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

Vascular malformations caused by genetic PI3K pathway activation are highly debilitating and hard to treat. Here, we present a PI3K inhibitor-free protocol to differentiate arterial and venous endothelial cells from human induced pluripotent stem cells (iPSCs) in 2D and 3D formats. We describe steps for iPSC culture, cell seeding on a synthetic matrix, defined media preparation, magnetic-activated cell sorting, and endothelial cell maturation under shear stress. We then detail procedures for determining cell density and culture duration for robust functional phenotyping.