Cells differentiated for 24, 54 and 100 days were purified by fluorescence activated cell sorting (FACS) and analyzed by RNA-Seq for validation. Analysis of key genes revealed that the in vitro generation of MGE-like cells replicates the temporal progression of gene expression observed in vivo. For descriptive purposes, we considered log10(TPM+1) values of <1 to be low expression, 1 – 2 to be moderate, and values above 2 to be high levels of expression. Forebrain patterning genes (OTX2, FOXG1, LHX2, SIX3, SOX2) were highly expressed in progenitors, with OTX2 and FOXG1 expressed in Cit+ cells at moderate levels (Figure 2A)(Acampora et al., 1995; Dou et al., 1999; Ferri et al., 2013; Gestri et al., 2005; Kelly and Moon, 1995; Matsuo et al., 1995; Monuki et al., 2001). PAX6 was expressed at moderate levels in Cit− cells at D24, but low levels thereafter (Briscoe et al., 1999). Pallial markers (EMX1) or midbrain/hindbrain markers (PAX2, IRX3, EN2 and GBX2) were expressed at low levels or absent. These results suggest this in vitro protocol correctly specified cells as ventral telencephalic progenitors.
