Bipotent differentiation potential of CD34^brightCD31⁺Flk1⁺ cells. (A) Flow cytometric analysis of hESCs allowed to differentiate on S17 cells for 14 days and sorted for CD31⁺Flk1⁺ cells demonstrates high level of CD34 coexpression. (B) Endothelial differentiation of CD34^brightCD31⁺Flk1⁺ cells in EGM2 medium supplemented with 10 ng/mL VEGF. Brightfield images (magnification: top panel, 100×; bottom panel, 200×) and flow cytometry analysis of cells cultured for 14 days in endothelial differentiation are shown. (C-F) Endothelial cells derived from CD34^brightCD31⁺Flk1⁺ cells analyzed for expression of (C) CD31, (D) VWF, (E) dil-acLDL uptake, and (F) tube formation. (G) Hematopoietic differentiation of CD34^brightCD31⁺Flk1⁺ cells by coculture with AFT024 stromal cells in medium supplemented with 10 ng/mL SCF, FL, and Tpo. Flow cytometry after 14 days of culture in hematopoietic differentiation identifies CD45⁺ hematopoietic cells. Representative image of clusters of hematopoietic-like cells is shown (magnification 100×). Similar results were observed for 3 independent experiments. Brightfield images were acquired on Olympus CKX41 microscope with 2×/0.05, 10×/0.25 PhP, and 20×/0.40 PhP objectives using Olympus DP2 camera. Fluorescent images were acquired with a Zeiss (Thornwood, NY) model Axiovert 200M microscope with a 20×/0.30 Ph1 objective at room temperature and photographed with Axiovision v4.3 (Zeiss). All images were processed using Adobe Photoshop 7.0 (San Jose, CA).