Regenerative medicine is one of quickly developing and promising areas of medicine in which there is essentially new approach to restoration of damaged organs by stimulation and Cor) use of stem (progenitor) cells for acceleration of regeneration. To realize this approach, it is necessary to know: what are stem cells? What are regional stem cells? What are their phenotype and potencies? Stem cells are already identified for a number of organs and tissues (epidermis, skeletal muscle) and their niche is determined. However liver, the organ whose regenerative abilities are known since antique times, has not opened its main secret yet - the secret of a regional stem cell. In this review on the basis of our own and literature data we discuss our hypothesis that perisinusoidal stellate liver cells can be liver stem cells.
Suppression of calcinosis and degeneration of heart valve transplants is important aim for improving their durability. It was shown previously that well-known methods of decellularisation including treatments with enzymes or some detergents or with hypotonic solution didn't suppress calcification of implanted aorta. In the present study we showed potential in prevention of calcinosis by a treatment, which induced death of donor cells without their total destruction before implantation. This method of anticalcinosis devitalisation is based on a hypothesis of mechanism of calcinosis initiation in heart valve transplants, which was earllier proposed in our group.
Efficacy of critical limb ischemia gene therapy can be improved by application of novel plasmid vectors with higher transgene expression. The goal of this study is to evaluate in vitro and in vivo expression of angiogenic growth factors after gene transfer using a novel plasmid vector PC4W. Plasmid constructs with genes of human VEGF185 CpC4W-hHGFopt), HGF CpC4W-hHGFopt) and angiopoietin-1 (pC4W-hAng-1optJ were tested in vitro in a HEK293T cell culture. Cells were subjected to calcium-phosphate transfection and conditioning medium samples were assayed for transgene levels using Western blot and ELISA. Results were compared with commercially available pcDNA3 based vectors encoding the same growth factors. Reverse transcription PCR was used to assay transgene expression in BALB-c mice ischemic muscle. ELISA and Western blotting data suggest that PC4W based constructs give a higher protein output of about 2-2,5 fold compared with pcDNA3 based plasmids. Optimization of nucleotide sequence in growth factors cDNA results in additional increase in transgene expression. RT-PCR data shows that expression of human HGF persists in murine ischemic skeletal muscle up to 14 days after gene transfer. Our results indicate that novel plasmid constructs for angiogenic growth factors expression have a good efficacy in vitro and in vivo and can be used for VEGF1B5, HGF and angiopoietin-1 expression in human cell culture and in experimental animals' tissue. At the moment all developed constructs pass through a series of experiments in animal ischemia models and will be used for combined gene therapy development.
Skin tissue reaction to implantation of synthetic material «TIOPROST» have been examined in 150 mice by morpho-histochemical analysis. Fast wound healing and absence of appreciable inflammatory reaction were observed in tissues surrounding the material during 3-40 days post implantation. The porous structure and chemical properties of the material modulated development of inflammation and provided formation of «a scaffold» for vessel growth inside the implant. Our study showed that the matrix based on «TIOPROST» is capable to fill in volume defects, to improve differentiation of fibroblastic cells, and to induce active migration of low-differentiated cells from stem cell niches, their proliferation and differentiation. The elaborated synthetic material «TIOPROST» can serve as osteoplastic material and the carrier of cells for development of tissue engineering constructions.
Despite the initial indications of positive therapeutic effects in cell therapy there are still limitations in numbers of autologous cell populations available without significant ex vivo expansion. Autologous adipose stromal cells CASCJ transplantation due to sufficient cell numbers, their multipotency and the ability to secret angiogenic growth factors may become an alternative tool to treat cardiovascular diseases. In this study we Investigated the ability to efficiently transfer gene into such cells using plasmid and recombinant adeno-associated virus (rAAVJ. Human ASC were isolated from adipose tissue obtained from different donors during surgical operations. Low passaged cells were transduced using gene delivery system CStratagene] based on recombinant adeno-associated virus (rAAVJ serotype 2 encoding human vascular endothelial growth factor (VEGF) or green fluorescent protein (GFP). Transduction efficiencies and transgene expression level in ASCs were analyzed by quantitative flow cytometry and ELISA. ASC population was analysed for heparan sulfate proteoglycan expression, the main cellular AAV binding receptor. It was found that 55-65% of human ASC population express this receptor. The efficiency of ASC transduction using AAV delivery system was found to be 60+7%. GFP expression was visible during a month. Relative to control, cells transduced by VEGF rAAV vector increased VEGF secretion level by at least 20-30 fold as compared to unmanipulated ASC. Recombinant adeno-associated virus provides efficient tools for ex vivo modification of human ASCs.
A set of composite films based on chitosan or chitosan-collagen mixtures are developed to be used as the resorbable matrices for fibroblastes and keratinocytes cultivation, and optimal conditions of their formation are precised. 3D porous matrices are obtained to insure the simultaneous cultivation of both types of cells. The composite materials prepared by this method in forms of films and sponges were characterized and then tested as the matrices for cell cultivation to obtain the transplantats for wound healing. Both in vitro and in vivo tests of all materials under study have evidenced the absence of any toxic actions on the cultivated cells and on the tissues of organism. The matrices are stable in the conditions of cultivation and resorb completely in in vivo conditions in 10 days after the transplantation.
Treatment of complications of portal hypertention at liver cirrhosis also includes as preventive maintenance of varieces bleedings of a gullet various ways, and as treatment of hepatocellular insufficiency. The result of complex treatment with application of surgical methods and to stimulation of liver regeneration transplantation fetal tissue for correction of hepatocellular insufficiency are submitted.