Proliferation phenotype of NUP98HOXA10hd–expanded HSCs. (A) Representative profile of viable, Lin⁻NUP98-HOXA10hd– and GFP-transduced day 10 cells assessed for Sca-1 and CD43 expression and further analyzed for the level of surface Mac1 protein expression. Viable, Lin⁻ transduced cells expressing both Sca-1 and CD43 also were confirmed to express the high levels of c-kit protein.³¹  (B) Empty bars indicate the average number (expressed as the means ± SD) of bulk or phenotypically defined NUP98-HOXA10hd–transduced cells at the end of 10-day culture. TNCs indicates total nucleated cells. Filled bars indicate the total number of HSCs generated by day 10 in cultures of NUP98-HOXA10hd–transduced cells and the HSC content of phenotypically defined subsets (Lin⁻Sca-1⁺c-kit⁺CD43⁺Mac1^dim; Lin⁻Sca-1⁺c-kit⁺CD43⁺Mac1⁻; Lin⁻Sca-1⁺c-kit⁺CD43⁻Mac1^bright), as determined by limiting dilution CRU assay. Values shown are the means ± SEM. (C) Distribution of the total NUP98-HOXA10hd–transduced HSCs generated in 10-day culture according to indicated phenotypes: Lin⁻Sca-1⁺c-kit⁺CD43⁺Mac1^dim; Lin⁻Sca-1⁺c-kit⁺CD43⁺Mac1⁻; Lin⁻Sca-1⁺c-kit⁺CD43⁻Mac1^bright. Each subset was isolated by FACS and assayed for the HSC content by limiting dilution. Black area represents the HSCs (< 10% of the total) undetected by indicated phenotype(s). (D) On the left, proportion of peripheral blood leukocytes produced more than 20 weeks after transplant by 5 Lin⁻Sca-1⁺c-kit⁺CD43⁺Mac1^dimNUP98-HOXA10hd–transduced and expanded cells. Each triangle represents an individual recipient/mouse. On the right, bars indicate the mean percentage of lineage (Ly6G and Mac1 [black]; B220 [dark gray]; and CD4 and CD8 [light gray]) contribution to white blood cells (WBCs) of mice reconstituted with the limited number (5 cells/mouse) of NUP98-HOXA10hd–transduced and expanded cells, repurified for Lin⁻Sca-1⁺c-kit⁺CD43⁺Mac1^dim phenotype (shown on the left). Error bars indicate the SD of the mean.