Extended Data Fig. 9: Trophectoderm state is crucial for interaction with endometrium during implantation.
From: Human blastoids model blastocyst development and implantation
a. Representative images of human blastoids shortly after attachment to an OFEL. Dotted line outlines the inner cluster of blastoids that were formed using GFP+ naive hPSCs (top, also see Supplementary Video 3). Immunofluorescence stainings for NR2F2 (Magenta) and OCT4 (Yellow) in blastoids shortly after attachment to an OFEL (bottom). b. Immunofluorescence stainings for NR2F2 (Magenta) and OCT4 (Yellow) and respective fluorescence intensity profiles of representative blastoids immediately after attachment onto OFEL. Profiles were measured perpendicular to the plane of attachment (right). Line width, 10 µm. Y axis shows normalized intensity. c. Quantification of the distance between the first peak of fluorescence intensity profiles of NR2F2 and OCT4. n=10 attached blastoids. mean± S.D. d. Pseudotime analysis of human pre-implantation development showing the expression of IL6, IL6R, GP130 and STAT3. Gene expression analysis is performed by using the public data analysis tool (https://bird2cluster.univ-nantes.fr/demo/PseudoTimeUI/). e. Quantification of the dose dependent effect of LIF on the yield of blastoids. n=2 (without Lif) and n=3 (all other conditions) independent experiments. mean± S.D. f. Immunofluorescence staining for NANOG (Yellow) and CDX2 (Cyan) (left), OCT4 (Yellow) and GATA3 (Cyan) (middle) and CDX2 (Cyan) and NR2F2 (Magenta) (right) in representative trophospheres formed from a blastoid exposed to SC144. Scale bar: 50 μm. g. Immunofluorescence staining for NANOG (Yellow) and CDX2 (Cyan) (left), OCT4 (Yellow) and GATA3 (Cyan) (right) in representative trophospheres formed from a blastoid exposed to XMU-MP-1. Scale bar: 50 μm. h. Heatmap of key lineage specific genes differentially expressed in bulk transcriptome of the trophectoderm of blastoids (TROP2 positive cells), trophospheres (SC144 or XMU) and TSCs (2D or 3D) compared to naive hPSCs. i. PCA plot computed using bulk transcriptome of blastoid cells, hPSCs (naive, primed or blastoid rederived naive cell lines), TSCs (bTS5, blastocyst rederived lines or human stem cell derived TSC like cells) and pluripotent stem cell derived primitive endoderm like cells (RACL or NACL cells). j. Immunofluorescence stainings for CDX2 (Cyan) (left) and CK7 (Magenta) and GATA3 (Cyan) (right) in aggregates formed from bTS5 hTSCs. Counterstain with Hoechst marking DNA. Scale bar: 50 μm. k. Representative phase contrast images of aggregates of naive hPSCs, deposited onto stimulated OFELs. Scale bar: 100 μm. l. List of selected putative ligand-receptor pairs involved in cross-talk between polar trophectoderm and endometrial epithelial cells. The list was generated by in silico ligand receptor analysis of genes enriched in polar trophectoderm and stimulated OFEL, using Cellinker47.
