Extended Data Fig. 9: Characterization of hypoblast-lake layer and extraembryonic mesoderm-like cells in human SEMs.

a, representative immunofluorescence image of day 6 SEM showing apical polarity of the visceral and parietal hypoblast-like layers (SOX17, yellow); aPKC (heat gradient), F-ACTIN (white). Scale bar, 50 µm; zoom, 10 µm. See also Supplementary Video 6. b, representative immunofluorescence image of day 6 SEM showing OCT4 (cyan), GATA4 (red), SOX17 (yellow), and nuclei (DAPI). Right, zoom on ExEM-like cells expressing GATA4, but not SOX17. YS, yolk sac-like. Scale bar, 50 µm; zoom, 10 µm. c (left), zoom into the SEM example with adequate morphology and structure (see Methods) aggregated from RUES2 reporter hESC line showing epiblast-like (SOX2-Citrine, yellow), hypoblast-like (SOX17-tdTomato, red), and trophoblast-like (CK7, magenta) compartments with 0.08% efficiency (N = 3 across 409, 295, and 528 aggregates); nuclei (DAPI, white); scale bar, 50 µm. c (right), representative large field of view immunofluorescence image of day 6 SEM aggregates, the zoom on the left is outlined; scale bar, 500 µm. d, representative immunofluorescence image of day 6 SEM showing mesenchymal-like cells underneath the yolk sac-like structure; OCT4 (cyan), F-ACTIN (red), and nuclei (DAPI, white). Right, zoom into the region underneath the yolk sac-like structure, shown in different Z-planes (number 50, 60, and 70). Arrows point at the cells between yolk sac-like and the trophoblast-like compartments. Scale bar, 50 µm; zoom, 25 µm. See also Supplementary Video 4. e, representative immunofluorescence images of day 6 SEMs showing chorionic-like cavity surrounded by ExEM-like cells (outlined), negative for SOX17 (yellow), but expressing BST2 (top, red) and GATA6 (bottom, red); OCT4 (cyan), nuclei (DAPI, white). Scale bar, 50 µm. f, heatmap of GATA6 and SOX17 gene expression across extraembryonic tissues in marmoset, corresponding to the Carnegie stages (CS) 5-7), extracted from previously published gene expression dataset⁸. Am, amnion; Tb, trophoblast; VE, visceral endoderm; SYS, secondary yolk sac; ExEM, extraembryonic mesoderm. g (top), representative immunofluorescence image of Z slice from day 8 SEM and the zoom into the ExEM-like region showing VIM (red) expression and nuclei (DAPI, white). g (bottom), merged brightfield and maximum intensity projection showing VIM (red) expression. Scale bar, 50 µm; zoom, 10 µm. h (top), representative immunofluorescence image of day 6 SEM from aggregation with trophoblast-like cells (control). The arrows in the zoom below point at the apical surface of the visceral hypoblast-like layer (SOX17, yellow); aPKC (green), F-ACTIN (red). Scale bar, 50 µm; zoom, 10 µm. h (bottom), representative immunofluorescence image of day 6 aggregate from experiment where trophoblast-like cell fraction was omitted (no BAP(J)) condition (related to Fig. 4k). The arrows in the zoom below point at the apical surface of the PrE/hypoblast-like cells (or layer) surrounding the aggregate (SOX17, yellow); aPKC (green), F-ACTIN (red). Scale bar, 50 µm; zoom, 10 µm.