Effect of Storage Conditions on the Integrity of Human Umbilical Cord Mesenchymal Stromal Cell-Derived Microvesicles.

We studied the effect of storage conditions on the safety of microvesicles produced by human multipotent umbilical cord mesenchymal stromal cells into the conditioned medium. It was found that microvesicles can be stored without serious degradation for up to 1 week at 4°С, but were almost completely destroyed during freezing and thawing cycles irrespective of the storage temperatures (-20°С, -70°С, or -196°С). Similar results were obtained for lyophilized medium conditioned by human multipotent umbilical cord mesenchymal stromal cells.

Comparative Analysis of Secretome of Human Umbilical Cord- and Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells.

Production of cytokines and growth factors by cultured human umbilical cord tissue- and bone marrow-derived multipotent mesenchymal stromal cells was measured by multiplex analysis. In most cases, the concentrations of bioactive factors in the culture medium conditioned by umbilical cord-derived cells was ten- to hundred-times higher than in the medium conditioned by bone marrow-derived cells. These results suggest that both multipotent mesenchymal stromal cells from the umbilical cord and cell-free products can have more pronounced therapeutic effect in comparison with mesenchymal stromal cells obtained from "adult" sources.

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Microvesicles Express Surface Markers Identical to the Phenotype of Parental Cells.

Production of microvesicles in culture of human umbilical cord multipotent mesenchymal stromal cells was studied and comparative analysis of the expression of some surface molecules (clusters of differentiation, CD) was performed. It was found that the mesenchymal stromal cells produce microvesicles in the amount sufficient for their detection by flow cytometry. Parallel analysis of the phenotypes of maternal mesenchymal stromal cells and secreted microvesicles revealed identical expression of surface molecules CD13, CD29, CD44, CD54, CD71, CD73, CD90, CD105, CD106, and HLA-I.

Human Umbilical Cord Mesenchymal Stromal Cells Support Viability of Umbilical Cord Blood Hematopoietic Stem Cells but not the "Stemness" of Their Progeny in Co-Culture.

Cell-cell interactions and the ability of mesenchymal stromal cells to support the expansion of hematopoietic progenitor cells were studied in co-culture of human umbilical cord tissue-derived mesenchymal stromal cells and nucleated umbilical cord blood cells. It was found that hematopoietic stem cells from the umbilical cord blood are capable to adhere to mesenchymal stromal cells and proliferate during 3-4 weeks in co-culture. However, despite the formation of hematopoietic foci and accumulation of CD34 and CD133 cells in the adherent cell fraction, the ability of newly generated blood cells to form colonies in semi-solid culture medium was appreciably reduced.

Cytokine Production in Mixed Cultures of Mesenchymal Stromal Cells from Wharton's Jelly and Peripheral Blood Lymphocytes.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.

Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

Expression of Surface Molecules in Human Mesenchymal Stromal Cells Co-Cultured with Nucleated Umbilical Cord Blood Cells.

We studied the expression of different classes of surface molecules (CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR) in mesenchymal stromal cells from human umbilical cord and bone marrow during co-culturing with nucleated umbilical cord blood cells. Expression of the majority of surface markers in both types of mesenchymal stromal cells was stable and did not depend on the presence of the blood cells. Significant differences were found only for cell adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) responsible for direct cell-cell contacts with leukocytes and only for bone marrow derived cells.

Changes in Cell Composition of Umbilical Cord Blood and Functional Activity of Hematopoietic Stem Cells during Cryogenic Storage and Repeated Freezing/Thawing Cycles.

We analyzed changes in cell composition of umbilical cord blood and functional activity of hematopoietic stem cells during cryogenic storage and after repeated freezing/thawing cycles. It was found that repeated freezing/thawing cycles performed according to the optimal programmable freezing protocol did not significantly affect viability and functional activity of hematopoietic stem cells. When fast freezing program was used, the cells completely lost their capacity to form colonies in semisolid medium, despite high viability parameters in the test with 7-AAD.

Isolation of Multipotent Mesenchymal Stromal Cells from Cryopreserved Human Umbilical Cord Tissue.

