Coculture of fetal liver HSCs with fetal liver CD3⁺ cells increases in vivo repopulating stem cells. (A) In vivo limiting dilution analysis of the repopulating ability of day-15 fetal liver Lin^-Sca-1⁺Kit⁺ cells before and after in vitro culture with day-15 fetal liver CD3⁺ cells. Freshly isolated day-15 fetal liver Lin^-Sca-1⁺Kit⁺ cells were mixed with the same number of freshly isolated day-15 fetal liver CD3⁺ cells. Half of the mixed cells were used for direct transplantation (* and solid line). The other half were cultured for 3 days in a volume of 30 μL followed by transplantation of the entire culture (▪ and dashed line). In addition, 100 freshly isolated day-15 fetal liver Lin^-Sca-1⁺Kit⁺ cells were cultured alone without CD3⁺ cells for 3 days followed by transplantation (▴). The medium contained fetal calf serum supplemented with SCF, FL, and IL-6. Irradiated CD45.1 congenic mice were injected with 2 × 10⁵ CD45.1 bone marrow cells and the indicated numbers of freshly isolated CD45.2 Lin^-Sca-1⁺Kit⁺ fetal liver cells or their progeny after culture. 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 initial Lin^-Sca-1⁺Kit⁺ cells; note that the abscissa is presented as the number of LSK cells initially added to the culture. The curve was anchored by the 0 cells/100% negative mice point. Injection of the culture of 50 or 100 initial Lin^-Sca-1⁺Kit⁺ cells with CD3⁺ cells resulted in repopulation of 100% of the mice. A data point of 0% negative mice cannot be plotted on a logarithmic axis; so to be conservative we assume and plot that only 90% of the mice injected with the culture derived from these 50 input LSK cells are positive, or 10% negative. Dotted lines show the determination of the CRU frequency values by the method of maximum likelihood (at 37% negative mice).¹¹  (B) Details of a typical competitive repopulation by 100 CD45.2 fetal liver Lin^-Sca-1⁺Kit⁺ cells cultured alone or with fetal liver CD3⁺ cells (as shown in panel A) 4 months after transplantation into lethally irradiated CD45.1 recipient mice. Data shown are representative FACS plots of peripheral blood mononuclear cells from 1 mouse in each group. To assess the repopulation frequencies of the donor cells, cell isolates were double stained with antibodies against CD45.2 (donor) and CD45.1 (recipient), Thy1.2 (T cells), B220 (B cells), Mac-1 (macrophages), and Gr-1 (granulocytes). As illustrated in the top panels, Lin^-Sca-1⁺Kit⁺ cells cultured in the absence of CD3⁺ cells yielded no reconstitution of any of the lineages analyzed.