Differentiation of Mouse Bone Marrow Precursor Cells into Neutrophil Granulocytes by an Activity Separation from WEHI-3 Cell-Conditioned Medium

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Colonies comprised exclusively of neutrophil granulocytes have been obtained by growing mouse bone marrow cells in nutrient semisolid agar cultures. A stimulator of predominantly granulocyte colony formation was present in the breakthrough fraction of preparations of colony-stimulating activity separated on DEAE-Sephadex A. The source of colony-stimulating activity was concentrated conditioned medium of a murine myelomonocytic cell line (WEHI-3), which unfractionated stimulated the growth of colonies of granulocytes, macrophages, megakaryocytes, as well as mixed colony types. After stepwise column chromatography of the conditioned medium, the breakthrough fraction was shown to stimulate predominantly granulocyte colony formation, and the fraction eluted with 1 M NaCl was found to induce primarily macrophage colony growth. Colony morphology was independent of the concentration of eluate used. The morphology of colonies varied with increasing concentrations of the breakthrough fraction. At low concentrations, granulocyte colony formation was almost exclusively observed. With increasing concentrations of this fraction, an increasing proportion of the colonies were found to contain macrophages. The effect of concentration of this activity was in marked contrast to previous findings where the incidence of granulocyte colony formation was inversely related to the concentration of colony-stimulating activity. This differential responsiveness of cell to stimulus has previously been interpreted as low concentrations of a growth and differentiation factor being required for macrophage production and high concentrations of the same factor required for granulocyte formation. Separation of these activities by DEAE Sephadex chromatography, and alteration of the dose-response curve, such that granulocyte colony formation varies directly with the amount of stimulator, indicates that the differentiation of these two cell blood lineages may be controlled by separate entities.