Culture with IGF-2 increases in vivo repopulating activity of fetal liver HSCs. (A) Culture of day-15 fetal liver Lin^-Sca-1⁺Kit⁺ cells with 500 ng/mL IGF-2 enhances in vivo repopulating stem cell activity. A representative experiment of the 3 performed is illustrated. Fifty freshly isolated day-15 CD45.2 fetal liver Lin^-Sca-1⁺Kit⁺ cells were cultured for 3 days in medium containing FBS and supplemented with SCF, FL, and IL-6 (i); in medium containing FBS and supplemented with SCF and TPO (ii); in medium containing FBS and supplemented with SCF, FL, IL-6, and 500 ng/mL IGF-2 (iii); and in medium containing FBS and supplemented with SCF, TPO, and 500 ng/mL IGF-2 (iv). Together with 2 × 10⁵ competitor CD45.1 bone marrow cells, the entire culture derived from these initial 50 Lin^-Sca-1⁺Kit⁺ cells were coinjected into CD45.1 recipients (n = 4 to 6). Peripheral blood cells from mice undergoing transplantation were analyzed for the presence of CD45.2⁺ cells in lymphoid and myeloid compartments at 3 months after transplantation. Each ▴ represents the percentage of population from a single recipient mouse. Horizontal bars represent the average of repopulation percentage in each group. Note that data points below zero represent no detectable population. ^*Significantly different from panel i value; P < .05. ^**Significantly different from lane ii value; P < .005. (B) In vivo limiting dilution analysis of the repopulating ability of day 15 fetal liver Lin^-Sca-1⁺Kit⁺ cells before and after in vivo culture with IGF-2. Freshly isolated day 15 CD45.2 fetal liver Lin^-Sca-1⁺Kit⁺ cells were transplanted directly (* and solid line) or cultured in medium containing FBS together with SCF, TPO, and 500 ng/mL IGF-2 (▪ and dashed line) followed by transplantation (with 2 × 10⁵ CD45.1 competitor bone marrow cells per mouse) into CD45.1 congenic recipients. Plotted is the percentage of recipient mice containing less than 1% CD45.2 lymphoid (B220⁺) and myeloid (Gr-1⁺/Mac-1⁺) subpopulations in nucleated peripheral blood cells 4 months after transplantation versus the number of fetal liver Lin^-Sca-1⁺Kit⁺ cells initially injected or cultured. The curve is anchored by the 0 cells/100% negative mice point. Dotted lines show the determination of the CRU frequency values by the method of maximum likelihood (at 37% negative mice).¹¹