Fig. 1

Derivation and characterization of human blood outgrowth endothelial cells. a Workflow illustrating the generation of BOECs. Images show BOEC colonies emerging during days 7–14 post-seeding of PBMCs, followed by characteristic cobblestone-like endothelial cells after passaging of colonies (scale bar, 100 μm). b Number of BOEC colonies per million PBMCs obtained from normal controls and PCV patients. c Proliferation dynamics of BOEC lines measured by cell doubling duration over passages. d Flow cytometry characterization of BOECs for endothelial, leukocyte, and progenitor cell markers (gray—isotype control; red/blue—cell lineage marker staining). e Immunostaining of endothelial markers VWF and CDH5 in BOECs. HeLa cells and HUVECs are negative and positive controls respectively. Scale bars, 50 μm. f Tube formation assay with representative images of tube formation ability of BOECs at 4 h. Bottom panel shows quantification of junctions, loops (tubes), and total branching length (total length of loops and branches) over time (quantified from n = 12 optical fields per timepoint from each cell line). Data are mean ± s.e.m. Scale bars, 200 μm. g Fibrin gel bead sprouting assay of BOECs at 24 h. BOECs were immunostained for F-actin (red) and DAPI (cyan). Bottom panel shows measurements of relevant sprouting parameters (quantified from n = 10 beads from each individual, BOECs from 3 PCV and 3 normal individuals). Data are nested graphs showing mean ± s.e.m. of each individual donor. No significant difference was found between PCV and Normal (two-tailed t-tests with Welch’s correction). Scale bars, 100 μm