VEGF enhances transient appearance of CD34⁺cells during differentiation of ES cells in liquid culture. (A) Surface marker analysis of differentiating ES cells. Undifferentiated A3-1 ES cells (day 0), as well as cells from EBs cultured for 5 days and 7 days in the presence of 100 ng/mL SCF, 10 ng/mL IL-3, and 1 U/ml EPO were stained with anti-E-cadherin, CD34, CD44, and CD45 monoclonal antibodies. Results of the corresponding isotype control are shown in gray. Positive cell population (region indicated with ⊢⊣) on day 0, day 5, and day 7 for E-cadherin are 98.4%, 26.4%, and 0.3%, for CD34 are 0.2%, 1.4%, and 6.7%, for CD44 are 4.4%, 58.4%, and 68.7%, and for CD45 are 0.4%, 0.1%, and 1.0%, respectively. (B) Effect of VEGF on the CD34⁺ cell generation. The day 8 EBs derived from A3-1 ES cells cultured in the absence of cytokine (−), and in the presence of 20 ng/mL VEGF (+) were harvested and analyzed for CD34 expression. Staining results of the gate R1 (top dot plot) are shown in histograms. Background staining is indicated in gray. Positive cell population (⊢⊣) is 3.9% (−) and 8.5% (+). (C) Kinetics of CD34⁺ cell generation in EBs cultured in various growth factors. A3-1 ES cells (1) and J7 ES (2, 3) cells were induced to differentiate in liquid culture in the absence of cytokine (○), and in the presence of 100 ng/mL SCF (▵), 20 ng/mL VEGF (□), 100 ng/mL SCF, and 1 ng/mL TGF-β1 (•), 100 ng/mL SCF, 10 ng/mL IL-3, and 1 U/mL EPO (┌), and 100 ng/mL SCF and 20 ng/mL VEGF (▪). From day 6 to day 18, EBs were harvested and analyzed for CD34 expression. For A3-1, only day 6 and day 8 data were average values of duplicated samples, and for J7, all but “no cytokine” and “SCF + TGF-β1” were average values of duplicated samples. The vertical bar indicates standard deviation.