To further authenticate the generation of DG granule neurons using this protocol, we assessed the EBs at various time points to track the development of these neurons during the differentiation process. Previous studies of both embryonic and adult neurogenesis in the DG have revealed a number of transcription factors, as well as their expression patterns, that are important for directing neuronal fate specification and lineage commitment during hippocampal neurogenesis (Hodge et al., 2012; Hsieh, 2012). We examined the expression dynamics of these key markers to determine the extent to which our protocol for in vitro differentiation recapitulated the in vivo neurogenesis process. Following 10 days of differentiation, we observed upregulated expression levels of EMX2, FOXG1, and PAX6, which are markers found in hippocampal NPCs (Pellegrini et al., 1996; Shinozaki et al., 2004; Shen et al., 2006; Osumi et al., 2008), 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
