Fig. 2

Endothelial differentiation of hPSC-EB-CD144⁺. A Representative phase-contrast images of endothelial colonies formed from single CD144⁺ cells at day 0 (left), day 7 (middle) and at day 15 (right). Scale bar 100 µm. B Confocal microscope images of endothelial colonies from the colony-forming assay. hPSC-ECs were labeled against CD31 (green), CD144 (red) and with DAPI for the nuclei (blue). Scale bar 500 µm. C Representative phase-contrast images of endothelial colony-forming cells (ECFC) and hPSC-derived endothelial cells (hPSC-ECs) from CD144+-EBs. Scale bar 500 µm. D Cumulative population doubling (CPD) of hPSC-ECs along passages. E Endothelial phenotype was analyzed along the passages by flow cytometry for hPSC-EC from A29 (left), SA01 (center) and H1 (right) cell lines. hPSC-ECs derived from the three cell lines kept a stable phenotype characterized by the expression of CD309, CD144, CD31, CD34 and CD143 (see also Additional file 2: Fig. S2). The expression of hematopoietic markers was negligible at every passage. F Confocal microscope images of hPSC-ECs and ECFC labeled with antibodies against von Willebrand Factor (vWF, red) and CD31 (green). Nuclei stained with DAPI (blue). Scale bar 500 µm. G–I RTqPCR analysis of arterial (NRP1 and EFNB2), venous genes (NRP2, EPHB4 and COUPTFII) and the pan-endothelial gene CDH5 (CD144) in hPSC-ECs after treatment with an antagonist of Notch (DAPT) during the EC differentiation, n = 5 independent experiments for every cell line. *P value < 0.05; **P value < 0.005 and ***P value < 0.0005. Data are represented as mean ± SEM