BECs were induced by having ECs interact with pericytes, astrocytes, and neurons. To promote this interaction, we prepared a co-culture system with hiPSCs-derived ECs, pericytes, astrocytes, and neurons. Astrocytes and neurons (total 3.5 × 106 cells) from day 90 to day 120 after differentiation were co-cultured on differentiated ECs and pericytes (about 3.5 × 106 cells) at day 7 in SFM (Life Technologies) supplemented with 20 ng/mL bFGF (Wako), 10 ng/mL EGF (Life Technologies), and 10 μg/mL human plasma fibronectin (Life Technologies) for 5 days. The population of the co-culture at day 7 included about 49.5% CD31-positive ECs, 0.5% PDGFRβ-positive pericytes, 20% TUJ1-positive neurons, and 22.5% GFAP-positive astrocytes. We refreshed the medium every 3 days. The population of the co-culture at day 12 included approximately 10% CD31-positive ECs, 5% PDGFRβ-positive pericytes, 20% TUJ1-positive neurons, and 45% GFAP-positive astrocytes (Figures 1 and S1). ciBECs and ECs were purified with FACS AriaII (Becton Dickinson) using anti-CD31 (PECAM1) conjugated with APC at 12 days after differentiation. The ciBECs were used to form the BBB model.
