Extended Data Fig. 8: Characterization of the epiblast-like structure in human SEM.
From: Complete human day 14 post-implantation embryo models from naive ES cells
a, representative immunofluorescence image of day 6 SEM showing aPKC (green), OCT4 (cyan), F-ACTIN (red), nuclei (DAPI, white). Scale bar, 50 µm; 12.5 µm (zoom, right). b (left), immunofluorescence image of day 6 SEM showing OCT4 (cyan), F-ACTIN (red), and phERM (green); alignment of Epi-like cells in a single 2D plane is marked with dashed lines; asterisk, pro-amniotic-like cavity. Scale bar, 25 µm. b (right), quantification of the angle between the epiblast-like cell axis and the pro-amniotic-like cavity; the plot shows the radial histogram of the angle values and indicates predominant alignment of epi-like cells towards the center of the emerging cavity (n = 32). c, representative immunofluorescence image of day 6 SEM showing T/BRA expression (red); OCT4 (cyan), nuclei (DAPI, white). Right, zoom into the posterior epiblast-like structure, arrows mark individual T/BRA-positive cells. Scale bar, 50 µm, 12.5 µm (4x zoom in, right). d, representative immunofluorescence image of day 6 SEM showing CER1 (green) localization inside the intracellular vesicles; OCT4 (cyan), nuclei (DAPI, white). Right, zoom into hypoblast-like compartment, arrows mark apical side of the visceral endoderm-like cells with CER1 vesicles. Scale bar, 50 µm, 12.5 µm (4x zoom, right). e (left), immunofluorescence image of day 6 aggregates showing T (red), OCT4 (cyan), CER1 (green), and nuclei (DAPI, white); scale bar, 500 µm. e (right), zoom into the SEM with expression of T in epiblast-like compartment and CER1 in hypoblast-like compartment at the opposite sides (defined as AP-axis, see XZ cross section below) observed in 1.02% of total number of starting aggregates on day 0. N = 2 across 1251 and 700 aggregates. Scale bar, 50 µm. f, representative immunofluorescence image of day 6 SEM showing F-ACTIN (white) and nuclei (DAPI, blue). Right, zoom into epiblast-like structure with squamous cells (sEpi) in the top and cylindrical/columnar cells (cEpi) in the bottom parts of the epiblast-like compartment. Scale bar, 50 µm; zoom, 25 µm. g, representative immunofluorescence image of day 6 SEM showing F-ACTIN (white) and OCT4 (cyan). Right, zoom into epiblast-like compartment with squamous cells in the top and cylindrical cells in the bottom parts of the epiblast-like compartment. Scale bar, 50 µm; zoom, 25 µm. h, representative brightfield and immunofluorescence images of day 8 SEM showing NANOG (cyan), SOX17 (yellow), and nuclei (DAPI, white). Right, zoom into PGC-like cells in day 8 human SEMs, co-expressing NANOG and SOX17 (cells marked with arrows). Scale bars, 50 µm.
