CD41⁺c-kit⁺ EB-derived cells contain all hematopoietic colony forming potential and are enriched for hematopoietic gene expression. OP9–colony-forming potential (A) and hematopoietic progenitor activity (B) of day 6 EB-derived cells fractionated with respect to c-kit and CD41 expression by FACS. OP9–colony-forming potential is presented as the average colony number/1000 cells plated for 9 independent experiments. Error bars represent the standard error. The data presented in panel B represent the pooled total number of colonies detected for each compartment in 6 independent experiments per 50 000 cells plated. Not all compartments were assessed in each independent experiment, but each were assessed a minimum of 4 (A) or 2 (B; CD41⁻c-kit⁻ cells) times. (C) Quantitative real-time RT-PCR of hematopoietic gene expression in CD41⁺ and CD41⁻ day 6 EB-derived cells. (D) CD41⁺ cells were significantly enriched in Scl, LMO2, Runx1, and Gata-2 gene expression relative to CD41⁻ cells. Normalization to expression levels of the housekeeping gene β-actin was performed for each sample. Results represent the average of 3 independent biologic experiments. CD41⁺ day 6 EB cells were further fractionated with respect to c-kit expression, and analyzed by quantitative real-time RT-PCR for the expression of hematopoietic genes. Data are presented relative to expression level of each gene in CD41⁺c-kit⁻ cells, after individual normalization of each sample to β-actin. CD41⁺c-kit⁺ cells were significantly enriched for Runx1, Gata-3, and PU.1 gene expression. For panels C and D, P values calculated using nonpaired, 2-tailed Student t test are depicted over bars.