In Vivo Studies of a Liquid Embolic Compound.

A composition in the form of liquid polymer substance intended for embolization procedures was studied in in vivo experiment. The preparation was injected to rabbits into the femoral artery and abdominal aorta. The polymer composition exhibited properties previously demonstrated in vitro: strong adhesion to the vascular wall, high plasticity sufficient for embolization of the blood vessels, distal distribution, and the absence of toxic effects. The contrast substance remained in the embolus, which simplified its further localization. The agent underwent nether resorption nor organization. Injection of the agent in a volume of 0.1 ml was sufficient for embolization of an artery with a diameter of 0.1 cm. The polymer composition completely obstructed the vessel without inducing perforation of its wall. During the first day of the experiment, a slight infiltration of surrounding tissues with lymphoid cells was observed. By day 7, total dry necrosis of pelvic limb distal to the injection site was diagnosed. Inflammation of the surrounding tissues was shown histologically and was considered as the body response to impaired circulation and necrosis.

Experimental Study of the Effects of Nanodispersed Ceria on Wound Repair.

We studied the effects of nanodispersed ceria on wound healing in vitro and in vivo. It was found that cerium dioxide stimulated wound healing, which manifested in shrinkage of burn wound area (by 1.5 times) and intensification (by 2.4 times) marginal epithelialization.

[CO-CULTURE OF BOAR SPERMATOGONIAL CELLS WITH SERTOLI CELLS].

In the present study, we developed in vitro culture conditions using co-culture of boar spermatogonial cells with Sertoli cells. Testes from 60-day-old crossbred boar were used. A spermatogonia-enriched culture was achieved by enzymatic digestion method and purification by density gradient centrifugation using a discontinuous Percoll gradient and differentiated adherence technique. Lipid drops were detected in isolated Sertoli cells by Oil Red O staining. We have found that the cultivation of boar spermatogonia in the presence of Sertoli cells (up to 35 days) leads to their differentiation as well as in vivo in testis. Association of cells in groups, formation of chains and suspension clusters of the spermatogenic cells were observed on the 10th day. Spermatogonial cellular colonies were noted at the same time. These cellular colonies were analyzed for the expression of genes: Nanog and Plzf in RT PCR. The expression of the Nanog gene in the experimental cellular clones obtained by short-term culture of spermatogonial cells in the presence of Sertoli cells was 200 times higher than the expression of this gene in the freshly isolated spermatogonial cells expression was found in freshly isolated germ cells and in cellular clones derived in vitro. We have found that, in the case of longer cultivation of these cells on Sertoli cells, in vitro process of differentiation of germ cells and formation of single mobile boar spermatozoa occurs at 30-33 days. Cellular population is heterogeneous at this stage. Spermatogenic differentiation in vitro without Sertoli cells stays on the 7th day of cultivation. The results show that co-culture of boar spermatogonia-enriched cells with Sertoli cells can induce their differentiation into spermatozoa in vitro and facilitate obtaining of porcine germ cell culture.

[Mouse embryonic stem cells - a new cellular system for studying the equine infectious anemia virus in vitro and in vivo].

The complexity of the pathogenesis and insufficient knowledge about the slow retroviral infections, which include equine infectious anemia, necessitates finding an adequate laboratory model for the study of the infection process and immunogenesis to create means of prevention and treatment of diseases. Data about strains and cellular tropism of the virus are discussed. It was shown that mouse embryonic stem cells (ESCS) exhibited unique properties and characteristics. In contrast to fibroblasts and other cell types, these cells can be considered as a new cell system for studying EIAV in vitro and in vivo. Under differentiation-inducing conditions they are able to reproduce in vitro embryogenesis cells and form cells of three germ layers. Differentiation of mouse ESCs in the direction of hematopoiesis could contribute new knowledge and understanding of viral tropism EIAV in vitro. ESC can be returned back to the early pre-implantation embryo. Once in the germ cell environment, they participate in the formation of tissues and organs of the developing fetus. Thus, the adaptation of the mouse ESC to the equine EIAV through genetic transformation makes it possible to get closer to the creation of a laboratory model for the study of the in vivo immune response in the lentiviral infection.

[Change of expression of integrins on multipotent mesenchymal stromal cells isolated from human adipose tissue during long cultivation].

