PI3K inhibitor-free differentiation and maturation of human iPSC-derived arterial- and venous-like endothelial cells

Abstract Congenital vascular malformations are commonly caused by aberrant, genetic activation of class I phosphoinositide 3-kinase (PI3K) signalling. Advances in mechanistic understanding and therapeutic targeting of these disorders will be accelerated by high-fidelity, human disease models. Building on a previously optimised differentiation strategy, we present a validated workflow for PI3K inhibitor-free generation of arterial- and venous-like endothelial cells from human induced pluripotent stem cells (iPSCs) under defined, xeno-free conditions. We further report experimental analyses of endothelial maturation under flow, culture duration-dependent stability, and downstream molecular and phenotypic characterisation. By providing a reproducible human system for mechanistic and translational studies, this platform will enable disease-relevant modelling of PI3K-driven vascular malformations, including PIK3CA -related overgrowth spectrum (PROS) and PTEN hamartoma tumour syndrome (PHTS).