The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

The purpose of the study was the morphological and histochemical characteristics of differentiation of tumors developed after transplantation of GFP-positive mesenchymal bone-marrow stem cells (MSC) of transgenic mice C57BL/6 into M. quadriceps femoris of mdx mice. The tumors occurred only after transplantation of MSCs of 43-45th passages and did not arise after transplantation of MSCs of the 15th passage. No tumors developed also after transplantation of MSCs of 43-45th passages into muscle of C57BL/6 mice. The average weight of tumors appeared in 4 mdx mice studied was 1.3 +/- 0.5 g. All four tumors were classified as mesenchymomas because they originated from mesenchymal stem cells. Most of the periphery of the tumors was classified as fibrosarcomas with mitotic index 0.9 +/- 0.1%. The central parts of tumors had areas with epithelial like morphology of cells. Such cells showed positive reactivity for alcyan blue staining at pH 2.5, which indicated chondrocyte nature of the cells. No mitosis was observed in epithelial like cells. In the tumors, there were also areas with bone trabeculae containing megacaryocytes and foci of myeloid and erythrocyte hematopoiesis. There were also areas with neuronal and glial cells, and accumulations of adipocytes. One of the tumors was classified as a round cells sarcoma. The observed types of tumor cell differentiation in vivo were in accordance with described in literature types of MSCs differentiation after induction in vitro with special inductors. The spectrum of in vivo differentiation of transgenic GFP-positive MSCs after transplantation to mdx mice was broader than the spectrum of in vivo differentiation of transfected or transformed in vitro adult MSCs after transplantation to immunodeficient mice and mdx mice.

Development of reconstructive therapy of the urinary tract using pluripotent and somatic stem cells, for example mesenchymal stem cell (MSCs), recently goes through the stage of experimental studies. These studies include investigation of the main functions of MSCs and urothelium lining from inside the organs of the urinary tract. An important role in the regulation of proliferation and differentiation of urothelium belongs to EGF and Wnt-beta-catenin signaling pathways which activity may be accessed by the level of Her-4 and Tcf3,4, accordingly. We found here that MSCs labeled by transgenic green fluorescence protein (GFP) did not produce in vitro Her-4 and Tcf3,4 but activated their production after transfer into cryoinjured bladder of the syngenic mouse. After MSCs transplantation, GFP was detected in the bladder by RT-PCR and was colocalized with Her-4 or Tcf3,4 in a few urothelium cells detected by immunohistichemical staining with specific antibodies. These results suggest that MSCs labeled by GFP may be used as a good model to study transdifferentiation of somatic cells into urothelium.

It has been shown that, upon incubation of mouse bone marrow stem cells (BMSC) in vitro with the nanoparticles of perfluorocarbon (PFC) emulsion stabilized by proxanol 268, these nanoparticles penetrate into cells and stay there for a long time (up to 20 days of observation). It has been found that, under in vitro conditions, mouse BMSC loaded with the nanoparticles of both the original emulsion and the emulsion preliminarily incubated with radachlorine do not differ from control stem cells in the rate of division, stretching on a plastic support, and the formation of a monolayer. It has been shown that the exposure to laser radiation of BMSC incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine under in vitro conditions leads to the death of these cells due to the destruction of the cell membrane. The treatment with laser radiation of BMSC incubated with the nanoparticles of the starting PFC emulsion (without preliminarily incubation with radachlorine) causes no death of these cells. It has been shown in in vivo experiments that, when transplanted to the organism of a recipient mouse, BMSC of a donor mouse incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine retain their functional activity, in particular the ability to migrate in the animal body. In this case, radachlorine contained in these stem cells retains its major function, to induce the death of stem cells by the action of laser radiation due to the destruction of the cell membrane. The observation period after the transplantation was 5-7 days.

