Culture with IGF-2 increases in vivo repopulating activity of adult bone marrow HSCs. (A) About 50% of adult bone marrow Lin^-Sca-1⁺ cells specifically bind IGF-2–hFc. Adult bone marrow cells were incubated at 4°C for 30 minutes with conditioned medium from control transfected 293T cells (iii) or cells transfected with the IGF-2–hFc expression vector (iv,v); the latter medium contained about 1 μg/mL IGF-2–hFc. Cells were then stained with antihuman IgG1-PE, followed by the biotinylated Lin⁺ antibody cocktail, streptavidin-APC, and anti-Sca-1–FITC. Total bone marrow cells and Lin^- cells were then stained, gated (i,ii), and analyzed as in Figure 3B to quantify the binding to IGF-2–hFc and the level of Sca-1. (B) All repopulating HSCs in the total population of bone marrow cells bind IGF-2–hFc. Of the 42.0% of total CD45.2 bone marrow cells that bind the IGF-2–hFc fusion protein (panel Aiv), the top 30% and the lower 70% were arbitrarily sorted into the ++ and + fractions, respectively. Similarly, of the 58.0% that cannot bind the IGF-2–hFc fusion protein (as shown in panel A), the top 70% and the lower 30% were arbitrarily sorted into the - and - - fractions, respectively. From each group of total bone marrow cells, 10⁴ cells were transplanted together with 105 CD45.1 competitor cells into lethally irradiated CD45.1 mice (n = 4 to 5). Peripheral blood cells were analyzed for the presence of CD45.2⁺ cells in lymphoid and myeloid compartments at 3 weeks and 6 months after transplantation. (C) All repopulating HSCs in the Lin^-Sca-1⁺ subpopulation of bone marrow cells bind IGF-2–hFc. Fifty CD45.2 bone marrow Lin^-Sca-1⁺IGF-2–hFc⁺ and 100 Lin^-Sca-1⁺IGF-2–hFc^- cells, sorted as depicted in panel Av, were transplanted together with 2 × 10⁵ CD45.1 competitor cells into lethally irradiated CD45.1 mice (n = 4 to 5). Peripheral blood cells were analyzed for the presence of CD45.2⁺ cells in lymphoid and myeloid compartments at 6 months after transplantation. Error bars represent SEM. (D) Culture of adult bone marrow SP cells with IGF-2 enhances in vivo repopulating stem cell activity. Fifty freshly isolated adult CD45.2 bone marrow SP cells were transplanted directly (with 2 × 10⁵ CD45.1 competitor bone marrow cells per mouse, n = 9) into CD45.1 congenic mice (i). From the same isolation 50 SP cells were cultured 3 days in medium alone (ii) or in medium with 1 μg/mL IGF-2 (iii). Serum-containing medium supplemented with SCF, FL, and IL-6 was used. Together with 2 × 10⁵ competitor CD45.1 bone marrow cells, the entire culture derived from these initial 50 SP cells was coinjected into CD45.1 recipients (n = 8 to 9). Peripheral blood cells from mice undergoing transplantation were analyzed for the presence of CD45.2⁺ cells in lymphoid and myeloid compartments at 3.5 months after transplantation. ^*Significantly different from panel i and ii values; P < .05.