Analysis of APLNR- and EPCAM-sorted cells from differentiated EBs. Paired samples of (A) posterior mesodermally (BVS) and (B) anterior mesendodermally (BloVSAhi) differentiated day 3 EBs were flow sorted based on the expression of APLNR and EPCAM expression (boxed areas in the panel). The same gates were used to sort cells differentiated under both protocols to ensure that phenotypically similar populations were compared. (C) Hematopoietic colony-forming potential of sorted cell populations indicating enrichment of hemangioblast colony-forming cells in the APLNR+ fractions (Ep+AR+ and Ep−AR+). Colony frequency was markedly diminished in the anterior mesendoderm differentiations. Error bars represent SEM for n = 3 independent sorting experiments. (D-E) Comparison of microarray-derived gene expression profiles of EPCAM+APLNR− (Ep+AR−) and EPCAM−APLNR+ (Ep−AR+) cells sorted from EBs generated under conditions that favor the formation of (D) posterior mesoderm (BVS) or (E) anterior mesendoderm (BloVSAhi). (F) Heatmap comparing the expression profiles of differentially expressed genes in sorted cell fractions generated under posterior mesodermal (BVS) and anterior mesendodermal (BloVSAhi) conditions, showing similar patterns of expression in both sets of differentiations. (G) Microarray comparison of EPCAM−APLNR+ (Ep−AR+) cell fractions from BVS and BloVSAhi cell fractions representing the most posterior mesoderm and anterior mesendoderm committed fractions. Blue and green dots indicate probes differing by ≥ 3.0-fold from the mean. The fold change of several key genes is indicated for each plot.
