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Vol 7, No 4 (2012)

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Artificial chromosomes for gene therapy and tissue replacement

Likovykh M., Kouprina N., Larionov V., Tomilin A.


For more than 20 years artificial chromosomes (ACs) are considered as a promising alternative to other vector systems, such as viral and transgene. Acs have almost unlimited capacity and can be stably maintained as separate chromosomes, thus, do not carry the risk of insertional mutagenesis. However, until recently, Acs have not widely spread due to the uncertainty of their composition and lack of. The situation has changed recently, when the method of de novo construction of ACs, based on defined DNA sequences, was suggested. The review is focused on the most advanced types of ACs and their perspectives in tissue replacement and gene therapy of human diseases.
Genes & Cells. 2012;7(4):8-20
pages 8-20 views

Characterization and ex vivo expansion umbilical cord bloodhematopoietic stem and progenitor cells

Ufimtceva A.I., Kanov E.V.


Umbilical cord blood as an alternative source of hematopoietic stem and progenitor cells is widely used in clinical practice. Thereby it is necessary to characterize hematopoietic progenitors and to define among them the most «primitive» cells with the greatest repopulating potential. In addition absolute number of hematopoietic stem and progenitor cells is limited, so it is important to find ways of increasing their ex vivo. Currently several methods of in vitro and in vivo characterize hematopoietic progenitors and of ex vivo expansion of these cells have been developed. In this review we describe this methods, as well as problems with their translation to the clinical transplantation.
Genes & Cells. 2012;7(4):21-27
pages 21-27 views

Structural dynamics of adhesive bone marrow cells by cultivation: primary passage (part 1)

Omelianenko N.P., Ilyina V.K., Kovalev A.V., Kalsin V.A., Rodionov S.A.


Structural dynamics of heterogenic adhesive human bone marrow cells was studied using primary cultures of donor's bone marrow cells. Various light microscopic techniques including Hoffman modulation contrast and Nomarski differential interference contrast were used to study cell cultures. Automatic interval photography of cell cultures with constantly maintained focusing under conditions of culture incubator that was integrated with inverted microscope was performed. Four degrees of cell passage density were used. Marked polymorphism of passaged, adhered and subsequently cultured cells was demonstrated. Four groups of adhesive cells with different morphology and structural dynamics were isolated. The majority of adhesive cells showed no marked proliferative activity and only their insignificant part possessed high proliferative potentialities. Several ways for compact monolayer formation depending on primary culture cell passage density were determined. Achieved data broaden the notion of the nature and structural dynamics of human bone marrow adhesive cells in primary culture in vitro.
Genes & Cells. 2012;7(4):28-37
pages 28-37 views

Optimal decellularization of rat hearts and diaphragms and morphological evaluation

Gubareva E.A., Sotnichenko A.S., Gilevich I.V., Macchiarini P.


Tissue engineering involves the design, evaluation, modification and maintenance of living cells or tissues embedded in biological (natural) or artificial scaffolds. Biological scaffolds need to be decellularized to become completely non-immunogenic while preserving the tissue structure and extracellular matrix. The primary goals of the present paper were to investigate and optimize different decellularization protocols of rats heart and diaphragm and establish the most reliable technique (paraffin vs. cryosections) to evaluate the morphology of the decellularized tissues. Hearts and diaphragm were decellularized with detergent-enzymatic based protocols, including deoxycholate and DNAse. Compared to published decellularization protocols, our was able to reduce exposure time (for heart: up to 3 hours — deoxycholate and 1 hour — DNAse; for diaphragm: up to 6 hours — deoxycholate and 2 hour — DNAse) of detergents and length (up to 24 hours). Results of morfological studies showed the absence of cells and preservation of the extracellular matrix. DNA quantification showed that about 81% and 74% of heart and diaphragm nuclear material, respectively, was removed by the decellularization process. Our results suggest that the investigated decellularization protocol was superior to others in removing DNA content and preserving the ECM of rats hearts and diaphragms.
Genes & Cells. 2012;7(4):38-45
pages 38-45 views

Adipose tissue-derived mesenchymal stem cells of and fibroblast's cultures - choice of biological transplants cellular component

Bogdan V.G., Zafranskaya M.M., Gain Y.M., Demidchik Y.E.


