In order to derive a mixed culture of human GABAergic and glutamatergic neurons, hESCs and hiPSCs (S1 Fig) were neural induced into primitive rosette-shaped hNES cells as described previously [25]. Between passages 0 to 10 hPSC-derived hNES cell cultures were characterized by morphology (Fig 1F and S2F Fig) and expression of hNES cell markers [14, 28]. Immunocytochemical analysis showed that hNES cells homogeneously expressed the rosette marker PLZF, the neural precursor marker PAX6 and the stem cell marker SOX2 (Fig 1A–1E and S2A–S2E Fig). RT-PCR indicated the expression of rosette specific markers PLZF, DACH1, ZNF312, HES5, stem cell marker SOX2, and neural stem cell markers Nestin and PAX6 (Fig 1G and S2G Fig). To generate a mixed glutamatergic and GABAergic network of neurons, a ventral GABAergic fate was partially induced using SHH [29, 30] and VPA [20, 31]. hNES cells were treated with 400ng/ml of SHH from day 0–4 followed by VPA from day 5–7 (Fig 1H). Abundant neurite outgrowth was observed at 8 days (Fig 1I). Between days 8–18 precursor cells were supplemented with growth factors BDNF, GDNF, IGF1
