Culture of human cord blood CD133⁺ cells in the presence of Angptl5 and IGFBP2 stimulates ex vivo expansion of SRCs. (A) Culture of 10⁴ cryopreserved human cord blood CD133⁺ cells was initiated in serum-free STF medium (containing SCF, TPO, and FGF-1), or in STF medium supplemented with 500 ng/mL human Angptl5 and 500 ng/mL human IGFBP2, at 5% O₂. Total cell numbers were counted during the 11 days of culture period. (B) Extent of human chimerism in the bone marrow of NOD/SCID mice that received a transplant of 8000 or 15 000 uncultured human cord blood CD133⁺ cells, or the progenies of 8000 initial CD133⁺ cells cultured in STF medium with or without Angptl5 and IGFBP2 for 11 days. Each symbol represents the engraftment of a single mouse that underwent transplantation assayed at 2 months after transplantation (n = 7-8). *Significantly different from lanes 1-3. Student t test, P < .05. (C) Representative fluorescence-activated cell sorting (FACS) plots of bone marrow cells from one mouse at the condition represented by lane 1 of panel B (after thaw), or at the condition represented by lane 4 of panel B (cultured in STF medium containing IGFBP2 and Angptl5) at 2 months after transplantation. Percentages of cells in each quadrant are listed. (D) Summary of multilineage reconstitution from mice in lanes 2 and 4 of panel B (n = 8, respectively). Some mice that received a transplant of uncultured cells had 0% donor repopulation and these data points are not plotted. *Values are significantly different from values of the uncultured cells. Student t test, P < .05. (E) Bone marrow cells collected from mice represented by lane 4 of panel B were transplanted into secondary recipients; bone marrow aspirate from one hind leg from a primary recipient was transplanted into 2 secondary recipients. Multilineage engraftment in secondary NOD/SCID recipients was assayed at 5 to 8 weeks after transplantation (n = 12 mice underwent transplantation).