[Mesenchymal stromal cells synchronize the rhythm of protein synthesis under the effect of an exogenous signal].

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.

[The sensitivity of mesenchymal stromal cell subpopulations with different times of adhesion property manifestation and derived from hemopoietic organs to growth factors EGF, bFGF, and PDGF].

The action of three growth factors (EGF, bFGF, and PDGF) on mesenchymal stromal cell (MSC) subpopulations from mature bone marrow (BM) and rat embryo liver (EL) was investigated. These cells are plastic-adhesive and have different rates of adhesion (AC1-AC4 subpopulations). The efficiency of colony-formation, the size of colonies, and the number of early osteogenic progenitors with alkaline phosphatase activity in colonies and induced osteogenesis were analyzed. It was shown that EGF increased the number of bone marrow (BM) MSC colonies, but it had no influence on osteogenic differentiation. bFGF suppressed colony formation, but it stimulated both early and late stages of steogenesis. PDGF increased the size and the number of colonies in AC2 and AC3 subpopulations, but it stimulated only the ostegenesis terminal stage. The distinction between MSC subpopulations from two organs were found: MSC from EL had small osteogenic capacities and low sensitivity to grow factors; MSC from BM had no such characteristics. MSC subpopulations with different adhesion properties and from different tissues had compatible sensitivity to growth factors. Thus, these cells have no parent-progeny relationship.

[Influence of primary adhesive interactions with fibronectin on clonal growth and osteogenic potential of rat mesenchymal stromal cells].

Interactions with extracellular matrix including fibronectin (Fn) play an important role in regulation of cell growth and differentiation. Influence of Fn and its individual domains on adhesion and osteogenic potencies of rat mesenchymal stromal cells (MSCs) was estimated. Investigation of bone marrow or fetal liver MSCs adhesion dynamics showed that after 7 days of cultivation on Fn the number of adhered clonogenic cells derived from both sources was comparable to their number observed on plastic but their content in suspension was commonly decreased. Population of fetal liver MSCs differed from bone marrow-derived population by greater fraction of cells that adhered for the first 7 days. Bone marrow MSC cultures on Fn were characterized by reduced activity of alkaline phosphatase as compared with cultivation on plastic; furthermore, they deposed significantly smaller amount of calcium salts under cultivation in osteogenic medium. Cultivation of MSCs on Fn fragments demonstrated the primary role of its cell-binding domain in the inhibition of osteogenesis.

[Analysis of sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to 5-fluorouracil].

The sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to the cytotoxic effect of 5-fluorouracil (5-FU) was compared in vivo and in vitro. Cells from both tissues demonstrated a similar resistance to 5-FU in vitro; however, stromal stem cells from fetal liver proved notably more sensitive to 5-FU compared to marrow CFU-f in vivo. Cells forming colonies of different size were identified in stem cell populations from both tissues. Cells giving rise to small colonies had a higher resistance to 5-FU both in vivo and in vitro.

[Alkylating damage by dipin of hematopoietic and stromal cells of the bone marrow].

Effect of alkylating agent dipin was studied on hematopoietic (CFU-S) and stromal (CFU-F) progenitor cells. Single administration of dipin (0.06 mg/g) to adult (CBAxC57Bl/6) F1 hybrid mice induced a long-term (2 years) oscillations in the numbers of day 7 CFU-S and day 11 CFU-S in the bone marrow and spleen. Dipin also damaged the hematopoietic stroma as indicated by decreased numbers of CFU-F which remained low for at least a year. The capacity of stromal cells to form ectopic hematopoietic foci was considerably inhibited and also remained low for 10 months. The obtained data suggest high dipin sensitivity of the earliest hematopoietic and stromal cells. The dynamics of CFU-S numbers in the hematopoietic organs supports their functioning on the basis of clonal succession (Kay, 1965).

Decrease in the number of progenitors of fibroblasts (CFUf) in bone marrow of rats after a 14-day flight onboard the Cosmos-2044 biosatellite.

A decrease in the number of progenitors of fibroblasts (CFUf) was found in bone marrow of rats that underwent a 14-day flight in the state of weightlessness onboard the Cosmos-2044 biosatellite, immediately after flight, when compared with rats maintained in control conditions of terrestrial gravitation. These changes may be explained by the action of specific factors of microgravitation.

Studies on clonogenic hemopoietic cells of vertebrate in space: problems and perspectives.