The estimation of proliferative potential, morphological and phenotypic characteristics of mesenchymal origin cultures for the purpose of a choice of the difficult multicomponent biological transplants cellular component for reconstructive- regenerative surgery was done. It's determined that the primary culture of adipose tissue-derived multipotent mesenchymal stromal cells (adMSCs) is superior to skin fibroblasts and aponeurosis cultures in proliferative capacity. adMSCs, skin fibroblasts, the aponeurosis and the postnatal fibroblasts (Foreskin line) cultures are characterized by identical morphological features with the high viability and resistance to environmental factors. Primary cultures of adMSCs are characterized by heterogeneous structure with the presence of cells with hemopoietic CD34, CD31, CD45 markers. Cultures from the 1 till 3 passages assumed cellular phenotype as CD90+/CD105+/CD44+/CD119+/CD34 -/CD45 -/ CD31 - characteristic for MSCs. Level of ССЙ7 expression was high and did not change while passaging that possible could be one of the specific for adMSCs phenotype features. High proliferative potential, expressed migratory ability, morphological and phenotypic similarity with the fibroblasts cultures, the elimination from adipose tissue well-tested protocol indicates the advantages of adMSCs application as a cell-component to build biological transplants.
Genes & Cells. 2012;7(4):48-54
pages 48-54 views

Characteristics of placental multipotent mesenchymal stromal stem cells

Shablii V., Kuchma M., Kyryk V., Onishchenko G., Areshkov P., Skrypnyk N., Lukash L., Lobyntseva G.


In this paper we have shown the presence of population of cytokeratyn-7-producing cells in culture of multipotent mesenchymal stromal cells (MMSC] from placenta with immunophenotype CD90 +/CD73 +/CD105 +/HLA -ABD ow/CD34 -/ CD45 -/CD133 -/CD14 -. It was shown by immunocytochemistry of placental MMCS a significant decrease of the population of cytokeratyn-7 positive cells during three passages in vitro. A chimerism of mother cells in cell culture of placental MMSC was established. PCR analysis has detected expression of transcription factors genes which are typical for embryonic and heart stem cells OCT-4 and NKX2-5, and at first the expression of genes sppl, col2a1 and ppar-y2 in cell cultures of placental MMSC with adipogenic, osteogenic and chondrogenic potential in vitro was described. Also we showed the necessity of individual selection of marker genes for differentiation of MMCS from various sources, depending on their initial level of expression.
Genes & Cells. 2012;7(4):55-61
pages 55-61 views

Properties of tissue-engineering polycaprolactone matrices impregnated by VEGF and bFGF growth factors

Sevostyanova V.V., Elgudin Y.L., Wnek G.E., Lubysheva T., Emancipator S., Golovkin A.S., Barbarash L.S.


A contemporary approach to small vascular conduits design for bypass surgery is growing them in vivo using tissue-engineered biodegradable polymer scaffolds. The study assessed the possibility to use grafts made by two-phase electrospinning out of polycaprolactone with impregnated VEGF and bFGF. The scaffolds were tested for the alterations in physical, mechanical and biological properties after their impregnation with the growth factors. An increase in polymer graft strength was observed following their impregnation with VEGF and bFGF. ELISA showed a prolonged biomolecule release out of the scaffold within up to 3 weeks. The results of subcutaneous implantation of the scaffolds to Wistar rats demonstrated that the biological activity of VEGF and bFGF is preserved after their release into the surrounding tissues. Thus, the study showed that there is a possibility to use PCL scaffolds with VEGF and bFGF to design small vascular tissue- engineered grafts.
Genes & Cells. 2012;7(4):62-67
pages 62-67 views

Significance of human herpesvirus detection in multipotent mesenchymal stromal stem cells for clinical practice

Astrelina T.A., Yakovleva M.V., Shahpazyan N.K., Gomzyakov A.E., Skorobogatova E.V., Karpova E.E., Kobzeva E.V.


