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Fig. 4. Kinetic analyses of VEGF function. (A) Scl^+/CD4 ES cells were differentiated in serum-free conditions in the presence of BMP4 and VEGF (upper panel) or BMP4 alone (lower panel). When ES cells were differentiated in BMP4 and VEGF, the factors were removed on day 3 or 4 from the medium by washing the EBs several times and returning them to fresh EB differentiation medium without BMP4 and VEGF. When ES cells were differentiated with BMP4, VEGF was added to the differentiation medium on day 3 or 4 of differentiation at 10 ng/ml. At day 5-6, the cells were subjected to FACS analysis for FLK1 and CD4 expression. Numbers in a given box indicate the percentage of cells that are FLK1⁺CD4^– (lower right), FLK1⁺CD4⁺ (upper right) or FLK1^–CD4⁺ (upper left). (B) Scl^+/CD4 ES cells were differentiated in serum-free conditions in the presence of BMP4 and VEGF. The factors were washed off on day 3 or day 4, and cells expressing Ter119 or CD31 were measured on day 6.

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