Induction of Expression of CD271 and CD34 in Mesenchymal Stromal Cells Cultured as Spheroids.

Cultured mesenchymal stromal cells (MSCs) are cells that can be used for tissue engineering or cell therapies owing to their multipotency and ability to secrete immunomodulatory and trophic molecules. Several studies suggest that MSCs can become pericytes when cocultured with endothelial cells (ECs) but failed to use pericyte markers not already expressed by MSCs. We hypothesized ECs could instruct MSCs to express the molecules CD271 or CD34, which are expressed by pericytes in situ but not by MSCs. CD271 is a marker of especial interest because it is associated with multipotency, a characteristic that wanes in MSCs as they are culture expanded. Consequently, surface expression of CD271 and CD34 was detected in roughly half of the MSCs cocultured with ECs as spheroids in the presence of insulin-like growth factor 1 (IGF-1). Conversely, expression of CD271 and CD34 was detected in a similar proportion of MSCs cultured under these conditions without ECs, and expression of these markers was low or absent when no IGF-1 was added. These findings indicate that specific culture conditions including IGF-1 can endow cultured MSCs with expression of CD271 and CD34, which may enhance the multipotency of these cells when they are used for therapeutic purposes.

Cytotoxic, genotoxic and mutagenic evaluation of surface waters from a coal exploration region.

Coal mining generates a considerable amount of waste, which is disposed of in piles or dams near mining sites. As a result, leachates may reach rivers and streams, promoting the wide dispersion of contaminants in solution and as particulate matter. The present study evaluated the cytotoxic, genotoxic, and mutagenic action of surface waters collected around a thermoelectric power plant and the largest mining area in Brazil (Candiota). Four sites in Candiota stream were selected, and samples were collected in winter and summer. Water samples were analyzed using the comet and CBMN assays in V79 and HepG2 cells. Furthermore, genotoxicity of water samples was evaluated in vivo using the SMART in Drosophila melanogaster. In addition, polycyclic aromatic hydrocarbons and inorganic elements were quantified. The results indicate that water samples exhibited no genotoxic and mutagenic activities, whether in vitro or in vivo. On the other hand, surface water samples collected in sites near the power plant in both summer and winter inhibited cell proliferation and induced increased frequencies of V79 cell death, apoptosis, and necrosis. The cytotoxicity observed may be associated with the presence of higher concentration of inorganic elements, especially aluminum, silicon, sulfur, titanium and zinc at sites 1 and 2 in the stream, as well as with the complex mixture present in the coal, in both seasons. Therefore, the results obtained point to the toxicity potential of water samples with the influence of coal mining and combustion processes and the possible adverse effects on the health of exposed organisms.

Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro.

Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.

Mesenchymal stem cells and their relationship to pericytes.

Our body contains cells that can be propagated in vitro and give rise to cells with mature mesenchymal phenotypes. These cells are interesting not only because of their differentiation capability, which could be used for tissue engineering, but also because they secrete molecules which have trophic, chemoattractant, and immunomodulatory properties. Along decades of study, these cells have been referred to as fibroblastic cells, stromal cells, or mesenchymal stem cells. There is evidence that pericytes, cells that wrap endothelial cells in blood vessels, behave as stem cells in the tissues, and give rise to these progenitor cells when removed from the body and expanded in culture - a process that may reflect changes that occur in vivo under injury conditions. Here, we discuss the evidence that favors this thesis, and discuss culture methods, clinical and preclinical applications of mesenchymal stem cells under this perspective.

Stability of Reference Genes during Tri-Lineage Differentiation of Human Adipose-Derived Stromal Cells.

Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), down-regulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable.

Genotoxicity testing of combined treatment with cisplatin, bleomycin, and 5-fluorouracil in somatic cells of Drosophila melanogaster.

