Agar Colony Formation Research Articles

Chromosomal mosaicism associated with prolonged remission in chronic myelogenous leukemia

The nature of the relationship between normal stem cells and the leukemic cells of chronic myelogenous leukemia (CML) is of considerable theoretical and practical importance. We studied a patient with CML who has maintained a complete hematologic remission for eight years without therapy. Presently, the only documentable abnormality is mosaicism for the Philadelphia (Ph1) chromosome. Studies of his bone marrow in vitro revealed normal colony formation in agar and normal cellular proliferation and differentiation in liquid culture. Observations in this patient indicate that a Ph1-positive clone may coexist with normal stem cells during prolonged remission in CML and the Ph1-positive cells may not always have a growth advantage over normal cells. The culture studies further suggest that the diminished proliferative capacity of the leukemic cells was not due to in vivo host suppressive factors.

The relationship between tumorigenicity, growth in agar and fibrinolytic activity in a line of human osteosarcoma cells.

The TE-85 osteosarcoma cell line has several of the in vitro properties of malignant cells, including colony formation in agar, but has low extracellular fibrinolytic activity and no capacity to form tumors in ATS-treated hamsters. Some TE-85 cells clones (clones 2, 4 and 6) have increased fibrinolytic activity but do not form tumors in hamsters. TE-85 cells infected with mammalian transformation-defective viruses have low (FeLV) or increased (RD-114 virus) levels of fibrinolytic activity and do not form tumors in hamsters. TE-85 cell either nonproductively infected with Ki-MSV or productively infected with M-MSV (RD-114), have fibrinolytic activity and can form tumors in hamsters. The MSV gene(s) but not colony formation in agar or extracellular fibrinolytic activity appears to be capable of rendering TE-85 cells tumorigenic in ATS-treated hamsters.

Hemopoietic colony forming cells in regenerating mouse liver.

Cells from regenerating mouse liver removed 2-25 days post 68% hepatic resection have been assayed for in vivo colony forming capacity in soft agar (CFU-C), proliferative capacity in liquid culture, and in vivo spleen colony forming capacity (CFU-S). These studies demonstrated low concentrations of CFU-C and CFU-S in normal and sham operated liver, with an appreciable increase of both in regenerating liver, reaching maximum values in tissue removed 5--7 days post hepatic resection. Colony formation in agar by regenerating liver cells occurred in the absence of exogenous colony stimulating factor. Separation of liver cells on the basis of adherent properties prior to culture indicated concentration of CFU-C in the non-adherent fraction, while cells producing colony stimulating factor were concentrated in the adherent fraction. Foci of actively dividing cells of themacrophage and granulocyte series arose in liquid culture from preparations of sham operated and regenerating liver, although total cell formation was greater with regenerating liver. A small proportion of the colonies formed in agar from regenerating liver consisted of cords of epithelioid cells, which resembled hepatocytes and differed from the macrophages or granulocytes found in the majority of colonies, raising the possibility that regenerating hepatocytes form colonies in agar culture.

Growth of B lymphocyte colonies in vitro from mouse lymphoid organs.

TECHNIQUES now exist for obtaining clonal growth in semi-solid agar of mouse neutrophil, macrophage, eosinophil, erythropoietic and megakaryocytic colonies1–5. Previous attempts to obtain colony formation by mouse lymphoid cells, however, were unsuccessful and cultured lymphocytes died very quickly, even in the presence of known lymphocyte mitogens. Mercaptoethanol and related substances have been shown to increase lymphocyte proliferation in liquid cultures6 and colony formation in agar by mouse plasmacytoma cells7. Based on these observations, attempts were made to obtain clonal growth of mouse lymphoid cells in agar medium supplemented with 2-mercaptoethanol.

