The proliferation and migration of cells on plain and laminin-coated surface with or without EGF were measured via image processing. To understand the molecular mechanism of laminin and EGF synergy, muscle cells cultured on different conditions were exposed with the Rho kinase inhibitor, Y-27632, and EGF-receptor (EGF-R) inhibitor, gefitinib. Myoblast migration and proliferation was enhanced significantly by synergistic stimulation of laminin and EGF compare to that by laminin alone, while no enhancement observed by EGF alone. However, no changes in proliferation and migration were observed for fibroblasts among the culture conditions. Inhibition of Rho kinase resulted in the increase of the myoblast migration on the laminin-coated surface with EGF condition. Compared to the untreated conditions, myoblasts cultured on the laminin-coated surface and EGF demonstrated elongated morphology, and average cell length increase significantly. In contrast, inhibition of EGF-R resulted in the decrease of myoblast migration and cell length on the laminin-coated surface with EGF supplemented condition in comparison to the untreated control. In conclusion, laminin and EGF preferentially enhance the proliferation and migration myoblasts, and Rho kinase and EGF-R play a vital role in this synergistic effect. These results will be beneficial for the development of skeletal muscle tissue substitutes for clinical applications.