Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor
TL;DR: An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro, finding that colony formation was shown to be related to the concentration and not the total amount of CSF.
Abstract: An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.
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TL;DR: A liquid culture system is described whereby proliferation of haemopoietic stem cells, production of granulocyte precursor cells (CFU‐C), and extensive granulopoiesis can be maintained in vitro for several months.
Abstract: 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.
2,178 citations
TL;DR: Several properties of lymphoid dendritic cells in situ have been determined, and contrasted to information previously established for lymphocytes and mononuclear phagocytes as mentioned in this paper, such as the mature splenic population does not actively divide (pulse labeling index with [3H]thymidine of 1.5-2.5%), but does turnover at substantial rate, 10+% of the total pool per day.
Abstract: Several properties of lymphoid dendritic cells in situ have been determined, and contrasted to information previously established for lymphocytes and mononuclear phagocytes. Dendritic cells are not found in newborn mice, and their concentration in both spleen and mesenteric lymph node does not reach adult levels until 3–4 wk of age. Dendritic cells largely disappear from adherent populations following administration of steroids (2.5 mg hydrocortisone acetate s.c.) and ionizing radiation (Do of 100 rads for Co60). Splenic dendritic cells can originate from precursors located in both bone marrow and spleen itself, probably the red pulp. The mature splenic population does not actively divide (pulse labeling index with [3H]thymidine of 1.5–2.5%), but does turnover at substantial rate, 10+% of the total pool per day. The influx of new cells appears to be derived from a proliferating precursor compartment, but the mechanism for efflux or turnover is not known. Dendritic cells in spleen and node undergo little or moderate increase in numbers during development of a primary immune response. These in vivo characteristics, taken together, further distinguish dendritic cells as a novel cell type, distinct from mononuclear phagocytes and lymphocytes.
967 citations
TL;DR: It is shown that the death of haemopoietic precursor cells on withdrawal of the relevant CSF is due to active cell death5, or apoptosis, indicating that CSFs promote cell survival by suppression of the process of apoptosis.
Abstract: The survival, differentiation, proliferation and development of haemopoietic precursor cells and the functional activity of mature blood cells are all influenced by colony stimulating factors (CSFs). As haemopoietic cells rapidly die in the absence of appropriate CSF, the promotion of cell survival mediated by CSFs, or growth factors, is fundamental to all the other effects exerted by these factors. This enhancement of cell survival is distinct from the stimulation of proliferation. Here we show that the death of haemopoietic precursor cells on withdrawal of the relevant CSF. is due to active cell death, or apoptosis, indicating that CSFs promote cell survival by suppression of the process of apoptosis. The existence of a positive control mechanism regulating precursor cell survival has important implications both for the regulation of normal haemopoiesis and for tumorigenesis.
956 citations
TL;DR: A method is described for the production of a highly purified and homogeneous population of adherent bone marrow-derived macrophages that are devoid of CSF-1-producing cells, and the method may also be used to obtain nonadherent precursors of the mononuclear phagocytic series.
653 citations
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TL;DR: A simple in vitro technique for the growth of colonies from single cell suspensions of mouse bone marrow involves the plating of marrow cells in agar on feeder layers of other cells, those from 8-day-old mouse kidney and 17th day mouse embryo being shown to be the most efficient types of feeder layer.
Abstract: A simple in vitro technique is described for the growth of colonies from single cell suspensions of mouse bone marrow. The system involves the plating of marrow cells in agar on feeder layers of other cells, those from 8-day-old mouse kidney and 17th day mouse embryo being shown to be the most efficient types of feeder layers.
1,903 citations
TL;DR: 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 and all colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells.
Abstract: 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.
202 citations
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147 citations
TL;DR: 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 were mixtures of granulocytic and mononuclear cells.
Abstract: 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.
144 citations