[Effects of media conditioned with bone marrow cells on proliferation of stromal clonogenic cells in vitro].
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Biomedical subjects
Publications and source records attributed to A Ia Fridenshteĭn.
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Purified mouse IL-1 at doses 15-100 mu/ml inhibits the growth of stromal clonogenic cells /CFU-f/ both in full bone marrow cell cultures /F-cultures/ and in adherent bone marrow cell cultures /A-cultures/. Rec. human TNF-alpha inhibits growth of these cells at doses greater than 50 u/ml, but stimulates it /in 1.5 fold increase/at low doses /0.1-20 u/ml/ in cultures of both types. Rec. mouse IL-3 at doses 0.8-50 mu/ml slightly increases/in 1.6 fold increase/the in vitro growth of CFU-f and inhibits it at low doses in F-cultures. In A-cultures this factor stimulates CFU-f growth at all doses tested, but this stimulating effect takes place if only explantation density of mouse bone marrow cells in sufficiently high.
In 24 hours adherent marrow cell cultures (AMCC) were represented by single stretched fibroblasts. In non-feeder-supplemented AMCC most of the CFU-f remained single fibroblasts or passed through 1-3 cell doublings [correction of dudlings]. The colony stimulating activity of irradiated marrow cells was found to be diffuse across the Millipore filter, which seems to indicate that haemopoietic marrow cells produce a colony stimulating factor which is required for triggering the CFU-f from the Go-period of the cell cycle into cell proliferation.
CFU-f-derived stromal colony formation was accomplished in adherent marrow cell cultures (AMCC) with serum-rich medium. It turned out to require additional stimulation by hemopoietic feeder cells: by irradiated marrow cells and spleen cells if they possess megakaryocytes and platelets or by platelets from the blood. PDGF, EGF and IL-3 did not substitute the colony stimulating activity of feeder cells. Thymus, lymph node cells and blood leucocytes had no colony stimulating activity. At low oxygen concentrations which improve colony formation the stimulating activity of hemopoietic feeder cells was expressed, as well. Thus, CFU-f colony formation depends on stimulation by hemopoietic cells in addition to serum growth factors. In full populations of marrow cells the CFU-f colony formation is stimulated by marrow cells which accompany the CFU-f.
Colony-forming fibroblast precursors were detected in circulating blood of adult guinea pigs by CFUf in vitro colony assay. SFU amount to 0.9 +/- 0.2 (M +/- m) per 10(5) explanted leucocytes ranging from 0.04 X 10(-5) to 3.0 X 10(-5) for individual donors. The presence of collagen type I and lack of factor VIII antigen and of FC-receptors proved that CFU-derived colonies in the blood cultures were composed of fibroblasts.
Bone formation in adult human bone marrow organ cultures is described. When culturing marrow fragments, thick bone lamina is formed. It has well-mineralized trabecular bone matrix with bone cells incorporated and is lined with osteoblast-like cells. In cultures of marrow deaggregated cell suspensions thin layers of the bone are only formed. Osteoclast-like cells develop in the cultures.
Disaggregated cell suspensions obtained by mouse bone marrow fermentative digestion as well as stromal tissue obtained by marrow mild mechanical destruction were explanted. Both methods yield the cultures in which the hematopoiesis duration is comparable with dexter cultures. Adhesive cells from all of these three culture types were resuspended and in the porous gelatin sponges heterotopically transplanted under the kidney capsule of syngenic recipients. In the transplantation site there develops the hemopoietic organ containing reticular stroma, hemopoietic cells, and in most cases the well developed bone tissue. Thus, the adherent layers of mouse bone marrow dexter and similar cultures contain for a long period (not less than 2-3.5 months) the stromal fibroblast population which maintains its osteogenic and hemopoietic microenvironment transfer capacities.
