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 ± 5.14% (n=10 hMGEOs, mean ± SD) of cells dissociated from hMGEOs expressed NKX2-1-GFP, whereas NKX2-1-GFP+ cells only account for 2.59 ± 1.17% (n=9 hCOs, mean ± SD) of total cell population from hCOs (Figure 1C). At this stage, 93.97 ± 4.78% of hMGEOs cells were FOXG1 positive, and 79.85 ± 5.67% of FOXG1+ cells were also NKX2-1-GFP positive (n=10 hMGEOs, mean ± SD) (Figure 1D). Meanwhile, 94.78 ± 3.56% of cells from hCOs were PAX6 and FOXG1 double positive (Figure 1E), confirming that hCOs underwent efficient telencephalic development. However, we rarely detected PAX6+ cells in dissociated hMGEO culture (Figure S1A), consistent with the expression patterns of PAX6 in developing subcortical domains (Hansen et al., 2013; Ma et al., 2013). Histological analysis revealed neural rosette-like structures that resemble the proliferative regions of the human VZ in both hMGEOs and hCOs (Figure 1F). Both hMGEOs and hCOs gradually gained size under spinning culture, and after 2 months of culture, hMGEOs and
