a, Second reversion of control and TFAP2C−/− cells. Bright-field images of cells are shown. Cells were then sorted and TRA-1-85+ (human) cells were gated into SSEA4- negative and positive populations. In this reversion many SSEA4- cells were apparent, but subsequent RNA-seq indicated that they showed low expression of pluripotency genes and elevated neural gene expression, indicating that they were differentiated rather than naive. Scale bar indicates 100 μm. b, Expression (RPKM) of pluripotency markers, naive-specific markers, and primed markers of control and TFAP2C−/− cells relative to primed controls. Ratio is shown for n = 2 biologically independent replicates (Control cells, TFAP2C−/− SSEA4-) or n = 3 replicates TFAP2C−/− SSEA4+ over n = 2 primed control biological independent replicates. c, Principle component analysis of RNA-seq from all pluripotent datasets. Embryoid body differentiation, day 5 5iLAF TFAP2C−/− cells, and 5% O2 reversion 2 TFAP2C−/− SSEA4- cells were excluded because they diverged strongly due to loss of pluripotency. Blue dots: TFAP2C−/− cells in 5% O2 show a shift toward primed-like gene expression relative to control cells. Brown dots: no shift toward naive identity is observed in cells which overexpress TFAP2C in primed media conditions. Green dots, rescue of TFAP2C−/− cells with doxycycline-inducible TFAP2C is indicated by partial shift toward naive identity. d, e, Average RPKM in the indicated cell type is plotted for all genes previously determined6 to be downregulated (d) or upregulated (e) upon the transition from pre-implantation to post-implantation epiblast in primates. The box and whiskers are plotted by the Tukey method. f, Immunofluorescent images of KLF17 and TRA-1-60 staining in control and TFAP2C−/− cells cultured in t2iLGöY. Data represent 1 of 2 independent experiments with similar results. Scale bars, 20μm. g, Ratio of KLF17 expression in t2iLGöY/primed conditions for WT control and TFAPC2−/− cells. Data are for n = 2 control and n = 3 TFAP2C−/− biologically independent replicates in both naive and primed conditions. Source data for b is in Supplementary Table 8.