Induction of hematopoietic cell surface markers during hESC differentiation. Human ES cells formed into embryoid bodies were cultured in differentiation medium alone (control, n = 12; panels A and E) or induced toward hematopoietic fate by culturing in differentiation medium supplemented with hematopoietic cytokines (cytokines, n = 7; panels B and F), cytokines and BMP-4 (cytokines + BMP-4, n = 13; panels C and G), or BMP-4 alone (BMP-4, n = 6; panels D and H). The cell surface phenotype of differentiated viable hESCs after 15 days of differentiation was examined by multiparameter flow cytometry for expression of SSEA-4 (A-D), CD45, and CD34 (E-H). Values in each quadrant are based on mean ± SEM from H1 and H9 cell lines. Histogram markers (horizontal bars) and dot plot quadrants were set based on their isotype controls, shown as open histograms (A-D) or insets (E-H). (I) Summaries of total percentage of CD45⁺ cells and percentage of CD45⁺CD34⁺ cells for each induction treatment. (J) Total number of differentiated hESCs (n = 6-13 for each treatment) and percentage of total live cells (inset) within treated cultures. (K) Total number of CD45⁺ cells (n = 6-13 for each treatment) from dissociated EBs after 15 days under various hematopoietic treatments. Error bars represent SEMs. In panels I and J, P < .02.