Extended Data Fig. 5: Comparison of molecular properties between MTSCs, TSCs, and TESCs.

(a) Clone formation and cell proliferation from 32-cell stage embryo cultured in the TESC induction medium for 6 days. The red circles indicate TESC clones; green arrows indicate trophoblast giant cells (TGCs). (b) Immunostaining for CDX2 of MTSCs, TESCs, and TSCs. BF, Bright field. (c) Analysis of fluorescence intensity of CDX2 positive cells in TSCs, TESCs, and MTSCs. (d) mRNA expressions of Tead4, Klf5, Cdx2, Eomes, Ly6a, Gost1, and Sox2 in TSCs, TESCs, MTSCs, and Diff-TSCs detected by qRT-PCR. diff-TSC, TSCs after removal of FGF4 for 2 days. (f) mRNA expressions of Gcm1, Ctsq, Tpbpa, Prl3d1, Krt8, and Krt18 in TSCs, TESCs, MTSCs, and Diff-TSCs were detected by qRT-PCR. diff-TSC, TSCs after removal of FGF4 for 2 days. (g) Transcription analysis of differentially expressed genes between MTSCs, TESCs, and TSCs. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. blue bar, up-regulated genes; orange bar, down-regulated genes; green, total differentially expressed genes. (h) Immunostaining for CDX2, YAP, and E-CAD of TESCs and MTSC cultured in the TESC medium for 48 h. (i) Heatmap analysis of differentially expressed genes between TESCs, MTSCs, TSCs, and differentiated TSCs (Diff-TSC). “Diff-TSC” refers to TSCs after FGF4 removal for 2 days. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. Experiments were repeated three times (a, b, g, h) with similar results. Data are shown as mean ± SEM from three independent experiments (d, e). Numerical data are available as source data. Scale bars: 50 μm in a; 20 μm in b, g, and h.