BMP4 induces a wave of GFP expression in differentiating MIXL1^GFP/w HESCs. (A) Time course of GFP expression determined by flow cytometric analysis of differentiating MIXL1^GFP/w HESCs shows the transient appearance of mesendodermal progenitors in response to 50 ng/mL BMP4. Note the absence of GFP⁺ cells in cultures differentiated in SFM alone (top panel). The proportion of GFP⁺ cells for each time point is indicated. (B) GFP expression is also induced in day 5 MIXL1^GFP/w EBs formed in SFM supplemented with either 100 ng/mL BMP4 or 50 ng/mL Activin A (Act A) but not with 100 ng/mL FGF2. BF indicates bright field. (C) Flow cytometric analysis substantiates the capacity of BMP4 and Activin A, but not FGF2, to induce GFP expression in MIXL1^GFP/w EBs (left panels). Intracellular flow cytometric analysis of endogenous MIXL1 protein shows that GFP mirrors MIXL1 expression (right panels). (D) Time course analysis of GFP (MIXL1), E-CAD, and PDGFRα expression in MIXL1^GFP/w HESCs differentiated in SFM containing BMP4, VEGF, and SCF, shows the transit of cells from undifferentiated E-CAD⁺GFP⁻PDGFRα⁻ HESCs toward GFP⁺PDGFRα⁺ mesoderm. This latter population gives rise to CD34⁺ cells (bottom panel). As expected, cells differentiated in FGF2 did not express GFP or PDGFRα. Region statistics relating to each population were calculated as described in Figure S4A. GFP⁺ cells are shown in red in all plots. The proportion of the population expressing E-CAD or PDGFRα is shown above the line, and percentages of negative cells are shown below the line. In all instances, the proportion of cells expressing GFP is shown in red. CD34⁺ cells are boxed with the GFP⁺ and GFP⁻ portions indicated with red and black type, respectively.