Extended Data Fig. 6: Characterization of epiblast-, hypoblast-, and trophoblast-like lineages in SEMs.

a (left to right), representative merged brightfield (BF) and immunofluorescence images of day 3 SEMs showing three lineages, epiblast-like (OCT4, cyan), hypoblast-like (SOX17, yellow), and trophoblast-like lineage (CK7, magenta) merged with a nuclei channel (DAPI, white). b, representative merged brightfield and immunofluorescence images of multiple day 6 SEMs, showing epiblast-like (OCT4, cyan), hypoblast-like or hypoblast/ExEM-like (SOX17 or GATA6, respectively, in yellow), and trophoblast-like (SDC1 and GATA3, magenta) compartments merged with a nuclei channel (DAPI, white). c (left), representative immunofluorescence images of day 6 SEMs showing epiblast-like (OCT4, cyan), hypoblast-like (SOX17, yellow), and trophoblast-like (CK7, magenta) compartments; nuclei (DAPI, white). Left panel represents a wide field image with adequate SEMs (outlined in green) and mis-developed structures (outlined in red); scale bar, 200 µm. Right panels zoom into the examples. c (from left to right), representative immunofluorescence image of the SEM developed according to the three success rate criteria (outlined in green, see Methods). Representative mis-developed day 6 SEMs (outlined in red) which do not fall within the success rate criteria: not surrounded by trophoblast (Tb)-like cells (I), without epiblast (Epi)- and hypoblast (Hb)- like cells (II), without the amniotic cavity (AC)-like structure and the yolk sac cavity (YS)-like structure and bilaminar Epi/Hb-like structure (III). Scale bars, 50 µm.