Virus susceptibility of mouse hemopoietic cells in vitro: inhibition of granulocyte-macrophage precursor cells by Newcastle disease virus

Normal mouse bone marrow cells were exposed to encephalomyocarditis virus (EMC), reovirus type 3 (REO3), influenza virus (FLU), and Newcastle disease virus (NDV) then assayed for granulocyte-macrophage precursor cells by the technique of colony formation in agar. Exposure to EMC, REO3, and FLU caused a slight but variable loss of colony-forming potential, whereas exposure to NDV caused a very marked loss. NDV acted directly on the cells, not indirectly through release of colony-inhibiting factors or destruction of colony-stimulating factor. Experiments with NDV inactivated by heat, ether, or ultraviolet irradiation indicated that colony inhibition was associated with fully infective virus, even though some of the inactivated preparations had retained full hemagglutinin, neuraminidase, or hemolytic activity.

Purine and pyrimidine modification of growth and surface properties of baby hamster kidney cells (BHK21/C13)

Hypoxanthine or adenosine and thymidine added to soft agar medium stimulated the growth of non-transformed BHK21/C13 cells. Increases in concanavalin A agglutinability and synthesis of a plasminogen activator also occurred. Thymidine was required for colony formation in agar but not for increased lectin agglutinability or activator activity.

Thymidine suicide in vivo and in vitro of spleen colony forming and agar colony forming cells of mouse bone marrow.

ABSTRACTThe ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed.Following 3H‐TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with 3H‐TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide.The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ‐irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells.It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.

Separation of agar colony forming cells from rat bone marrow.

SummaryRat bone marrow cells formed colonies in agar after stimulation by colony stimulating factor (CSF) obtained from endotoxin-injected rats.Separation of rat agar colony forming cells (CFCs) was performed on a discontinuous bovine serum albumin density gradient (BSA). Cell fractions obtained between 26.5 and 29.0% BSA showed enrichment for rat agar colony forming cells when cultured with CSF. Although the cell separation has not been complete the present studies support the contention that a subpopulation of cells intermediate between small lymphocytes and “blast” cells and resembling “transitional cells” are responsible for the agar colonies produced.

Some Biophysical Characteristics of the Agar Colony Forming Cell in Man

Some Biophysical Characteristics of the Agar Colony Forming Cell in Man

A comparison of the effect of cytotoxic agents on agar colony forming cells, spleen colony forming cells, and the erythrocytic repopulating ability of mouse bone marrow.

AbstractThree assays for bone marrow progenitor cells have been used to determine the effect of single doses of two cytotoxic agents, cyclophosphamide and vinblastine. The assays employed were the agar colony forming and spleen colony forming assays and the crythroid repopulating ability.In normal mice, there was little difference between the response of the progenitor cells assayed by the three methods, following cyclophosphamide: and no detectable difference following vinblastine.Bone marrow from continuously irradiated mice and bone marrow regenerating seven days following transplantation was also studied: in both these situations the proliferation rate of the progenitor cells is increased. Cyclophosphamide was found to be only slightly proliferation dependent with each assay. However, vinblastine was strikingly proliferation dependent. In irradiated mice and also in regenerating marrow the agar colony forming cells were many times more sensitive to this agent than were the other progenitor cells.These results show that under some but not all circumstances the agar colony forming and spleen colony forming cells behave similarly in C57BL mice, but are not a single population of cells.

Inhibition of haemopoietic colony growth by interferon preparations from different sources.

INTERFERON preparations have been shown to inhibit the multiplication of tumour cells in vitro1–4 and more recently comparable preparations have also been shown to inhibit normal cell division. Thus, inhibition has been observed in %of mouse embryo fibroblasts5, of weanling mouse kidney5, of stimulated mouse lymphocytes6 and of the formation of granulocyte/macrophage colonies derived from mouse and rabbit bone marrow7–9. In the course of investigating the effect of mouse and rabbit interferons from different sources on bone marrow colony formation in agar, it became apparent that a strict correlation did not always exist between the antiviral titre and the colony inhibiting titre of interferon preparations derived from cell culture interferon, whereas it did exist when interferon preparations derived from mouse serum or brain were used. The development of granulocyte/macrophage colonies in agar is dependent upon the presence and concentration of colony stimulating factor (CSF)10, so the most likely explanation for the lack of correlation observed in testing some interferon preparations was the presence of significant amounts of CSF in these preparations. The results of the experiments presented here support this hypothesis and suggest the possibility that both CSF and interferon may be mutually antagonistic regulators of cell division.

Colony formation in agar by murine plasmacytoma cells: potentiation by hemopoietic cells and serum.

