Long-term ex vivo–expanded RUNX1a progenitor cells are capable of differentiating into multiple lineages. (A) Progenitor cells (CD34⁺CD45⁺) from vector or RUNX1a-transduced H9 cells are expanded ex vivo for 7 days in liquid culture. Cell numbers are monitored every day. (B) Wright-Giemsa staining of 3- or 10-day ex vivo–expanded progenitor cells from vector or RUNX1a-transduced H9 cells. Scale bar, 50 μm. (C) Representative FACS analysis of CD235a/CD45 expression on ex vivo–expanded progenitor CD34⁺CD45⁺ cells from vector or RUNX1a-transduced H9 cells in liquid culture. (D) CFU assay on progenitor cells from vector or RUNX1a-transduced H9 cells. (Left) Numbers of colonies formed from 10 000 cells. (Right) Scoring of CFUs reveals differences in CFU subtypes. BFU, burst-forming unit; E, erythroid; G, granulocyte; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM, granulocyte-macrophage; M, macrophage. (E) Expression of β-globin is detected in HPCs differentiated from RUNX1a EBs after 14-day expansion by FACS analysis or western blot. Kasumi-1 is used as a negative control. Peripheral blood is used as a positive control. GAPDH is used in western blot as a loading control.