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Vol 6, No 4 (2011)


Ot redaktsii

Kozlov V.A.
Genes & Cells. 2011;6(4):6-8
pages 6-8 views

Mneniya spetsialistov

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опыт зарубежных стран, в частности, сша, где совсем недавно был зарегистрирован клеточный препарат на основе стволовых клеток пуповинной крови hemacord, третью фазу клинических испы- таний проходит инновационный лекарственный препарат prochimal, в котором в качестве дей- ствующего начала используются живые клет- ки, вынуждает формулировать представления о «клеточных препаратах», а, следовательно, решать вопрос о пересмотре существующих клас- сификаций лекарственных средств, дополнений в фармакопею, выработки правил проведения доклинических и клинических исследований с этой категорией препаратов (в частности, иссле- дование фармакокинетики, фармакодинамики, а при выпуске дженериков - биоэквивалентности), создании производственных регламентов и др. по вашему мнению, может ли такой подход решить спорные вопросы о стандартизации и регламентации в создании и применении алло- генной клеточной трансплантации, например, при негематологических показаниях?
Genes & Cells. 2011;6(4):9-13
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Modern strategies of regenerative therapy and safety of allogenous human umbilical cord blood stem cellsadministration for neurodegenerative diseases

Smolyaninov A.B., Hurtsilava O.G., Tyrenko V.V., Novitsky A.V., Ivolgin D.A.


Biotechnological market in harvesting and application of umbilical cord blood stem cells, is permanently developing in the world. Efficiency of regenerative therapy, based on use of cord blood stem cells depends on the number and diversity of HLA-typed cord blood units. Human umbilical cord blood is a genuine source of the stem cells rich biological material, important for human. 114 patients with neurodegenerative diseases were transplanted with allogenic umbilical cord blood. Cord blood stem cells were used without selection by HLA antigens, but only by ABO blood group and Rh factor. The possibility of using of allogenic umbilical cord blood stem cells in regenerative therapy, and the lack of immunosupression will overcome significant obstacles existing in cell therapy to date. In practical terms, its becoming clear that extensive application of allogenic umbilical cord blood stem cells regenerative therapy will seriously changes approaches to the development and delivery of umbilical blood stem cells-based drugs existing nowadays and approved by many generations of doctors.
Genes & Cells. 2011;6(4):14-20
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Еpidermolysis bullosa: approaches of gene and cell therapy

Shved A.D., Tourovets A.N.


Dystrophic epidermolysis bullosa (EB) is an inherited skin fragility disorder in which blistering occurs in the sublamina densa zone at the level of anchoring fibrils of the dermoepidermal junction. EB result from mutations in the type VII collagen gene (COL7A1) and the nature of these mutations and their positions correlate with the severity of the resulting phenotypes. Presently there are not methods of therapy with the expressed curative effect. It is known that the development of approaches to the treatment of EB is based mainly on the protein, gene and cell therapy. In mouse model it was shown that protein therapies of EB is promising since intradermally injected purified human C7 collagen corrected the EB phenotype and markedly prolonged survival of the animals. For gene therapy approaches to EB, the use of various vectors have been developed. In such a way scientists efficiently delivered cDNA or full-length type VII collagen into keratinocytes or fibroblasts and achieve correction of the genetic defect and persistent synthesis and secretion of functionally active collagen in transduced cells. The use of stem cell therapy looks like considerable progress in solving the problems of EB. For this purpose were found suitable the bone marrow mesenchymal cells as they were capable to give rise to functional development of skin cells, including keratinocytes and dermal fibroblasts. Some investigators are looking at the potential of human umbilical cord cells as a source of epithelial stem cells with appropriate ability for epidermal reconstitution. It is also shown that stem cells from human umbilical cord blood in in vitro experiments can differentiate into keratinocytes, so, cord blood is a good source of cells for ex vivo expansion and transplantation in patients with large defects of the skin. An international team of researchers recently conducted an initial phase of clinical trials of donor bone marrow stem cells in patients with the most severe form of EB. Despite the modest initial successes, the results showed that treatment with stem cells in the future will bring hope to extend and improve the quality of life of patients with EB.
Genes & Cells. 2011;6(4):21-25
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The role of thyroid hormones in the regulation of the angiogenesis and cells proliferations

Glushakov R.I., Proshin S.N., Tapilskaya N.I.


