Colonies In Diffusion Chambers Research Articles

Radioprotective potential of primitive hematopoietic precursors forming colonies in diffusion chambers in mice

The purpose of this study was to determine the radioprotective ability of primitive hematopoietic precursors which form colonies in diffusion chambers in mice (CFU-D). Thirty-two lethally irradiated female ICR mice were injected with 5 to 7 male ICR mouse bone marrow-derived CFU-D colonies each. Fourteen of these mice survived over 30 days and were sacrificed at intervals up to a year. As a control, 20 lethally irradiated female ICR mice received cells from intercolony areas. All of these mice died before day 20. DNA samples obtained from hematopoietic organs and liver from 8 sacrificed mice were analyzed for the presence of CFU-D colony-derived cells. Only in 1 ICR mouse was CFU-D colony origin DNA detected by Southern analysis in all hematopoietic organs: bone marrow, spleen, thymus and lymph nodes. In 6 mice, only selected hematopoietic organs were repopulated by CFU-D colony-derived cells as judged by Southern analysis. In some of these mice, the remaining hematopoietic organs contained small CDU-D-derived cell populations which could be detected by more sensitive polymerase chain reaction (PCR). In 1 mouse, the presence of CFU-D-derived cells in all hematopoietic organs was only demonstrated by PCR. These findings suggest that lethally irradiated mice can be rescued by CFU-D-derived daughter cells. They appear to have the potential to give rise to clones containing lymphoid and myeloid cells in all hematopoietic organs, at least temporarily. Thus, it can be concluded that CFU-D represents a very primitive hematopoietic precursor cell with radioprotective capability.

Potentiality of the Murine Colony-Forming Cells Detected by an In Vivo Diffusion Chamber Culture System

AbstractCulture of a mixture of bone marrow cells with and without T6 chromosome marker in diffusion chambers in mice yielded colonies (CFU-DG) containing cells of a single karyotype, suggesting clonality. Injection of individual CFU-DG colonies into lethally irradiated mice resulted in increased spleen colony formation on day 12 (CFU-S). The possibility of endogenous origin was excluded by demonstrating the presence of T6 marker in both CFU-DG and CFU-S colonies in karyotypically normal hosts. These findings suggest that the cells giving rise to granulocytic colonies in diffusion chambers also can give rise to multipotential hemopoietic cells.

Potentiality of the murine colony-forming cells detected by an in vivo diffusion chamber culture system

Culture of a mixture of bone marrow cells with and without T6 chromosome marker in diffusion chambers in mice yielded colonies (CFU- DG) containing cells of a single karyotype, suggesting clonality. Injection of individual CFU-DG colonies into lethally irradiated mice resulted in increased spleen colony formation on day 12 (CFU-S). The possibility of endogenous origin was excluded by demonstrating the presence of T6 marker in both CFU-DG and CFU-S colonies in karyotypically normal hosts. These findings suggest that the cells giving rise to granulocytic colonies in diffusion chambers also can give rise to multipotential hemopoietic cells.

Separation by velocity sedimentation of human haemopoietic precursors forming colonies in vivo and in vitro cultures.

Cells which give rise to granulocyte-macrophage colonies under the influence of peripheral blood white cells (CFU-c (WBC] and Mo T cell conditioned medium (CFU-c (Mo] sedimented at a faster rate than the cells which form mixed erythroid-granulocytic colonies in methylcellulose in vitro (CFU-mix) and granulocytic (CFU-dg) and megakaryocytic (CFU-dm) colonies in diffusion chambers in mice. Despite identical peak sedimentation rate for the two CFU-c populations, sedimentation profiles suggest that they are heterogeneous with respect to size. A proportion of CFU-c (Mo) may be identical with CFU-dg and CFU-mix. Sedimentation profiles for cells which give rise to mixed colonies in vitro (CFU-mix) and to granulocytic colonies in diffusion chambers in cyclophosphamide pretreated mice (CFU-dg (CY] and in Mo conditioned medium treated mice (CFU-dg (Mo] were similar. On the average CFU-dm sedimented somewhat slower than CFU-dg. These and other observations suggesting a close relationship between CFU-dg and multipotential haemopoietic precursors are discussed.

Density distribution of chronic myeloid leukaemia and normal colony-forming cells in diffusion chambers (CFU-D) and agar (CFU-GM).

