Abstract
The role of mechanical regulation in driving human induced pluripotent stem cell (hiPSC) differentiation has been minimally explored. Although endothelial cell (EC) fate from hiPSCs has been demonstrated using small molecules to drive mesoderm induction, the effects of substrate stiffness with regard to EC differentiation efficiency have yet to be elucidated. We hypothesized that substrate compliance can modulate mesoderm differentiation kinetics from hiPSCs and affect downstream EC commitment. To this end, we used polydimethylsiloxane (PDMS)-a transparent, biocompatible elastomeric material-as a substrate to study EC commitment of hiPSCs using a stepwise differentiation scheme. Using physiologically stiff (1.7 MPa) and soft (3 kPa) PDMS substrates, compared to polystyrene plates (3 GPa), we demonstrate that mechanical priming during mesoderm induction activates the Yes-associated protein and drives Wnt/β-catenin signaling. When mesoderm differentiation was induced on compliant PDMS substrates in both serum and serum-free E6 medium, mesodermal genetic signatures (T, KDR, MESP-1, GATA-2, and SNAIL-1) were enhanced. Furthermore, examination of EC fate following stiffness priming revealed that compliant substrates robustly improve EC commitment through VECad, CD31, vWF, and eNOS marker expression. Overall, we show that substrate compliance guides EC fate by enhancing mesoderm induction through Wnt activation without the addition of small molecules. These findings are the first to show that the mechanical context of the differentiation niche can be as potent as chemical cues in driving EC identity from hiPSCs.