Effect of alpha-fetoprotein on the count of bone marrow and splenic stromal precursor cells and proliferation of their cultural descendants.

The efficiency of cloning of stromal precursor cell increased more than 2-fold in splenic cultures and more than 3-fold in bone marrow cultures 24 h after injection of Profetal preparation to mice in vivo. The number of nucleated cells did not change in the bone marrow and slightly increased in the spleen. Addition of Profetal in vitro 2-fold decreased the efficiency of stromal precursor cells colony formation in mouse splenic cultures and dose-dependently decreased this process in bone marrow cultures derived from these animals, the maximum (5-fold) inhibitory effect was observed in a dose of 50 microg/ml. Addition of Profetal to cultured human bone marrow fibroblasts did not change the content of stromal fibroblasts in cultures. These data indicate the possibility of indirect effect of alpha-fetoprotein on the number of stromal precursor cell in hemopoietic and lymphoid organs.

[Analysis of the changes of stromal precursor cell numbers in the thymus and the spleen of animals of different age groups].

The objective of this study was to analyze the species differences in the numbers of stromal precursor cell (CFU-f), their cloning efficiency (CFE-0 and their dynamics in different organs during aging, using the mathematical gradient decrease method. Age changes of CFU-f numbers and of their CFE-f were studied in the thymus and the spleen of mice and guinea pigs. The study was performed using CFU-f cloning in monolayer cultures. CFU-f numbers and CFE-f were found to decrease with aging both in the thymus and the spleen of mice and guinea pigs. However these changes were different in each species and were variable in different organs of the animals of the same species, which, probably was associated with the physiological characteristics and aging peculiarities of the animals of different species and with the functional role of organs studied. The process of reduction was more significant in the thymus of guinea pigs and mice - the numbers of CFU-f were decreased 75- and 12-fold, respectively. Since it is known that the population of CFU-f in the thymus and the spleen includes inducible osteogenic precursor cells, the data obtained indicate the possibility of a reduction in numbers of this category ofstromal precursors, that could be one of the reasons of osteoporosis of aging. The application of a mathematical analysis using the gradient decrease method allows to predict the time-course of age changes and to evaluate the dynamics of CFU-f numbers and of CFE-f in association with organism aging.

[Age changes in the numbers of stromal precursor cells in the animal bone marrow]. / Vozrastnye izmeneniia kolichestva stromal'nykh kletok-predshestvennikov v kostnom mozge zhivotnykh.

Age changes of stromal precursor cell (CFU-f) numbers and their cloning efficiency (CFE-f) in monolayer cultures were evaluated using the mathematical gradient decrease method in the bone marrow of mice, guinea pigs and rats. It was found that aging was accompanied by a decline of CFE-f and CFU-f numbers in all animal species studied. However, these changes were variable in different species, that could possibly be explained by species-related physiological characteristics and aging peculiarities. Since the population of bone marrow CFU-f is known to include committed osteogenic precursor cells, these findings indicate the possibility of age-related decline in the numbers of this cell type, which could be one of the reasons of osteoporosis of aging. The application of a mathematical gradient decrease method allows to predict the course of age changes and to evaluate the dynamics of CFU-f numbers and of CFE-f in association with organism aging.

[Proliferative and differentiation potential of individual clones derived from bone marrow stromal precursor cells]. / Proliferativnye i differentsirovochnye potentsii individual'nykh klonov stromal'nykh kletok-predshestvennikov kostnogo mozga.

We present the results of a study on the proliferative and differentiation potential of individual clones of stromal fibroblasts growing in monolayer cultures of bone marrow cells. Each precursor cell yielding a large colony in primary culture is capable of up to 34 doublings in vitro. The transplantation of clones or monoclonal strains of stromal fibroblasts into the open system results in the formation of microenvironment consisting of the bone and reticular tissue and is suitable for the differentiation of all three lines of hemopoiesis. Evidence has been obtained that, in a closed system, individual clones are capable of differentiation into the bone, cartilaginous, and reticular tissues. In other words, the adult organism has a common cell precursor for these tissues.

