The regulatory region of the bovine alphaS1 casein gene was used to obtain two genetic constructs for expression of human lactoferrin in the mammary gland of transgenic animals. Several transfected mouse embryonic stem cell (ESC) lines and primary transgenic mice were generated with these constructs. Recombinant lactoferrin was not detected in milk of transgenic mice by Western blotting. However, a recombinant transcript was found in RNAs isolated from mammary glands of transgenic females during lactation and from transfected ESC lines.

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.

We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

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.

Nestin is a protein that belongs to a family of intermediate filament proteins which are typical for undifferentiated neural stem and progenitor cells. In this work nestin expression was studied in the hippocampus obtained from patients with epilepsy. Immunohistochemical investigation demonstrated five types of nestin-positive cells, differing in morphological and immunological phenotype. These included cell with a radial glia phenotype, bipolar cells, small dendritic cells, subependymal and astrocyte-like cells. Two types of these cells: radial glia of dentate gyrus and bipolar NG2+ cells can be considered as neural progenitor cells possessing different degrees of commitment.

The radioprotective and antistressful activities of L-arginine and the "Pronumol" preparation, in which L-arginine is contained in the complex of proteins with nucleic acids, were studied. In mice repeated peroral intake of L-arginine and "Pronumol" partially prevented radiation-induced and stress-induced lipid peroxidation and DNA degradation in thymus, increased hemopoietic stem cell survival, and prevented an increase in chromosome aberration frequency in bone marrow cells of irradiated mice. When repeatedly administered per os before irradiation, "Pronumol" increased survival of intestinal stem cells in irradiated mice and prevented thymus cell devastation induced by radiation and stress.

Changes in the distribution of mitochondria in the two-cell mouse embryos preceding the developmental arrest in vitro, caused by a genetically determined "two-cell block in vitro" or genisteine treatment, were examined vitally using the mitochondrial-specific probe rhodamine 123 and conventional fluorescence microscopy. In the former case, serious disturbances in the localization of mitochondria appeared already from the middle of two-cell stage, long before the time corresponding to the 2nd cleavage division. Comparison of the behavior of mitochondria in the embryos successfully developing between the one- and two-cell stages and that in the embryos that ceased to cleave suggests that the developmental arrest was accompanied by aggregation of the mitochondria into clusters. There are many such clusters unlike in the cytoplasm of normally developing embryos. Intracellular localization of clusters observed in the genisteine-treated embryos differed radically from that observed in the embryos blocked in vitro at the two-cell stage.

Ability of mononuclear and adhesive cells to form differentiated elements of various tissues and organs in tissue culture in vitro has been studied in experiments on CBA mice. It has been established that adhesion mesenchymal stem cells are present in bone marrow. These cells are able to generate muscular, nervous, endothelioid, epithelioid, reticular, fibroblastoid, chondrogenic, osteogenic, and other types of cells. Mesenchymal islets have been frequently found both as parts of colonies and as independent units. Potential developmental flexibility of mesenchymal cells allows to suppose their high efficacy in pathogenetic cell therapy of heart diseases when besides damaged muscular elements vascular and nervous elements should be also restored. Mononuclear cells have not demonstrated this ability. They formed mainly monocyte-macrophagal and lymphoid cells.

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC have the ability to differentiate into osteoblasts, chondroblasts and adipocytes, neurons, glia and also into cardiomyocytes. The problem of MSC use in cell therapy of various diseases and in myocardial infarction therapy is widely discussed at present. The experiments were carried out on the inbred line Wistar--Kyoto rats. Myocardial experimental infarction (EI) was induced by left descending coronary artery ligation. MSC were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the structure of injured myocardium in experimental group significantly differed from that in control group. MSC transplantation led to inflammatory process acceleration and to increased angiogenesis in the damaged myocardium; also, live cardiomyocyte layers were detected in the scar. As a result, ventricular dilatation and overload of the border zone of infarct region decreased, no features of infarction relapse were shown in the border zone.

To study expression of ribosomic cistrons (RC) of bone marrow hemopoietic elements (BMHE) in bronchial asthma (BA).

A stable and easily reproducible model of experimental retinopathy was constructed in rabbits. The influence of transplanted neural stem cells (NSC) on the functional activity of the retina was studied. Retinopathy was provoked by 0.04 mg of kainic acid introduced intravitreously. The cultivated NSCs were transplanted into the vitreous bodies of the right (experimental) eyes, physiological solution was administered into the left (control) eyes. Clinical examinations of the eyeball in the experimental group showed less pronounced proliferative changes and a lack of gliosis, whereas, in the control group, the retina looked like tissue with fibrous changes and glial bars. An evaluation of the functional activity of the retina denoted a reliably better function of rod bipolars and Muller glias in the eyes with transplanted NSCs during the whole follow-up.

An attempt was undertaken in the last decade of the 20th century to use a principally new approach to the treatment of neurological diseases--cell therapy. Main efforts were focused on developing a method related with replacement of neurons dying in neurodegenerative pathology, primarily, in Parkinson disease (PD). Outlined below are the key elements of the technology:--ensuring, in experiment, of a prolonged therapeutic effect in transplantation, to the affected part, first of embryonic neurons of the animal of the same species (allografting) and then of homologous embryonic neurons of man (heterografting);--obtaining, standardization and preparation (for transplantation) of embryonic nervous tissue of man; transplantation of embryonic nervous tissue of man to the brain of patient and evaluation, in situ, of the functional activity of its neurons; and evaluation of the therapeutic effect of grafting. Cell suspension of meseencephalon of 6-9 week human fetus containing around 10% of differentiating dopaminergic neurons was used for grafting in PD. Embryonic dopaminergic neurons, administered stereotactically into the striatum of patient, established synaptic links with neurons of the recipient, which was accompanied by the onset of synthesis and reverse uptake of dopamine (DA) as well as by the onset of spontaneous and stimulated release of DA. Neurografting ensured a temporary improvement of the condition in a part of PD patients but did not cure them. Moreover, such positive therapeutic effect was registered only in patients with the akineticorigid but not trembling variation of the disease. Hence, although there was a certain progress in clinical neurografting, the approach cannot be now recommended for introduction in neurology and neurosurgery. The limited therapeutic effect of the treatment method is primarily explained by a low rate of survival of transplanted dopaminergic neurons and, consequently, by the persisting DA deficit in patient's body. Therefore, the outlooks for perfecting the cell technology are related with increasing the survival rate of implanted dopaminergic neurons and with stimulating the innervation of target neurons in patient's striatum as well as with using the neural (glia) and non-neural (fibroblasts, myoblasts) cells with modified gene and stem cells. Finally, despite a certain progress of advancing the cell technology in neurology the approach still needs more research, which would enable further clinical trials.

The formation of blood system adoptive reaction in many cases is defined by the type of action and the condition of hemopoietic inductive microenvironment: hemodynamics of hemopoietic tissue, functional conditions of bone marrow macrophages, mast cells and glycosaminoglycans content. The shift in hemodlobin fraction occurs in extreme conditions requiring an increased gas transport by the blood. In case of tissue lesion, lymphocytes stimulate their regeneration. Morphogenetic function of lymphoid cells may be alerted by immunomodulators. Blood cells participate in angiogenesis, and this property may be used for vessel grafts production.

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.