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.

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

Purification and properties of colony-stimulating factor from mouse lung-conditioned medium.

The concept that the continuous formation of blood cells might be under the control of specific humoral regulatory factors received early support from the demonstration that a humoral factor, termed erythropoietin, was involved in the regulation of erythropoiesis (Carnot and Deflandre, 1906). However it was not until the introduction in 1966 of methods for growing colonies of normal hemopoietic cells in semisolid culture, that progress was made in identifying and characterizing corresponding specific regulators for other hemopoietic populations.

The hemopoietic colony stimulating factors

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells.



Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells.



The active factor in serum was filterable, non-dialysable and heat and ether labile.

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

An analysis has been made of cell colonies developing in agar cultures from mouse bone marrow cells following stimulation either by neonatal kidney cell feeder layers or AKR lymphoid leukemia serum. Colonies arose by cell proliferation and were mixtures of granulocytic and mononuclear cells.



Colonies stimulated by kidney feeder layers reached a mean size of 2000 cells by day 10 of incubation and remained predominantly granulocytic in nature. When bovine serum was substituted for fetal calf serum, cell colonies grew to a smaller size and lost their granulocytic nature, finally becoming almost pure populations of mononuclear cells.



Colonies stimulated by AKR leukemic serum reached a mean size of 350 cells by day 10 of incubation. Although these colonies initially were granulocytic in nature, they finally became almost pure populations of mononuclear cells.



The colony mononuclear cells actively phagocytosed carbon, and contained metachromatic granules probably derived from ingestion of agar.



The mononuclear cells in these colonies may not have been members of the original colony, but may have been incorporated in the colony as it expanded in size, subsequently proliferating in the favourable environment of the colony.

W. Robinson

Papers

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum