Force As a Regulator of Mesodermal Differentiation

Multiple groups have shown that the physical microenvironment, including applied tension, compression, and shear stress, as well as the stiffness of the underlying substrate, can promote differentiation towards specific phenotypes. For example, we have previously shown [Wolfe Integrative Biology 2012 & Wolfe Biotech Bioeng 2013] that fluid shear stress, when applied to cells on a stiff 2D glass surface, can promote differentiation towards the mesodermal lineage, and particularly towards endothelial and hematopoietic differentiation. Yet mechanotransduction, the conversion of external forces into intracellular signals, is still poorly understood in differentiated phenotypes and even more so in stem cell populations. In differentiated cells, it has been shown that externally applied forces are counterbalanced in part by intracellular tension generated by actin-myosin motors. So recently we have chosen to explore the role that intracellular tension may play in differentiation of pluripotent cells. During both early (Day 2 to Day 4) and later (Day 4 to Day 7) embryoid body culture of mouse embryonic stem cells, we perturbed actin polymerization and actin-myosin binding using the inhibitors cytocholasin-D and blebbistatin. We found that the induced decrease in intracellular tension had little effect during the early time point but decreased gene expression of mesodermal markers during later differentiation. In particular, markers for both the intermediate (PAX2) and lateral (FLK1) mesodermal plates were downregulated. This later time point overlaps with in vitro germ lineage specification, which is analogous to gastrulation-related germ layer separation in vivo. Taken together with the previous findings with applied external force, these results indicate that the ability to transduce external force into intracellular force may inherently regulate specification to the mesoderm. Thus, further mechanistic studies into the conversion of external force into regulation of transcription factors in stem cells may help inform strategies to promote efficient differentiation towards mesodermal phenotypes, including cardiovascular and orthopaedic phenotypes, for tissue engineering and regenerative medicine therapies.