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Background: Bone marrow mesenchymal stem cells [BM-MSCs] have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs [ESC-MSCs] as an alternative for BM-MSCs. Some of the therapeutic effects of the ESCMSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome.Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs [hESC-MSCs] spheroids on secretion of IL-1Beta, IL-10, and tumor necrosis factor Alpha [TNF-Alpha] from lipopolysaccharide [LPS]-induced peripheral blood mononuclear cells [PBMCs]
Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were nonadherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation
Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-Alpha [301.7 +/- 5.906, p < 0.0001] and IL-1 Alpha [485.2 +/- 48.38, p < 0.001] from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome [TNF-Alpha : 166.6 +/- 8.04, IL-1Beta: 125.2 +/- 2.73]
Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs
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Background: Mesenchymal stem cells [MSCs] are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells [ESCs] and bone marrow cells after hypoxia and normoxia preconditioning
Methods: ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs [bone marrow-derived mesenchymal stromal cells], which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell [PBMC] assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs
Results: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium
Conclusions: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BMMSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor alpha, which needs further investigation
Тема - темы
Humans , Human Embryonic Stem Cells , Culture Media, Conditioned/pharmacology , Cell Hypoxia/physiology , Blood Cells , Leukocytes, Mononuclear/physiology , Interleukin-10/metabolismРеферат
Background and Objectives: Targeting transgene carriers and vectors to individual cells and tissues is one of the most important goals of gene therapy. Bacteriophages are of appropriate transgene carriers and there are different methods for their targeting to target cells. Present study reports preparation of targeted M13-based bacteriophage particles by a chemical coupling strategy
Materials and Methods: First, the pCMV-Script-GFP construct was produced via in vivo excision protocol from lambda-GFP Phage particles using ExAssist helper phage and XLOLR as specific host. Then, M13 phage particles bearing GFP [M13-GFP] were obtained by single stranded rescue using R408 helper phage. The human holotransferrin molecules were then coupled to the surface of phage particles by reductive amination chemistry. Transferrin molecules bind to the surface of phage particles were studied by phage-ELISA
Results: Phage-ELISA tests showed that holotransferrin molecules were coupled to the surface of M13 phage particles in a correct way and the transferrin-targgeted M13 phage particles were prepared. Further analysis showed that about 485 transferrin molecules coupled per phage particle
Conclusion: The results show that chemical coupling might be considered as a suitable strategy for targeting of M13 particles via coupling of targeting molecules in high density to the phage surface
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The aim of this study was to investigate relation between H-ras T81C polymorphism and some of the important risk factors in gastric adenocarcinoma [GA]. GA is one of the leading causes of cancer death in most countries. RAS gene is an important member in the PI3K-AKT signaling and the single nucleotide polymorphism at H-rasc DNA position 81 has been demonstrated has an important role in tumor genesis. In this study, we carried out single-nucleotide polymorphism analysis in an Iranian population. A total of 100 patients with gastric adenocarcinoma and 100 controls were examined for the presence of T81C H-ras polymorphism using PCR- RFLP assay. Statistical analysis revealed no relationship significant between TT, TC, CC and risk of GA, but, there was a poorly relation between male patient with C-carrier genotype and increasing risk of GA [P=0.07]. Also, we investigate effect of four important risk factors for GA. There was a statistically significant difference between increasing of age and susceptibility for GA [OR-1.106, 95%CI=1.073-1.139, P<0.001]. We observed a statistically significant between smoking and T81C polymorphism C-carrier genotypes [ORX3.98, 95%CI=1.831-8.68, P<0.001] as this individual had three-time risk for GA. We did not show a significant association between three main genotypes and H. pylori infection for risk of GA. These results suggested that there is no relationship between T81C-HRAS polymorphism and gastric cancer risk in Iranian patients. But, gender [male in our study] and the other risk factor described above have an important role in developing of GA
