The BBB is composed of specialized BECs surrounded by pericytes, astrocytes, and neurons. Thus, we hypothesized that BECs are generated by cell-cell interactions with the other three lineages and created a co-culture system with the four cell populations derived from hiPSCs described above. Astrocytes and neurons from 90 to 120 days after differentiation were recultured on differentiated cells with the EC and pericyte differentiation system at day 7 after differentiation (Figure 2A). Immunostaining showed that TUJ1-positive neurons and GFAP-positive astrocytes were approximately 40% and 45% of the total population from 90 to 120 days after differentiation (Figure S1A). The endfeet of astrocytes elongated to the ECs, while neurons also interacted with ECs during the co-culture (Figures 2B and 2C). After co-culture with the four lineages derived from hiPSCs for 5 days, we purified ciBECs by FACS and analyzed their properties. Notably, 21 of the 22 BBB transporters and receptors analyzed in this study tended to have higher expressions in ciBECs compared with normal ECs, which were not co-cultured with astrocytes and neurons. Of the BBB-specific transporters analyzed, six, including cationic