An expression ofintegrins on multipotent mesenchymal stromal cells (MMSCs), isolated from human subcutaneous adipose tissue during long cultivation was studied. Results of the comparative analysis of MMSCs on the 2nd and 17th passages of cultivation revealed considerable distinctions in expression of α1, α4 and α6 integrins on these cells. Strong decrease for 87.2 and 11.2%, quantities of the cells which have been positively stained by AT against CD49a (α1 integrin) and CD49d (α4 integrin), respectively was observed. The share of the cells which have been positively stained by AT against CD49f (α6 integrin) and CD49b (α2 integrin) on the contrary was increased during long cultivation by 9.9 and 2.3%, respectively. The high expression of ß1 integrin (98%) on MMSCs, wasn't changed significantly in the course of long cultivation. As a result of induction of MMSCs to a differentiation in the osteogenic direction it was revealed that cells on the 17th passage of cultivation concede by efficiency of formation of cells of bone tissue and extracellular matrix in vitro to cells which induced on the 2nd passage. Thus, long cultivation of MMSCs which is required for building of cellular population after isolation, influences on a cytoskeleton and adhesive abilities of cells that it is necessary to consider when using them for cellular technologies, including for modeling of this or that tissue in three-dimensional scaffolds.

[Human subcutaneous adipose tissue subjected to cold shock as a source of viable cellular population with characteristics of multipotent mesenchymal stromal cells].

Cellular population with characteristics of multipotent mesenchymal stromal cells (MMSCs) was isolated from subcutaneous adipose tissue frozen without any cryoprotectant at -70 degrees C. Under critical for the adipose tissue condition, the cells retained their viability in vitro and ability of adhesion to plastic. Cellular population was homogeneous and represented by small cells (d - 7 microm) with fibroblast-like morphology. Cells were positively stained with Abs for the Abs: CD29, CD44, CD49a, b, d, CD73, CD90, CD105, CD166, HLA ABC. Cells were negative for CD34, CD45--markers of hematopoietic cells, CD31--marker of endothelial cells, Stro-1, as well as for HLA DR, DP, DQ (flow cytometer analysis). Being induced to differentiate in vitro, the cells were able to differentiate into cells similar to cells of bone, adipose and cartilage tissue. Karyological assay of the cells isolated from human adipose tissue subjected to cold shock revealed diploid set of chromosomes, 46, XX, without aneuploidy and structural reconstructions of chromosomes. Thus, it has been established that, under extreme condition for the organism, the population of cells with a phenotype similar to miltipotent mesenchymal stromal cells is preserved in subcutaneous adipose tissue.

[Osteogenic differentiation of human multipotent mesenchymal stromal cells of bone marrow and adipose tissue].

Cellular populations with phenotype similar to multipotent mesenchymal stromal cells have been isolated from two different sources: a human bone marrow and adipose tissue. The comparative analysis of differentiation efficiency of these cells in the direction of osteogenesis revealed morphological changes confirmed by Alizarin red and von Kossa staining in bone marrow cells on the 14th day, and in adipose tissue cells on the 28th day of culturing in the medium with inductors. The analysis of osteopontin, osteocalcin and bone sialoprotein gene expression in RT-PCR reactions detected essential distinctions in the potency of these cells to differentiate into bone tissue cells.

[Differentiation of multipotent mesenchymal stromal cells of bone marrow into cells of cartilage tissue by culturing in three-demential OPLA scaffolds].

Bone marrow multipotent mesenchymal stromal cells represent a perspective material for engineering of human three-dimensional transplants of cartilage tissue. We are demonstrated the opportunity of the directed differentiation of BM MMSC in cells of cartilage tissue by culturing them in three-dimensional scaffolds, presented by polymer OPLA in medium with inductors of chondrogenesis. For loading cells in porous scaffolds used method which essence consist in saturation of polymeric blocks by cellular suspension with the subsequent centrifugal force of cells in scaffolds and culturing of engineering constructs for 28 days in chondrogenic medium. Histological analysis derived in vitro of three-dimensional transplants showed uniform distribution of cells in the matrix with morphologically distinct chondrocytes-like cells of hyaline cartilage. Immunohistochemical analysis detected aggrecan and collagen type II within the extracellular matrix. Preclinical the researches lead on a livestock of immunodeficient mice have shown not toxicity of the engineering constructs.

[Cultivation and transplantation of boar type A spermatogonia].

A cell population enriched with type A spermatogonia has been isolated from the boar testes. Cell types occurring during isolation were morphologically characterized, factors maintaining the cultured spermatogonia in the undifferentiated state were studied, and these cells were transferred to sterile recipients preliminarily treated with busulfan. The cells of spermatogenic epithelium cultivated in vitro for 24 h were used for transfer experiments. The transfer efficiency was estimated within 27 and 29 days according to the histological picture of the testes and the isolated cultures. Spermatogenic cells at various developmental stages and a few Sertoli ells and spermatozoa were found on sections and in cell suspensions. Sperm samples could be taken from recipient boars within nine months after the transfer. Microsatellite analysis of DNA showed the endogenous pattern of spermatogenesis. Thus, it was shown that spermatogenic donor cells can restore and maintain spermatogenesis of a recipient for at least 30 days. However, the donor cells were fully forced by the recipient reserve cells, type A0 spermatogonia, within eight to nine months.

