UC Riverside

The derivation of human induced pluripotent stem cells (IPSCs) has revolutionized the field of personalized medicine. With their ability to self-renew and differentiate to all cell types of the adult body, their therapeutic potential for multiple diseases is invaluable. Albeit their therapeutic potential is promising, they pose the risk of uncontrolled differentiation or tumorigenesis. Physical forces in the developing embryo have been shown to play a critical role in driving lineage commitment to regulate tissue morphogenesis. In this regard, the current work focused on examining the effects of the mechanical microenvironment, using electrospun scaffolds, on the cellular behaviors of human IPSCs. We examined the role of scaffold stiffness on the (1) self-renewal and (2) directed differentiation of IPSCs. Our findings demonstrate that the mechanical microenvironment contributes to the development of distinct IPSC colony morphology. Under proliferation conditions, a two-dimensional (2D) colony morphology on stiff scaffolds enhances proliferation and minimizes spontaneous differentiation of IPSCs. In contrast, the development of three-dimensional (3D) colonies on soft scaffolds results in increased spontaneous differentiation towards ectodermal lineage. Additionally, the development of distinct colonies, directed differentiation to mesendodermal lineage is enhanced on stiff scaffolds while ectodermal differentiation is enhanced on soft scaffolds. Furthermore, we demonstrate that modulation of the RhoA signaling pathway by scaffold stiffness or using a ROCK inhibitor can prime IPSCs to differentiate towards a mesendodermal lineage. We further demonstrate that actin and E-cadherin/β-catenin clustering mediates structural pre-stresses imposed on the cells, which may delay activation of the Wnt signaling pathway, as a result of developing 3D colony morphologies. Finally, we demonstrate that modulation of the scaffold stiffness, at each developmental stage of differentiation towards three germ layer derivate cell phenotypes, can enhance the differentiation of IPSCs. Overall, these findings demonstrate that IPSCs are mechano-responsive and that the mechanical niche can be designed to modulate IPSC behaviors for the control of differentiation or to study developmental processes in vitro.