FIGURE 2.

Expression of KO in human hematopoietic stem cells and in their descendant cells. A and B, expression of KO in CD133 (+) cord blood cells is shown. CD133 (+) cells were purified with magnetic beads conjugated with anti-CD133 antibody (Miltenyi Biotech). The cells were infected with SeVdp(Bsr/ΔF/KO) at a multiplicity of infection of 4 at 37 °C for 2 h. The cells were then cultured for 3 days (A) and 10 days (B) and examined using fluorescence and phase-contrast microscopy (A) and with flow cytometry using a FACSCalibur (BD Biosciences) (B), respectively. C–F, expression of KO in descendant colonies differentiated in vitro is shown. Cells infected with SeVdp(Bsr/ΔF/KO) as described above were cultured on OP9 cells in a 96-well plate for 5 weeks for lineage commitment. The cells in each well were then harvested, cultured in semisolid medium for 2 weeks, and examined for the expression of KO using fluorescence microscopy. C, the ratio of KO-positive colonies; 2931 differentiated colonies were examined. KO (+++), colonies expressing KO strongly; KO (+), colonies expressing KO weakly or heterogeneously; KO (−), colonies with no detectable KO expression. D–F, fluorescence and phase-contrast micrographs of typical colonies representative of each lineage. D, CFU-G, CFU-granulocytes. E, CFU-M, CFU-macrophages. F, BFU-E, burst-forming unit-erythroid cells. Scale bar, 100 μm.