Extended Data Figure 8. Molecular deconvolution of vascular-niche-derived angiocrine factors during stepwise differentiation of endothelial cells into rEC-HSPCs and rEC-HSCs.

a, Adult lung mECs (VEcad⁺CD31⁺CD45⁻) were isolated from Runx1-IRES-GFP mice. Human-derived VN-ECs were discriminated from Fgrs-transduced Runx1-IRES-GFP adult lung endothelial cells by anti-human CD31 (hCD31). Fgrs-transduced Runx1-IRES-GFP endothelial cells and their progeny were gated as hCD31⁻. Flow cytometry plots showing the expression of VEcad and CD45 in mouse hCD31⁻ Fgrs-transduced endothelial cells and derivatives over the course of endothelial to haematopoietic cell reprogramming. b, Quantification of mouse hCD31⁻ Fgrs-transduced endothelial cells and their derivatives over the course of reprogramming. Data represents mean ± s.e.m. (n = 5). c, Colony number arising in methylcellulose from Fgrs-transduced endothelial cells and derivatives (gated on human hCD31⁻ to exclude VN-EC feeder). n = 4 independent experiments are shown and each condition performed in triplicate. Data represents mean ± s.e.m. (n = 5); two-tailed unpaired t-test. d, Adult mouse endothelial cells were treated with different small molecules at their known IC₅₀ (CXCR4 antagonist, AMD3100, 44 µmol l⁻¹; CXCR7 agonist, TC14012, 350 nmol l⁻¹; BMP antagonist, Noggin, 0.5 µg ml⁻¹; TGF-β antagonist, SB431542, 10 µmol l⁻¹). Representative flow cytometry plots of apoptosis assays are presented. e, Quantification of apoptotic cells following treatment with each small molecule tested. Data represents mean ± s.e.m. (n = 3); two-tailed unpaired t-test. f, Quantification of hCD31⁻ Fgrs-transduced endothelial cells and their derivatives over the course of reprogramming in presence of CXCR4 inhibitors (AMD3100, AMD3100, 44 µmol l⁻¹), CXCR7 agonists (TC14012, 350 nmol l⁻¹), BMP inhibitor (Noggin, 0.5 µg ml⁻¹), TGF-β/ALK5 inhibitor (SB431542, 10 µmol l⁻¹). Data represents mean ± s.e.m. (n = 5); two-tailed unpaired t-test. g, Quantification of hCD31⁻ Fgrs-transduced endothelial cells and their derivatives over the course of reprogramming in presence of VN-ECs (HUVEC-E4ORF1) overexpressing mouse CXCL12 (n = 5 independent experiments, 3 technical triplicates each). Data represents mean ± s.e.m.; two-tailed unpaired t-test.