[Population dynamics of clonogenic stromal precursor cells in hemopoietic and lymphoid organs during bone marrow regeneration]. / Dinamika chislennosti klonogennykh stromal'nykh kletok-predshestvennikov v krovetvornykh i limfoidnykh organakh pri regeneratsii kostnogo mozga.

This paper describes our study on the regeneration of hemopoietic and stromal components of bone marrow after mechanically emptying the medullar cavity of the guinea pig tibia. The intensity of hemopoiesis was determined from the number of hemopoietic cells, while the concentration and total number of stromal precursor cells were used to estimate the ability of the bone marrow to produce stromal structures, including its ability to restore a specific microenvironment. We found that there was no direct correlation between the recovery characteristics of hemopoietic and stromal cells. An increase in the population size of stromal precursor cells takes place early after curettage, and stromal fibroblasts become phosphatase-positive according to Gomori, which is characteristic of osteogenic tissue. We have also demonstrated that curettage of 3-5 tubular bones results in the growth of this cell population in the bone marrow of nonoperated bones and even in the spleen, which in guinea pigs participates only in lymphopoiesis.

[The humoral nature of the colony-stimulating action of bone marrow cells on stromal colony formation in bone marrow cultures]. / Gumoral'naia priroda koloniestimuliruiushchego vozdeistviia kostnomozgovykh kletok na obrazovanie stromal'nykh kolonii v kul'turakh kostnogo mozga.

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.

[The dependence of the formation of stromal CFU-f colonies on the stimulating action of hematopoietic cells]. / Zavisimost' obrazovaniia stromal'nykh KOKf-kolonii ot stimuliruiushchego vozdeistviia gemopoéticheskikh kletok.

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.

[Clonal nature of fibroblast colonies formed by stromal bone marrow cells in culture]. / Klonal'naia priroda kolonii fibroblastov, obrazuemykh stromal'nymi kostnomozgovymi kletkami v kul'turakh.

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.

[Clonogenic stromal cell count in the bone marrow of mice]. / Soderzhanie stromal'nykh klonogennykh kletok v kostnom mozge myshei.

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.

[The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form]. / Soderzhanie stromal'nykh kolonieobrazuiushchikh kletok (KOKf) v kostnom mozge myshei i klonal'naia priroda obrazuemykh imi kolonii fibroblastov.

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.

[Effect of bone marrow trypsinization on the efficiency of fibroblast colony formation in monolayer cultures]. / Vliianie tripsinizatsii kostnogo mozga na éffektivnost' obrazovaniia kolonii fibroblastov v monosloinykh kul'turakh.

Stromal bone marrow mechanocytes responsible for microenvironment transfer during heterotopic transplantation of hemopoietic tissue from fibroblast clones in monolayer cultures. Fibroblast colony forming cells (FCFC) are readily released from tissue structures together with hemopoietic elements and are contained in bone marrow cell suspension at a concentration of 1 x 10(-5)-10 x 10(-5). Pretreatment with trypsin of bone marrow fragments increases the content of FCFC in bone marrow cell suspension. Trypsin releases from the bone marrow and additional fraction of FCFC (trypsin-dependent FCFC), which are more strongly bound to tissue structures and are likely to be injured in the course of routine suspension of hemopoietic tissue fragments. The ratio of the number of trypsin-independent and trypsin-dependent FCFC is approximately 1 : 10.

[Formation of hematopoietic territories in heterotopic bone marrow transplantation]. / Obrazovanie krovetvornykh territorii pri geterotopnoi peresadke kostnogo mozga.

The possibility of the formation of a new bone marrow organ from the initially isolated bone marrow cells has been proved for the first time. Suspensions of the bone marrow cells (5 x 10(6)) placed on millipore filters were grafted under the renal capsule in mice. As a result, the bone formed and the medullary cavity developed which was populated by hemopo etc cells. These processes were closely followed during 9 weeks. To compare the sizes of hemopoietic territories formed by grafting of different bone marrow fragments, the number of bone marrow cells in the heterotopic bone marrow was estimated. The bone marrow organs formed under the renal capsule after grafting bone marrow from the whole femur or its half contained practically the same number of hemopoietic cells. It was also true for the subcutaneous transplants of bone cylinders of the same size but with different amount of bone marrow. Hence it follows that the amount of hemopoietic territory grafted depends to a great extent on the transplant form and, therefore, does not reflect the number of initially grafted microenvironment-forming cells.

