Fig. 2: Generation of inducible post-implantation human embryoids.
From: Pluripotent stem cell-derived model of the post-implantation human embryo
a, Overview of the protocol used to generate inducible human embryoids by combining wild-type RSeT human ES cells with inducible GATA6-SOX17 and GATA3-AP2γ cells. Extraembryonic-like cells were induced for 3 days before aggregation at day 0. KSR, knockout serum replacement. b, Size of cell aggregates between days 1 and 3 post-aggregation. Day 1: n = 175, day 2: n = 171 and day 3: n = 91 structures from 5 independent experiments. All individual embryoid lengths are plotted. Each symbol (orange cross, orange triangle, green triangle, blue circle, purple square) represents an independent experiment. c, At 96 h post-aggregation, structures demonstrate clear self-organization. d, Quantification of embryoid formation across starting pluripotency states. RSeT: n = 952, mTeSR: n = 30 and PXGL: n = 207 structures from 5 independent experiments. One-way ANOVA with Holm–Šídák multiple comparisons test. RSeT versus mTeSR: P = 0.0347; RSeT versus PXGL, P = 0.0283. Unmarked pairwise comparisons are not significant (P > 0.05). e, Proportions of cell types in correctly organized embryoids. n = 16 embryoids from 3 independent experiments. f, Representative image of an in vitro cultured human embryo at 9 dpf showing a clear lumenized SOX2 domain surrounded by a layer of GATA6-positive cells. A subset of GATA6-positive cells expresses the anterior hypoblast marker CER1. Representative of three independent experiments. g, The hypoblast-like domain expresses N-cadherin (N-cad), SOX17 and GATA4, and the epiblast-like domain maintains expression of the pluripotency factors SOX2, OCT4 and NANOG. Cells derived from GATA3-AP2γ cells (which express GFP) show clear outer localization. Representative of two experiments each. E-cad, E-cadherin. h, Inducible human embryoids demonstrate clear apicobasal polarity. Right, quantification of inducible human organization. Top right, n = 506 structures from 3 independent experiments for lumen and extracellular matrix (ECM) efficiency. Bottom right, n = 27 embryoids from 2 independent experiments for lumen number. g,h, Inner domains of embryoids are surrounded by a dashed line. d,e,h, Data are mean ± s.e.m. Scale bars, 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant.
