Here, we present a differentiation paradigm that produces an enriched population of hippocampal DG granule neurons using key developmental cues that specify the hippocampal DG identity. Using two differentiation approaches based on free-floating embryoid bodies (EBs) and neural progenitor cell (NPC) monolayers, we generated neurons expressing PROX1 and TBR1, markers found in mature DG granule neurons (Hodge et al., 2012; Iwano et al., 2012). The differentiation process recapitulates expression patterns of key developmental genes, including NEUROD1 and DCX, that are critical for hippocampal neurogenesis (Miyata et al., 1999; Liu et al., 2000; Schwab et al., 2000; Gao et al., 2009). Furthermore, these neurons formed functional networks characteristic of granule neuron maturation and are able to functionally integrate into the endogenous DG upon in vivo transplantation. To test the effectiveness of this protocol to uncover functional defects in human diseased neurons, we used this differentiation paradigm on SCZD patient hiPSCs and control hiPSCs as a proof-of-principle application. We found deficits in the generation of hippocampal granule neurons from SCZD hiPSC-derived hippocampal NPCs with reduced levels of PROX1 and TBR1. Furthermore, SCZD