Treatment of malignant tumors using radiotherapy, chemotherapy, immunosuppressive drugs required to recover bone marrow transplant by the donor bone marrow or purified adult stem cells. During the next 1-15 years of follow up of these patients compared with healthy individuals of the same age increases the risk of multiple malignancies. It used to be attributed to the influence of therapeutic effects. However, it is revealed that some of the cells and the stroma of the secondary tumors are composed of descendants of transplanted stem cells. This indicates the important role of stem cells in tumor growth. This is also evidenced by numerous studies showing that adult stem cells from both mice and humans multiplied in vitro, after transplantation into the body give the sarcoma, cancer foci and other types of malignant growth. In this, malignant growth is most intense in the presence of focal chronic inflammation. No less than the experimental data, including those obtained in humans suggest that the transplanted stem cells actively colonize stroma of tumor tissue, stimulating the growth of the tumor and its metastasis. The human condition of survival is the presence of rigid homeostatic control mechanisms of low numbers of stem cells in the body and the limit their division, even in areas of regeneration. After the transplantation of stem cells their number in the bloodstream and, consequently, in the pathological foci of regeneration, increases in many dozens of times--this level can not be achieved by the organism itself. This leads to a sharp increase in the rate of regeneration of tissues, which creates conditions for amplification of malignant growth.

Intensification of cycle polychemotherapy in disseminated tumors considerably improves the efficiency of complex treatment. Reinfusion of peripheral blood stem cells as a factor of replacement treatment during hemopoietic suppression or disorders is now becoming more and more promising.

We studied subpopulation structure of rat cord blood and its effects on regeneration of the bone tissue. In cord blood leukocytes, cell fractions with phenotypes CD45+/CD90-, CD45-/CD90+, CD34+/CD45+ were determined. The cells adhered to plastic during culturing and had a fibroblast-like morphology. Transplantation of rat cord blood cells into modeled femoral bone defects stimulated regeneration of the bone tissue and led to recovery of its anatomical integrity, which was confirmed by X-ray examination and histological analysis. No complete recovery of the bone structure was observed in controls (without cell implantation).

It is demonstrated that the output optical signal of MTT test is directly proportional to the number of viable cells in the primary culture of mesenchymal cells (skin fibroblasts and mesenchymal stem cells from the bone marrow, placenta, and umbilical cord). The slope of the best curve in coordinates "cell number - optical signal" reflecting specific productivity of MTT-formazan characterizes mean dehydrogenase activity of cells and their physiological activity. It was found that in vitro dehydrogenase activity of primary cultures of mesenchymal cells increased during the first 3-5 passages and then tended to decrease. The variant of MTT method presented here can be used for standardization of cell materials.

Neoangiogenesis after transplantation of auto- and allogenic mononuclears and multipotent stromal cells from the bone marrow was studied on the model of inflammatory angiogenesis. Transplanted auto- and allogenic cells stimulate the formation of new blood vessels in the granulation tissue, this manifesting in an increase in the quantity and volume density of blood vessels. The most pronounced angiogenesis was observed after transplantation of allogenic mononuclears and multipotent stromal cells. It was associated with intense inflammatory infiltration, with less numerous and mature collagen fibers in the granulation tissue. Injection of allogenic cells led to stimulation and chronization of inflammation, infiltration with inflammatory and poorly differentiated cells, and more pronounced and lasting angiogenesis. However, neither auto-, nor allogenic transplanted labeled cells were detected in the walls of new blood vessels. Hence, it seems that bone marrow mononuclears and multipotent stromal cells stimulated angiogenesis mainly at the expense of production of angiogenic factors, and after transplantation of allogenic cells also by stimulating the inflammation.

Experiments were performed on the model of cyclophosphamide-induced myelosuppression. We showed that regeneration of the granulocytic hemopoietic stem is related to activation of multipotent, granulocyte-erythroid-macrophage-megakaryocyte, and granulocyte-macrophage precursors. The division and maturation of granulocyte colony-forming cells and significant decrease in the number of these cells in the bone were suppressed under these conditions. The granulocytopoiesis-stimulating effect of granulocyte CSF during myelosuppression was associated with an increase in functional activity of multipotent and granulocyte-erythroid-macrophage-megakaryocyte precursors (primarily of differentiation). In the period of regeneration, this effect was attributed to activity of granulocyte precursors.

To study the quality of life (QOL) patients with inflammatory diseases (IBD), gender and age characteristics of QOL, as well as its dynamics under the influence of biological and standard therapy.

Out of 28 patients with inflammatory bowel disease in 11 (39.3%) revealed the presence of autoantibodies to gastric parietal cells. Appointment of MSC and infliximab did not lead to a reduction in antibody levels, in fact, in 6 (21.4%) patients had a further increase in the content of mentioned autoantibodies. Identification of autoantibodies to gastric parietal cells is considered as adherence to IBD autoimmune gastritis (formation of a systemic process) that requires use of corticosteroids. Transplantation of mesenchymal stromal cells in IBD reduces enhanced circulation of autoantibodies against antigens of neutrophils cytoplasmic structures, thereby reducing the severity of autoimmune reactions. Transplantation of MSCs reduces autoaggression in patients with ulcerative colitis, reducing the autoreactive clone of B lymphocytes (CD19+CD5+). Analysis effectiveness of the therapy. Transplantation of MSCs in IBD has a systemic immunoregulatory effect: on the one hand, stimulates oppressed cytokine synthesis, on the other--reduses the intensity of the autoimmune reactions and activity of pathological processes. Infliximab selectively blocks TNF-alpha, without affecting other proinflammatory cytokines.

Despite combination therapy with immunosuppressive agents, 32.5% of patients with IBD showed the formation of antibodies to infliximab. Simultaneous study of the concentration of the drug (infliximab), TNF-alpha and antibodies to it in the blood serum allows to judge not only on the effectiveness of anticytokine therapy, but also on the advisability of further conducting therapy. Elevated levels of AINF may lead to infusion reactions, reducing the effectiveness and duration of response to this therapy. Transplantation of MSCs reduces the level of antibodies to infliximab, but in 2 (5%) patients noticed a gradual increase of these antibodies. After infliximab infusion from 4 to 8 weeks the level in serum increased up to 45 mg/ml and higher, further serological concentration of infliximab is gradually reduced and then falls below the-horn. High concentrations of infliximab (> 45 mkg/ml) in blood samples at combined immunosuppressive therapy (infliximab + glucocorticoids + cytotoxic agents) should be considered as a sign of potential complications. The absence of antibodies to antigens of HLA I and class II after systemic transplantation of allogeneic mesenchymal stromal cells (MSCs) of bone marrow demonstrates not only the effectiveness but also the safety of transplantation of allogeneic MSCs, in relation with that the special selection of donors for transplantation of allogeneic MSCs is not required.

The processes of the repair of the damaged mandibular bone in rats were studied using light microscopy and x-ray densitometry at various time intervals after the local injection of the platelet-rich fibrin clot (PRFC), autologous mesenchymal (stromal) stem cells of bone marrow origin (AMSCBMO) or AMSCBMO, adsorbed on PRFC, into the damaged site. The best results were obtained after the application of PRFC with AMSCBMO. One week after the operation, the mandibular bone defect was largely filled with the newly formed bone tissue. It seems most probable that in this case the effects of fibrin and stem cells on the damaged bone were summarized or even amplified. Bone formation in these cases appeared to begin in the center, but not at the edges, of the defect. AMSCBMO were distributed over the whole volume of PRFC, filling all the defect more or less uniformly. As a result, maximally fast and successful restoration of bone tissue was reached in the area of the defect.

Using the model of the rat spinal cord dosed contusion injury at T8 level, cross sectional area of the pathological cavities was measured and the number of myelinated nerve fibers was calculated in the outer zones of white matter after immediate single injection in the damaged area of human umbilical cord blood mononuclear cells (UCB-MC) transfected with plasmid with vegf and fgf2 genes. UCB-MC transfected with pEGFP-N2 plasmid with egfp gene of enhanced green fluorescent protein were injected into the rats of control group under similar conditions. By Day 30 after the injection of UCB-MC transfected with vegf and fgf2 genes, total cross-sectional area of the cavities in outer zones of white matter at a distance of 3 mm caudally from the epicenter of the injury was reduced more than twice as compared with that found in control group. Number of myelinated nerve fibers in the same zones of white matter at the same distance from the epicentre in rostral and caudal directions, was increased by 20% on the average as compared with control, and at a distance of 5 mm in rostral direction--by 40 to 70%. Thus, the delivery to the injury region of the therapeutic genes vegf and fgf2 reduced cavitation, restrained the processes of secondary degeneration and supported the number of myelinated fibers in the injured spinal cord.

