[BDNF secretion in human mesenchymal stem cells isolated from bone marrow, endometrium and adipose tissue].

The ability of mesenchymal stem cells (MSCs) to differentiate into neuronal lineage determines the potential of these cells as a substrate for a cell replacement therapy. In this paper we compare the neurogenic potential of MSCs isolated from bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was found that the native eMCSs, BMSCs and AD MSCs express neuronal marker ß-III-tubulin with a frequency of 90, 50 and 14%, respectively. We also showned that eMSCs have a high endogenous level of brain-derived neurotrophic factor (BDNF), whereas the BMSCs and the AD MSCs are characterized by low basal BDNF levels. As induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMF caused changes in the cells morphology, the increased expression of ß-III-tubulin, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. BDNF secretion during differentiation was significantly enhanced in the BMSCs and decreased in the eMSCs cultures. However, no correlation between the basal and induced levels of the neuronal markers expression and BDNF secretion in the studied MSCs has been established.

[Generation of dopamine neurons from human embryonic stem cells in vitro].

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

[Novel human embryonic stem cell lines C612 and C910].

Novel human embryonal stem cell lines C612 and C910 have been established from hatching blastocytes. Cells were cultivated in mTeST medium on mouse fibroblast feeder-layers. They express common pluripotent markers such as alkaline phosphatase, Oct 3/4, SEEA-4, Nanog, Rex1. Immunophenotyping of these cells by flow cytometry revealed expression of CD90 (Thy-1) and CD117 (c-kit) antigens and weak or no expression of CD13, CD34, CD45, CD130, HLA class I and HLA class II antigens. This pattern of surface antigen expression is common for human embryonic stem cells. G-banding assay of C612 and C910 metaphase plates showed that karyotypic structure of these cells was normal both in chromosome number and structure. The cells are pluripotent because of their capability to generate embryoid bodies, undergo spontaneous differentiation and express markers of all germ layers: nestin, keratin, vimentin (ectoderm), alpha-fetoprotein (entoderm), and muscle alpha-actinin (mesoderm). Thus, C612 and C910 cells have all attributes of typical human embryonic stem cells (diploid, capable of self-renewal, express pluripotent markers and differentiate into three germ layers) and may be of potential use for fundamental and regenerative medicine researches.

[Induction of apoptosis in murine myeloma cells NS0/1, transfected with the gene for the basic heat shock protein HSP70i]. / Induktsiia apoptoza v kletkakh myshinoi mielomy NS0/1, transfitsirovannoi genom osnovnogo belka teplovogo shoka BTSh70i.

Murine myelomas are rare cell variants deficient in inducible isoform of Hsp70 that protects cells from injury. In these cells Hsp70 is absent and is not induced under stress conditions. In this study myeloma cells NS0/1 were transfected with hsp70, and their susceptibility to apoptosis was challenged by serum deprivation or hydrogen peroxide. Expression of Hsp70 in NS0/1 cells made them more resistant to apoptosis in serum-free medium but did not affect their response to hydrogen peroxide. Hsp70 involvement in the protection of myeloma cells from apoptosis caused by different agents is discussed.

Accumulation of major stress protein 70kDa protects myeloid and lymphoid cells from death by apoptosis.

The major heat shock protein, hsp70, is known to contribute to the mechanisms of cell protection against a variety of stress and cytotoxic factors, providing an increase of cell survival. Whether hsp70 could be implicated in the rescue of cells from stress-induced death proceeding on apoptotic pathway is not well established. Here we report that susceptibility of myeloid and lymphoid cell lines to apoptosis induced by heat shock or ethanol coincides with hsp70 content and can be modulated by changes in expression of this protein. Cells of lymphoid and myeloid lines differing in basal and inducible level of the protein were tested. The cells containing higher amounts of hsp70 (U937, Jurkat, Molt4) were more resistant to the apoptosis-inducing stimuli then cells which accumu-late lower amounts of the protein (HL60) and especially those lacking the protein (NSO). Inhibition of hsp70 accumulation by quercetin made cells more susceptible to the same apoptotic inducer. Enhancement of hsp70 expression by previous heating or by liposomal delivery of the exogenic protein to the cells lacking hsp70 made them more resistant to apoptosis. The possible mechanisms of the hsp70 protective effect in apoptosis are discussed.