A series of reports about pro-angiogenic and procancerogenic activity of thyroids hormones (TH) and its analogues sold through nongenomic action. The nongenomic actions of TH require a plasma membrane receptor or nuclear receptors located in cytoplasm. The plasma membrane receptor is located on integrin бVв3 at the Arg-Gly-Asp recognition site important to the binding by the integrin of extracellular matrix proteins. TH differ in their ability to influence the бVв3: L-thyroxine (T4), linking through the main site of the receptor, it activates mainly mitogen-activated protein kinase (MAPK; ERK1/2) and 3,5,3-triiodo-L-thyronine (T3) binding by other site affects to phosphatidylinositol-3- kinase (PI-3-K). This messengers transduce the hormone signal into complex cellular/nuclear events including increased expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hypoxia-induced factor 1-б (HIF-1б) and consequent leads to activation of angiogenesis and cell proliferation. Treatment of cell with thyroid hormones caused expression of inflammation-associated genes: cyclooxygenase-2, matrix metalloproteinase-9. Tetraiodothyroacetic (tetrac) blocks thyroid hormone effects on angiogenesis and cancer cell proliferation.
Genes & Cells. 2011;6(4):21-33
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Regulation development of bone marrow postnatal stem cells by specific xenogenic immune globulins

Selivanov, E.A., Rugal V.I.


The regulation development of haemopoietic stem cells by rabbit anti-human CD34+ stem cells immune globulin has been established. It was showed by experimental investigation on rats with cytostatic haemo-immunosuppression and in culture human CD34+ stem cells. Thus, in animals which received anti-CD34+ immune globulin the recovery of blood and immunity values occurred earlier than in the control animals. The experimental animals recovered haemolymphocytopoiesis after one month while control group normalized haemopoietic function after two months. In vitro colony-forming ability human haemopoietic stem cells are increased in 30 per cent by the addition specific CD34+ immune globulin in counditioned medium. Our preliminary date suggest that rabbit anti-human mesenchymal stem cells immune globulin can stimulate the differentiation of mesenchymal cells in osteoblasts and endotheliocytis the cells that form haemopoietic niche.
Genes & Cells. 2011;6(4):34-38
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DNA damage in human multipotent mesenchymal stromal cells at different terms of cultivation

Chausheva A.I., Nikitina V.A., Zhanataev A.K., Durnev A.D., Bochkov N.P.


The levels of DNA-damage and 8-oxiguanine were estimated using the comet assay in multipotent mesenchymal stromal cells (MSC) on different passages. Twenty eight cultures MSC from bone marrow of healthy donors have been analyzed. The level of DNA-damages in MSC, estimated as percent of DNA in comet tail (%DNA in comet tail), didnt change in the process of cultivation (3,9±0,4 % on 3-4 passages and 3,8±0,6 % on 10-12 passages). No significant differences in the content of 8-oxiguanine in the DNA of cells at different stages of cultivation (1,9±0,24 p.u. 3-4 passages and 2,1±0,22 p.u. on 10-12 passages) havent been revealed. The higher levels of DNA damage and apoptotic comets observed in two cultures of MSC.
Genes & Cells. 2011;6(4):39-41
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Investigation of telomere length and umbilical cord blood hematopoietic stem cells colony-formingpotential in cryoconservation

Karpova, N.S., Abdulkadyrov, K.M., Selivanov, E.A., Балашова В.А.


The study was focused on proliferative capacities of human hematopoietic cord blood stem cells at two different methods of cryopreservation. The proliferative potential was determined by measurement of telomere length and colonyforming cell assay. The telomere length was estimated by technique that combined flow cytometry and fluorescence in situ hybridization on Beckman Coulter Cytomics FS-500. We used the Telomere PNA Kit/FITC for Flow Cytometry for measuring telomeric sequences as well as MethoCult® GF H4435 in colony-forming cell assays. The programmed Planer Cryo freezer and direct transfer into liquid nitrogen vapour were used for cryopreservation of samples. The correlation between telomere length and capacity of cells to forming colony after thawing were established. The programmed freezing showed advantage over freezing by direct transfer cells into liquid nitrogen vapour by better keeping of proliferative capacity hematopoietic cells.
Genes & Cells. 2011;6(4):42-47
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Experimental treatment with allogeneic multipotent mesenchymal stromal cells pulmonary emphysema in rats

Averyanov A.V., Konoplyannikov A.G., Chernyaev A.L., Petrov V.N., Konoplyannikova O.A., Agaeva E.V., Zib, A.F., Bruhovetsky A.S., Kulagina N.S., Trusov A.E., Kuzovlev O.P.


