together with NEUROD1, an indispensable transcriptional activator that regulates neuronal differentiation of granule neurons in the hippocampus (Miyata et al., 1999; Liu et al., 2000; Schwab et al., 2000; Gao et al., 2009) (Figure 1F). These hippocampal NPCs further matured into neuroblasts and postmitotic granule neurons, as indicated by the increasing levels of DCX and TBR1, together with PROX1, after 20 days of differentiation (Figures 1E and 1G). Taken together, these data indicate that we were able to obtain PROX1+ hippocampal granule neurons from hESCs and that these neurons were generated via a process that recapitulated important aspects of hippocampal neurogenesis in vivo. We then dissociated the EBs after 40 days of differentiation and coculture with human hippocampal astrocytes to allow formation of neural networks and facilitate further neuronal maturation. As a result, we observed neurons that had extensively branched dendritic arbors and were triple positive for PROX1, MAP2AB, and NEUN, indicative of postmitotic hippocampal granule neurons (Figures 1H and 1I).
