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.

Conditions controlling the proliferation of haemopoietic stem cells in vitro.

A technique for growing human bone marrow cell colonies in agar-gel medium is described. “Feeder layers” containing 1 × 106 normal human peripheral white blood cells are used as the stimulus for colony growth. Human bone marrow aspirates are collected in heparinized syringes and plated as 2 × 105 cells on “feeder layers.” Normal human bone marrow yields 32–102 colonies per 2 × 105 cells plated. Colonies are almost exclusively granulocytic. Growth rate of colonies is slower than with mouse bone marrow but colonies reach a comparable size (500–1500 cells) at days 12–16.

Human bone marrow colony growth in agar-gel.

Summary. The mouse yolk sac has been shown to contain in-vivo colony forming cells capable of producing granulocytic, megakaryocytic and erythroid spleen colonies; in-vitro colony forming cells producing granulocytic and mononuclear-macrophage colonies in agar; and cells capable of repopuiating the lymphoid and myeloid tissue of lethally irradiated hosts. Similar haemopoietic precursor cells were also demonstrated in the blood at the time of initiation of the circulation and in the early embryonic liver.



Organ cultures of 7 day embryos with intact yolk sacs, and embryos or yolk sacs after separation have shown the autonomous nature of the development of haemopoiesis in the yolk sac and the dependence of intra-embryonic haemopoiesis, particularly in embryonic liver, on colonization by yolk sac haemopoietic cells.



Both in-vivo and in-vitro colony forming cells have been involved in the first migration stream, between yolk sac and embryonic liver, and evidence has been presented for the role of local environmental factors in controlling the differentiation of these cell types.



These results support the view that development of haemopoietic organs in both embryo and adult is dependent on colonization by circulating cells and that these circulating stem cells originate initially in the yolk sac. This indicates that the yolk sac is the only site of genuine de novo formation of haemopoietic stem cells.

Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo.

Assay of red cell progenitors by colony formation in culture is expected to allow study of early events in erythroid differentiation in animal models and in man. In this communication, a simplification of the culture method for mouse bone marrow cells originally reported by Stephenson et al. ('71) is described. In the modified procedure, plasma clot is replaced by methyl cellulose, and scoring of erythroid colonies is done directly in the plates without staining. With additional modification the system proved applicable to the culture of erythroid colonies from human bone marrow as well. The development of both mouse and human erythroid colonies was dependent on “erythroid colony stimulating activity” (E-CSA) supplied by extracts of anemic sheep plasma, or by extracts of urine from anemic patients. Over a certain range, the number of colonies was a function of dose of E-CSA.

In addition to E-CSA, these extracts also possessed erythropoietin activity as measured in plethoric mice. The possible chemical equivalence of urinary E-CSA and erythropoietin was suggested by their similar behavior on both gel filtration and affinity chromatography on agarose-concanavalin A. The finding further suggests that the culture method might prove useful for the bioassay of erythropoietin.

Granulocyte colony stimulating activity (G-CSA) was also present in the urine extracts, as detected in cultures of mouse bone marrow. Virtually all of this activity was bound on agarose-con A, whereas only a small fraction of E-CSA was retained on this material. Agarose-con A may thus be useful for the purification of erythropoietin.

Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A.

Factors regulating macrophage production and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells.

Stimulation of mouse bone marrow colony growth in vitro by conditioned medium.

The characteristics of stimulation of colony formationin vitro from cells of mouse haemopoietic tissues has been briefly reviewed. Mouse kidney or embryo feeder cells, media conditioned by the cells from these tissues, normal or leukemic mouse sera, sera from leukemic or infectious mononucleosis patients, human urine and mouse embryo extracts are all sources of colony stimulating activity and their properties have been described. All sources of colony-stimulating activity produce clones of cells of the granulocyte series. In tritiated thymidine treated mice injection of preparations rich in colony-stimulating activity has been shown to produce a neutrophil leucocytosis and accelerate the rate of accumulation of labelled neutrophils in the blood. It is suggested that thein vitro assay can detect factors capable of stimulating granulocyte development.

T. R. Bradley

Papers

Stimulation of mouse bone marrow colony growth in vitro by conditioned medium.

Characteristics of in vitro colony formation by cells from haemopoietic tissues