Relative quantitative distribution of all the associative and descending efferent fibers and the ultrastructural organization of the terminals of the parietal cortex areas 5 and 7 in the caudate (NC) and red nucleus (NR) in the cat were analyzed after a local, pointed destruction of the cortex of these areas. The maximal numbers of the associative fibers were found to project to the fundus areas of the motor cortex and to the area of Clare-Bishop; moderate projections were detected to the areas 31, 19 and single degenerating fibers were registered in the areas 1,2, 3a, 3b, 30, and 23. The descending efferents were maximally projecting to NC, NR, reticular nuclei of the thalamus, midbrain, and pons, in all of which, according to the immunocytochemical studies, GABA-ergic terminals are prevalent. On the basis on the electron microscopical studies, it was suggested that the influence of the parietal cortex is mediated by the axo-spinal synapses of the medium shortaxonal spiny cells of the dorsolateral part of NC caput and by the axo-dendritic synapses of Golgi II cells of the parvocellular part of NR. On the basis of the maximal involvement of the fundus areas of the motor cortex, as well as of the inhibitory subcortical (NC) and stem nuclei (NR, reticular nuclei of the thalamus, midbrain, and nuclei pontis), it is suggested that these structures serve as the morphological substrates for the realization of the inhibitory, integrative function of the parietal cortex.

Cultures of mesenchymal cells from human decidual tooth pulp were derived. The phenotype and capacity to osteogenic and adipogenic differentiation of these cells are close to those of bone marrow mesenchymal stem cells. Decidual tooth pulp mesenchymal cells populate biodegraded polylactide scaffolds and hence, can be used for the creation of tissue engineering transplants for bone defect repair. Storage of decidual tooth pulp mesenchymal cells in the stem cell cryobanks together with umbilical blood will appreciably extent the periods of age for collection of juvenile autologous stem cells for use throughout the life span.

We studied the biocompatibility of porous polylactide carrier matrices obtained by means of surface selective laser sintering. Carrier matrices had no cytotoxic activity, but maintained adhesion and proliferation of cells. Subcutaneous transplantation of tissue engineering constructions from these carriers and bone marrow-derived multipotent stromal cells did not cause the inflammatory response and pathological changes in rats. The conditions for organotypic regeneration were provided at the site of transplantation (high degree of blood supply and considerable amount of immature precursor cells).

Studies of the probability of regulating the regenerative and reparative processes in pathologically modified tissues are reviewed. A ready cell system, providing the realization of reparative and regenerative processes in all organs (cell sources of regeneration), exists in all organisms. The authors suggest that active bioregulators, presented in this paper, are involved in the mechanisms of tissue regeneration by modulating the cell sources of regeneration.

We studied the effects of recombinant granulocytic CSF on heart remodeling in BALB/c mice after cryodestruction. Administration of granulocytic CSF was started 1 day after cryodestruction (subcutaneously, 10 μg/kg/day, for 4 days). As early as after the first injection, leukocytosis in the peripheral blood started to develop, leukocyte count peaked on days 4-6 and returned to normal on day 14. Treatment with granulocytic CSF significantly increased the content of progenitor cells in the bone marrow and led to rapid development of the inflammatory reaction and myocardium infiltration with mononuclear cells. Injections of granulocytic CSF did not reduce scar area, but provided significantly less pronounced heart hypertrophy, which attests to its better functional properties. By day 30 after cryodestruction, control animals and animals receiving granulocytic CSF exhibited similar morphological picture at the site of damage. Thus, our regimen of granulocytic CSF administration produced a mobilizing effect on bone marrow progenitor cells and postinfarction heart remodeling. Direct effects of granulocytic CSF on the heart have to be established for its use in the treatment of myocardial infarction.