Inhibition of canonical Wnt signaling reduces hematogenic endothelium cell population. (A) Evaluation of control- and DKK1-conditioned medium in 293 cells (left) and hESCs (right) transfected with a β-catenin–responsive reporter system as a measure of Wnt activity. (B-E) hESCs were differentiated by stromal cell coculture in control medium (○) or medium containing dickkopf1 (DKK1) (■). Results from cocultures with S17 and M210B4 stromal cells were combined for the analysis (n = 3 experiments). Cells were analyzed by flow cytometry for presence of (B) CD34⁺, (C) CD34⁺CD31⁺, (D) CD34⁺Flk1⁺, and (E) CD31⁺Flk1⁺ cells over a time course of differentiation. Error bars indicate plus or minus SEM of 3 independent experiments. (F) Effect of DKK1 on formation of more committed hematopoietic progenitors was evaluated by flow cytometry for presence of CD34⁺CD45⁺ cells (n = 1 experiment). (G) Development of CFCs was evaluated for hESC-derived cells cultured with M210 stromal cells in control medium () or DKK1-conditioned medium () for the indicated number of days (n = 1 experiment). ***P < .001; **P < .01; *P < .05.