We generated hMGEOs and hCOs based on the developmental principles that anterior neuroectodermal fates can be achieved by dual SMAD-inhibition (Chambers et al., 2009), and activation of SHH signaling pathway induces ventral fates (Maroof et al., 2013; Nicholas et al., 2013). Canonical Wnt signaling was also inhibited in order to enhance the neural induction (Maroof et al., 2013; Nicholas et al., 2013) (Figure 1A, see STAR methods for details). The MGE of developing ventricular zone in the human brain is marked by the expression of NKX2-1. To facilitate the identification of this domain, we used a previously established and widely adopted NKX2-1-GFP reporter hESC line (HES-3 NKX2-1GFP/w) (Germain et al., 2013; Goulburn et al., 2011; Maroof et al., 2013; Nicholas et al., 2013). We observed robust NKX2-1-GFP induction that marked the whole organoid by day 18 using hMGEO protocol (Figure 1B). In contrast, organoids generated from hCO protocol showed a limited cluster of NKX2-1-GFP+ cells, indicating spontaneous production of MGE progenitors inside hCOs (Figure 1B). In total, we produced over 400 hMGEOs and over 300 hCOs. By day 21, 82.40
