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The complexity of the pathogenesis and insufficient knowledge of the equine infectious anemia virus (EIAV) make it necessary to find an adequate laboratory model for studying the infectious process and the immune response. One of the ways to solve this problem is the production of cell cultures with the desired properties using genetic transformation. The aim of the present study was to obtain mouse embryonic fibroblasts with genes of the receptor of the equine infectious anemia virus (ELR1) and cyclin T1 (eCT1). For this purpose, cells were transfected with pcDNA3.1-ELR1 and pcDNA3.1-cycT1 plasmids containing nucleotide sequences of the target genes using the calcium-phosphate method. Eight stable genetically transformed clones of STO cells with [ELR1eCT1neo]+ phenotype were obtained using the selection of cells on G418. The frequency of genetic STO transformation was 2.8 × 10–5. The integration of genes into the cell genome was detected by PCR using gene-specific primers with subsequent nucleotide sequencing using DNA isolated from cells as the matrix. The expression of equine ELR1 and eCT1 genes in mouse fibroblasts was confirmed at the transcriptional level by the presence of the mRNA of the target genes in the RT-PCR reaction. The obtained stable STO cell line was deposited in the cell culture collection of the All-Russia Scientific Research Institute of Experimental Veterinary Medicine Federal Scientific Center.

Multipotent mesenchymal stromal cells (MMSCs) isolated from human adipose tissue have been subcultured for over 50 population doublings. In three cultures that experienced more than 50 cellular doublings, there appeared cells lacking contact inhibition upon reaching the monolayer. Karyotype analysis (GTG banding) showed a normal diploid karyotype, and aneuploidy and restructurings were not registered. Flow-cytometric analysis of 20 surface antigens on MMSCs in early and late passages revealed changes in the share of cells positively stained with antibodies against CD10 (a zinc depended metalloproteinase); CD34 (sialomucin); CD49 a, d, f, (α1, α4, α6, integrins); and CD71 (a transferrin receptor). Long-term cultivation influenced cell adhesion to proteins of the extracellular matrix (ECM), such as fibronectin and laminin (ligands to α, integrins), as well as the functional abilities of MMSCs to form cells of adipose and bone tissues in vitro. These findings extend our knowledge of cell behavior in culture and allow us to get closer to a deeper understanding of the processes happening to precursor cells in vitro.

Properties and characteristics of multipotent mesenchymal stem cells (MMSCs) isolated from equine umbilical cord’s blood in vitro have been studied. The cells had morphology similar to fibroblast, high adhesive capabilities, and immunophenotype CD31–, CD34–, CD90+. Analysis of sensitivity of MMSCs to the equine infectious anemia virus (EIAV) was conducted. The immunocytochemical analysis detected expression of the p26 antigen in MMSCs that are being infected with EIAV. The results obtained show that MMSCs isolated from equine umbilical cord blood can be applied in veterinary virology.

Macrophages derived from pluripotent stem cells represent an attractive cellular system for the study of lentiviruses. The possibility of obtaining cells with a phenotype similar to macrophages in the process of differentiation of mouse embryonic stem cells (ESCs) in the presence of the conditioned medium (CS) from bone marrow-derived stromal cells or interleukin 3 (Il-3) and macrophage colony-stimulating factor (M-CSF) was demonstrated. The results of flow cytofluorimetry showed that the replacement of CS with recombinant cytokines in induction media contributed to an increase in the number of cells positively stained antibodies (AB) against AG CD34 by 8% and CD45 by 20%. When seeding such cells in a methylcellulose medium supplemented with CS or a mixture of Il-3 and M-CSF, the formation of colonies with different morphology, including monocyte/macrophage, was observed with an efficiency of 0.11 % and 0.33%, respectively. The percentage of positively stained cells AB against F4/80 AG was 8±0.02 %. The cells demonstrated the ability to non-specific phagocytosis of latex particles. Thus, it is shown that it is possible to obtain cells with a phenotype similar to macrophages from mouse ESCs during differentiation, and the advantage of using two recombinant cytokines for this purpose is established.

Integrin expression during long-term cultivation in multipotent mesenchymal stromal cells (MMSCs) isolated from human subcutaneous adipose tissue was studied. It was found that expression of α1, α4 and α6 integrins at the 2nd and 17th passages was different. The cell number positively stained with antibodies against CD49a (α1 integrin) and CD49d (α4 integrin) dropped to 87.2 and 11.2%, respectively. On the contrary, the cell number stained with antibodies against CD49f (α6 integrin) and CD49b (α2 integrin) increased during long-term cultivation by 9.9 and 2.3%, respectively. The high expression of β1 integrin (98%) did not significantly change during MMSC cultivation. Induction of MMSC to osteogenic differentiation (formation of bone tissue cells and extracellular matrix) was more efficient at the 2nd than the 17th passage. Thus, long-term MMSC cultivation required for cell-mass accumulation affects the cell functions. It should be considered for their application in cellular technology, including tissue modeling in the three-dimension scaffold.

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 β1 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.

A correct formulation of the problem of the structure and stability of relaxation waves is given for arbitrary Lewis (Le) and Prandtl (Pr) numbers. The high activation-energy approximation in which the reaction to heating zone length ratio is assumed to be small is analyzed in detail.