Fig. 1: A 3D system from hEPSCs to mimic embryo-like events.

A Scheme for natural human pre/peri-implantation embryo development. B A representative dome-shaped naïve pluripotent hEPSC colony in 2D culture. Representative of at least 10 independent experiments. C Left: Schematic of an AggreWell and 3D aggregation protocol with hEPSCs. Right: Representative examples of cystic structures generated from a typical experiment after 4 days demonstrate three lineages, resembling blastocyst stage natural embryo. Representative of at least 3 independent experiments. Lineage markers: SOX2, yellow; GATA3, magenta, and SOX17, cyan. n = 10 experiments. D Representative phase-contrast images of hEPSC multicellular aggregates in AggreWell at the indicated time points during 3D culture. E Phase-contrast images of cystic structures from hEPSCs at D6. (top) and natural human blastocysts at D6 (bottom). Representative of at least 3 independent experiments. F A representative cystic structure generated from RUES2 hEPSC line with SOX2-flourescent reporter. Representative of at least 2 independent experiments. G A representative structure immunostained for SOX2 in magenta, KRT18 in yellow to label inner compartment and outside epithelium, respectively. DAPI is shown in blue. Maximum projection image is shown on the right. n = 50 structures, 3 experiments. H Efficiency quantification showing the number of structures with a cavity and identifiable lineage segregation. Present EPI-like and HYPO-like cells were determined by positive expression of SOX2 and SOX17, respectively, within inner compartment as seen by IF staining. Present TE-like cells were determined by positive expression of GATA3 or KRT18 in outer cells observed by IF staining. n = 186 structures, 2 experiments. I Quantification for cell numbers in individual cystic structures generated from two established hEPSC lines, ESI017 (n = 11), RUES2 (n = 18). J Measurements of axial diameters in cystic structures from ESI017 (n = 11), RUES2 (n = 18) hEPSC lines. Illustration on right shows the two axes measured. All scale bars in the figure indicate 20 µm.