Fig. 1. BMP4 induces FLK1⁺ cells. (A) ES cells were differentiated in serum (FCS) (upper panel) or in serum-free conditions (SR) with BMP4 (5 ng/ml), BMP2 (5 ng/ml), bFGF (10 ng/ml), activin A (2 ng/ml) or TGFß1 (1 ng/ml). At day 2.75-3 of differentiation, EB cells were FACS analyzed for FLK1 expression. Numbers in insets indicate the percentage of FLK1⁺ cells. 2AB indicates cells stained with secondary antibody alone. (A') Results from four independent experiments are shown as a percentage of BMP4 control. Error bars indicate s.e.m. (B) ES cells were differentiated in serum (FCS) alone or with Noggin (upper panel). Alternatively, ES cells were differentiated in serum-free conditions (SR) with BMP4 alone or with BMP4 and Noggin (lower panel). At day 2.75-3 of differentiation, EB cells were FACS analyzed for FLK1 expression. Numbers in parenthesis indicate Noggin concentration (µg/ml). IgG1 isotype antibodies were used as control. Numbers in insets indicate the percentage of FLK1⁺ cells. (B') Results from three (FCS) or five (serum-free conditions) independent experiments are shown as a percentage of serum alone (FCS) or BMP4 (SR) control. Error bars indicate s.e.m. (C) ES cells were differentiated in serum-free conditions, and then BMP4 was added at different days (D0, D1, D2, D3 or D4). At day 6 of differentiation, EB cells were FACS analyzed for FLK1 expression. Numbers in insets indicate the percentage of FLK1⁺ cells. (C') Results from three independent experiments are shown as a percentage of BMP4 (D0) control. Error bars indicate s.e.m.