IMR90-c4 induced pluripotent stem cell line was obtained from the WiCell cell repository (WiCell, Madison, WI, USA). iPSCs differentiated into brain microvascular endothelial cells (BMECs) as per the established protocol [
50,
51]. Briefly, undifferentiated stem cells were maintained on Matrigel (C-Matrigel; Corning, Corning, MA, USA) coated 6-well plate in Essential 8 medium (E8 Thermo Fisher, Waltham, MA, USA) containing 10 μM Y-27632 (Tocris, Minneapolis, MN, USA) for 3 days before differentiation. Then, differentiation was initiated using an unconditioned medium [UM: Dulbecco’s modified Eagle’s medium/F12 with 15 mM HEPES (Thermo Fisher, Waltham, MA, USA), 20% knockout serum replacement (Thermo Fisher, Waltham, MA, USA), 1% non-essential amino acids (Thermo Fisher, Waltham, MA, USA), 0.5% Glutamax (Thermo Fisher, Waltham, MA, USA) and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA)] and maintained for 6 days. On day 6 of differentiation, cells were incubated for 2 more days in EC
++ media [human serum-free endothelial medium (hESFM, Thermo Fisher, Waltham, MA, USA) supplemented with 1% bovine platelet-poor plasma-derived serum (PDS, Alfa Aesar, Ward Mill, MA, USA), 10 ng/mL bFGF and 10 μM retinoic acid (Sigma- Aldrich)]. On day 8 of differentiation, cells were enzymatically dissociated using Accutase
® (Corning) and seeded as single cells on 12-wells transwell systems (polyester, 0.4 μm pore size; filter area 1.1 cm
2, Corning) pre-coated with a solution of collagen from human placenta (Sigma-Aldrich) and bovine plasma fibronectin (Sigma-Aldrich) (400 μg/ mL collagen IV and 100 μg/mL fibronectin) at a density of 1,000,000 cells/cm
2 [
52]. Please note that higher seeding density is necessary to compensate for the much lower rate of proliferation and higher cell losses of iPSC-BMECs compared to immortalized and primary brain microvascular endothelial cells. 24 h after seeding, media was replaced with EC
−− (EC medium supplemented with 1% platelet-poor derived serum). Purified endothelial monolayers were formed on day 10 of the experiment, and barrier integrity tests were performed 48 h after seeding on the transwell system.