To develop a differentiation paradigm that enriches for hippocampal granule neurons, we first utilized free-floating EBs, which provide 3D spatial cues that have been shown to simulate aspects of pregastrulation development and early gastrulation (Kopper et al., 2010). The differentiation approach was optimized based on key morphological cues that specify the hippocampal DG identity during development. Mimicking the developmental signals that generate the anterior-posterior patterning of the forebrain, we first treated the EBs with a cocktail of anticaudalizing factors (DKK1, noggin, and SB431542) blocking the WNT, bone morphogenetic protein (BMP), and transforming growth factor-β (TGF-β) pathways to obtain telencephalic neural precursors (Watanabe et al., 2005). In addition, we applied cyclopamine, an antagonist of the Sonic Hedgehog pathway, to enrich for dorsal forebrain progenitors (Gaspard et al., 2008) (Figure 1A). After 20 days, the mature EBs were treated with Wnt3a, a WNT protein previously shown to be important for the maintenance of hippocampal progenitors and their differentiation into DG granule neurons (Lee et al., 2000; Machon et al., 2007; Kuwabara et al., 2009; Wexler et al., 2009; Karalay et al., 2011),
