Limiting-dilution analysis of human cord blood CD133⁺ cells transplanted into NOD/SCID mice after culture at normal or low oxygen levels. (A,B) Culture of 2 × 10⁵ cryopreserved human cord blood CD133⁺ cells was initiated in serum-free STF medium supplemented with 500 ng/mL human Angptl5 and human 100 ng/mL IGFBP2. The numbers of total cells (A) and CD34⁺ cells (B) were counted. (C) Amount of human chimerism in the bone marrow of NOD/SCID mice that received a transplant of the indicated numbers (5000, 10 000, 20 000) of postthaw uncultured human cord blood CD133⁺ cells, or the progenies of 1667, 5000, or 10 000 CD133⁺ cells cultured in STF medium with Angptl5 and IGFBP2 in ambient oxygen (lanes 4-6) or 5% oxygen (lanes 7-9) for 10 days. Each symbol represents the engraftment of a single mouse that underwent transplantation assayed at 2 months after transplantation. Horizontal dotted lines represent arbitrary cutoffs of 0.1% and 1% reconstitution. (D,E) Limiting-dilution analysis of the repopulating ability of cells before culture (D) and after culture for 10 days in serum-free STF medium containing 500 ng/mL Angptl5 and 100 ng/mL IGFBP2 at normal or low O₂ levels (E). Plotted is the percentage of recipient mice containing less than 1% human hematopoietic populations in recipient mouse bone marrow 6 to 8 weeks after transplantation versus the number of input or input-equivalent cells injected. The progenies of 10 000 input cells cultured at normal or low O₂ repopulated all recipients and these data points (0% negative mice) are not plotted. (F) Multilineage engraftment in NOD/SCID recipients that received a transplant of 20 000 postthaw uncultured CD133⁺ cells (n = 8) or cultured progenies of 5000 initial CD133⁺ cells at normal O₂ (n = 10). Some mice that received a transplant of uncultured cells had 0% donor repopulation and these data points are not plotted.