Detection of the granulocyte colony-stimulating factor receptor using biotinylated granulocyte colony-stimulating factor: presence of granulocyte colony-stimulating factor receptor on CD34-positive hematopoietic progenitor cells

Granulocyte colony-stimulating factor (G-CSF) was linked to NHS-biotin to yield biotinylated G-CSF (b-G-CSF), which retained the ability to stimulate colony formation by normal bone marrow (BM) cells in methylcellulose. The use of streptivadin-phycoerythrin conjugate in conjunction with flow cytometry demonstrated that the binding of biotnylated G-CSF to its receptor is saturable, competitive, and specific. A 100-fold molar excess of unlabeled G-CSF almost completely inhibited the binding of the biotinylated G-CSF to the human leukemia cell line U937, which is known to posses the G-CSF receptor. G-CSF receptors were clearly detected by flow cytometry on adult human peripheral granulocytes and monocytes, but not on lymphocytes. Using this method, the expression of G-CSF receptors on hematopoietic progenitor cells in bone marrow and umbilical cord blood, detected as CD34-positive (CD34⁺) cells, were examined. A small but significant number of CD34⁺ cells were detected among the bone marrow mononuclear cells and umbilical-cord-blood mononuclear cells (4.28%±0.31%, 1.09%±0.20%, respectively). The percentage of CD34⁺ BM mononuclear cells was significantly higher than for cord blood mononuclear cells (P<0.01). These CD34⁺ cells were then analyzed by biotinylated G-CSF binding. CD34⁺ cells from bone marrow contained 25.8%±7.9% G-CSF receptor positive cells and those from cord blood possessed 29.2% ±7.0% of G-CSF receptor-positive cells. The difference was not statistically significant.