Extended Data Fig. 7: Removal of VEGF at day 7 of differentiation accelerates the loss of arterial endothelial markers.

(a) Swirling embryoid body (EB) differentiation protocol outlining the VEGF (V) titration. Numbers represent VEGF concentration in ng ml^-1. Partially created using BioRender.com. (b) CD34 expression in RM TOM cells by flow cytometry correlated with VEGF concentration. Error bars, mean ± s.e.m., n = 3. One-way ANOVA test for CD34 linear trend with VEGF, P < 0.0001. (c) Percentage of RM TOM CXCR4⁺CD73^lo/+ arterial cells, subsetted from CD34⁺ cells, by flow cytometry correlated with VEGF concentration. Error bars, mean ± SEM, n = 3. One-way ANOVA test for CXCR4⁺CD73^lo/+ linear trend with VEGF, P < 0.0001. Comparison of d8 and d9 samples continuing VEGF with d3-7 VEGF, both P < 0.0001, one-way ANOVA with Sidak’s multiple comparisons test. (d) CD34 expression in PB5.1 cells by flow cytometry correlated with VEGF concentration. Error bars, mean ± s.e.m., n = 3. One-way ANOVA test for CD34 linear trend with VEGF for day 5 and day 7, P = 0.0105. (e) Percentage of PB5.1 CXCR4⁺CD73^lo/+ arterial cells, subsetted from CD34⁺ cells, by flow cytometry correlated with VEGF concentration. Error bars, mean ± s.e.m., n = 3. One-way ANOVA test for CXCR4⁺CD73^lo/+ linear trend with VEGF, P < 0.0001. Comparison of d9 and d12 samples continuing VEGF with d3-7 VEGF, both P < 0.0001, one-way ANOVA with Sidak’s multiple comparisons test.

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