Characteristics of human neural stem cells in vitro and after transplantation into rat brain.

We studied the effect of culturing conditions on the fate of human neural stem cells after transplantation into rat brain. Human neural stem cells cultured in the presence of mitogens without LIF migrated along the ependyma and cerebral vessels of recipients, but to a great extent degenerated by the 20th day after transplantation. Neural stem cells cultured with LIF migrated, apart from the above mentioned pathways, in the cortex and hippocampus, well survived; proliferating cells were retained 30 days after transplantation.

[Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro]. / Ekspressiia reguliatornykh genov Oct-4, Pax-6, Prox-1, Ptx-2 na nachal'nykh stadiiakh differentsirovki émbrional'nykh stvolovykh kletok 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-4 involved 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.

[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.

[Hematopoiesis on cellulose acetate membranes in the presence of lipopolysaccharides]. / Krovetvorenie na atsetat-tselliuloznykh membranakh pod vliiniem lipopolisakharida.

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.

[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.

[A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines]. / Issledovanie gemopoc'za na podsle peritoneal'nykh kletok pod vliianiem krovetvornykh tsitokinov.

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.

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.

[Localization of hematopoietic tissue in sturgeon]. / Issledovanie lokalizatsii krovetvornoi tkani u osetrovykh ryb.

Morphological and autoradiographic studies on various hemopoietic tissues in sturgeon are presented. The classic hemopoietic organs characteristic of lower vertebrates, such as the kidney and spleen, are studied, as well as unique hemopoietic structures described only in the evolutionarily most ancient fish species (hemopoietic tissues of cartilaginous skull capsules and epicardium). The intensity of cell divisions in hemopoietic foci has been characterized by autoradiography. The results obtained provide a basis for the revision of traditional views about the phylogeny of hemopoiesis. They provide evidence that the osteogenic gravitation of hemopoietic tissue shows up in evolution alongside the appearance of the inner skeleton.

[Use of the copolymer ethylenevinylacetate for studying the effect of hemopoietic cytokines]. / Ispol'zovanie kopolimera étilenvinilatsetata dlia issledovaniia éffekta krovetvornykh tsitokinov.

Copolymer ethylenevinylacetate is known as a polymeric carrier, which ensures a lasting release of biologically active substances. An attempt has been made to use it for testing the effects of hemopoietic cytokinins. It was shown that erythropoietin, embedded in this polymer, induces formation of erythropoietic foci on acetate-cellulose membranes in the peritoneal cavity of mice.

[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 NADP-diaphorase reaction as a neurohistologic method]. / NADF-diaforaznaia reaktsiia kak neirogistologicheskii metod.

We present evidence about the possible use of histochemical NADP diaphorase reaction to visualize elements of the nervous system.

[NO synthetase in the cells of different tissues and organs of the mouse]. / Issledovanie NO-sintetazy v kletkakh razlichnykh tkanei i organov myshi.

Cellular NO synthase was assayed in bone marrow, spleen, thymus, lymph nodes, epidermis, hypodermic connective tissue, small gut, peritoneal exudate, liver, kidney, and heart by cytochemical and immunofluorescent methods. As a rule the enzymatic activity was found in differentiated cells that finished proliferation (mature hemopoietic cells, epithelium of gut villi, etc.). The enzyme activity was undetectable in actively reproducing low-differentiated cells (basal layer of the epidermis, crypt cells of the small gut, or blast hemopoietic forms). NO is proposed to participate in regulation of tissue-specific terminal differentiation (maturation) of the cells.

Experimental analysis of cell interactions during hemopoiesis.

The principal approach was to study hemopoiesis on different stromal cell underlayers (fibroblasts or fibroblast-like cells covering a foreign body implanted into the peritoneal cavity of mice or other rodents) after intraperitoneal transplantation of syngeneic, allogeneic, and xenogeneic hemopoietic cells. The data obtained are compared with the results of experiments on repopulation of ectopic hemopoietic territories (under the mouse kidney capsule) by syngeneic and xenogeneic hemopoietic cells. Competitive cell interactions are described that occur during repopulation of the hemopoietic stroma or formation of the hemopoietic foci on cellulose acetate membranes (CAMs) in the peritoneal cavity of irradiated mice by genetically different hemopoietic cells transplanted to these animals (multicomponent radiation chimeras). The model of xenogeneic and multicomponent radiation chimeras was reproduced in long-term bone marrow cultures, where hemopoietic cells of different genotypes coexisted, without any competitive cell elimination. The second part of this review deals with hemopoiesis on stromal cell underlayers, formed by cells of different origin, different stages of development, and obtained from other sources. These underlayers were formed on CAMs in vitro and then transferred into the peritoneal cavity of irradiated mice, which subsequently received intraperitoneal injections of donor hemopoietic cells. Specific features of hemopoiesis on stromal underlayers formed by the following cell types are described: (1) fibroblasts from mouse embryos at different developmental stages; (2) fibroblasts from the skin, liver, and bone marrow of 17-day mouse fetuses and newborn mice; (3) fibroblasts from the monolayer cultures of mouse and rat bone marrow; (4) 3T3 cell line; (5) hepatocytes of 17-day mouse fetuses or sexually mature rats; (6) newborn mouse kidney cells; and (7) cells transgenic for the erythropoietin gene. The phenomena observed in these experiments and their probable mechanisms are discussed.

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.