To characterize human interneuron development, we developed a protocol to generate MGE-like cells in vitro based on previous methods (Maroof et al., 2013). Telencephalic neural induction of hESCs was initiated with dual SMAD inhibition, combined with a small-molecule inhibitor of the WNT pathway (Figure 1A)(Chambers et al., 2009). On day 10 (D10), cells were ventralized using sonic hedgehog (Shh) and purmorphamine for 8 days (orange bar). On D24, the ventralized cells were seeded at low density to induce neuronal differentiation (green bar). Immunofluorescence at D10 indicated widespread SOX2 expression, and MKI67-positive proliferating cells, indicating that these cultures were correctly specified as neuronal progenitors (Figure 1B). We assessed high (100 ng/ml Shh and 1 μM purmorphomine) and low (50 ng/ml Shh and 0.5 μM purmorphamine) Shh treatment conditions to determine the effects on MGE subtype specification (Xu et al., 2010). With high levels of Shh signaling factors, 74.5 ± 9.4 % of cells were NKX2-1+ compared with 65.2 ± 6.2% in cultures treated with low levels of Shh, though this difference was not statistically significant (Figure 1C). Telencephalic specification was equivalent
