Fig. 3

Differentiation of HLA-I-modified iPSCs to endothelial cells. A Schematic diagram of the experimental protocol for EC differentiation. B The typical phases of EC differentiation shown by phase-contrast imaging. iPSC stage on Day 1, mesoderm stage on Day 4, EC stage on Day 6 and EC expansion stage on Day 10. Scale bars, 200 µm for Day 1 & 10, 100 µm for Day 4 & 6. C Representative FCM assay for the KDR and CD144 expression on iPSC-ECs differentiated from C55-A11 iPSCs. Isotype control (left panel) and KDR/CD144 staining (right panel). D Histograms displaying the differentiation efficiency of iPSC-ECs from C55-A11. Columns show mean ± SD of 5 independent experiments. ECs were counted by CD144/KDR double positive staining. Isotype staining was used as negative control. E Representative immunofluorescent assay for expression of the EC marker CD31 on iPSC-ECs from C55-A11 (upper panel) and positive control HUVECs (lower panel). Left panel: CD31 immunofluorescent staining, right panel: merged imaging of CD31 and DAPI staining. Scale bars, 50 µm. F In vitro tube formation assay of iPSC-ECs (left) and HUVECs (right) on Matrigel for 18 h. Images are representative of 5 independent experiments. Scale bars, 100 µm. G, H In vivo vascular formation in nude mice. Matrigel plugs from C55-A11 iPSC-ECs and HUVEC were stained by H&E dyes (G) and anti-CD31 antibody (H). Scale bars of G and H, 100 µm (low power) for left panel, 50 µm (high power) for right panel