AbstractClonal growth in semisolid agar medium was obtained using cells from 19 of 25 transplanted murine plasmacytomas when the medium was supplemented by whole mouse blood or washed red cells. With different tumors cloning efficiency ranged from 0.01% to 21.6%. With two exceptions, mouse blood did not potentiate colony formation in agar by cells from transplantable myelomonocytic, myeloid, and lymphoid leukemias, reticulum cell sarcomas and fibrosarcomas. The clonal growth of some plasmacytomas was also potentiated by syngeneic thymic, spleen or bone marrow cells. Plasmacytoma colony growth was not stimulated by normal mouse serum but serum from mice injected with endotoxin or polymerised flagellin stimulated colony growth by some plasmacytomas. The active serum factor was not the colony stimulating factor (CSF) and its appearance after antigenic stimulation was not T cell‐dependent. Preimmunised mice failed tq respond to antigenic stimulation. Whole body irradiation did not induce a rise in the capacity of serum to stimulate colony formation by plasmacytoma cells.

PRODUCTION OF COLONY-STIMULATING FACTOR BY HUMAN MACROPHAGES

The monocyte is the cell present in human peripheral blood which produces a factor stimulating bone-marrow colony formation in agar. Monocytes give rise to macrophages, both in vivo and in vitro. Therefore, to define a possible physiological role for the tissue macrophage in human leucopoietic regulation, monocyte-derived macrophages and human pulmonary macrophages were tested for colony-stimulating activity. Macrophages and conditioned media prepared from macrophage cultures were highly active in stimulating human bone-marrow granulocyte and mononuclear cell proliferation in vitro. These observations suggest that tissue macrophages may be involved in the control of leucopoiesis and that these cells may function both as phagocytes and recruit new granulocytes and monocytes for host defence.

Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue.

AbstractHemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.

Colony formation in agar: in vitro assay for haemopoietic stem cells.

ABSTRACTUsing a method in which embryo fibroblasts were used as feeder layers, the colony forming capacity in agar of a variety of mouse haemopoietic suspensions was compared with their CFUs content. A striking parallelism between the results of the two assays was found. In addition, under certain conditions higher numbers of CFUs could be retrieved from 5‐day‐old agar colonies than were originally plated, indicating that the CFCa (Colony Forming Cell agar) may fulfil the requirements of pluripotency as well as of self‐renewal, both prerequisites for any haemopoietic stem cell candidate. Although our data by no means provide direct proof that the CFCs and the CFCa are identical, they certainly support such a concept. the contradictory findings by others that CFUs and CFUc (Colony Forming Unit culture) can be separated on a velocity gradient is attributed to different culture conditions, in other words, that their CFUcè are not identical with our CFUa.Our findings also indicate that for mouse cells our soft agar colony assay meets the criteria of a quantitative assay for haemopoietic stem cells and that extension of this technique to bone marrow of primates including humans seems to be justified.

Inhibition of bone marrow colony formation in vitro by dialysable products of normal and neoplastic haemopoietic cells.

SummaryDialysates of tissue culture medium conditioned by normal cells (bone marrow, thymic, spleen or lymph node) or by leukaemic cells (myelomonocytic, lymphoid or plasma cell tumour) inhibited colony formation in agar by mouse bone marrow cells. The dialysable inhibitors were not species specific, did not alter colony morphology and were heat‐ and ether‐resistant. Leukaemic cells were as effective in producing inhibitors as normal cells, and colony formation in vitro by leukaemic cells was as susceptible to inhibition as was colony formation by normal cells. No dear evidence was obtained for the presence of similar dialysable inhibitors in the serum of normal or experimental mice, and the real biological function of these dialysable inhibitors remains in doubt.

Colony production in vitro by regenerating marrow.

SummaryA study has been made of agar colony forming capacity of marrow during regeneration after localized depletion. From the second week of regeneration agar colonies (in vitro CFU) of marrow are readily observed. In vitro CFU then increase linearly with hemic cellularity with a slope of about one. If the in vitro CFU represents a granulocyte precursor, the data suggest that recovery of these cells requires a sustained influx from ancestral cells.