The brain vasculature sprouts into the neuronal tube and elongates toward the ventricular zone to form vascular networks that are surrounded by neuroepithelial cells, radial glia, neuroblasts, and neurons at around embryonic day 9.5 (E9.5) in mouse embryo (Engelhardt, 2003). The vascular permeability decreases at E16 as it interacts with BECs, neurons, and radial glial cells (Risau et al., 1986). Our ciBEC specification process seems to involve Notch signaling in response to neuron-derived Dll1 (Figure 4C), which may recapitulate the in vivo microenvironment for BBB development. We found that mature barrier characteristics of the BBB were induced by crosstalk between iPSC-derived astrocytes (Figure 5C). Several astrocytic factors for BBB maturation, including transforming growth factor, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), interleukin-6, angiopoietin-1 (Ang1), and hydrocortisone, are capable of glial-mediated barrier induction (Abbott, 2002, Lee et al., 2003). Indeed, hiPSC-derived astrocytes highly expressed GDNF, bFGF, EGF (epidermal growth factor), and Ang1 (Figure S6D). Further analyses are needed to identify the BBB maturation factors from hiPSC-derived astrocytes. In addition, previous anatomical examination of the brain microvasculature showed that
