A synthetic hydrogel niche reveals the interplay of matrix adhesion and mechanics in human epiblast model development

• MMP-cleavable starPEG-GAG hydrogels enable orthogonal stiffness/adhesion control. • DoE analysis reveals that vitronectin-mediated adhesion dominates iPSC survival. • Adhesion, stiffness and ROCK inhibition drive lumenogenesis and polarity. • Epiblast models maintain pluripotency and trilineage differentiation capacity. • Defined, Matrigel-free platform for the mechanobiology studies of early development. Investigating the role of physical and adhesive matrix cues in human peri -implantation development is hampered by insufficiently defined culture systems, limiting mechanistic understanding. Here, we use a rationally designed matrix metalloproteinase (MMP)-cleavable glycosaminoglycan-based hydrogel system with orthogonally tunable stiffness and adhesiveness to systematically dissect the exogenous control of epiblast morphogenesis in an induced pluripotent stem cell (iPSC)-based model system. A Design of Experiments (DoE) approach reveals that matrix adhesiveness is the primary factor governing iPSC survival, outweighing the influence of stiffness. We further reveal that adhesive ligand presentation, matrix stiffness, and prolonged Rho-associated protein kinase (ROCK) inhibition cooperatively drive the self-organization of iPSCs into lumenized structures with apicobasal polarity. The resulting epiblast models maintain pluripotency and trilineage differentiation potential. Importantly, pluripotency maintenance and morphogenetic competence are partially decoupled, with matrix properties more strongly influencing morphogenesis than pluripotency among viable cells. In sum, our work establishes a defined platform to decouple the interplay of matrix mechanics and adhesion, revealing how their coordination with intracellular signaling controls early epiblast development and providing design principles for engineering synthetic microenvironments that recapitulate developmental processes.