IGF-2 produced by the fetal liver CD3⁺Ter119^- cells is the key factor that supports cultured HSCs. (A) FACS analysis of surface expression of CD3, CD4, CD8, Thy1.2, and Ter119 in murine fetal liver and adult spleen cells. Cells freshly isolated from day-15 fetal liver (i-vi) or from adult spleen (vii-xii) were stained with the isotype control (I, vii), anti-CD3–FITC (ii, viii), anti-CD3–FITC and anti-CD4–PE (iii, ix), anti-CD3–FITC and anti-CD8–PE (iv, x), anti-CD3–FITC and anti-Thy1.2–PE (v, xi), and anti-CD3–FITC and anti-Ter119–PE (vi, xii), respectively. (B) RT-PCR analyses of expression of IGF-2 and IGF-2 receptors in fetal liver cells. On the left, RT-PCR was performed on cDNAs isolated from FACS-sorted murine day-15 fetal liver Lin^-Sca-1⁺Kit⁺ (FL LSK) cells and CD3⁺ cells to determine expression of IGF-2, IGF type 1 receptor (IGF1R), IGF type 2 receptor (IGF2R), and insulin receptor (IR) as indicated. On the right, RT-PCR was performed on cDNAs isolated from FACS-sorted murine day-15 fetal liver CD3⁺Ter119⁺ and CD3⁺Ter119^- cells to determine expression of IGF-2. Expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was measured as an indicator of the amount of RNA. (C) Fetal liver CD3⁺Ter119^- cells secrete more IGF-2 than do CD3⁺Ter119⁺ cells when cocultured with LSK cells. FACS-sorted fetal liver CD3⁺Ter119^- cells, CD3⁺Ter119⁺ cells, and LSK cells were cultured for 2 days in 100 μL serum containing medium supplemented with SCF, FL, and IL-6 as follows: 7 × 10⁴ CD3⁺Ter119^- cells (i), 7 × 10⁴ CD3⁺Ter119⁺ cells (ii), 2 × 10³ LSK cells (iii), 7 × 10⁴ CD3⁺Ter119^- cells and 2 × 10³ LSK cells (iv), or 7 × 10⁴ CD3⁺Ter119⁺ cells and 2 × 10³ LSK cells (v). ELISA was then performed to determine the level of IGF-2 in the medium. Error bars represent SEM. (D) Fetal liver CD3⁺Ter119^- cells supported cultured HSCs by producing IGF-2. Fifty freshly isolated day 15 CD45.2 fetal liver Lin^-Sca-1⁺Kit⁺ cells were cultured 3 days in 30 μL medium alone (i), with 50 fetal liver CD3⁺Ter119⁺ cells (ii), with 50 fetal liver CD3⁺Ter119^- cells (iii), or with 50 fetal liver CD3⁺Ter119^- cells and 20 μg/mL anti–IGF-2 antibody (iv), respectively. The medium contained fetal calf serum supplemented with SCF, FL, and IL-6. Together with 2 × 10⁵ competitor CD45.1 bone marrow cells, the total culture, derived from 50 initial Lin^-Sca-1⁺Kit⁺ cells, was coinjected into CD45.1 recipients (n = 4 to 5). Peripheral blood cells from mice undergoing transplantation were analyzed for the presence of CD45.2⁺ cells in lymphoid and myeloid compartments at 4 months after transplantation. Each ▴ represents the percentage of repopulation from a single recipient mouse. Bars represent the average percentage of repopulation for each group; data points below zero represent no detectable repopulation. ^*Significantly different from lane 1 and 2 values; P < .05. ^**Significantly different from lane 3 value; P < .005.