During many decades the development of effective methods of wounds treatment has been and still remains to be one of the most actual problems of surgery. Interest and constant attention to wounds speaks first of all that simultaneously with development of medicine, cellular and molecular biology, molecular genetics and immunology consideration of wounds processing is constantly vary. New prospects and opportunities of effective treatment of wounds are opening. We develop a new original method of treatment full-thickness wounds by a method of transplantation fibroblasts and stem cells on the basis of collagenic matrix. The presented method of treatment of full-thickness wounds provides the most perfect realization of stimulating action of cultures of cells on the processes of regeneration of tissue. Application of fibroblasts and stem cells on the basis of collagenic matrix promotes restoration of normal structure of a skin and allows reducing terms of healing considerably. Stem cells are integrated into three-dimensional tissue structures of an organism and promote histotopically to restoration of defects of a skin covering. Here it is necessary to note, that transplanted skin fibroblasts synthesize and allocate in an environment a plenty of biologically active substances among which distinguish epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), and also various components of extracellular matrix.

To study effects of parathyroid hormone (PTH) used in therapy of osteoporosis on hemopoiesis in long-term culture of human bone marrow (LCBM) in terms of its potential influence on stem hemopoietic and stromal cells.

The problems of a low efficiency of mammalian cloning are discussed with emphasis on the necessity of the expertise of each step of single cell reconstruction, beginning with microsurgical manipulations. The fact of cell content leakage when the cell is held during microsurgery or microinjections with the help of the conventional method using negative pressure in the holding micropipette was demonstrated in experiments on murine embryos. It was shown that the rate of cell content efflux depends on the value of negative pressure generated in the holding micropipette, and is directly proportional to the dimensions of its orifice and the duration of micromanipulations. An alternative method of cell fixation using the capillary forces of the holding micropipette was proposed. The method optimizes the process of cell fixation, reducing the holding effort by two orders of magnitude. As a result, 92% of embryos remain viable after fixation of embryo, as compared with 39% in the conventional technique. In order to diminish the cell damage produced by the tip of a microinstrument, a new technique of fabricating micropipettes was proposed. The improved method of filling the micropipette with viscous liquids, including DNA, which is described in details in the paper, enabled constant (non-stop) microinjection of more than 1000 cells by hand, without any special automatic device.

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.

Results of studies of properties of adipose tissue stromal cells are summarized in this review. It contains data on separation and cultivation of these cells as well as description of their antigenic characteristics, ability to differentiate into cells of mesenchymal and nonmesenchymal origin, angiogenic potential, and potential for transfection and transfer of therapeutic genes. Analysis of perspectives of clinical use of adipose tissue stromal cells in therapy of cardiovascular and other diseases is also presented.

Mechanisms of myelotoxic action of Doxorubicin associated with changes in NAD(+)-glycohydrolase/CD38 expression and activity have been assessed. During the acute and subacute exposure of bone marrow cells to Doxorubicin in vivo, expression of CD38 is decreased corresponding to elevation of intracellular and extracellular NAD+ concentrations. These changes are associated with increased susceptibility of hemopoietic cells to the apoptogenic action of Doxorubicin. Possible role of NAD(+)-glycohydrolase/CD38 in the regulation of sensitivity of the cells to the cytotoxic effects of the xenobiotic is discussed.

In vivo and in vitro experiments on the application of cell technologies to tissue defect closure were conducted; autologic mesenchymal stem cells on 3-demensional matrices were used. The authors analyze the results of the application of bioengineering tissue equivalents for the closure of soft tissue and upper airway defects after extensive resections performed in 52 oncological patients. Tissue equivalents with stem cells provide engraftment and long-term graft functioning; they also modify wound surface, thus stimulating wound epithelization. In this study the application of tissue equivalents led to wound healing and functional recovery in 87% of patients.

The authors analyzed the heterogenicity of a cell population in dysplasia of varying degrees and gastric cancer from the expression of the proliferation marker Ki-67 and apoptosis activator p53 gene. Twenty-four stomachs (surgically removed for cancer) and biopsy materials (4 cases of moderate dysplasia, 4 cases of severe dysplasia) were studied. Four samples from clinically healthy individuals served as a control. The labeling index of Ki-67 in third-degree dysplasia was 37.9 +/- 2.5% and that in cancer 61.6 +/- 3.8%. There was no expression of the p53 gene in the controls. In three-degree dysplasia and cancer, the expression was 17.7 +/- 5.7% and as high as 69%, respectively. The densitometric characteristics of the labeled cells were of great diagnostic importance. Gastric cancer was represented mainly by 2 cell populations: (1) a stem cell line located in G1 with a low labeling index and a low optical density of Ki-67 and p53 and (2) an aggressive cell clone located in the S and G2 phase with marker hyperexpression.

We have optimized lentiviral vector constructs and cassettes for expression of short hairpin RNAs (shRNAs) in order to create genome-wide library capable of inhibition of full variety of human mRNAs. The vector optimization has resulted in 15-20-fold improvement in virus stock titers. We found that in the context of lentiviral vector the most effective structure for the shRNA is simple hairpin with 21 nucleotide stem. The shRNA-expressing lentiviral constructs contain choice of puro(R), copGFP or H-2K(k) selective markers. The efficiency of the optimized library was evaluated in experiments on screening of shRNAs that reactivate oncosuppressor p53 in HeLa cells. The cells contained reporter construct with p53-dependent expression of a fluorescent protein, which allows cytofluorimetric isolation of cell population with reactivated p53.

