Our ciBEC generation method is amenable to investigating the underlying mechanisms of ciBEC specification. We first attempted to investigate the mechanisms of ciBEC specification by a pharmacological approach. We treated the cells with six inhibitors that are associated with various signaling pathways, such as Notch signaling and Wnt signaling, from the beginning of the co-culture of ECs, pericytes, astrocytes, and neurons. Interestingly, treatment with DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a γ-secretase inhibitor which prevents Notch signaling, inhibited the upregulation of BBB-specific transporters in ECs co-cultured with astrocytes and neurons (Figure 3A). Similarly, LY294002, a phosphoinositide 3-kinase inhibitor, specifically inhibited the upregulation of BCRP in the co-culture system. Wnt inhibitors, including XAV939 and IWP4, did not have significant effects on the expression of BBB transporters (Figure 3A). We next examined Notch activation in ciBECs by immunostaining for Notch intracellular domain (NICD). NICD was highly expressed in the nuclei of ciBECs compared with ECs, and this localization was prevented by DAPT treatment (Figure 3B). These results suggested that Notch signaling is involved in ciBEC generation.
