Activation of Wnt signaling results in accelerated differentiation of hESCs. (A) Evaluation of β-catenin activity in 293 cells (left) and undifferentiated hESCs (right) transfected with TopFlash plasmid and cultured on stromal cells expressing either GFP (control), Wnt1, or Wnt5. (B-E) hESCs were differentiated by coculture with stromal cells expressing GFP only as a control (○) or Wnt1 (■) over a defined time course. Cells were analyzed by flow cytometry for CD34⁺, CD34⁺CD31⁺, CD34⁺Flk1⁺, or CD31⁺Flk1⁺ cells, as indicated. Results from cocultures with S17 and M210B4 stromal cells were combined for the analysis (n = 6 experiments, except for CD31⁺Flk1⁺ cells where n = 3). P > .05 for all cell populations. (F) Change in CD34⁺ (■), CD34⁺CD31⁺ (▴), CD34⁺Flk1⁺ (○), and CD31⁺Flk1⁺ (◇) cell populations of hESCs cocultured with Wnt1-expressing cells compared with GFP control cells. Error bars indicate SEM. A significant increase in CD34⁺CD31⁺ cells at day 7 of differentiation and CD34⁺Flk1⁺ at day 10 of differentiation was found for Wnt1 compared with control stromal cells (P < .05). (G) Effect of Wnt1 on generation of CD34⁺CD45⁺ cells was analyzed by flow cytometry. Error bars indicate SEM of 2 independent experiments. (H) CFCs derived at the indicated days of differentiation with control stromal cells () or Wnt1-expressing stromal cells (). *P < .05.