The aim of the work was to study the effectiveness of using human embryo fibroblast culture in complex treatment of trophic ulcers of venous etiology in 23 patients with trophic ulcers of lower extremities. The cause of the appearance of ulcers was postthrombophlebitic disease in 16 patients and varicose disease in 7 patients. A control group consisted of 25 patients (postthrombophlebitic disease in 18 patients and varicose disease in 7 patients). The human embryo cell culture grown on the wound cover "Foliderm" was used at the stage of epithelization in the main group, while in the control group the modern alginate, collagen, hydrogel, polyurethane and hydrocolloid covers Suprosorb--Suprosorb A, Suprosorb C, Suprosorb G, Suprosorb F and Suprosorb H were used. Healing of the varicose trophic ulcers in the control group was achieved in 86% of patients, of post-thrombophlebitic--in 78% of patients. The average period of healing was 3.6 and 3.9 months respectively. Healing of trophic ulcers in the main group took place in 100% of patients. The average period of healing was 1.5 week for varicose and 3.2 weeks for postthrombophlebitic ulcers. The cell therapy was shown to be a highly effective method in treatment of venous trophic ulcers.

280 epithelial tumors of the pancreas including 72 exocrine tumors (50 adenocarcinomas, 10 mucin-cystic, 6 intraductal, 6 serous cystic), 202 endocrine tumors and 6 undifferentiated neoplasms and surrounding parenchyma outside of the tumors were studied. Tumors arise against the background of pronounced proliferation of polypotent cells of the ducts. In case of tumors of ductal origin hyperplasia of ductal epithelium and acinar- ductal transformation of surrounding parenchyma are observed while in tumors with neuroendocrine differentiation there is mucous-papillary hyperplasia of ductal epithelium with proliferation in them of endocrine precursors, less frequently- acinar-endocrine transformation of surrounding parenchyma.

Much effort has been made in searching for multipotent cell types with high therapeutic potentials for repair of damaged tissue. Through enzymatic digestion of fat tissue, it is possible to obtain a large number of stromal cells. Isolated cells show a high proliferate capacity in culture. All this makes adipose stromal cells (ASC) promising candidates for their use in cell therapy. This review is focused on analyzing the surface antigen profile of isolated population of ASC, expression of angiogenic factors by these cells, as well as on their differentiation potential. A high percentage of ASC population initially express the progenitor cell marker CD34, but during culturing, cells exhibit a mesenchymal cell phenotype and express CD29, CD105, CD106, CD166. Culturing ASC in specific differentiation media induces expression of early markers of differentiated mesenchymal cells, such as adipocytes, chondrocytes and osteoblasts, as well as myoblasts, cardiomyocytes and neural cells. It has been also shown that ASC have a strong pro-angiogenic potential, they are able to secret growth factors, such as VEGF, HGF, bFGF and others, which stimulate survival and proliferation of endothelial cells. In addition, systemic or local delivery of ASC to mice with hindlimb ischemia stimulates recovery of injured tissue and blood flow. Potential clinical uses of ASCs are discussed in the review.

Autotransplantation of splenic tissue in two patients after total duodenopancreatectomy and splenectomy for trauma of the duodenum and pancreas has demonstrated positive results and stable compensation of insulin insufficiency in two cases. Experimental studies of autotransplantation of splenic tissue carried out on 20 cats and 100 rats with pancreatectomy- and alloxan-induced diabetes demonstrated good results too. This method may be regarded as the variant of repair therapy with stromal stem-cells of the spleen.

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.

Two cell populations with a phenotype similar to that of mesenchymal stem cells (MSC) with different characteristics for expression of surface antigene CD34 were derived from subcutaneous fat. CD34-positive cells were derived from subcutaneous fat of the inferior eyelid obtained during transconjuctival blepharoplasty. CD34-negative cells were derived from adipose tissue obtained during lipoaspiration from the same patients. These cells displayed common characteristics for morphology and expression of basic markers characterizing them as mesenchymal stem cells. On being induced for differentiation, these two cell populations were able to differentiate to cells of adipose (adipocytes), bone (osteoblastes, osteocytes), cartilage (chondroblasts, chondrocytes), and nervous (neurons, astrocytes and oligodendrocytes) tissues.

Cytogenetic anomaly frequencies were analysed in three sublines of ES R1 line in its five clonal sublines, obtained from two cell colonies after transformation of ES R1 cells by plasmid with gene lif. Cell transformation did not increase cytogenic anomalies, however, the initial sublines of ES R1 line, as well as its transformed clonal descendants bore a redundant quantity of the chromosome 8 material within the structure of various Robertsonian translocations even in cells with diploid chromosome quantity (2n = 40). In the initial sublines ES R1 and its clonal descendants a common Rb (8; 15) was revealed. It was supposed that selection for the increase in ES cell sensitivity to cytokines (in particular, LIF) under cultural conditions was accompanied by an increase in chromosomal copies, carrying genes of mapk andjak/stat, through which downstream effectors of cytokine signals for preservation of cell pluripotention and propagation are realized. Genes of chromatid separation and chromosome segregation control (for example, separase gene Esp1 in chromosome 15) may be passively involved in this process, thus promoting acceleration of karyotype evolution in ES cells.

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.