We next induced astrocytes and neurons from hiPSCs. We established a differentiation method by modifying previous procedures (Kondo et al., 2013) (Figure 1I). After plating embryoid bodies, almost all differentiated cells were positive for NESTIN, a neural progenitor marker, and some cell populations started to differentiate into class III β-tubulin (TUJ1)-positive neurons at stage 2 (Figure 1J). TUJ1-positive neurons were dramatically induced by changing the medium at stage 3 (Figure 1K). Glial fibrillary acidic protein (GFAP)-positive astrocytes were increased after neural maturation at stage 4, which was followed by brain development in vivo (Figure 1L). An astrocyte-enrichment culture showed a high population of GFAP-positive astrocytes at stage 5 (Figure 1M). qPCR clearly showed the induction of the neural progenitor marker Pax6 at stage 2, the mature neuron marker MAP2 at stage 3, and the astrocyte marker GFAP at stages 4 and 5 (Figure 1N). Taken together, our two systems efficiently differentiated hiPSCs into the four BBB components.
