Hematopoietic derivatives of transgenic hESCs express the CD41a-GFP and originate from VPr-mOrange⁺ hemogenic ECs. (A) hESCs that were transduced with the vascular specific reporter transgene (VPr-mOrange) as well as a hematopoietic specific transgene (CD41a-GFP) were placed in serum-free differentiation coculture with E4ORF1⁺ ECs (see “Methods”). (B) At day 14 of differentiation, cells were observed that expressed the VPr-mOrange (green) and CD41a-GFP (red) transgenes, and were labeled by an allophycocyanin-conjugated CD43-specific antibody (blue). (C) At day 14 of differentiation, cultures were labeled for flow cytometry with antibodies specific for CD31 and CD43; scatter plots showing the distribution of CD41a-GFP⁺/CD43⁺ (red), CD41a-GFP⁻/CD43⁺ (blue), and remaining VPr-mOrange⁺ (green) cells are shown. (D-E) CD43⁺ derivatives were isolated at day 12 and cultured for an additional 5 days in the presence of hematopoietic cytokines, followed by labeling with an antibody specific for CD41a; a scatter plot of CD41a-GFP versus CD41a antibody identifies derivatives that dimly (blue) or strongly (red) expressed the CD41a-GFP transgene. (F) The VPr-mOrange/CD41a-GFP hESC line was differentiated on E4ORF1⁺ ECs for 11 days, and then time-lapse confocal microscopy was performed over the next 4 days. Panels shown are time 0, the first indications of VPr-mOrange expression in an isolated EC (22 hours), when the VPr-mOrange⁺ cell noted at time 0 began to display discernible expression of the CD41a-GFP transgene (36 hours), and late time points at 61 and 86 hours as the double-positive population expands. (G) Lineage dendrogram of hemogenic EC progression with the onset of VPr-mOrange expression (green) and the onset of CD41a-GFP expression (red); cell divisions are marked by circles. (H-J) At day 14 of differentiation, hESC-derived ECs were isolated based on a VPr-mOrange⁺VE-cadherin⁺CD43⁻CD73⁻ phenotype (red in panels H-I) and continuously monitored by confocal time-lapse microscopy for > 60 hours. (J) Zero-, 12-, 24-, and 48-hour time points with arrowheads indicating an EC undergoing EHT. (B,D,J insets) Magnified views. Scale bars represent 100 μm.