Fig. 3: Specification of blastocyst lineages.

A Bulk qRT-PCR analysis of blastocyst lineage marker genes in EPSCs in 2D, and multicellular aggregates at day 4, 5, 6 formed in 3D represented as a heatmap of global ΔΔCt (fold-change) to GAPDH. 20 cystic structures were pooled per group from 3D culture and a minimum of 10 K hEPSCs were collected from the 2D culture. B Immunofluorescence staining of structures generated from hEPSCs at day 5 for OCT4 (green), KRT18 (white) and SOX17 (red). Zoom image on the right shows cells with KRT18 expression. DAPI is shown in blue. n = 30 structures, 3 experiments. C A representative structures generated from hEPSCs at day 5 stained for SOX2 (green) and FOXA2 (red) to suggest Epi/Hypo-like inner compartment (zoom on the right). Image presented as maximum projection. n = 20 structures, 2 experiments. D Immunofluorescence staining of structures generated from hEPSCs at day 4 for OCT4 (green) and GATA3 (red). DAPI is shown in blue. n = 10/23 structures, 2 experiments. E Left: Immunofluorescence staining of GATA3 (green) and E-CADHERIN (magenta) in a representative structure at day 6. Right: Quantification shows frequency of structures at day 6 of 3D culture showing Gata3 nuclear expression (57.03%, 77/135 structures scored); Gata3 cytoplasmic expression (39.25%, 53/135 structures scored); no detectable Gata3 expression (3.70%, 5/135 structures scored). F Quantification shows frequency of cavitated structures in control and WNT3A-supplemented culture. WNT3A is applied in either at 25 or 50 ng/mL concentration. One-way ANOVA with multiple comparisons, p = 0.8473. ns, not significant. n = 450 for control; n = 483 for 25 ng/mL WNT3A; n = 419 for 50 ng/mL WNT3A (see source data). 3 independent experiments. Error bars show S.E.M. All scale bars in the figure indicate 20 um.