The aim of the study was to asses morphologic and morphometric lung tissue changes and peritoneal macrophages activity (MA) after allogeneic multipotent mesenchymal stromal cells (MMSC) systemic transplantation in acute elastase model of pulmonary emphysema(PE) in rats. Methods. Forty 3-months old Wistar rats were randomized into 4 groups. Control group (1 group) was injected intratracheally 0,4 ml of normal saline, other animals (2-4 groups) received one intratracheal injection of 20 units (U) porcine pancreatic elastase in 0,4 ml of saline. Next day (3 group) and 7 day (4 group) rats were intravenously injected 2106 autologous MMSC in 0,5 ml of saline. 2 group was used as emphysema control. Before euthanizing at the 21st day rats were undergone peritoneal lavage with analysis of hemi-luminescent macrophages activity in the obtained fluid. Results. The lungs of 2-4 groups had various degrees of PE. The imean linear intercept in group 2 experimental emphysema increased by 231% versus the control group. The transplantation of MSCs in a day after elastase decreased mean linear intercept by an average of 50.3% compared to the control of experimental emphysema. In animals MMSC injected at the 7 th day of study, this sizes decreased more greater - by 64,5%, but was higher by 40.7% compared to the first control group. Another quantitative measure of PE alveolar index was significantly increased by 149% in group 2. The transplantation of MMSC at 1-st and 7-th day of experiment leaded to alveolar index decreasing, respectively on 163.4 and 237% . The peak Index of hemi-luminescent macrophages activity (Mv/106 cells) has made 28,8+1,1 (1 group), 57,3+1,3 (2 group), 35,8+1,6 (3 group), 31,9+1,9 (4 group). Conclusions. Our study confirmed the possibility of regenerative lung tissue effect of autologous MMSC intravenous injected in experimental rat models of PE
Genes & Cells. 2011;6(4):48-53
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Comparative investigation of polyhydroxyalkanoate scaffolds with various chemical compositions

Nikolaeva E.D., Shishatskaya E.I., Mochalov K.E., Volova T.G., Sinsky A.J.


The authors have constructed and characterized a series of membranes based on resorbable polyhydroxyalkanoates of different compositions. Five PHA types have been studied: a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids, 3-hydroxybutyric and 3-hydroxyvaleric acids, 3-hydroxybutyric and 3-hydroxyhexanoic acids. Scanning electron microscopy and atomicforce microscopy were used to examine the microstructure of membrane surfaces, showing that membranes based on the copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate had the roughest surface, while membranes based on the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate had the smoothest surface. The contact angle for water in air was smaller and hydrophilic properties better in the copolymer membranes than in the membranes based on the high-crystallinity homopolymer of 3-hydroxybutyric acid. The culture of mouse fibroblast cell line NIH 3Т3 was used to test PHAbased membranes; results of fluorescent probes of DNA DAPI and the MTT assay show that membranes based on studied PHAs are not cytotoxic on direct contact with cells and are highly biocompatible; their adhesive properties and ability to maintain fibroblast proliferation are similar to those of polystyrene and better than those of polylactic acid membranes.
Genes & Cells. 2011;6(4):54-63
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Regeneration of rat cardiac myocytes in vitro: the proliferative activity of neonatal cardiac myocytes

Golovanova, T.A., Belostotskaya G.B.


In mammalian heart, cardiac myocyte division ceases within the first week of postnatal life posing one of the most intriguing questions of evolution. Stimulation of cardiac myocyte proliferation during postnatal period would have profound impact on cardiac regeneration in humans. It has been shown that the primary culture of cardiac myocytes obtained from newborn rat could serve as an appropriate model for the investigation of the processes taking place in the heart during early postnatal ontogenesis. Similarly to the in vivo situation, the increased mitotic activity observed during the first 2-4 days after birth is diminished in cardiac myocyte culture within 4-5 days of culturing. Besides, 60% of cultured cells underwent mitotic division followed by increase in their volume which also closely resembles the process of cardiac myocyte hypertrophy in vivo. The cardiac myocyte volume was progressively increased during culturing and equaled to 81968, 1532212, and 3246190 ƒm3 on the 1st, 3rd, and 6th day of culture, respectively. Furthermore, the rate of cardiac myocyte growth in culture was similar to the pattern of myocyte hypertrophy observed in the in vivo settings. Myocyte hypertrophy in culture was associated with the formation of polyploid and multinucleated, most commonly binucleated, cells which is generally analogous to the in vivo myocyte hypertrophy. The analysis of myocyte volume distribution suggested that during cultivation nearly 60% of cells are switched from hyperplasia to hypertrophy, stopping at the G2/M boundary of the cell cycle. The results obtained indicate that cell growth patterns in primary cardiac myocyte culture share a lot of similarity with the in vivo cardiac myocyte growth. Primary cardiac myocyte culture is a valuable tool for investigation of the processes responsible for cessation of cardiac myocyte division in adult mammals.
Genes & Cells. 2011;6(4):66-70
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Hepatic stellate cells stimulate hepatocyte differentiation of rats bone marrow derived mesenchymalstem cells in vitro

