To create a BBB model with hiPSC-derived ciBECs, we seeded ciBECs purified from the co-culture with pericytes, neurons, and astrocytes by FACS onto Transwell filters coated with fibronectin and grew them to confluence for 7 days (Figure 5A). Immunostaining of ZO-1 and CLAUDIN5 showed the maintenance of continuous cell-cell contacts between ciBECs on the Transwell filter (Figure 5B). Western blotting with membrane protein showed that CLAUDIN5 was highly expressed in ciBECs compared with ECs (Figure S4B). A hallmark of the BBB is high transendothelial electrical resistance (TEER) due to tight junctions between ciBECs. As expected, ciBECs showed a significant increase of TEER compared with ECs. When we co-cultured the cells with astrocytes from days 125–180 after differentiation on the bottom side of the Transwell, the TEER of ciBECs, but not of ECs, was dramatically enhanced (Figure 5C), indicating ciBECs had a brain-like phenotype. To confirm the existence of tight junctions between ciBECs, we analyzed the cells by transmission electron microscopy. ciBECs co-cultured with astrocytes clearly formed tight junctions (Figure 5D). To further validate the barrier function in our iPSC-derived BBB
