UC San Diego

Contractile vascular smooth muscle cells (VSMCs) sourced from donors are limited in supply for vascular tissue research and autologous transplantation therapies. Protocols using biochemical signaling for the derivation of functional, contractile SMCs from human induced pluripotent stem cells (hiPSCs) and mesenchymal stem cells (MSCs) have recently been explored, but they do not induce the full SMC phenotype. Biochemical cues are just one type of signaling that occurs in vivo and more thorough differentiation protocols could be developed through understanding the biomechanical effects of external forces resulting from shape modulation and surface topography. The goal of this study is to delineate the induction mechanism of biomechanical shape modulation and identify the key post-transcription regulators through the study of microRNAs and how they affect differentiation pathways in response to external stimuli. Of particular interest are miR-27 and miR-145, whose roles in MSC to VSMC differentiation are not yet fully established. To study the roles of miR-145 and miR-27 in regulating the shape modulation-induced MSC-to-VSMC differentiation, MSCs were cultured onto 10-μm wide microgrooves fabricated through soft lithography over a 3-day period with TGF-β supplemented growth media. The synergistic SMC inductions of both biochemical and biomechanical signals were validated through the increases of SMC marker expression and contractile phenotype, which were quantified by Western blot, RT-qPCR, and cell traction force microscopy. Finally, the changes of myogenesis-related miRNAs were profiled by miRNA RT-qPCR, and gain-of-function and loss-of-function experiments on miR-27 and miR-145 were performed to evaluate their roles in the biomechanical induction of VSMC differentiation. These studies were conducted to elucidate roles of miR-27 and miR-145 in modulating SMC phenotype in response to biomechanical and biochemical cues and advance our ability to control stem cell (SC) induction into VSMCs. The findings have implications in a broad range of bioengineering applications, including vascular pathophysiology and graft tissue engineering .