Hematopoietic progenitors enriched in CD34⁺ C-Kit⁺ population. (A) Magnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) selection of day 7 EB cells by CD34 and C-Kit. The expression of CD34 and C-Kit in unselected cells (1), in MACS-selected CD34⁻ cells (2), in MACS-selected CD34⁺ cells (3), and in FACS-selected CD34⁺C-Kit⁺ cells (4) was examined by flow cytometric analysis. Numbers represent percentage of total cells. (B) Gene expression of selected and unselected cells was assessed by real-time PCR. Relative gene expression was determined by normalization to that in unsorted cells. Similar results were produced by 3 independent experiments with 3 parallel samples. The data were from one experiment, and data represent mean ± SEM (C) Hematopoietic progenitor potential of MACS-selected and unselected cells were tested by methylcellulose hematopoietic colony assay. Error bars indicate mean ± SEM (n = 3). Each sample was in triplicate. (D) MACS-selected cells were cultured as aggregates for 7 days with serum-free expansion medium supplemented with 10% FBS, SCF, Flt-3L, IL-3, and IL-6. The hematopoietic potential of selected and unselected cells was assessed by CD45 expression determined by flow cytometric analysis. Error bars indicate mean ± SEM (n = 3). (E) Quantification of the percentage of CD34⁺ C-Kit⁺ hESC-derived hematopoietic progenitors expressing a given marker by flow cytometry.