Matrix Stiffness Regulates Mechanotransduction and Vascular Network Formation of hiPSC-Derived Endothelial Progenitors Encapsulated in 3D Hydrogels

Abstract Matrix mechanics are central to vasculogenesis, yet stiffness is often confounded with ligand density or degradability in common hydrogel platforms. Here, we use norbornene-modified hyaluronic acid (NorHA) hydrogels to independently tune stiffness within a sub-kilopascal range (190–884 Pa) relevant to embryonic vascular development and assess how stiffness regulates self-assembly of human iPSC-derived endothelial progenitors (hiPSC-EPs) into vascular networks, focusing on temporal dynamics of cellular mechanotransduction and contractility, leading to matrix displacement. EPs in intermediate-stiffness hydrogels (∼551 Pa) displayed the highest cell volume and surface area, increased nuclear Yes-associated protein (YAP), and elevated upstream phosphorylated focal adhesion kinase (pFAK), consistent with enhanced mechanotransduction signaling. However, despite high vinculin and pFAK expression, cells at this stiffness condition failed to form a robust, interconnected plexus. In contrast, the most compliant hydrogels (∼190 Pa) supported the most interconnected plexus assembly despite lower nuclear YAP, vinculin, and pFAK expression. ROCK inhibition reduced nuclear YAP, vinculin, and pFAK, confirming tension-dependent regulation, but did not rescue vasculogenesis at 551 Pa and instead impaired network formation at 190 Pa. Importantly, 3D traction microscopy revealed that hiPSC-EPs displayed the highest matrix displacement at this lowest storage modulus, suggesting that cell-generated traction forces and matrix deformability synergistically facilitate vascular morphogenesis. These results suggest that the plexus formation is promoted by matrix compliance that permits matrix displacement, rather than by elevated YAP-mediated mechanotransduction alone. Together, these findings establish a mechanically tunable framework to guide the design of next-generation hydrogels for therapeutic vascular regeneration.