Differentiation of striatal projection neurons from NPCs. (A): Outline of the spontaneous differentiation protocol. (B): Outline of the two-step differentiation protocol. (C): Outline of the three-step differentiation protocol. (D): SOX2, Nestin, PAX6, NKX2.1, FOXG1, MASH1, and MEIS2 immunostaining of NPCs. The scale bar corresponds to 50 μm on the specimen level. (E): Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis of EMX2, PAX6, NKX2.1, GBX2, MEIS2, and MASH1 expression in NPCs. For NKX2.1, no amplification was observed in the qRT-PCR analysis (no Ct value). Abbreviations: B-27, B-27 Supplement XenoFree CTS; D, day; DAPI, 4′,6-diamidino-2-phenylindole; DMEM/F12, Dulbecco’s modified Eagle’s medium nutrient mixture F12; GF, growth factors; IGF-1, insulin-like growth factor 1; MO, morphogens; NPC, neural progenitor cell; NT, neurotrophins; VPA, valproic acid.