Umbilical cord stroma is an easily available, convenient, and promising source of multipotent mesenchymal stromal cells for regenerative medicine. Cryogenic storage of umbilical cord tissue provides more possibilities for further isolation of multipotent mesenchymal stromal cells for autologous transplantation or scientific purposes. Here we developed a protocol for preparation of the whole umbilical cord tissue for cryogenic storage that in combination with the previously described modified method of isolation of multipotent mesenchymal stromal cells allowed us to isolate cells with high proliferative potential, typical phenotype, and preserved differentiation potencies.

Optimized Protocol for Isolation of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord.

Extraembryonic tissues, in particular, umbilical cord stroma are promising sources of multipotent mesenchymal stromal cells for regenerative medicine. In recent years, methods for isolation of mesenchymal stromal cells from different compartments of the umbilical cords based on enzymatic disaggregation of the tissue or on tissue explants have been proposed. Here we propose a protocol of isolation of multipotent mesenchymal stromal cells from the whole umbilical cord that combines the advantages of each approach and ensures sufficient cell yield for further experimental and clinical applications.

Effect of γ-irradiation on expression of tight and adherens junction protein mRNA on in vitro blood-brain barrier model.

We studied the effect of γ-irradiation on HUVEC endothelial cells co-cultured with allogeneic astrocytes. This 2D in vitro model of the blood-brain barrier has the same parameters as cerebral microvascular endothelial cells forming the blood-brain barrier and allows reproducing its functions in vivo. Dose-dependent changes in cell morphology and violation of monolayer integrity were observed.

Generation of recombinant extracellular fragment of vascular endothelial growth factor receptor 2 and specific monoclonal antibodies to this receptor.

cDNA extracellular Ig-like domains I-III of vascular endothelial growth factor receptor 2 (VEGFR2) was cloned in an expressing vector pET_32a. Western blotting showed immunochemical identity of recombinant VEGFR2I-III produced by prokaryotic expression system to the native receptor. BALB/c mice were immunized with VEGFR2I-III for obtaining specific antibodies to VEGFR2.

Prevalence of borreliosis, anaplasmosis, ehrlichiosis and Dirofilaria immitis in dogs and vectors in Voronezh Reserve (Russia).

Most of the dogs studied for the prevalence of CVBD have previously received acaricidal and insecticidal treatments. In the present work, a very specific population of dogs (Group 1) that had never been treated against ticks and mosquitoes was studied. Moreover, the territory occupied by this population has also never been treated, because it is a protected area--Voronezh Natural Reserve.

Computer evaluation of drug interactions with P-glycoprotein.

The (Q)SAR models for evaluating the structure-property relationships, fit for prediction of drug interactions with P-glycoprotein as inhibitors or substrates, were constructed using PASS and GUSAR software. The models were constructed and validated on the basis of information on the structure and characteristics of 256 and 94 compounds used as P-glycoprotein substrates and inhibitors, respectively. The initial samples were divided 80:20 into training and test samples.

Expression of tight junction proteins by umbilical vein epithelial cells co-cultured with allogenic astrocytes.

We studied expression of tight junction proteins and formation of the barrier properties in the culture of umbilical vein endothelial cells under conditions of co-culturing with allogenic GFAP-positive astrocytes. This culturing significantly increases of expression of tight junction proteins (claudin-5, occludin, and ZO-1). The formation of tight junctions significantly increased transendothelial resistance and reduced permeability for sodium fluorescein.

[Fundamental and applied aspects of the hematoencephalic barrier research].

The results of fundamental and applied studies of blood-brain barrier had been conducted by authors during the last 10 years are summarized in the publication. The molecular anatomy of barrier microvessels, as well as promising markers of BBB and other proteins involved in barrier functions are discussed. Via in vitro experiments with endothelial cells of cerebral microvessels we characterized the basic conditions required for adequate BBB modeling.

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells.

Activation of expression of brain-derived neurotrophic factor at the site of implantation of allogenic and xenogenic neural stem (progenitor) cells in rats with ischemic cortical stroke.

Ischemic stroke was modeled in the sensorimotor zone of the brain cortex in adult rats. Rat embryonic nervous tissue, neural stem cells from human olfactory epithelium, and rat fibroblasts (cell control) were implanted into the peri-infarction area of rats of different groups immediately after stroke modeling. Expression of BDNF mRNA was analyzed 7 days after surgery by real-time PCR.