[Phagocytic capacity of hematopoietic tissue stromal precursor cells]. / Izuchenie fagotsitarnoi sposobnosti stromal'nykh kletok-predshestvennikov krovetvornoi tkani.

A method of removal of phagocytising elements from the cell suspension by means of iron powder in magnetic field was used. Clonogenic fibroblast precursors of the hemopoietic tissue not belonging to histiocytes-macrophages, but referred to mechanocytes, were characterised by high phagocytic activity. Following Fe treatment less than 1% of clonogenic fibroblast precursors which gave rise to fibroblast colonies under conditions of monolayer cultures remained in the bone marrow cell suspension, and about 10%--in the spleen and the peritoneal exudate cell suspension.

[Number of clonogenic stromal precursor cells in the hematopoietic organs of guinea pigs at different ages]. / Soderzhanie klonogennykh stromal'nykh kletok-predshestvennikov v krovetvornykh organakh morskikh svinok raznogo vorzrasta.

The content of clonogenic stromal precursors in the femoral bone marrow, spleen and the thymus of guinea pigs of different age was studied by the in vitro colony assay method (monolayer cultivation of hemopoietic and lymphoid tissue). The concentration and the total amount of the stromal precursor cells was shown to be age-dependent. In the femoral bone marrow and in the spleen the amount of fibroblast precursors was maximal by the age of two months; then, with ageing, it remained at the former level in the spleen, but decreased essentially in the bone marrow. The number of stromal precursors was rather constant in the thymus during the period of its active functional activity, but these cells were absent in the thymus of old animals.

[Origin of stromal mechanocytes in the bone marrow cell cultures]. / Proiskhozhdenie stromal'nykh mekhanotsitov v kul'turakh kostnogo.

The method of indirect immunofluorescence with the use of the isolinear antiserum was applied to the study of the linear reference of fibroblasts growing in the monolayer bone marrow cultures from the semisyngenous heterotropic transplants and radiochimerae. As shown, fibroblasts from the bone marrow of complete radiochimerae were of the recipient origin, whereas the histiocytes-macrophages (in the same cultures) - of donor origin. All the fibroblasts in the bone marrow cultures of the heterotropic transplant belonged to the initial donor, whereas 80% of the histiocytes were recipent's. This is a direct proof of the fact that stromal mechanocytes and hemopoietic cells, and also macrophages belonged to different cell lins.

[Self-maintenance of induced bone tissue]. / O samopodderzhanii indutsirovannoi kostnoi tkani

The capacity for self-maintenance of the bone marrow osteogenic precursor cells from the skeletal bones and from the bones induced by implantation of decalcified bone matrix is compared. Transplantation in diffusion chambers is employed as the test system. Osteogenesis in the bone marrow transplants isolated from the skeletal bone lasts several months, whereas osteogenesis in the bone marrow transplants isolated from induced bone stops after the second month. Fibroblasts arising in the monolayer cultures of the skeletal bone marrow retained their osteogenic potencies after repeated passages. On the contrary, fibroblasts from the monolayer cultures of induced bone marrow lost their osteogenic capacity after the second passage. Thus, contrary to osteogenic precursors of the skeletal bone, osteogenic precursors of induced bone tissue had a very limited self-maintaining capacity after the cessation of induction.

[Proliferative activity of stromal cells--bone marrow precursors possessing clonogenic properties]. / Proliferativnaia aktivnost' stromal'nykh kletok--predshestvennikov kostnogo mozga, obladaiushchikh klonogennymi svoistvami

Proliferative activity of bone marrow clonogenic stromal precursor cells was studied by using the thymidine "suicide" method in vitro. Crude bone marrow stromal clonogenic cells failed to proliferate in vivo. But 24 hours after the explantation they entered the S-period and formed colonies of fibroblasts in monolayer cultures. 39+/-4% of these clonogenic cells were subject to thymidine "suicide". It follows that, in difference from cells-precursors in the crude bone marrow, clonogenic stromal cells were actively proliferating in monolayer cultures.