Lineage potential of hemogenic ECs varies with the stage of hESC differentiation. (A) Single time points from live confocal image capture shown in SV7 and 8 are shown at 30-hour intervals for type I and type II hemogenic conversion. (B) At the end of image capture, the time-lapse cultures shown in supplemental Videos 7 and 8 were labeled with antibodies specific for CD41a and CD43; staining of type I and type II hemogenic derivatives is shown. (C-E) VPr⁺CD73⁻CD34⁺ ECs were isolated from differentiation of VPr-mOrange/ CD41a-GFP hESCs at day 11 and day 18 and cultured in parallel under continuous observation by confocal microscopy (SV10); after 5 days, cultures were labeled with fluorophore-conjugated antibodies to identify CD41a and CD43 cells. (D) The relative surface area of total CD43⁺ cells (black) and the proportion of CD41a⁺CD43⁺ (green) to CD41a⁻CD43⁺ (blue) populations were calculated using ImageJ Version 1.43 software. (E) Cultures that were recorded in the time-lapse video were also labeled with antibodies to CD15 (green) and CD45 (red); arrows indicate CD15⁺ cells; and arrowheads, CD43^dimCD45⁺ cells. (F-G) CD43⁺ cells were isolated from parallel VPr-hESC differentiation cultures at multiple 12-, 15-, and 18-day time points and expanded in coculture with hESC-derived ECs for an additional 5 days. Cells were labeled each day with antibodies to identify Lineage⁻ and Lineage⁺ populations, and their distribution was quantified at each day during expansion. (H) Hematopoietic progenitors were isolated from hESC differentiation cultures at variable time points and expanded for 5 days; after expansion, CD71⁺ cells were isolated, and expression of ϵ, γ, and β globins was measured by quantitative PCR. (I) A schematic representation of distinct early and late phases of EHT during hESC differentiation. (D) Error bars represent SD between duplicate samples. (F,H) Error bars represent SD among triplicates. Scale bars represent 100 μm.