D Metcalf

Summary A simple in vitro technique is described for the growth of colonies from single cell suspensions of mouse bone marrow. The system involves the plating of marrow cells in agar on feeder layers of other cells, those from 8‐day‐old mouse kidney and 17th day mouse embryo being shown to be the most efficient types of feeder layers. Approximalely 400 colonies per 1 × 10 6 nucleated marrow cells were grown, using kidney cell feeder layers. A linear relationship between the number of cells plated and the number of colonies developing was demonstrated. In comparison with the marrow cells, lymph node or thymus cells did not form colonies, but a small number of colonies was formed using spleen cells. Early in the development of the colonies the dominant cell type was a large mononuclear cell with cytoplasm filled with granules staining metachromatically with toluidine blue. With growth of the colony, cells with ring or horseshoe‐shaped nuclei appeared, and a progression with further colony growth to smaller cells with segmented nuclei similar to polymorphonuclear blood cells was observed.

Rapid progress has occurred recently in characterizing the molecular nature of the specific glycoprotein colony-stimulating factors (CSFs) controlling the proliferation; and some functional activities of granulocytes and monocyte-macrophages. All four known murine CSFs have been purified, and cDNAs for two have been cloned and expressed by mammalian and bacterial cells. Similarly, three human CSFs have been purified, and cDNAs for two cloned and expressed. This work has opened up the exciting prospects of testing the effects of these recombinant CSFs on hematopoiesis in vivo. Each CSF has a broader range of hematopoietic target cells than previously suspected, and it is now clear that the CSFs are not simply proliferative stimuli but can also regulate the functional activity of mature cells. There are increasing reasons to believe that these CSFs will be useful therapeutic agents in stimulating hematopoietic regeneration in leukopenic states and the functional activity of granulocytes and monocytes in infections.

Cell lines have been produced from long-term cultures of mouse bone marrow that require a factor, present in WEHI-3 conditioned medium (CM) or in spleen CM, for their sustained growth. The cell lines were obtained from nonvirus-treated cultures, are nonleukemic, maintain a normal karyotype, and form colonies showing granulocyte maturation when plated in soft agar. Granulocyte/macrophage (GM) colony-stimulating factor is not the inductive moiety involved in the maintenance of proliferation of these cells. It is suggested that the cell lines represent a self-renewing population of cells ancestral to GM colony-forming cells, which may be responding to a hitherto unrecognized regulator.