Fig. 1

hPSC form asymmetric epithelial cysts in a 3D amniogenic culture system. a Cartoon of an implanting human embryo. The amniotic ectoderm, amniotic cavity, and epiblast constitute the amniotic sac. b Schematic of the 3D culture system and the formation of asymmetric cysts from hPSC. The culture substrate is made of a thick layer of Geltrex^TM (with a thickness >100 µm). The ECM overlay was applied from day 1 onward to provide the 3D culture environment. Asymmetric cysts were consistently observed on day 5 in all (n > 18) independent experiments. c Representative confocal micrographs showing an asymmetric cyst on day 5, stained for βCAT (green) and ECAD (red). HOECHST (blue) counterstains nuclei. n = 3 independent experiments. Scale bar, 50 µm. d Carnegie stage (CS) 5a-2, 5b, and 5c human embryo sections, showing prospective and definitive amniotic ectoderm (Am.) and epiblast (Epi.). Phase contrast image shows a representative asymmetric cyst with a distinct bipolar morphological pattern. Scale bars, 30 µm