Subset specification of primitive streak/mesendoderm cells. BMP4 and Wnt3a-induced EBs (day 1.5) were cultured with different induction factors for 2.5 days. Numbers represent percentage of total cells. The characteristics of day 4 EBs were analyzed by flow cytometry with KDR and PDGFRα (A) and by reverse transcription PCR (B). (C) Images of blast colony derived from day 4 EB cells (left) and hematopoietic and endothelial cells derived from this single blast colony (right). Images were captured under culture mediums of the cells and are shown at 100× original magnification. (D) Hematopoietic and endothelial potentials of day 4 EB cells under different conditions were assessed by blast colony-forming assay. Error bars indicate mean ± SEM (n = 3). Each sample was in triplicate. BW indicates BMP4 and Wnt3a; NC, no cytokine; V, VEGF; F, bFGF; L, LE540. Images were recorded with the use of NIS-Elements F 2.30 in the Nikon ECLIPSE TE2000-U microscope system and processed with the Adobe Photoshop CS software. Scale bar, 200 μm.