Hemato-endothelial differentiation of hESCs in serum-free conditions is augmented by coculture with vascular feeder cells. (A) hESCs that were transduced with a vascular specific reporter transgene, VE-cadherin-GFP/mOrange (VPr-hESCs), and clonally selected were placed in serum-free differentiation coculture with primary ECs (HUVECs) that had been transduced with E4ORF1 cDNA and expanded. (B) Cocultures were sequentially stimulated with a series of cytokines and a small-molecule inhibitor of TGF-β signaling; a schematic view of the differentiation protocol is shown. (C) VPr hESCs were differentiated on E4ORF1⁺ ECs from 14 days followed by confocal microscopy; a wide-field view of vascular colonies is shown. (D-F) hESCs that were differentiated in coculture with E4ORF1⁺ ECs were fixed on days 3, 5, and 7 and stained for the markers Oct3/4 (red, D-E), Brachyury (green, E-F), and VEGFR2 (blue, F); E4ORF1⁺ ECs were labeled by CD31 (white). (G) The crude hESC-derived population on days 3-8 of differentiation was separated from feeder cells by flow cytometry, and relative levels of the transcripts Oct3/4, Brachyury, and VEGFR2 were measured. (H) Surface expression of VEGFR2 and SSEA3 was measured on hESC derivatives at multiple time points during differentiation on EC feeders (solid area curve), as well as hESCs differentiated in feeder-free conditions (black line), and hESCs differentiated on non-EC bone marrow stromal cells (bar graph, measured at day 8). (I-J) VPr-hESCs were clonally labeled with PGK-BFP so that differentiated derivatives could be distinguished from feeder cells (I). (J) Flow cytometry identified a significant portion of the BFP⁺ population that was positive for the VPr transgene and CD31. (K) VPr-hESCs that were clonally labeled with PGK-BFP were isolated at multiple time points during differentiation, and the percentage of hESC derivatives expressing both the VPr transgene and CD31 was measured. (L) Endothelial cells were FACS isolated from hESC differentiation cultures based on expression of the VPr-GFP transgene and plated at a density of 5000 cells/cm² on gelatin-coated tissue culture plastic. Time-lapse microscopy was performed over 5 days; 0-, 24-, 48-, and 72-hour time points are shown. (M) Commencing at day 13 during differentiation of VPr-hESCs on E4ORF1⁺ ECs, time-lapse confocal microscopy was performed over 3 days with addition of anti CD43-allophycocyanin antibody at 48 hours; 0-, 24-, 48-, and 50-hour time points are shown. (G-H) Error bars represent SD of experimental values performed in triplicate. (K) Error bars represent at least 6 replicates for each time point. (C-F) Lower panels: Higher power views of the dashed stroke box in the upper panel. (C,E insets) Bright-field view. (M insets) Magnified views of the stroke boxes. Scale bars represent 100 μm.