Overexpression of HOXA9 during EB development leads to an increased hematopoietic differentiation. (A) Schematic of the EB-based hematopoietic differentiation and endpoint analyses (left panel). More than 90% of the cells within both empty vector (EV)-hESC/EB and HOXA9-hESC/EBs cultures were transduced (GFP⁺; middle panel). Representative flow cytometry displaying how HEPs (CD34⁺CD31⁺CD45⁻) and blood cells (CD45⁺) are identified (right panel). (B) HOXA9 robustly promotes differentiation into HEPs, primitive blood cells (CD34⁺CD45⁺), and total blood cells (CD45⁺). (C) Hoxa9-expressing blood progeny displays threefold higher clonogenic potential (left panel). Colonies retained GFP expression after the CFU assay, and scoring of CFU reveals a skew toward G-CFU in Hoxa9-transduced progenitors (middle and right panels). (D) Quantitative PCR analysis demonstrating that exogenous levels of Hoxa9 remain high throughout EB differentiation. Data represent mean ± standard error of the mean for 6 independent experiments.