Differentiation of iPSC-derived Brain Microvascular Endothelial Cells

iPSC-derived brain microvascular endothelial-like cells (iBECs) were differentiated as previously described [33 (link), 44 (link)–46 , 48 (link)]. Briefly, iPSCs were seeded from a single cell suspension onto Matrigel coated cell culture flasks (Sarstedt) at a density of 1 × 10⁴ cells/cm2 and expanded in StemFlex medium for 3 days with daily medium changes. Following iPSC expansion, the cells were differentiated in unconditioned medium [UM; DMEM/F-12 (Gibco), 20% Knockout serum replacement (Gibco), 1% minimal essential medium-nonessential amino acids (Gibco), 0.5% GlutaMAX (Gibco), and 0.07% beta-mercaptoethanol (Sigma)] for 6 days, changing medium daily. After 6 days, the medium was changed to basic EC medium [human endothelial cell serum-free media (Gibco) and 1% platelet-poor plasma derived serum (Fisher) or 1% B-27 supplement (Gibco)], supplemented with 20 ng/ml basic fibroblast growth factor (bFGF, ReproTech), and 10 μM all trans-retinoic acid (RA, Sigma), for 2 days. Finally, the differentiated iBECs were purified onto collagen IV (Sigma) and Fibronectin (Sigma) coated plates and transwells inserts (Corning; Greiner). Basic fibroblast growth factor and retinoic acid were removed one day after iBEC purification. Quality of differentiated iBECs was assessed via TEER measurements and immunofluorescence staining of characteristic BEC markers.