Conditions controlling the proliferation of haemopoietic stem cells in vitro

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 vitro 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°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.