Extended Data Figure 9. Organ-specific adult endothelial cells are amenable to hierarchical Fgrs-mediated reprogramming to rEC-HSPCs.

a, Left, Fgrs-transduced liver endothelial cells were directly co-cultured with VN-ECs. Graph indicates absolute quantification (cell number) of CD45⁺ rEC-HSPC (n = 3 independent biological replicates, 3 technical replicates each). Right, Quantification of phenotypically marked CD45⁺ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represents mean ± s.e.m. b, Left, Fgrs-transduced lung endothelial cells were directly co-cultured with VN-ECs. Graph indicates absolute quantification (cell number) of CD45⁺ rEC-HSPC Right, Quantification of phenotypically marked CD45⁺ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represents mean ± s.e.m. (n = 3 independent biological replicates, 3 technical replicates each). c, Left, Fgrs-transduced brain endothelial cells were directly co-cultured with VN-ECs. Graph indicates absolute quantification (cell number) of CD45⁺ rEC-HSPC (n = 3 independent biological replicates, 3 technical replicates each). Right, Quantification of phenotypically marked CD45⁺ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represents mean ± s.e.m. d, Left, Fgrs-transduced kidney endothelial cells were directly co-cultured with monolayers of confluent VN-ECs. Graph indicates absolute quantification (cell number) of CD45⁺ rEC-HSPCs. Right, Quantification of phenotypically marked CD45⁺ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represents mean ± s.e.m. (n = 3 independent biological replicates, 3 technical replicates each). e, rEC-HSPC-derived cells were purified and co-culture on VN-ECs in presence of doxycycline for 28 days. Cells were transplanted into lethally CD45.1⁺ irradiated recipients in absence of doxycycline. Data represents individual data points for donor contribution to peripheral blood at indicated time point after transplant (n = 3 biological replicates). f, Experimental model for hierarchical differentiation of endothelial cells into rEC-HSPCs. D8 VEcad⁺RUNX1⁺CD45⁻ cells were purified by flow cytometry and replated on inductive vascular niche in presence or absence of doxycycline. Subsequently, at day 15 VEcad⁺RUNX1⁺CD45⁻ cells haemogenic-like cells were purified by flow cytometry and replated on the inductive vascular niche in presence (Dox-on) or absence (Dox-off) of dox. g, Flow cytometry quantification of cell subsets during stepwise conversion in f. Data represents individual data points (n = 3 independent biological replicates, 3 technical replicates each).