Supplementary Figure 1: Supplementary analysis of an in vitro 3D model of a pre-gastrulation human epiblast.

(a–b) Spontaneous differentiation of spherical hESC epithelia grown in pure Matrigel from single cells. (a) Shown are IF of pluripotency markers of a colony grown in Matrigel (Mgel) for three days and in Matrigel and LDN (Mgel/LDN) for five days. Plots quantify the pluripotency loss where each colony is binned based on the fraction of its cells expressing the pluripotency marker (n = 31 independent colonies for Mgel; n = 28 independent colonies for Mgel/LDN). The spontaneous differentiation of Matrigel-grown hESC colonies is BMP4-dependent, since colonies remain pluripotent in the presence of the BMP inhibitor LDN. (b) After five days in Matrigel, cells lose structural integrity and appear to invade into the surrounding gel (representative of 12 independent colonies). Shown are two different examples. Top right shows a detail in the dotted rectangle 15 μm displaced in z from the image on the left, where these nuclei are in focus. (c) Day-10 in vitro attached human embryo. Left: DAPI of an entire embryo; right: magnified epiblast (NANOG+ and OCT4+) and primitive endoderm (GATA6+). Shown is a different example from Fig. 1 and representative of 4 embryos. (d) Additional data on human epiblast model (spherical hESC epithelia grown in hydrogel/Matrigel mix). Pluripotency markers in the human epiblast model grown for 3 (left) or 4 days (right). Left example shows a z-slice and the entire z-stack in DAPI. Both differ from Fig. 1 and are based on 51 epiblast models from the main figure. (e) Surface markers z-stacks in a human epiblast model. Shown is the same example as in Fig. 1. All scale bars, 20 μm. (f) Schematics of the RUES2-GLR line, expressing mCitrine-SOX2 (mCit-SOX2), mCerulean-BRACHYURY (mCer-BRA), and tdTomato-SOX17 (tdTom-SOX17). (g–h) Live-cell imaging of the human epiblast models made up of RUES2-GLR cells under pluripotency conditions, starting ~ 48 h after seeding. The quantification shown is for the single examples shown here. Imaging frequency: 1 h⁻¹. Shown examples are different from the one in Fig. 1 and representative of 39 model epiblasts that remained SOX2+ out of 48 total imaged independent model epiblasts. See also Supplementary Video 1. All scale bars, 20 μm. Numerical source data are available in Supplementary Table 2.