Тема - темы
Humans , Female , Male , Stomach Neoplasms/genetics , Adenocarcinoma , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthРеферат
Salivary gland tumors [SGT] are rare lesions with uncertain histopathology. One of the major signaling pathways that participate in the development of several tumors is protein kinase A. In this pathway, glycogen synthase kinase [3 [GSK3p] and cAMP responsive element binding protein [CREB3] are two genes which are supposed to be down regulated in most human tumors. The expression level of the genes was evaluated in SGT to scrutinize their possible under expression in these tumors. Methods: Forty eight fresh tissue samples were obtained from patients with benign and malignant SGT, including pleomorphic adenoma, warthin's tumor, mucoepidermoid carcinoma [MEC], salivary duct carcinoma and carcinoma ex pleomorphic adenoma. Eight normal samples were used as controls. Quantitative real-time PCR was used to analyze the expression level of interest genes. Data was analyzed by statistical methods. GSK3P was downregulate in all samples and all results were statistically significant [P<0.05]. CREB3 did not show a significant decrease or increase in its mRNA expression, but the results were significant in MEC and salivary duct carcinoma. GSK3p down regulation has been reported in many human tumors. This gene stimulates CREB3, inducing cell proliferation and oncogenesis. Our findings showed GSK3P down regulation; however, CREB3 expression level was close to normal group. No association between CREB3 expression and inactivated GSK3P could be postulated in SGT
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The purpose of this study was to investigate the possible influence of age and gender on association between - 765G > C COX-2 genetic polymorphism and gastric adenocarcinoma risk in Iranian patients. The promoter polymorphism of COX-2 gene -765G>C has been described to play an important role in many cancers such as gastric cancer. We carried out single-nucleotide polymorphism analysis in Iranian samples including 91 patients and 91 control normal using PCR-RFLP technique. Statistical analysis revealed no significant association between GG, GC and CC genotypes and risk of gastric adenocarcinoma. However differences were considered significant [P=0.043] for female subjects with C carrier genotypes [GC and CC] and gstric adenocarcinoma when compared with male patients [P=0.645] and control groups [P=0.653]. Also, there was a statistically significant difference between increasing of age and susceptibility for gastric adenocarcinoma [Odd Ratio = 1.125, 95% CI=1.089-1.162]. These results suggested that Iranian C carrier females can be more susceptible for gastric adenocarcinoma in comparison with control group. Also increasing of age should be considered as a risk factor for this disease
Тема - темы
Humans , Male , Female , Age Factors , Sex Factors , Polymorphism, Genetic , Stomach Neoplasms/genetics , Case-Control Studies , Risk FactorsРеферат
Increased pressure of oxygen in cell culture condition in comparison with in vivo, leads to changes in expression of some genes and subsequently abnormality in some functions of cells. The aim of this study was to investigate the effect of in vitro%1 acute hypoxia on the expression of connexin 43 and CXCR4 in human bone marrow derived mesenchymal stem cells. After culturing, a group of stem cells were treated under%1 acute hypoxia for 4, 8, 16, 24 and 48 hours. In another group [Hypoxia/Re-oxygenation], the cells were exposed to normoxia condition for 8 hours following mentioned hypoxia treatment. In each case, RNA was extracted and subsequently cDNA synthesised and then the expression of connexin 43 and CXCR4 genes were compared with normoxia group using real-time PCR technique. Connexin 43 gene expression significantly increased at 4, 8 and 16 hours of hypoxia. This increase in CXCR4 expression was observed in only 16 hours hypoxia. In Hypoxia/Re-oxygenation group, although Connexin 43 gene expression was greatly increased at 4 and 8 hours, CXCR4 gene expression showed no significant change compared with normoxia group. Based on these results, O2 concentration, time of exposure to O2 and and type of it's administration play important roles in the expression of CXCR4 and Connexin 43 in human bone marrow derived mesenchymal stem cells. Regarding the main role of oxygen in expression of these genes, it would be necessary to optimise the O2 concentration to reach the maximum expression in each gene in these cells
