Fig. 5: scRNA-seq analysis of hEPSC-derived cystic structures.

A Uniform manifold approximation and projection (UMAP) grouped by cell group: natural human embryos at D5/D6 (n = 542 cells), hEPSCs grown in 2D (n = 228 cells, 2 replicates), D5 cyst structures (n = 2013 cells, 3 replicates), and D6 cyst structures grown in IVC for 24 h (n = 2057 cells, 3 replicates). B. UMAP showing lineage scoring of all cell groups into EPI-like cells (ELCs, teal) and EPI (dark teal), HYPO-like cells (HLCs, yellow) and Hypoblast (dark yellow), TE-like cells (TLCs, magenta), and TE (dark magenta). Undefined cells appear in grey. C Pie charts showing the distribution of lineage assignments for natural embryo, D5 structures, and D6 structures. D. Heat maps showing the expression of canonical TE markers enriched in natural cluster, as well as a subset of D5 TLCs (top row), and genes shown to be enriched exclusively in D5 TLC cluster (bottom row). A circle with a dotted line denotes D5 TLC cluster. E Collection of violin plots showing the relative expression of certain key lineage markers in ELCs, HLCs, and TLCs. For EPI-related genes: SOX2, NANOG, POU5F1. For HYPO-related genes: GATA4, PDGFRA, and GATA6. For TE-related genes: GATA3, GATA2, and KRT18. F Violin plots showing the expression of canonical markers for HYPO-related genes (yellow), EPI-related genes (teal) and TE-related genes (magenta) in Day 5 hEP-structures, D6 hEP-structures, and the natural embryo. (*p < 0.05, **p < 0.01, ***p < 0.001; (two-sided ad-hoc Dunn’s multiple comparison test applied to an ANOVA), exact p-values are listed in Supp. Data 2). For D5 structures: HLCs, n = 445 cells; ELCs, n = 235 cells; TLCs, n = 50 cells. For D6 structures: HLCs, n = 586 cells; ELCs, n = 144 cells; TLCs, n = 73 cells. For natural blastocyst, HYPO, n = 11 cells; EPI, n = 32 cells; TE, n = 484 cells. All genes were taken from Liu et al. 2021⁴⁹, and are listed in Supp. Data 1.