Fig. 3: Stabilization of a naïve-like pluripotent state with a KLF2/ERAS/PRMT6 gene cocktail.

a Phase-contrast and confocal images of B19_VAL before and after single, double, and triple transfection with PiggyBac plasmids expressing KLF2-V5 (K), HA-ERAS (E), and flag-PRMT6 (P). Immunostainings for CD75, H3K27me3 and H2AK119Ub is shown. Scale bars: phase contrast, 100 µm; CD75, 50 µm; histone modifications, 20 µm. b Graphical representing the distribution of fluorescence intensities (between 0 and 200 arbitrary units) for CD75 immunostaining. Source data are provided in the Source Data file. c Percentage of cells with nuclear foci of H3K27me3 and H2AK119Ub across control, single-, double-, and triple-transfected cell populations. Data are from three independent replicates, with each dot representing one replicate. “n” indicates the number of cells analyzed per condition. Bars and error bars represent mean ± SEM. A two-sided Welch’s t-test revealed significantly lower percentages of cells with nuclear foci in KEP_VAL compared to all other conditions (p \(\le \,\)0.01). Source data are provided in the Source Data file. d Immunostaining of KEP_VAL cells for CD75 and H3K27me3. Scale bar: 40 µm. e Experimental design for generating KEPc_KF and KEPc_VAL stable cell lines. f Phase-contrast and confocal images of control B19_KF, KEPc#37_KF, and KEPc#37_VAL cells stained for KLF2-V5, HA-ERAS, flag-PRMT6, H3R2me2/3, H3K27me3, and H2AK119Ub. Scale bars: phase contrast, 100 µm; immunostainings, 30 µm. g Quantification of cells with H3K27me3 and H2AK119Ub nuclear foci in B19 parental iPSCs, KEPc_KF, and KEPc_VAL cell lines. Values represent means ± SEM from at least 300 cells per condition (exact n provided), based on three independent replicates (additional replicates for parental B19 cells). Each dot indicates a replicate. Two-sided Welch’s t-test was used to compare KF and VAL conditions (p values indicated). Source data are provided in the Source Data file. h Two-dimensional PCA of transcriptomic profiles from five cell populations (B19, KEPc#37, #39, #42, and #44), cultured in two conditions (KF: square; VALGöX: circle), with three biological replicates each. i Histograms showing gene expression levels (RNA-seq) in B19_KF, B19_VAL, KEPc_KF, and KEPc_VAL lines. Mean ± SD are from three independent replicates. j Heatmap of differentially expressed genes among B19_KF and four KEPc_VAL lines (#37, #39, #42, and #44). Genes were selected from a set of 80 differentially expressed genes identified during embryonic development (inner cell mass at E3.5, early epiblast at E4.0, mid-epiblast at E5.0, and late epiblast at E6.0-E6.6²⁴).