Khrushchov Ng

Blood and clonogenic hemopoietic cells of newts after the space flight

Ribbed newts were used for studying the effect of space flight on board of the biosatellite (Cosmos-2229) on blood and clonogenic hemopoietic cells. In blood of newts of the flight group, the relative proportion of neutrophils increased, whereas that of lymphocytes and eosinophils decreased. Space flight did not result in loss of the ability of newt blood cells to incorporate H3-thymidine. Analysis of clonogenic hemopoietic cells was performed using the method of hemopoietic colony formation on cellulose acetate membranes implanted into the peritoneal cavity of irradiated newts. To analyze reconstitution of hemopoiesis after irradiation donor hemopoietic cells from flight or control newts were transplanted into irradiated newts whose hemopoietic organs were investigated. The newt can be considered an adequate model for studying hemopoiesis under the conditions of the space flight.

Comparative Investigation of Mesenchymal Stem Cells Isolated from the Bone Marrow and Fetal Liver of Mouse and Rat

We studied the properties of cells forming fibroblast colonies from the bone marrow and fetal liver of mouse and rat. Bone marrow and fetal 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 fraction of cells expressing alkaline phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the fetal liver included low proportions of such cells. The cell layers derived from the colony-forming fibroblasts of both bone marrow and fetal liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicated that these progenitor cells can form hematopoietic microenvironment.

Expression of Regulatory GenesOct-4, Pax-6, Prox-1, and Ptx-2 at the Initial Stages of Differentiation of Embryonic Stem Cells in vitro

The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

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.

Differentiation of Pluripotent Embryonic Stem Cells in the Peritoneal Cavity of Irradiated Mice

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies implanted in the abdominal cavity of irradiated mice were shown to form tumors containing the derivatives of all germ layers. Cells of the embryoid bodies form tumors two weeks after implantation, while undifferentiated embryonic stem cells form tumors only by week three.

DIFFERENTIATION OF EMBRYONIC STEM CELLS AFTER TRANSPLANTATION INTO PERITONEAL CAVITY OF IRRADIATED MICE AND THE EXPRESSION OF SPECIFIC GERM CELLS GENES IN PLURIPOTENT CELLS.

Permanent embryonic stem cell lines (ES cells) are considered as one of the most promising cellular sources for regenerative medicine. ES cells have a high proliferative potency and ability to differentiate into all kinds of somatic and germ cells. However, transplantation of undifferentiated ES cells into adult recipient tissue results in the formation of teratomas. To understand the mechanisms underlying self-renewal and determination of pluripotent cells, we investigated differentiation potencies of undifferentiated ES cells and differentiating embryoid bodies (EB). ES cells and EBs growing on acetate-cellulose membranes were transplanted into the peritoneal cavity of irradiated mice. Behavior and differentiation of transplanted cells were studied within 1, 2, 3, and 6 weeks after transplantation. No differences in the cell composition were found in the teratomas formed by ES cells and differentiating EBs. The pattern of expression of the genes specific for pluripotent and germ cells was studied in all types of experimental teratomas. The expression of oct4, stella, fragilis was detected in the teratomas, but nanog was not expressed. We conclude that pluripotent cells are retained in the experimental teratomas formed after transplantation of ES cells and EBs but the pattern of expression of the studied genes underwent changes.

Hemopoiesis on Acetate Cellulose Membranes in the Presence of Lipopolysaccharide

The formation of hemopoietic colonies on acetate cellulose membranes in the peritoneal cavity of mice was markedly enhanced after the injection of bacterial lipopolysaccharide. In addition to granulocytic-macrophagal differentiation, the foci of erythropoiesis appeared. The stimulating effect of lipopolysaccharide was not expressed in nonirradiated mice and during the formation of hemopoietic foci on acetate cellulose membranes in the subcutaneous connective tissue.

A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.

Hemopoiesis on Stromal Sublayers Formed by Constant Lines of Fibroblasts

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 γ-Irradiation at Low Doses on Clonogenic Hemopoietic (CFU-S) and Stromal (CFU-F) Cells of Bone Marrow

We studied the effects of low doses of continuous γ-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 γ-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.