Effect of RA on hematopoietic progenitor commitment from hemato-vascular precursors. Stage 2 EBs (BW+VF) were cultured for 3 days in the presence of different induction factors. BW indicates BMP4 and Wnt3a; NC, no cytokine; V, VEGF; F, bFGF; L, LE540. (A) Hematopoietic potential of day 7 EB cells under different conditions were assessed by methylcellulose hematopoietic colony assay. Error bars indicate mean ± SEM (n = 4). Each sample was in triplicate. (B) Day 7 EB cells generated under different conditions were cultured as aggregates for 7 days with serum-free expansion medium supplemented with 10% FBS, SCF, FLT-3L, IL-3, and IL-6. Expression of CD45 was determined by flow cytometric analysis. Errors bars indicate mean ± SEM (n = 4). (C) Gene expression analysis by real-time PCR of day 7 EB cells under different conditions. The relative gene expression was normalized to the cells in culture without any induction factors in stage 3. Similar results were produced by 3 independent experiments with 3 parallel samples. The data were from one experiment, and data represent mean ± SEM.