Extended Data Fig. 7: Identification of endothelial/hematopoietic populations in heX-embryoids.

(a) Violin plots showing the distribution of ECM genes as well as the yolk sac mesoderm marker gene BST2 in day 5 scRNA-seq. (b) Heatmap showing day 5 scRNA-seq populations compared to hematopoietic populations from the human E16-19 embryo via hypergeometric statistical comparison. This heatmap shows selected results from Extended Data Fig. 4c. We performed multiple test adjustments via Benjamini Hochberg (BH) correction in all comparisons. Tests were performed as one-sided tests. (c) Scatterplots showing the distribution of markers obtained via image analysis of 3 independent experiments at day 5. Percentages correspond to the fraction of cells recorded with corresponding marker expression levels above the thresholds represented by the dotted lines. (d) IF image showing cells expressing hematopoietic markers in heX-embryoid culture. Cells expressing the hematopoietic marker TAL1 (Scl) localize between the yolk sac endoderm-like compartment and the tissue culture dish and form arrangements of spindle-shaped cells. Orthogonal slice shows the position of spindle cells against the dish. Dashed line indicates the position from which the slice was taken. Arrow indicates the position of these cells in the EGFP channel, demonstrating that they were derived from the iGATA6 population. Scale bar = 100 µm. (e) IF image showing RUNX1 and TAL1 expressing cells positioned against the dish. Scale bar = 100 µm. (f) Image analysis of z-slices from 5 areas in 3 biological replicates. The peak of ERG expression is underneath the peak of EGFP expression, representing the iGATA6 endoderm-like layer. Dotted curves represent s.e.m. calculated at each point. (g) IF image showing cells expressing VE-cadherin and VEGFR2 positioned against the dish. Arrow indicates likely differentiating endothelial cell from the overlaying iGATA6 layer. Scale bar = 100 µm. (h) Results from image analysis demonstrating the change in area of CD34⁺ cells between day 5 and day 12 of the cultures, assessed via analysis of immunofluorescence Images. n = 3 biological replicates for D5 and D12 (mTeSR); n = 5 biological replicates for D12 (IMDM). **: P = 0.0055 (C.I. = 95%) P-value was calculated via a one-way ANOVA, using Dunnett’s multiple comparisons test. Error bars represent mean ± s.e.m. (i) Representative flow cytometry plot on day 5 and day 12 showing expansion of the CD34⁺ population by day 12, along with an unstained control (n = 3).

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