[A cytogenetic study of the Chinese hamster lung cell line V-79 infected with Mycoplasma arginini and decontaminated using ciprofloxacin]. / Tsitogeneticheskoe issledovanie linii kletok legkogo kitaiskogo khomiachka V-79, infitsirovannoi mikoplazmoi Mycoplasma arginini i dekontaminirovannoi s pomoshch'iu tsiprofloksatsina.

Karyotypic variability has been studied in a line of the Chinese hamster cells artificially contaminated with Mycoplasma arginini. The contaminated cultures differed from mycoplasma-free cells in cell distribution for chromosome number. The frequency of cells with modal chromosome number 21 decreased, while that of cells with 20 chromosomes increased. Decontamination of cell culture with ciprofloxacin (10 mg/ml) and a subsequent cultivation of cell in the antibiotic-free medium did not restore the original cell distribution for chromosome number. In-53--131 days after infection, the increased frequency of chromosomal aberrations was registered. Eradication of Mycoplasma by ciprofloxacin and a long-term cultivation in antibiotic-free medium restored the frequency of chromosomal aberrations to the control level corresponding to mycoplasma-free cultures. In contaminated cultures no cyto- or genotoxic effect of ciprofloxacin was observed. These data, together with the previous ones, enable the authors to recommend ciprofloxacin to make cell cultures free from mycoplasmas with minimal risk to change the original properties of cell lines.

[The chromosome variability of the Indian muntjac in somatic cell hybrids]. / Izmenchivost' khromosom indiiskogo muntzhaka v gibridakh somaticheskikh kletok.

Hybrids were produced between the Indian muntjak fibroblasts and rat Jensen sarcoma cell line (JF1) auxotrophic for asparagine. They were selected without cloning under conditions providing survival of parental Indian muntjak and hybrid cells. This allowed to compare the Indian muntjak chromosome variability in the parental cells and hybrids under identical culture conditions. The frequency of muntjak chromosome aberrations proved to de higher in the hybrids (up to 47%) than in the parental cells (6.5%). Predominant are chromosomal breaks and dicentrics. The latter are mainly formed by fusion of chromosomes 1 and 2. The most fragile are 1 and X-chromosomes. Chromosomal breaks are evenly distributed along chromosome 1, and "hot" points are observed in X-chromosome. Possible mechanisms of the Indian muntjak chromosome rearrangements induced by somatic cell hybridization are discussed.

[Monoclonal antibodies to the muscle isoform of alpha-actinin--a marker for the study of the differentiation of skeletal and cardiac muscles]. / Monoklonal'nye antitela k myshechnoi izoforme al'fa-aktinina--marker dlia izucheniia differentsirovki skeletnoi i serdechnoi myshts.

A battery of monoclonals to the rabbit skeletal muscle alpha-actinin has been produced. The majority of monoclonals proved to be species-specific by indirect immunofluorescence on the isolated rabbit skeletal myofibrils and on the differentiating cultures of chicken and rat skeletal muscles. One monoclonal, EA-53, reacts with the skeletal muscle alpha-actinin of various species (rat, rabbit, chicken) in immunofluorescence and immunoblotting. The monoclonal EA-53 recognizes also heart muscle alpha-actinin in cultured cardiomyocytes of human, rat and mouse origin. EA-53 does not stain alpha-actinin in myoblasts, fibroblasts, and endothelial cells. The monoclonal antibody EA-53 discriminating muscle and nonmuscle alpha-actinin isoforms could be used as a tool to study the mechanisms of skeletal and cardiac myogenesis.

[Intraspecies identification of cultured murine cells by using H-2 antigen typing]. / Vnutrividovaia identifikatsiia kul'tiviruemykh kletok myshi s pomoshch'iu tipirovaniia H-2-antigenov.

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.