Na-beta-glycerophosphate was added to the organ culture medium of mice marrow fragments. New bone ground substance is formed, its mineralization degree and morphology being highly dependent upon glycerophosphate addition and removal periods. Complete or partial ground substance mineralization occurs, in last case calcium insoluble salts may be found in young apical or in old basal parts only. Ground substance deposition and mineralization dynamics in vitro is discussed, paying attention to possibility of demineralization process.
In the presence of irradiated bone marrow cells the efficiency of stromal colony formation increases from 0.8 +/- 0.2 to 3.6 +/- 0.4 per 10(4) explanted bone marrow cells. The growth-stimulating activity of bone marrow cells on passaged bone marrow fibroblasts depends on growth conditions of passages to which irradiated bone marrow cells are added. The response of proliferating bone marrow fibroblasts to stimulating activity of bone marrow cells is low, while addition of bone marrow cells to fibroblast cultures stimulates their proliferation.
Adult mouse bone marrow cell suspensions prepared by trypsinization were cultivated in gelatin sponges on millipore filters. When HAWP filters were used, multilayer bone structure was formed. It contained mineralized ground substance, incorporated bone cells and osteoblast layer. With the use of AUFS filters, bone tissue developed not only on the top surface, but also inside the filter.
The clonal nature of CFUf-derived fibroblast colonies was tested in mixed cultures of CBA and CBAT6T6 bone marrow cells. Inoculation of marrow cell suspensions into flasks coated with poly-I-lysin has proved that no stromal aggregates were present among cells subjected to explantation. Marrow cell cultures depleted of macrophages and myeloid cells were used for chromosome analysis. The coincidence of karyotypes within a stromal colony was found in mixed cultures, which proves that CFUf-derived fibroblast colonies are cell clones.
The clonal nature of bone marrow fibroblast colonies derived from clonogenic bone marrow osteogenic cells (CFUf) was proved by the chromosome analysis. During subsequent passages of multi-colony derived bone marrow fibroblast strains there occurs a pronounced increase in the cell number and in the number of osteogenic units (tested by transplantation in diffusion chambers). Single colony-derived strains are capable of forming bone and cartilage simultaneously. It follows that CFUf or part of them are clonogenic cells with high proliferative potentials and are common precursors for bone and cartilage tissue. Thus, CFUf may be regarded as osteogenic stem cells.
The clonal nature of CFUf-derived fibroblast colonies was proved by chromosomal analysis of individual colonies and single-colony-derived fibroblast strains using mixed cell cultures from male and female rabbits. CFUf progeny, forming colonies composed of more than 10(3) cells was capable of 20-30 cell doublings during subsequent passages. When transplanted in diffusion chambers, single-colony-derived fibroblast strains formed bone and cartilage simultaneously. Thus, CFUf or part of them can be regarded as bone marrow osteogenic stem cells.
The number of fibroblast colonies in bone marrow cultures depends on FCFC concentration in explanted cells and FCFC cloning efficiency. For mouse bone marrow the efficiency of fibroblast colony formation increases in the presence of the feeder (irradiated bone marrow of spleen cells). Colony-stimulating feeder activity does not depend on the presence of phagocytic and stromal cells in the feeder cell population. Trypsinization of the bone marrow leads to the release of additional FCFC and the increase of their concentration in bone marrow cell suspensions.
Bone tissue composed of typical bone trabeculae containing ground substance with incorporated osteogenic cells and osteoblast layer was formed in organ cultures of bone marrow obtained from adult mice. Electron microscopic properties of the bone formed in vitro were identical to those of the bone tissue in vivo. The mineralization of the bone took place only in the presence of Na-beta-glycerophosphate in the culture medium.
Heterotopic transplantation of marrow fragments results in the formation of new bone marrow organs. The amount of hemopoietic cells populating these organs is connected nonlinearly with the volume of the grafted tissue or with the number of transplanted marrow cells. Statistical analysis points out that the number of the populating cells depends on the radius of the initial bone marrow transplant. Thus, the previous data on the radiosensitivity of the microenvironmental transfering stromal cells and on their concentration obtained by measuring the size of the heterotopic bone marrow organs have turned out incorrect.