T. M. Dexter

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S), production of granulocyte precursor cells (CFU-C), and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells, "epithelial" cells, and "giant fat" cells. The latter appear to be particularly important for stem cell maintenance and furthermore there is a strong tendency for maturing granulocytes to selectively cluster in and around areas of "giant fat" cell aggregations. By "feeding" the cultures at weekly intervals, between 10 to 15 "population doublings" of functionally normal CFU-S regularly occurs. Increased "population doublings" may be obtained by feeding twice weekly. The cultures show initially extensive granulopoiesis followed, in a majority of cases, by an accumulation of blast cells. Eventually both blast cells and granulocytes decline and the cultures contain predominantly phagocytic mononuclear cells. Culturing at 33 degrees C leads to the development of a more profuse growth of adherent cells and these cultures show better maintenance of stem cells and increased cell density. When tested for colony stimulating activity (CSA) the cultures were uniformly negative. Addition of exogenous CSA caused a rapid decline in stem cells, reduced granulopoiesis and an accumulation of phagocytic mononuclear cells.

Cell proliferation in the bone marrow and blood of two patients with metastatic breast cancer who were treated with granulocyte colony-stimulating factor was studied by using [3H]thymidine labeling and autoradiography. Additionally, the fate of neutrophils labeled with 99mTc-hexamethylpropyleneamineoxime was observed following granulocyte colony-stimulating factor infusion. Proliferation increased in all stages of granulopoiesis, but a significant amount of the increased production stemmed from a greater input to the myeloblast compartment. Changes in the myelogram combined with the increased labeling indicated a faster throughput of cells, which resulted in labeled cells appearing in the circulation within 1 day compared to the normal 4 or 5 days. The 99mTc studies demonstrated no sequestration of circulating neutrophils by spleen, lungs, or liver. The half-life of the circulating neutrophils was not significantly changed, and calculations from the flow of labeled cells to the peripheral blood indicated an increase of 3.2 extra amplification divisions during neutrophil development. The dramatic neutrophil response to granulocyte colony-stimulating factor can therefore be accommodated by a relatively modest increase in granulopoietic activity.