Hemopoietic tissues were studied in vertebrates launched aboard the Soviet (Russian) biosatellites ("Cosmos-1129, 1514, 1667, 1887 and 2044"; "Bion-10 and 11") between 1980 and 1996. In the bone marrow of rats exposed to spaceflight conditions, a statistically significant decrease in cell number was revealed in the progenitor cell compartment accounting for the compensatory response of granulocyte-macrophage (CFU-gm) and erythrocyte lineages (BFU-e and CFU-e) and in the compartment of multipotent hemopoietic stem cells (CFU-s), which is responsible for the permanent renewal of hemopoietic tissue. The number of stromal fibroblastic progenitors (CFC-f) in the bone marrow of these rats was also reduced. Apparently, changes in the hemopoietic stroma damage the hemopoietic microenvironment and, hence, may be responsible for changes observed in the hemopoietic tissue proper. Attempts were made to develop methods for analyzing morphologically indiscernible clonogenic hemopoietic cells of newts, and studies on the effects of spaceflight factors on these cells were performed. The results showed that the numbers of clonogenic cells in newts of the flight group newts were significantly lower than in control newts. The data obtained are used as the basis for formulating the problems to be studied, drawing up a program for further research on the effects of spaceflight factors on stem and other clonogenic hemopoietic cells, and developing new experimental models for analyzing stem cells, the state of the hemopoietic stroma, etc.

[Effects of 10-day long exposure to gamma irradiation at low doses on bone marrow cells in mice]. / Effekt 10-sutochnogo gamma-oblucheniia v nizkikh dozakh na kostnomozgovye kletki myshi.

Effects of ten day long exposure to gamma-irradiation at low doses (mean dose rate of 1.5-2.0 m Gy/day, total dose of 15 m Gy) on hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells from murine bone marrow were examined. The CFU-F content measured as in vitro fibroblastic colony number showed 1.5-4.5-fold increase. Additionally, the size of ectopic marrow transplants evaluated by counting myelokariocytes and CFU-S numbers also increased. No significant changes of CFU-S proliferation rate were found.

[Simple method of calculating the average duration of mitosis by using a radiation method]. / Prostoi sposob rascheta srednei prodolzhitel'nosti mitoza pri ispol'zovanii radiatsionnogo metoda.

On the mitotic index curve of the cellular population following radiation there is a linear part. The time interval between the initial decrease of the mitotic index and the extrapolation of the straight part of the curve to X-axes is the mean duration of mitosis. The values of this parameter for the culture of fibroblasts obtained by this method are practically identical with those obtained by a method based on determination of the mitotic index and the doubling time of the cell count.

[The functioning of mammalian mitochondria injected into fish embryos]. / Funktsionirovanie mitokhondrii mlekopitaiushchikh, in''etsirovannykh v zarodyshi ryb.

The possibility of mammalian mitochondria functioning in fish embryos has been studied. Suspension of mitochondria isolated from the mouse fibroblast B-82/cap (chloramphenicol-resistant) and B-82 (chloramphenicol sensitive) cell cultures, were injected into the fertilized loach eggs. These embryos with an artificially increased number of mouse mitochondria developed and lived till the larval stages. Activity of cytochrome oxidase in these embryos was 1.5-2 times that in the control several hours after the injection, decreased during development and reached the control level by the gastrula stage. If these embryos with artificially increased number of mouse mitochondria were incubated in presence of chloramphenicol, only embryos that contained mitochondria from chloramphenicol-resistant cells survived, thus suggesting that the injected mitochondria do not degrade but are preserved and function in the cytoplasm of developing loach embryos.

[Experimental research on the cellular interactions during hemopoiesis]. / Eksperimental'noe issledovanie mezhkletochnykh vzaimodeistvii pri gemopoéze.

We present a review of experimental studies performed at the Laboratory of Histogenesis of the Institute of Developmental Biology, Russian Academy of Sciences, on the problem of cell interactions during hemopoiesis. Special attention has been given to original experimental models, such as production of hemopoietic foci on underlayers of fibroblasts encapsulating a foreign body in the peritoneal cavity of rodents (after intraperitoneal transplantation of hemopoietic cells) and repopulation of ectopic hemopoietic territories under the kidney capsule of mice by syngeneic or xenogeneic hemopoietic cells. We describe the competitive interactions of genetically different hemopoietic cells after the transplantation of their mixtures to irradiated mice (multicomponent radiation chimeras). Xenogeneic and multicomponent chimeras have also been obtained in long-term bone marrow culture. We have examined characteristics of hemopoiesis on stromal cell underlayers produced by cells of various origins in vitro and then transplanted into the peritoneal cavity of irradiated mice. We discuss the results obtained and possible mechanisms of these phenomena.

