HOXA9 silencing abrogates hematopoietic differentiation of hESC. (A) Hematopoietic differentiation of hESCs transduced with an irrelevant short hairpin RNA sequence (scramble) or 3 different short hairpin RNA specific sequences for HOXA9 (shHOXA9). (B) CFU read out from day 15 EBs confirming greater than a twofold decrease of hematopoietic progenitor potential in short hairpin (shHOXA9) cells. Scoring of CFUs revealed a skewed differentiation toward erythroid lineage in shHOXA9 progenitors. (C) Quantitative reverse-transcription polymerase chain reaction analysis showing a highly reduced HOXA9 expression throughout the differentiation in shHOXA9 EBs. Data represent mean ± SEM for 3 independent experiments. (D) Quantitative reverse-transcription polymerase chain reaction showing the expression of the pluripotency factors OCT4, SOX2, and NANOG in scramble- and shHOXA9-hEBs after 15 days of hematopoietic differentiation (upper panel). Flow cytometry confirming that shHOXA9-EBs are composed by OCT4⁺ cells after 15 days of hematopoietic differentiation (lower panel). mRNA, messenger RNA.