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Considering the increasing use of mesenchymal stem cells isolated from various sources in the clinic, the basic studies needed to evaluate proliferation, differentiation and other biological characteristics of them done. In the present study, efficiency and power differentiation in five different categories mesenchymal stem cells are compared with each other. Bone marrow mesenchymal stem cells from rabbits, rats, C57 mice, chicken and mesenchymal stem cells derived from human knee synovium tissue were cultured with a density of 5000 cells/ cm2 in 12-chamber dishes. After the cells reached the appropriate level of growth, specific differentiation inducers were added. 21 days, differentiation of all stem cells to adipocyte, osteocyte and chondrocyte were evaluated and compared using specific staining methods. Our results indicated that all five mesenchymal stem cells were able to differentiate into osteocyte, adipocyte and chondrocyte. Moreover, the highest potentials for differentiation to adipocyte and osteocyte and chondrocyte were seen in mesenchymal stem cells derived from chicken bone marrow and human synovium, respectively. In this study, differences in the differentiation potential of mesenchymal stem cells isolated from different tissues and species were observed that could be caused by small differences in their environment in vivo. Understanding these differences can lead to more effective use of these cells in the clinic
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The aim of this study was optimization of the PolyFect gene delivery method of pcDNA3.1 expression vector transfected with the mouse pdx-1 gene in three different kinds of mesenchymal stem cells and Hepa cells as well as comparison of transfection efficiency leading to expression of the mentioned gene in the cell types used. Rat bone marrow-derived mesenchymal stem cells, C57 mouse bone marrow-derived mesenchymal stem cells, human synovium derived mesenchymal stem cells and Hepa cells were used in this study. After culturing of the mentioned cells, mouse pdx-1 gene were transfected into them using the Qiagen PolyFect kit. 72 hours later, the cells were treated with anti-mouse Pdx-1 antibody and immunocytochemically analyzed using a fluorescent inverted microscope. Transfection conditions were optimized in each of these cells by changing different lipofection parameters such as DNA concentration, PolyFect reagent concentration and cell density. The results demonstrated that for transfection of these cells, the best concentrations of DNA and PolyFect reagent are 400 ng/IL and 6000 ng/IL respectively. For maximum transfection efficiency, the best cell density in 12-well plates was 105 cells in Hepa cells, 1.3105 cells in rat bone marrow-derived mesenchymal stem cells, 1.5105 cells in human synovium-derived mesenchymal stem cells and 105 cells in C57 mouse bone marrow-derived mesenchymal stem cells. Under the mentioned optimized conditions, the maximum efficiency of transfection was determined to be 50% for Hepa cells, 40% for rat bone marrow-derived mesenchymal stem cells, 21% for human synovium-derived mesenchymal stem cells and 10% for C57 mouse bone marrow-derived mesenchymal stem cells. These findings implicate that the most important factor extremely influencing transfection efficiency in mesenchymal stem cells is the cell derivation origin. Results of this study can be used in basic and clinical studies dealing with gene therapy in mesenchymal stem cells
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Humans , Animals, Laboratory , Trans-Activators , Homeodomain Proteins , Transfection , Rats , Bone Marrow , MiceРеферат
Embryonic stem cells [ESCs] are pluripotent cells derived from the inner cell mass [ICM] of blastocysts. A feeder layer and cytokines are necessary for culture of these embryonic cells in most species. The aim of this study was application of rat mesenchymal stem cells [MSCs] as a feeder layer for the isolation and culture of mouse embryonic stem cells. Mesenchymal stem cells were isolated from rat bone marrow and cultured in DMEM [Dulbecco's modified Eagle's medium] medium supplemented with 10% FBS [fetal bovine serum]. To verify the isolated cells, they were affected by osteocyte differentiation inducer to become bone mass. After twenty-one days, the differentiation was evaluated by Alizarin red staining. Blastocysts were obtained from Balb/c pregnant mice and cultured on this MSCs feeder layer. Two days later; after hatching of blastocysts, the cells were trypsinized and the inner cell mass dissociated to the small cell clumps. These clumps were cultured on 12-well plates