Gumerova, A.A., Shafigullina, A.K., Trondin A.A., Gazizov, I.M., Andreeva, D.I., Kaligin, M.S., Rizvanov, A.A., Kiasov A.P.


Wide dissemination of chronic hepatitis and low efficiency of their medication require development of new approaches for treatment of this pathology. One of the most perspective search directions is connected with stimulation of mesenchymal stem cells differentiation into hepatocytes by applying fibroblast growth factor 4 (FGF-4) and hepatocyte growth factor (HGF), which determine primary differentiation of anterior gut epithelial cells into hepatocytes during prenatal ontogenesis in mammals. It is known that hepatic stellate cells (HSC) are the natural source of these growth factors in the liver and they are essential component of microenvironment for differentiating epithelial cells during liver ontogenesis and regeneration. The aim of this survey was to study the ability of stimulation of bone marrow derived multipotent mesenchymal cells (MMSC) hepatic differentiation by cultivation in the hepatic stellate cells conditioned media. MMSC were cultivated in various models: 1) in the media free of growth factors; 2) in the media by sequential exposure of growth factors; 3) in HSC conditioned media; 4) co-cultivation together with HSC separated by semipermeable membrane on the boyden chambers; 5) in the mixed co-culture. Results of the survey showed that factors and cytokines, produced by HSC in the media render significant effect on MMSC and lead to their differentiation into hepatoblasts and hepatocytes with stable expression (unlike expression of MMSC cultivated with growth factors) of epithelial markers and ƒ-fetoprotein. Direct intercellular contacts between MMSC and HSC in the mixed co-culture stimulate bulk and apparent expression of hepatic markers unlike in monoculter, but the phenomenon of cell fusion was not detected. Thus, results of this study confirm the hypothesis of the HSC role as an important element of microenvironment for MMSC hepatic differentiation in vitro.
Genes & Cells. 2011;6(4):72-81
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Obtaining and characteristics of human induced pluripotent stem cells from skin fibroblastsof patients with neurodegenerative diseases

Nekrasov E.D., Lebedeva O.S., Chestkov I.V., Syusina M.A., Fedotova E.Y., Lagarkova M.A., Kiselev S.L., Grivennikov I.A., Illarioshkin S.N.


Induced pluripotent stem cells (iPSCs) are analogous to embryonic stem cells in their properties, and might be derived from adult somatic cells. There exist two main approaches to practical application of iPSCs technologies - they are regenerative medicine and human diseases modeling. Nowadays the process of iPSCs obtaining is being investigated in many world laboratories, but there are no standards in the iPSC obtaining technology. This work was aimed to develop a simple, cheap and practical protocol for obtaining iPSCs and to establish a collection of iPCS lines derived from cells of patients with a Parkinson disease to make a cell model of this disease. The work resulted in a development of the protocol to produce iPSCs based on a lentiviral delivery of transgenes into cells. iPCS lines from 3 patients with inherited Parkinson disease forms have been obtained.
Genes & Cells. 2011;6(4):82-88
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Comparative investigation of reparative bone tissue regeneration while using a tissue-engineered matrix based on«TIOPROST» and «KollapAn-M» materials

Sakharov A.V., Glotova A.A., Makeev A.A., Prosenko A.E., Ryabchikova E.I.


Regeneration of the rat mandible bone tissue when using a novel osteoplastic material TIOPROST in comparison with the material «KollapAn-M» is studied by methods of light microscopy. The results obtained have shown that the inflammation rate of a bone defect volume applying TIOPROST surpasses bone tissue regeneration by the material «KollapAn-M» for more than one month. Having filled the bone tissue defect TIOPROST is determined to form a porous 3D construction which has a supportive function being a tissue-engineering matrix, have no pro-inflammatory properties. TIOPROST restrains the duration of the inflammation alteration and exudation stages in a wound. During biodegradation TIOPROST induces an active growth of blood vessels from a bone defect bed into tissue engineered matrix canals. The data obtained evolve the current knowledge on the role of free-radical processes in post-traumatic bone tissue regeneration mechanisms and substantiate the rationale for application of antioxidant compounds to optimize osteogenesis in bone tissue injury.
Genes & Cells. 2011;6(4):89-94
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Mesenchymal stromal cells in complex anti-inflammatory therapy of ulcerative colitisL.B.