The simultaneous treatment with the cross-linking agent cisplatin, the radiomimetic antitumoral drug bleomycin, and the anti-metabolite drug 5-fluorouracil has been used as a regimen to treat patients with squamous cell carcinoma of the head and neck. Considering that these drugs interact directly with DNA, one of the important late-occurring complications from treatment of primary malignancies is the therapy-related secondary cancers as a result of the genotoxic activity of the drugs on normal cells. In this sense, the genotoxicity of this combination was evaluated using the wing somatic mutation and recombination test in Drosophila melanogaster. The mutant spots observed in marker-heterozygous and balancer-heterozygous flies were compared in order to quantitatively and qualitatively estimate the genotoxic effect of these drugs. Cisplatin (0.003 and 0.006mM), bleomycin (0.005 and 0.01mM), and both combinations preferentially induced recombinational events, while mutation is the major event regarding the genetic toxicity of 5-fluorouracil (0.025 and 0.05mM). The combination of these drugs produced synergistic and antagonistic genotoxic effects, depending on the concentrations used, which could impose a higher risk of secondary effects associated with their genotoxic effects, emphasizing the importance of long-term monitoring in patients being treated with these drugs.

Evaluation of the genotoxicity of cisplatin, paclitaxel and 5-fluorouracil combined treatment in the Drosophila wing-spot test.

The somatic mutation and recombination test in Drosophila melanogaster was applied to analyze the mutagenic and recombinagenic activity of the chemotherapeutic drugs cisplatin, paclitaxel, and 5-fluorouracil, comparing the effects observed in combinatory treatments with those observed in single administrations. The results obtained in two different genotypes allowed to quantitatively and qualitatively estimate the contribution of genotoxic effects. The results obtained with the individual drug treatments showed that cisplatin and 5-fluorouracil were genotoxic, being able to increase the frequency of total spots on both genotypes. While cisplatin preferentially induced DNA damage of recombinational origin, all the damages induced by 5-fluorouracil were caused by gene and/or chromosome mutations, and the aneuploidogenic compound paclitaxel was not genotoxic. The combination of these drugs does not exert a synergist genotoxic effect in both genotypes compared to the single-agent administration. Instead, it was observed a modification in the proportion of mutation and recombination to the final genotoxicity observed. The antiproliferative activity of PAC could be responsible for the non-synergic genotoxic effect observed. Based on our results it is possible to suggest that cisplatin/paclitaxel/5-fluorouracil treatment regimen cannot impose a higher risk of the development of genotoxicity-associated secondary tumors in comparison to their individual applications.

Mutagenic evaluation of combined paclitaxel and cisplatin treatment in somatic cells of Drosophila melanogaster.

Recent studies have added paclitaxel (PAC) to traditional cisplatin (CIS) regimen to treat squamous cell carcinoma of the head and neck. The target of these antineoplastic agents is nuclear DNA for CIS and microtubules for PAC, although it is not restricted to malignant cells. In this study, the genotoxicity of the combined treatment of PAC and CIS was investigated using the standard version of the wing Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Quantitative and qualitative genotoxic effects of these compounds were estimated by comparing wing spot frequencies in marker-heterozygous to balancer-heterozygous flies. Two different concentrations of PAC (0.0025 and 0.005mM) and CIS (0.025 and 0.05mM) as well as combinations of them were employed. The results demonstrated that the spindle poison PAC alone was not genotoxic in this test system, while CIS was able to induce a high incidence of DNA damage in both genotypes, mainly related to somatic recombination. The data obtained for the combined treatments showed that its genotoxicity varied with the concentrations used. In small concentrations the number of total spots induced by combination was reduced in relation to CIS 0.025mM just for marker-heterozygous flies, showing that somatic recombination was the prevalent event involved. At higher concentrations the combined treatment showed significant reductions in the frequencies of large single spots, for both genotypes, and twin spots for marker-heterozygous flies, but did not significantly reduce the total spots frequency in either genotype. The data suggest that aneugenic activity of PAC could be responsible for the reduction in the genotoxicity of CIS.