Role of RhoA/Rho kinase signaling pathway in microgroove induced stem cell myogenic differentiation

In our previous report, the authors have demonstrated that direct laser machined microchannels would trigger upregulation of myogenic markers in human mesenchymal stem cells (hMSCs) through promotion of cell elongation. However, the molecular basis signaling pathways behind this observation remains unclear. In this work, three types of microchannels generated by femtosecond laser were utilized to investigate possible mechanisms behind the induction of hMSCs myogenesis by microchannels. The authors hypothesized that small G-proteins RhoA and Rac1 play a vital role on myogenesis of hMSCs through regulating cytoskeleton rearrangement, via cell tension signaling cascades. The RhoA and Rac1 activities were evaluated for cells cultured on the micropatterned substrates, using a flat unpatterned substrate as control. It was found that significant activation of RhoA GTPase was exhibited for cells cultured on narrow microchannels (20-20-20 and 30-30-20), while no obvious differences were obtained on wide ones (80-30-20). Meanwhile, no significant difference was found for Rac1 activities on all tested groups. To further deduce the role of RhoA signaling pathway in microchannel directed stem cell myogenesis, the effectors of Rho, Rho kinase (ROCK) was chosen to explore how cell shape regulate myogenesis of hMSCs cultured on laser micropatterned substrate. A pharmacological ROCK inhibitor, Y-27632, was used to treat the cells and the effect on RhoA activation was investigated. Our data on the role of RhoA/ROCK in regulating cell myogenic differentiation on lasered microchannels substrates may provide a mechanistic insight on hMSCs fate directed by substrate topography.