[Cytogenetic changes in an explanted mouse rhabdomyosarcoma during prolonged cultivation]. / Tsitogeneticheskie izmeneniia éksplantirovannoi rabdomiosarkomy myshi pri dlitel'nom kul'tivirovanii.

Quantitative and qualitative chromosome rearrangements, dynamics of distribution of double-minute chromosomes (DMs), and morphological characteristics of tumor rhabdomyoblasts MH-82 during explantation and following in vitro cultivation are analysed. Cells of the 13th and 27th passages of cultivation were characterized by the epithelial type of growth, although their form and size varied. Chromosome analysis of tumor rhabdomyoblasts was carried out on passages 4, 14, 20, 25 and 30 of in vitro cultivation. The modal class with 53-55 chromosomes was established within 20 passages. Heterogeneity of cell population in concern to the chromosome number and content of hypotetraploid cells (72-78) diminished during cultivation. Chromosome rearrangements (marker chromosomes) in hyperdiploid and hypotetraploid cell subpopulations differed. The number of cells with DMs and the number of DMs per cell decreased till the full disappearance by the 30th passage. It is concluded that the establishment of the MH-82 cell line was completed up to the 30th passage of cultivation.

[Mycoplasma contamination of cell cultures: methods for its detection and the possible routes of Mycoplasma infection spread]. / Kontaminatsiia kletochnykh kul'tur mikoplazmami: metody obnaruzheniia i vozmozhnye puti rasprostraneniia mikoplazma-infektsii.

83 continuous cell lines were screened for mycoplasma contamination by three methods: microbiological seeding for enriched media, staining with Hoechst-33258 stain, and autoradiography with 3H-thymidine incorporation. It has been shown that combination of different methods is necessary for the strict control of mycoplasma-contamination in cell cultures. Simultaneous manipulation with mycoplasma infected and pure cell lines leads to cross-contamination in 2-3 passages. Precautions are described to preclude mycoplasma-contamination during prolonged cell cultivation.

[Expression of the traits characterizing the cell surface in hybrids of malignant and nonmalignant cells]. / Ekspressiia priznakov, kharakterizuiushchikh kletochnuiu poverkhnost', v gibridakh malignizirovannykh i nemalignizirovannykh kletok.

Hybrids between non-malignant mouse 3T3 cells and highly malignant rat JF1 cells were selected in semisolid agar medium without asparagine: 3T3 cells do not grow in agar; JF1 cells required asparagine for their growth. Some surface properties: agglutination by Con A, saturation density, electrophoretic mobility and behaviour in the two-phase aqueous polymer system dextran-polyethylenglycol, -have been studied in the parental cells and in two their hybrids (H9-1 and H9-2). JF1 cells differ from 3T3 cells in their behaviour in two phase polymer system by lower electrophoretic mobility, by higher agglutinability with Con A and higher saturation density. H9-1 and H9-2 hybrids behave in two phase polymer system similar to 3T3 cells, they have low saturation density, intermediate electrophoretic mobility and intermediate agglutinability by Con A. These results indicate that in hybrids between non-malignant 3T3 cells and malignant JF1 cells selected under conditions (semisolid agar) favorite for the growth of oncogenic cells, surface properties of malignant parents are not dominated. Both hybrids are phenotypically similar with drastic genetical difference. By means of C-banding it has been shown that H9-2 hybrid contain approximately one 3T3 and one JF1 genome; H9-1 hybrid contains two JF1 genomes and about 15% of 3T3 genome.

[Characteristics of "photo depletion" of the green fluorescence of tumor cells]. / Ob osobennostiakh "fotovygoraniia" zelenoi fluorestsentsii nekotorykh opukholevykh kletok.

The green (oxidized flavoproteins) fluorescence intensity was found to increase during investigation of NADH and oxidized flavoproteins fluorescence with the use of optimal excitation of different fluorescence bands. This effect was observed under excitation with blue light (436 nm). It is suggested that in some malignant cells, the structure of flavoproteins (probably of mitochondrial ones) may be altered in the way of increasing the quantum yield under the action of light irradiation.