Small-molecule antagonism of AHR enhances early hematoendothelial development from hESCs. (A) Schema of hESC differentiation into early hematoendothelial cells as spin EBs. hESCs are made into spin EBs at day 0 and conditioned into mesoderm lineages for 6 days using defined cytokines (stage 1). At day 6, spin EBs are transferred into hematoendothelial culture media (stage 2) to promote endothelial and hematopoietic cell differentiation. For these studies, cells are treated with either 1 μM SR-1, 10 nM TCDD, or DMSO vehicle control beginning at day 6+0 with media exchanges and/or harvesting performed at day 6+3, day 6+6, and day 6+9. (B) Representative flow cytometry plots of 1 hESC differentiation. Both adherent and nonadherent cell fractions are harvested at day 6+3, day 6+6, and day 6+9 and assessed for endothelial cell (CD34⁺CD31⁺, CD34⁺CD144⁺) and hematopoietic progenitor cell (CD34⁺CD43⁺, CD34⁺CD45⁺) phenotype. (C) Fold change of the total percentage of each hematoendothelial phenotype for SR-1– and TCDD-treated hESCs normalized to matched DMSO-treated controls; n = 4-6, error bars represent standard error of the mean (SEM). *P < .05 as compared with DMSO-treated controls by the Student t test. N/A, not applicable due to absence appreciable of CD34⁺CD45⁺ populations at day 6+3 time point; TPO, thrombopoietin.