Journal: bioRxiv

Article Title: Capturing trophectoderm-like stem cells enables step-wisely remodeling of placental development

doi: 10.1101/2025.08.25.672082

Figure Lengend Snippet: (A) The morphology of ESCs, TBLCs and ESCs, TBLCs in TS medium after 3 days of induction. Scale bars, 250 μm. (B) FACS analysis of the percentage of CDX2 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using V6.5 cell line. (C) FACS analysis of the percentage of CD40 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using TC1 cell line. (D) FACS analysis of the percentage of CD40 + TELCs obtained from the TBLCs after induction with different molecules, including FGF4, Activin A, TGFβ1 and BMP4. The corresponding cell morphology is displayed in the lower panel. (E) Scatterplots displaying the transcriptome comparison of TELCs before and after CD40-based FACS using RNA-seq. Upregulated (FC>2) and downregulated (FC<0.5) genes are shown in red and blue, respectively. (F) The morphology of TBLCs of different passages and long-term culture in TX and TS medium, also the morphology of TBLCs after CD40 FACS after induction. Scale bars, 250 μm. (G) Western blotting was used to detect OCT4, CDX2 and EOMES in TELSCs from different passages. β-Tubulin was used as a loading control. (H) The morphology 8C embryos cultured in TX medium. Scale bars, 250 μm. (I) FACS analysis of the percentage of CD40 + cells in TELSC em s at different passages. (J) Immunofluorescence staining of TFAP2C and PEG10 in TBLCs, TELSCs and TELSC em s. Scale bars, 50 μm. (K) Cell cycle analysis of ESCs, TELSCs and TELSC em s. (L) Heatmap indicating the relative expression of TBLCs, TELSCs and TELSC em s. The representative genes and enrichment of GO terms of these genes is shown. (M) Heatmap indicating the relative expression of characteristic genes in TELSCs, TELSC em s and TSCs. Bubble chart showing the relative expression of these genes in mouse embryos. (N) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (O) The scatter plot displays differentially expressed genes between TELSCs and TSCs cultured in various media. The bar graph summarizes the number of differentially expressed genes identified under each comparison condition. (P) GSEA analysis of ESCs, TBLCs, TELCs and TELSCs based on “embryonic placenta development” and “placenta development” geneset. (Q) Heatmap indicating the differentially expressed genes in Hippo pathway of TELSCs and TBLCs. (R) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Bubble chart showing the relative expression of these genes in mouse embryos. (S) Phase contrast images of TBLCs cultured in TS medium for 24h supplemented with Verteporfin at the indicated concentration. Scale bars, 100 µm. (T) Heatmap indicating the differentially expressed genes of TELCs and TBLCs induction in TS medium plus verteporfin. Bubble chart showing the relative expression of these genes in mouse embryos. (U) GSEA analysis of TELCs, TBLCs induction in TS medium and in TS medium plus verteporfin based on TE geneset. (V) The morphology of TELSCs cultured in TS medium, TS medium plus ITS-X and TS medium plus TGFβ1. (W) Heatmap indicating the differentially expressed genes of TELSCs, TBLCs induction in TX medium withdraw ITS-X, in TS medium and in TS medium plus ITS-X. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (X) GSEA analysis of TBLCs induction in TX medium withdraw ITS-X and in TX medium based on “Positive regulation of stem cell proliferation” and “Positive regulation of cell cycle” geneset.

Article Snippet: All TSLs were cultured on Matrigel-coated plates, in 30% TS medium (RPMI 1640 (GIBCO, 11875119), 20% FBS, 1% GlutaMax (GIBCO, 35050061), 1% penicillin-streptomycin (GIBCO, 15140163), 1% sodium pyruvate (GIBCO, 11360070)) and 70% MEF-conditioned TS medium supplemented with 25 ng/ml human recombinant FGF4 (MCE, HY-P7014) and 1 μg/ml heparin (STEMCELL, 7980).

Techniques: Cell Culture, Comparison, RNA Sequencing, Western Blot, Control, Immunofluorescence, Staining, Cell Cycle Assay, Expressing, Concentration Assay