The density distribution in Percoll gradients of clonogenic cells forming colonies in diffusion chambers (CFU-D) or in agar culture (CFU-GM) was studied in chronic myeloid leukaemia (CML). The density distribution of CFU-GM in CML was homogeneous with peaks within 1.058-1.061 g/ml, which is slightly lower than normal. CFU-D, on the other hand, showed heterogeneous distributions both in CML and in normal controls. Two separable populations of CFU-D were recognized, one with the same or lower density than CFU-GM that formed almost exclusively neutrophilic colonies in diffusion chambers, and a second population concentrating in the density range 1.068-1.075 g/ml which primarily formed macrophage colonies. The second population contained colony-forming cells derived from both Fc-receptor positive and Fc-receptor negative precursor cells, suggesting that at least some colonies in diffusion chambers arise from Fc-receptor positive granulopoietic cells of intermediate maturity and/or monocytes. The concentration of CFU-D in peripheral blood was increased 40- to 100-fold in 2 patients currently off treatment who had increased WBC counts.

Effect of cooling rate on human and murine hemopoietic precursor cell recovery

The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me 2SO, a cooling rate of 7 °C/min from −4 to −30 °C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 °C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoietic precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 °C/min.

The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2–4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

The Role of Polyamine Biosynthesis in Hematopoietic Precursor Cell Proliferation in Mice

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2-4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU-C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

Expression of DR antigens on early human progenitors of neutrophilic and eosinophilic colonies in diffusion chambers (CFUd).

Incubation of the non-adherent low density cells from normal human bone marrow with an antiserum to DR antigens markedly inhibited their capacity to form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally into irradiated mice. The inhibitory effect of antiserum was abrogated after absorption of antiserum with B cells. Inhibition was obtained both with and without complement treatment. Neutrophilic colony formation exhibited a higher sensitivity to diluted antiserum to DR antigens as compared with eosinophilic colony formation. These results suggest that DR determinants are expressed on the neutrophilic and eosinophilic progenitor cells (CFUd) which give rise to colonies in diffusion chambers.

Oscillations of granulocytic and megakaryocytic progenitor cell populations in cyclic neutropenia in man.

The concentration of granulocytic progenitor cells in the bone marrow of 2 patients with cyclic neutropenia was assessed at intervals during the cycle. Syncroneous oscillations were observed for cells forming neutrophilic and megakaryocytic colonies in diffusion chambers in vivo (CFU-D), suggesting a close relationship between the two cell types. However, an almost reverse relationship was observed between CFU-D and cells forming colonies in agar culture in vitro (CFU-C), which cycled out of phase with CFU-D. This supports the concept that CFU-D and CFU-C denote different cell types.

Diversity of human granulopoietic precursor cells: separation of cells that form colonies in diffusion chambers (CFU-d) from populations of colony-forming cells in vitro (CFU-c) by velocity sedimentation

Normal human bone marrow contains cells that form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally in sublethally irradiated mice (CFU-d). A series of experiments was performed to determine the relationship between CFU-d and the cells that form colonies in agar culture in vitro (CFU-c). Low-density bone marrow cells ( 3 ) were separated by velocity sedimentation and colony-forming cells in each fraction assessed by culture in diffusion chambers and in soft agar. CFU-d sedimented more slowly than the vast majority of CFU-c, with a peak sedimentation velocity of 5.0 ± 0.4 mm/hr. Two different CFU-c populations could be distinguished. One was a rapidly sedimenting cell population (7.3 ± 0.9 mm/hr.) that formed colonies after 1 wk incubation in vitro. On day 14, however, colonies were derived from more slowly sedimenting cells that had a peak sedimentation velocity of 6.0 ± 0.5 mm/hr. Clusters (3-50 cells), scored on day 7 in agar ##

Diversity of Human Granulopoietic Precursor Cells: Separation of Cells That Form Colonies in Diffusion Chambers (CFU-d) From Populations of Colony-forming Cells in vitro (CFU-c) by Velocity Sedimentation

Diversity of Human Granulopoietic Precursor Cells: Separation of Cells That Form Colonies in Diffusion Chambers (CFU-d) From Populations of Colony-forming Cells in vitro (CFU-c) by Velocity Sedimentation

The effect of syngeneic peripheral blood cells on the formation of colonies by normal human bone marrow cells in diffusion chambers in mice.

