An ECM-driven mechanotransductive signaling module promotes NKX6.1-enriched human pancreatic progenitor formation

Efficient generation of insulin-producing beta cells from human pluripotent stem cells requires precise specification of PDX1+/NKX6.1+ pancreatic progenitors, a developmental bottleneck that limits lineage fidelity and reproducibility across differentiation platforms. Here, we identify a previously unrecognized extracellular matrix (ECM)-coupled mechanotransductive module that promotes an NKX6.1-enriched pancreatic progenitor state. Transcriptomic profiling and targeted perturbations revealed that isoxazole-9 (ISX-9) engages a collagen-TRPV-Ca²⁺-Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) axis, linking ECM sensing to NKX6.1 regulation. Collagen I/IV supplementation and TRPV agonists phenocopied ISX-9, whereas inhibition of integrin α1/α2, TRPV channels, or CaMKII abrogated its effect. Downstream of CaMKII, relief of histone deacetylase 4 (HDAC4)-mediated myocyte enhancer factor 2 (MEF2) repression contributed to NKX6.1 regulation, while increased cycling of NKX6.1+ cells accompanied expansion of the progenitor pool. Temporally restricted activation of this module by ISX-9 increased PDX1+/NKX6.1+ progenitors and yielded stem cell-derived islets (SC-islets) with enhanced glucose responsiveness. These findings provide a basis for optimizing microenvironmental cues to improve the generation of NKX6.1-enriched pancreatic progenitors and functional SC-islets.