Labeznik L.B., Knyazev O.V., Konoplyannikov A.G., Parfyenov A.I., Scherbakov P.L., Sagynbaeva V.E.


Прогресс в изучении патогенеза язвенного колита (ЯК) и болезни Крона (БК) и полученные данные о иммуноре- гуляторных механизмах, происходящих в организме чело- века, открыли новые направления в терапии воспалитель- ных заболеваний кишечника (ВЗК). Таким перспективным методом лечения является биологическая, в том числе клеточная терапия. Многочисленные работы показали, что мультипотентные мезенхимные стромальные клетки (ММСК) могут служить в качестве альтернативной тера- пии больных ВЗК, у которых терапия генно-инженерными моноклональными антителами оказалась неэффективна. Цель настоящей работы - разработка оптимальных схем введения культуры аллогенных ММСК для повышения эф- фективности противовоспалительной терапии ВЗК. Больных с хроническим непрерывным и хроническим рецидивирующим течением ЯК в зависимости от метода проводимой терапии распределили на 3 группы: I группа больных (n = 15), которым ММСК вводились трижды в течение месяца с интервалом 1 нед. и еще один раз че- рез 6 мес. с момента первого введения ММСК; больные II группы (n = 20) получали ММСК дважды в течение ме- сяца с интервалом 1 нед. и еще один раз через 6 мес. с момента первого введения ММСК; больным III группы (n = 20) ММСК вводились однократно; в IV группе больных (n = 20) применялась только стандартная консервативная противовоспалительная терапия. Эффективность лечения оценивали по индексам клинической, эндоскопической патогистологической активности, продолжительности ре- миссии. Результаты работы продемонстрировали, что наи- более эффективна трансплантация ММСК костного мозга с более частым введением - дважды и трижды в течение одного месяца с интервалом 7-10 дней и еще один раз че- рез 6 мес. Это позволяет снизить риск развития рецидива и усиления патологической симптоматики у больных хро- ническим рецидивирующим и хроническим непрерывным течением ЯК, увеличить продолжительность ремиссии, уменьшив тем самым частоту госпитализаций и улучшив качество жизни больных ВЗК.
Genes & Cells. 2011;6(4):95-103
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Complications, side effects and clinical application peculiarities of mobilized autologous hematopoietic stem cellsin complex therapy of patients with spinal cord injury

Avdeikin, S.N., Bryukhovetsky A.S.


Stem cell therapy finds ever-widening application in restoration of neural tissue, and particularly spinal cord injuries (SCI). Still, safety problems are far from being solved and adverse effects of the therapy remain understudied. The goal of the study is to evaluate safety and to detect complications and specific characteristics of clinical application of mobilized autologous hematopoietic stem cells (AHSC) in complex therapy of SCI. The complications have been studied at all stages of SCI therapy: at the stage of AHSC mobilization to peripheral blood, separation stage and cell therapy. It was detected that overall complication rate achieved 75%. Our study showed that AHSC therapy of SCI demands specific training of the medical staff and must be administered in multi-field hospital under the supervision of hematologist, intensivist and neurologist experienced in highly technological methods of treatment.
Genes & Cells. 2011;6(4):104-110
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Features of ethical expertise in planning and conducting clinical research in regenerative medicine

Macchiarini P., Kondratieva E.


Nowadays we are dealing with a new level of biomedical science, that is «regenerative medicine», which provides opportunities for new treatments and the prolongation of human life. It demands that new evaluative criteria, both ethic and regulatory should be worked out and the society, scientific institutions and local authorities should respond in a quick and adequate way. The authors, who are working in the frame of Megagrant Project of the Russian Government on creating Research, Education and Clinical Center of Regenerative Medicine, consider that one of the major tasks is regenerative medicine approaches to be governed by precise ethical guidelines, and social and cultural aspects. So, the first target of this collaboration is to define regulations and address ethical issues governing the field of bioengineered organs and tissues (simple or complex), both in the field of research and clinic. They present information about clinical application of tissue engineering in the world together with the summary of experience how regulations and authorities in Europe involving regenerative medicine approaches for Human Subjects. The authors presented the list of ethical rules for scientific research and clinical application in the field of regenerative medicine.
Genes & Cells. 2011;6(4):111-118
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