[Characteristics of human mesenchymal stem cells isolated from bone marrow and adipose tissue].

Populations of human mesenchymal stem cells were derived from bone marrow and adipose tissue. Here analysis of six individuals is represented. Cells were isolated, expanded and evaluated by the expression of surface antigens using flow cytometry. These cells displayed similar characteristics for many markers. Cells isolated from bone marrow and adipose tissue were found to be homogeneously positive for CD13, CD44, CD90, CD105, and negative for CD45, CD34, CD31 and CD117. Besides, differences in surface antigene CD10 expression between narrow and adipose tissue-derived cells were detected. All these findings indicate that both bone marrow and adipose tissue are important sources of mesenchymal stem cells, which could be used in cell therapy protocols.

[Comparative analysis of cell populations with a phenotype similar to that of mesenchymal stem cells derived from subcutaneous fat].

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.

[The use of pluripotent mouse embryo stem cells for the production of chimeric animals]. / Ispol'zovanie pliuripotentnykh émbrional'nykh stvolovykh kletok myshi dlia polucheniia khimernykh zhivotnykh.

Embryonic stem (ES) cells derived from pluripotent cells of the early mouse embryos provide a powerful tool for genome manipulation in mammals. Conditions for maintaining and preservation of pluripotent properties of embryonic stem (ES) cells in culture using different tests are described. A simple aggregation of pluripotent ES cells with morulae-stage embryos for derivation of chimaeras is considered.

[A comparative analysis of the development of rabbit preimplantation embryos in vitro on different somatic-cell monolayers]. / Sravnitel'nyi analiz razvitiia preimplantatsionnykh émbrionov krolika in vitro na razlichnykh monosloiakh somaticheskikh kletok.

Conditions for isolation of pluripotent inner cell masses (ICM) from rabbit blastocysts are described. The rabbit intact embryos were collected on day 4 post coitus, zona pellucida was removed from embryos by pronase treatment, and rabbit blastocysts were plated on different monolayers of somatic cells. The differences in attachment and proliferation of ICM of rabbit embryos were determined in dependence on the feeder layer type on their long culturing in vitro. Among the tested monolayers of somatic cells a feeder layer of rabbit embryonic fibroblasts, derived from fetuses on day 16-18 pregnancy, appeared more effective for isolation of ICM and for receipt of rabbit embryonic stem cells (with ES-like morphology).

Changes in expression of surface and core antigens of hepatitis B virus in different mutant clones of hepatoma PLC-PRF-5 cells.

Mutant clones of human hepatocarcinoma PLC-PRF-5 cells carrying a hepatitis B virus (HBV) genome have been obtained using selection for resistance to the toxic action of a variety of preparations to induce cell differentiation. The clones differed in various features such as expression of alpha-fetoprotein (AFP) and albumin as well as in growth rates, ability to grow in semisolid media and to be cloned in agar. Expression of the surface antigen (HBsAg) was significantly increased in mutant cells exhibiting differentiation features in contrast to the parental cells. In addition, the core antigen (HBcAg), which was silent in the original cells, was detected in some clones. There was no temporal correlation between the peak of enhanced expression of HBsAg and activation of HBcAg observed at different life periods of each clone. Evidence of cell fusion in cell culture such as premature chromatin condensation and increased numbers of binucleate cells was detected in clones with differentiation features and an increased level of viral gene expression. The approach used in this study can be used to develop cell lines of the same origin with different degrees of differentiation whilst maintaining HBV expression. This model system may be useful in the study of HBV.

[Changes in the gene expression of the hepatitis B virus in PLC-PRF-5 cells of human hepatocarcinoma during their acquisition of drug resistance]. / Izmenenie ékspressii genov virusa gepatita B v kletkakh gepatokartsinomy cheloveka PLC-PRF-5 v protsesse priobreteniia imi lekarstvennoi ustoichivosti.

Mutant clones of hepatocarcinoma cell line PLC-PRF-5 containing integrated hepatitis B virus genome were derived by multistep selection for resistance to some toxic agents. These clones were found to display various stages of cell differentiation according to growth rate, ability to synthesize alpha-fetoprotein, to grow in semisolid medium and to cloning in agar. In the majority of the clones with higher stages of differentiation the expression of HBsAg gene was demonstrated to be increased as compared to the original line, and in some of these clones the expression of silent HBcAg was activated.