The main requirement for the quality of cellular material for clinical use is to ensure that viral control in order to prevent infection of the recipient. The aim of the study was to evaluate the persistence and multiplication of human Herpesviridae in human bone marrow and placenta- derived multipotent mesenchymal stromal cells (MMSCs- BM and MMSCs-P), and their prevalence among donors MMSCs. Expansion MSCs-BM was held from of 6 donors and expansion MMSCs-P was held from of 25 donors for further clinical application of the standard method. We investigated the expression levels of cell surface markers of MMSCs. The quantitative detection of DNA viruses HHV-1, HHV-2, EBV, CMV, HHV-6 was investigated by PCR to the expansion and achieving a sufficient number of MMSCs for clinical use (2-5 passage). In 2 the bone marrow donors was found DNA EBV and HHV-6. These donors were diagnosed lymphohistiocytosis and biliary cirrhosis. In the placenta donors and MMSCs-P were detected DNA EBV and HHV-6 in 2 cases. DNA CMV, HHV-1 HHV-2 and were not detected in any of the investigated samples. The results showed that careful selection of donors, the algorithm reduces the risk of infection of MMSC, the risk of infection with samples of bone marrow donors is increased if the donor bone marrow are patients with severe chronic diseases; EBV and HHV-6 have a tropism to the MMSCs and are able to multiply in the process of expansion; PCR-investigation on the human Herpesviridae before expansion of cells and when a sufficient number of cells MMSCs for further clinical application allows you to quickly identify infected cell material, evaluation of the quality of MMSCs for further clinical application must include the compulsory study of PCR for the human Herpesviridae.
Genes & Cells. 2012;7(4):68-72
pages 68-72 views

Changes of angiogenic properties of adipose derived MMSC in patients with coronary heart disease with age

Efimenko A.Y., Dgoyashvili N.A., Kalinina N.I., Kochegura T.N., Achkurin R.S., Tkachuk V.A., Parfenova E.V.


Tissue regeneration is impaired in aged individuals. Adipose- derived multipotent mesenchymal stromal cells (adMMSC), a promising source for cell therapy, were shown to secrete various angiogenic factors and improve vascularization of ischemic tissues. We analyzed how patient age affected angiogenic properties of adMMSC. adMMSC were isolated from surgically obtained subcutaneous fat tissue of patients (n = 64, 43-77 years old) with coronary artery disease (CAD). adMMSC phenotype characterized by flow cytometry was CD90+/ CD73+/CD105+/CD45-/CD31- for all samples and cells were capable for adipogenic and osteogenic differentiation. adMMSC conditioned media stimulated formation of capillary-like tubes by endothelial cells (EA.hy926) and this effect significantly decreased with age of patients. There was no age-associated difference in angiogenic factors gene expression (evaluated by real-time PCR). Level of pro- angiogenic factors in adMMSC conditioned media measured by ELISA significantly declined with age of patients, but level of anti-angiogenic factors did not. Thus, angiogenic properties of adMMSC from aged patients with CAD decline due to the decreasing of pro- angiogenic factors secretion. Our data provide new insights into mechanisms of age-associated impairment of autologous adMMSC therapeutic potential.
Genes & Cells. 2012;7(4):73-82
pages 73-82 views

The First experience of quantitative evaluation of therapeutic angiogenesis results

Katelnitskiy I.I., Katelnitskiy I.L., Alexeeva G.A.


The goal of research is objective evaluation of effectiveness of therapeutic angiogenesis in patients with critical lower limb ischemia of the atherosclerotic etiology. It was shown that angiogenesis induction by drug Neovasculgen was manifested by good clinical effect in the nearest period and that was confirmed by growth of tissue blood flow according to laser Doppler flowmetry. Use of technetium-pertehnetate- macroaggregates angioscintigraphy with intra-arterial administration of the drug allowed to assess objectively the increase of blood flow to the increased volume of the vascular bed.
Genes & Cells. 2012;7(4):83-85
pages 83-85 views

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