(A) Expression of the Sox2-T2A-H2b-tdTomato (Sox2::HT) reporter from the endogenous Sox2 allele in targeted TST18 cells. TST18 cells cultured in LIF/FCS/GMEMβ were replated in LIF/2i/N2B27 or LIF/BMP4/N2B27 for four passages or in Activin/FGF/N2B27 (Activin/FGF) for nine passages, examined microscopically (top) and assessed by flow cytometry (bottom); E14Tg2a cells were represented as a grey dashed line. (B) Three gates (A, B, C) were used to purify cells for microarray analysis. Gate C captured the Sox2::HT level in LIF/2i cultured ESCs. Gate B captured the overlapping Sox2::HT level in LIF/FCS, LIF/BMP and Activin/FGF cultured cells. Gate A captured the lower Sox2::HT level in Activin/FGF. (C) Principal component analysis of cells in different culture conditions, either unsorted or sorted using the gates indicated by brackets. (D) Despite similar Sox2::HT levels, no differentially expressed genes (DEGs, FDR = 0.1) were common to LIF/FCS-low and Activin/FGF-high cell populations. (E) RT-qPCR analysis of the indicated transcripts in ESCs (LIF/FCS, red bars) and EpiSCs (Activin/FGF, cultured for 16 passages, blue bars). Transcript levels were normalized to Tbp and plotted relative to ESCs. Error bars represent standard error of the mean (n = 3 to 4).