Fig. 2: The spontaneous qESCs give rise to extraembryonic trophoblast stem cells.

a Left, immunofluorescence of OCT4 (red), CDX2 (green) and nuclei (DAPI blue) at day 7 post TSC differentiation. Scale bar, 200 µm. Right, quantification of CDX2⁺/OCT4⁻ cells. Data were presented as mean ± SEM from three independent experiments. P values were calculated using the two-sided Student’s t test. TSC trophoblast stem cell. b Scheme for TSC derivation. c Left, representative images of typical TSC-like colonies at day 14. The TSC-like colonies were only observed from low ΔΨ_m ESCs, but not from high ΔΨ_m ESCs. Scale bar, 200 µm. Right, efficiency of TSC derivation. The success rate was calculated by dividing the number of successfully derived iTSC lines from total number of picked colonies. d RT-qPCR analysis for indicated genes in ESCs, TSCs and iTSCs. Relative mRNA expression was normalized against that in naive ESCs. Data from two independent cell lines were presented as mean ± SEM in the form of Log₁₀ (fold change). e Immunofluorescence of indicated proteins in indicated cell types. Nuclei were co-stained with DAPI (blue). Scale bar, 200 µm. Images were representatives from two independent experiments. f Top, heatmap showing DNA methylation level at the upstream region of Elf5 promoter. The heatmap score (Log₂) was shown on the right. Each dot indicates a methylated CpG site. Dark arrowheads denote five CpG sites that exhibit substantial differences in methylation levels between ESCs and TSCs. Bottom, quantification of methylation level for five CpG sites shown from the top. Violin plots show the median, 1st and 3rd quartiles. Data from two independent cell lines were presented. P values were determined by a two-tailed, nonparametric Mann-Whitney test. g Representative images from two independent iTSC lines at day 6 of differentiation. The white arrow indicates the trophoblast giant cells. Scale bar, 200 µm. h RT-qPCR analysis for indicated genes in the iTSCs at day 0 and 6 of differentiation. Relative mRNA expression was normalized against that of β-actin. Data from two independent iTSC lines were presented as mean ± SEM. Source data are provided as a Source Data file.