This paper describes the influence of cells capable of releasing colony stimulating activity (CSA) in vitro on the formation of granulocytic colonies by normal human bone marrow in diffusion chambers in mice. A carbonyl iron method was used to remove phagocytic cells from normal human bone marrow. This treatment prevented spontaneous colony and cluster formation when the cells were cultured in agar in vitro at initial concentrations of 2-5 x 105 cells per ml. However, non-phagocytic bone marrow cells formed granulocytic colonies when inoculated into diffusion chambers at 105 cells per chamber and cultured in 450 R-irradiated or non-irradiated mice. The formation of granulocytic colonies by carbonyl iron treated marrow in diffusion chamber cultures was not consistently enhanced by the admixture of 1.4 x 105 1500 R-irradiated syngeneic light density blood cells (less than 1.077 g/ml) to the chamber inoculum. In contrast, these cells induced cluster or colony formation when added in the same proportion to the marrow cells in agar cultures in vitro. Addition of 1.4 x 105 1500 R-irradiated high density blood cells(greater than 1.077 g/ml) to the inoculum resulted in a slight, non-significant decrease in the number of colonies in diffusion chambers. The stimulating effect of host irradiation on neutrophilic colony formation was independent of the presence of CSA releasing cells in the chamber inoculum.

Human granulocytopoietic colonies in diffusion chambers in mice: growth of colonies and the effect of host irradiation.

Normal human non-separated bone marrow cells were cultured in fibrin clots in diffusion chambers implanted intraperitoneally in mice, and harvested at different intervals by a previously described chamber centrifugation technique. This method demonstrates the presence of cell aggregates in the diffusion chambers. When the chambers are implanted in irradiated mice (450 R) and retransplantated into newly irradiated mice every seventh day, a continous increase in number of cells per granulocytopoietic aggregate is observed from day 8 to day 21. This is compatible with the view that the aggregate is observed from day 8 to day 21. This is compatible with the view that the aggregates are colonies. The term 'colony forming unit diffusion chamber' (CFUD) is suggested to denote the ancestor(s) of the colonies. However, formal proof that one colony is derived from one cell is lacking. Preirradiation of mice with 450 R significantly increases the number of neutrophilic granulocytopoietic colonies at day 14, provided the chambers are retransplantated to newly irradiated mice at day 7, indicating that the neutrophilic colony forming unit or its progeny is involved as at least one of the targets of the stimulating effect of host irradiation. In contrast, no effect of host irradiation on the numbers of eosinophilic colonies was observed.

Ultrastructural Study of Bone Marrow‐Derived Granulocytic Colonies in Diffusion Chambers in Rats

An ultrastructural study of diffusion chamber granulocytic colonies is reported. The colonies were derived from normal rat bone marrow cells and the chambers were inserted into the peritoneal cavity of lethally irradiated rats. The colonies were processed en bloc for electron microscopy enabling observation of cellular relationship to be made. Ultrastructural analysis of colonies disclosed normal maturing cells at all stages of maturation, with the exception of occasional granulocytes on neutrophil type that presented narrow strips of chromatin ('nuclear blebs'). The cells in colonies generally occurred in close apposition to one another and to occasional macrophages. This organization possible enhanced transmission of factors of maturation. Advantages of diffusion chamber haematopoietic culture are discussed; among the most important are the occurrence of fibroblasts originating from implanted marrow cells and the local formation of fibrin within the chambers.

Effect of Cytosine Arabinoside Administration on Donor Rats

Ultrastructural observations of erythroid colonies in diffusion chambers implanted in lethally irradiated rats are reported. The donor cells were obtained from rats treated with cytosine arabinoside (250 mg/kg body weight) 6 h prior to seeding. The marrow of donor rats contained a high proportion of stem cells committed to the erythroid line. 9 days after implantation, vast numbers of erythroid colonies developed in the diffusion chambers. The erythroid cells were observed arranged around a central macrophage resembling erythroid islands in bone marrow. Erythroid colonies most probably differentiated from committed stem cells in the inoculum. It is postulated that development of erythroid colonies in diffusion chambers is conditioned by, among other factors, the presence of a special type of macrophages. These macrophages have the same function as those of central reticular cells (CRC) in bone marrow erythroid islands.