By day 18, over 85% of cells expressed PAX6, and more than 80% of these progenitors expressed FOXG1 (Fig. 1b; Supplementary Fig. 1). To promote differentiation of neural progenitors into neurons, FGF2 and EGF were replaced with brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) starting at day 25. From day 43 onward, only neural medium without growth factors was used for medium changes every 4 d. After ~7 weeks of differentiation in vitro, 78.8% ± 2.5 (n = 4 hCSs, mean ± s.e.m.) of the cells expressed the neuronal marker β3-tubulin (Fig. 1c). We also observed a small population of cells (7.6% ± 1.02, n = 6 hCSs, mean ± s.e.m. at day 76) expressing the astrocyte and radial glial marker GFAP. At this stage, 36.2% ± 3.6 (n = 3 hCSs, mean ± s.e.m.) of neurons expressed the mature neuronal marker NEUN, which is present in the human forebrain only after 20 weeks of gestation11. The hCSs grew in size to more than 300 μm in diameter by 2 weeks of culture and reached up to 4