[The 5-fluorouracil sensitivity of the hematopoietic stroma of the bone marrow and spleen]. / Chuvstvitel'nost' k 5-ftoruratsilu krovetvornoi stromy kostnogo mozga i selezenki.

We studied the effect of a single injection of 5-fluorouracil (5-FU) (150 mg/kg) on the numbers of stromal precursor cells (CFU-F) in the bone marrow and spleen and their capacity for formation of hemopoietic foci after ectopic transplantation under the renal capsule. 5-FU decreased the number of CFU-F in the bone marrow and spleen of mice and rats to 30 - 50% of the control values. The size of transplants, as estimated according to the number of hemopoietic clonogenic (CFU-S) and nucleated cells, diminished within the first two months after transplantation. Within four months after transplantation the size of the bone marrow transplants approached the control level, while that of the spleen transplants markedly exceeded the control level. Differences in the growth rate of regenerating transplants of the hemopoietic organs are discussed with special reference to the organ specific features of populations of the stromal cells.

[Comparative investigation of mesenchymal stem cells isolated from the bone marrow and embryonic liver of mouse and rat]. / Sravnitelnoe issledovanie mezenkhimnykh stvolovykh kletok, vydelennykh iz kostnogo mozga i embrionalnoi pecheni myshi i krysy.

We studied the properties of cells forming fibroblast colonies from the bone marrow and embryonic liver of mouse and rat. Bone marrow and embryonic liver cells formed colonies in vitro including fibroblasts as well as a considerable proportion of macrophages. The colonies formed from bone marrow and hepatic cells of rat differed from the murine ones by a higher proportion of fibroblasts. Most colonies derived from the bone marrow of both mouse and rat included a proportion of cells expressing acid phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the embryonic liver included low proportions of such cells. Cell layers derived from the colony-forming fibroblasts of both the bone marrow and embryonic liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicates that these precursor cells can form hematopoietic environment.

[Hemopoiesis on stromal sublayers formed by constant lines of fibroblasts]. / Krovetvorenie na stromal'nykh podsloiakh, obrazovannykh postoiannymi liniiami fibroblastov.

We studied the formation of hemopoietic colonies on an artificial sublayer in the peritoneal cavity of mice under the influence of stromal sublayers consisting of fibroblasts of six constant cell limes. All studied lines of stromal cells supported the formation of granulocytic foci and some of them supported the formation of erythroid foci as well. It was shown that hemopoiesis was preserved or, in some cases, enhanced on sublayers of fixed (metabolically inactive) cells. The treatment of fibroblasts by E. coli lipopolysaccharide did not lead, as a rule, to significant stimulation of hemopoiesis.

[Effect of continuous gamma-radiation at low doses on clonogenic hemopoietic (CFU-S) and stromal (CFU-F) bone marrow cells ]. / Effekt nepreryvnogo gamma-oblucheniia v nizkikh dozakh na klonogennye krovetvornye (KOE-S) i stromal'nye (KOE-F) kletki kostnogo mozga.

We studied the effects of low doses of continuous gamma-irradiation (Co60, 10 days, mean daily dose power 1.5-2.0 mGy, total dose 15 mGy) on hemopoietic and stromal progenitor cells of murine bone marrow. The content of hemopoietic clonogenic cells representing a "younger" (CFU-S-11) and more "mature" (CFU-S-7) categories in the compartment of stem cells was determined in the bone marrow. The state of bone marrow stroma was estimated by the method of in vitro cloning according to the number of progenitor cells that form colonies of fibroblasts (CFU-F) and by the method of ectopic transplantation according to the capacity of stroma of organizing and building new hemopoietic territories. Continuous gamma-irradiation at low doses, that were by one order of magnitude lower than those inducing hermesis, exerted a stimulating effect on both hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells. The number of CFU-S in the compartment of stem cells of the bone marrow markedly increased and they formed larger hemopoietic territories but these cells appeared to create a qualitatively different microenvironment, which stimulated the proliferation of CFU-S.

[Effect of a leukocytic serum preparation on hematopoietic cells in vitro]. / Vliianie preparata leikotsitarnoi syvorotki na krovetvornye kletki in vitro.

The effects of leucocyte serum (LS) on bone marrow cells (BMC), thymus and HL-60 human myeloid leukemia cells were studied in liquid suspension and agar cultures. LS increased 3H-thymidine incorporation in BMC and intensified the cloning efficiency of granulocyte-macrophage progenitor cells (CFU-GM) and human myeloid leukemia cells. No significant stimulatory effect on thymus cells was observed. It has been shown that LS prevents or markedly decreases the effect of granulocyte inhibitor (GI-3S2).