Time course of the differentiation of the hiPSCs and a schematic representation of the hydrogel model on transwell filters.

HiPSCs were maintained in mTeSR1 medium. When they had reached an optimum density of 3x10⁴/cm² they were switched to unconditioned (UC) medium for 6 days, followed by serum free endothelial cell (EC) medium containing B27, human basic fibroblast growth factor (hbFGF) and retinoic acid (RA) for 2 days. Strongly adherent cells were isolated by 1hour incubation on collagen IV/fibronectin-coated plates in Hanks balanced salt solution (HBSS). Adherent, endothelial-like cells were plated onto a 1cm² transwell insert that had a 0.3ml collagen I hydrogel and a fibronectin/collagen IV surface layer, prepared 24hours previously. The cells were cultured in EC medium for 24-48hours before use, and the inserts were usable for up to 7 days.