Fig. 3

hPSC-EB-CD144⁺-derived endothelial cells are functional in vitro and in vivo. A Endothelial activation by TNF-α assay. Representative histograms of flow cytometry analysis for ICAM-1 expression in hPSC-ECs and ECFCs (left panel) and quantification of ICAM-1^High cells (right panel) in the absence and presence of TNF-α. B Matrigel-based tube formation assay 8 h after seeding. Phase contrast images with scale bar 1000 µm (left panel) and cell network characterization based on the number of segments, meshes, branches and nodes (right panel). C Confocal microscopy images of hPSC-ECs and ECFCs after 4 h of incubation with acLDL-AlexaFluor-488. CD31 in red and nuclei in blue (DAPI). Scale bar 100 µm. D hPSC-ECs nitric oxide (NO) production was analyzed based on the expression of the endothelial nitric oxide synthase (eNOS) measured by flow cytometry (left histogram) and indirectly, by using the probe DAF-FM diacetate to detect NO in hPSC-ECs and ECFCs. DAF-FM fluorescence was measured by flow cytometry in cells that were not incubated with the probe (-DAF-FM) as a negative control, in cells incubated with the probe alone (+ DAF-FM) and in the presence of a S-nitrosothiol molecule (+ DAF-FM + SNAP) or in cells previously stimulated with lipopolysaccharides (+ DAF-FM + LPS), the two latter as positive controls, n = 5 independent experiments for every cell line. E Representative confocal image at day 19 post-injection of a mouse dorsal skinfold chamber without ischemia, injected with mcherry-hPSC-ECs. Vessel perfusion was observed thanks to the injection of dextran 70 kDa-FITC (green). Scale bar: 200 µm. F Percentage of new blood vessels formed at day 19 post-injection. n = 6 independent experiments. G Confocal images of the dorsal chambers analyzed post-mortem by immunofluorescence. Yellow arrows indicate human CD144⁺ ECs (green) localized at the wall of blood vessels. Scale bar: 100 µm. *P value < 0.05; **P value < 0.005 and ***P value < 0.0005. Data are represented as mean ± SEM