covered by the same MSCs without applying any cytokines or growth inducer. Two to three days after the passage, colonies appeared which were similar to embryonic stem cell colonies in morphology. These colonies were passaged two more times using the mentioned procedure and their identities were examined by morphological observation and alkalin phosphatase staining. In this study we could easily cultured MSCs using DMEM media. The mesenchimic origin of cultured cells, which showed fibroblastic morphology, was proved by differentiation to bone masses using osteocyte inducer and detection with Alizarin red. By applying DMEM media and MSCs cells, as feeder layer, we could culture ESC without any need to cytokines or growth factors. After passage to the inner cell mass colonies were formed. These colonies were formed in two more other passages. The colonies were verified with alkaline phosphatase assay. Results of this study showed mesenchymal stem cells isolated from rat bone marrow can differentiate to osteoblast line and can be used as feeder layer for isolation, culture and forming embryonic stem cells colonies. This method, by using MSCs as feeder layer and bypassing the need of cytokine and growth factors, seems to be a simple efficient method for culture and isolation of embryonic stem cells
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Humans , Animals, Laboratory , Embryonic Stem Cells , Pluripotent Stem Cells , Blastocyst Inner Cell Mass , Cytokines , Mice , Rats , Culture TechniquesРеферат
Mesenchymal stem cells [MSC] are a very promising transplantable stem cell source for a variety of cell replacement therapies. As the main source of MSC is bone marrow [BM], most of studies have been done on BM-derived MSC [BM-MSC]. Umbilical cord [UC]-derived MSC [UC-MSC] which are recently introduced, is one of the good alternative source for these cells. The objective of this study was to isolate and characterize UC-MSC from human UC veins and studying of their potential to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this way, a cell population was isolated from 20 UC veins using a solution of 0.1% collagenase type IV. After identification of isolated cells by immunocytochemical and flow cytometry methods, these cells were exposured with adipogenic, osteogenic, chondrogenic and neurogenic agents. Resulted differentiated cells were detected using specific staining for each lineage and room temperature [RT]-PCR. Immunophenotypically, this cell population was positive for CD73, CD 166, CD 105 markers and alpha-smooth muscle actin and negative for CD31, CD34, CD49d markers, von Willebrand factor and smooth muscle myosin. Exposure of these cells to adipogenic, osteogenic, chondrogenic and neurogenic agents resulted in morphological changes followed by lineage-specific staining for each differentiated cell type. RT-PCR analysis showed that these differentiated cells express fat, bone, cartilage and neuronal markers, respectively. Altogether, these findings indicate that UC-MSC possess morphological, immunophenotypical and cell differentiation capacities similar to BM-MSC and so they can be used more extensively in cell based therapy protocols and in vitro differentiation study models
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Humans , Umbilical Veins , Umbilical Cord , Cell Differentiation , Adipose Tissue , Bone and Bones , Cartilage , Neurons , Immunohistochemistry , Flow Cytometry , Reverse Transcriptase Polymerase Chain ReactionРеферат
On the basis of reports that mesenchymal stem cells [MSCs] can be isolated from the placenta/umbilical cord stroma, the present study was undertaken to isolate and characterize MSCs from the human umbilical cord veins. In this investigation, a cell population was isolated which was derived from the endothelium/subendothelium layers of 20 umbilical cord veins obtained from term deliveries using a solution of 0.1% collagenase type IV. Results suggest that these cells possess morphological, immunophenotypical and cell differentiation capacities similar to the bone marrow-derived mesenchymal stem cells [MSCs]. The isolated cell population has fibroblastoid morphology which upon proper stimulation gives rise to adipocytes, osteocytes and chondrocytes in culture. Immunophenotypically, this cell population is positive for CD54, CD29, CD73, CD49e, CD166, CD105, CD13, and CD44 markers and alpha-smooth muscle actin and negative for CD31, CD45, CD49d, and CD34 markers, von Willebrand factor [vWF] and smooth muscle myosin [MySM]. Altogether, these findings indicate that umbilical cord obtained from term deliveries is an important source of MSCs which could have an important application in cell therapy protocols