Figure 1

IGF-2 receptor expression in fetal liver cells. (A) Production and secretion of IGF2-hFc in transfected 293T cells. The upper panel shows a schematic of the plasmid expressing the human prepro-IGF-2 protein fused to a human IgG1 Fc fragment. The bottom panels show western Blots of conditioned medium collected 48 hours after transfection from 293T cells transfected with either control or Arg-IGF2-hFc vectors; blots were probed with antibodies against human IGF-2 (left) or human IgG1 (right). (B) Mouse fetal liver cells (FL) and adult bone marrow cells (BM) contain IGF-IR⁺ cells that bind to Arg-IGF-2-hFc as determined by flow cytometry. (C) Western blot of total fetal liver cells sorted based on binding of Arg-IGF2-hFc. Antibody against IGF-IR (Cell Signaling) was used to detect the expression of IGF-IR in flow cytometry-sorted Arg-IGF2-hFc⁺ and Arg-IGF2-hFc⁻ fetal liver cells. These results confirmed the specificity of Arg-IGF2-hFc binding to IGF-IR⁺ cells. Blotting with anti-beta-tubulin (Sigma) served as the loading control. (D) All repopulating HSCs in the total population of bone marrow (BM) or fetal liver cells (FL) bind Arg-IGF2-hFc. One thousand CD45.2 bone marrow or fetal liver Arg-IGF2-hFc⁺ cells and 1,000 Arg-IGF2-hFc⁻ cells were transplanted together with 2 × 10⁵ CD45.1 competitor cells into lethally irradiated CD45.1 mice (n = 5). Peripheral blood cells were analyzed for the presence of CD45.2⁺ cells at 6 months after transplant. (E) All the mouse bone marrow Lin⁻ Sca1⁺ Kit⁺ cells are IGF-IR⁺ cells as determined by flow cytometry. Bone marrow Lin⁻ cells were gated. (F) All repopulating HSCs in the Lin⁻Sca-1⁺Kit⁺ cells bind Arg-IGF2-hFc. Fifty CD45.2 bone marrow or fetal liver Lin⁻Sca-1⁺Arg-IGF2-hFc⁺ and 100 Lin⁻Sca-1⁺IGF2-hFc⁻ cells were transplanted together with 2 × 10⁵ CD45.1 competitor cells into lethally irradiated CD45.1 mice (n = 4-5). Peripheral blood cells were analyzed for the presence of CD45.2⁺ cells at 6 months after transplant.