In our protocol, we developed a hiPSC differentiation system to produce ECs that depends on cyclic AMP (cAMP). cAMP is crucial for enhancing the EC differentiation and arterial specification of mouse embryonic stem cells (Yamamizu et al., 2009, Yamamizu et al., 2010, Yamamizu et al., 2012a, Yamamizu et al., 2012b). To generate BECs, we co-cultured the ECs with pericytes, neurons, and astrocytes, which were also derived from hiPSCs. Culturally induced BECs (ciBECs) showed properties consistent of the BBB, including an enrichment of BBB-specific transporters, good barrier function, and the efflux of drugs. This method allowed us to investigate the mechanisms of BBB formation, which led to our discovery that ciBEC specification occurs through Notch signaling via Dll1 in neurons. Our model should be useful for the study of BBB development and homeostasis, and also for the discovery of CNS drugs that bypass the BBB.
