MiR-145 mediates cell morphology-regulated mesenchymal stem cell differentiation to smooth muscle cells

Abstract The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC phenotype remains to be a major challenge. Cell morphology has been shown to regulate MSC differentiation into various lineages, including SMCs. We engineered substrates with microgrooves to induce cell elongationto study the mechanism underlying the MSC shape modulation in SMC differentiation. In comparison to those on flat substrates, MSCs cultured on engineered substrates were elongatedwith increased aspect ratios for both cell body and nucleus, as well as augmented cytoskeletal tensions. Biochemical studies indicated that the microgroove- elongatedcells expressed significantly higher levels of SMC markers. MicroRNA analyses showed that up-regulation of miR-145 and the consequent repression of KLF4in these elongatedcells promoted MSC-to-SMC differentiation. Rho/ROCK inhibitions, which impair cytoskeletal tension, attenuated cell and nuclear elongationsand disrupted the miR-145/ KLF4regulation for SMC differentiation. Furthermore, cell traction force measurements showed that miR-145 is essential for the functional contractility in the microgroove-induced SMC differentiation. Collectively, our findings demonstrate that, through a Rho-ROCK/miR-145/ KLF4pathway, the elongatedcell shape serves as a decisive geometric cue to direct MSC differentiation into functional SMCs.