Purification of murine bone-marrow-derived granulocyte-macrophage colony-forming cells

Previous attempts to purify progenitor cells that form colonies and clusters of granulocytes and/or macrophages (CFU-GM) from adult murine bone marrow have had limited success because of the paucity of these cells. In the present paper we report studies with a rapid, reproducible method involving pretreatment of mice, three days prior to sacrifice, with 200 mg/kg of Cytoxan (cyclophosphamide), density separation on Ficoll-Hypaque, and counterflow centrifugal elutriation, that yielded highly enriched populations of CFU-GM. The peak CFU-GM-containing fraction (FR-28) eluted at a flow rate of 28 ml/min and contained very little contamination by other in vitro colony-forming cells (BFU-E, CFU-GEMM, CFU-MK). FR-28 contained 0.54% +/- 0.30% (16 experiments) of the unfractionated post-Cytoxan bone marrow nucleated cells and lacked significant contamination by lymphocytes and monocytes. The mean CFU-GM cloning efficiency of FR-28 was 44% +/- 9% in agar (11 experiments) and 75% +/- 10% in agarose (nine experiments). CFU-GM from FR-28 demonstrated linear plating characteristics even at very low cell density (25 cells), and formed colonies and clusters of granulocytes, macrophages, or both in the same proportions as did unfractionated post-Cytoxan or untreated bone marrow. Approximately 10% (assuming a seeding efficiency of 10%) of FR-28 cells were in vivo spleen colony-forming cells (CFU-S) measured at day 12. These results represent the highest degree of purity (up to 94%) of CFU-GM thus far reported and should prove useful in studies of this cell population.