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Sample records for fibroblastic cell lines

  1. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  2. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    PubMed

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-01-01

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. PMID:26840224

  3. Impact of intense pulsed light irradiation on cultured primary fibroblasts and a vascular endothelial cell line

    PubMed Central

    WU, DI; ZHOU, BINGRONG; XU, YANG; YIN, ZHIQIANG; LUO, DAN

    2012-01-01

    The aim of this study was to determine the effects of intense pulsed light (IPL) on cell proliferation and the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in human fibroblasts and vascular endothelial cell lines, and to investigate the effects of IPL on the mRNA expression levels of type I and III procollagens in cultured human fibroblasts. Foreskin fibroblasts and a vascular endothelial cell line (ECV034) were cultured and treated with various wavelengths and doses of IPL irradiation. After culture for 1, 12, 24 and 48 h following IPL irradiation, fibroblasts and the vascular endothelial cell line were harvested for investigation of morphological changes by light microscopy, cell proliferation viability by MTT assay, and VEGF and MMP secretions by ELISA. The mRNA expression levels of type I and III procollagens in the fibroblasts were detected by RT-PCR. No marked morphological changes were observed in the cultured fibroblasts compared with the control. Cell growth and cellular viability were increased in fibroblasts 24 and 48 h after IPL irradiation. The levels of type I and III procollagen mRNA expression in fibroblasts increased in a time-dependent manner. However, the IPL management had no impact on VEGF and MMP secretion levels in fibroblasts and the ECV034 cell line at any time-point after irradiation as well as cell morphology and cellular proliferation. IPL irradiation may induce cellular proliferation and promote the expression of procollagen mRNAs directly in cultured primary fibroblasts, which may primarily contribute to photorejuvenation. PMID:23170124

  4. Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

    PubMed Central

    Lu, Rong; Bian, Fang; Lin, Jing; Su, Zhitao; Qu, Yangluowa; Pflugfelder, Stephen C.; Li, De-Quan

    2012-01-01

    There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction. PMID:22723892

  5. Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

    PubMed

    Grześkowiak, Bartosz F; Hryhorowicz, Magdalena; Tuśnio, Karol; Grzeszkowiak, Mikołaj; Załęski, Karol; Lipiński, Daniel; Zeyland, Joanna; Mykhaylyk, Olga; Słomski, Ryszard; Jurga, Stefan; Woźniak, Anna

    2016-05-01

    The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation. PMID:27048425

  6. Deciphering the differential response of two human fibroblast cell lines following Chikungunya virus infection

    PubMed Central

    2012-01-01

    Background Chikungunya virus (CHIKV) is an arthritogenic member of the Alphavirus genus (family Togaviridae) transmitted by Aedes mosquitoes. CHIKV is now known to target non hematopoietic cells such as epithelial, endothelial cells, fibroblasts and to less extent monocytes/macrophages. The type I interferon (IFN) response is an early innate immune mechanism that protects cells against viral infection. Cells express different pattern recognition receptors (including TLR7 and RIG-I) to sense viruses and to induce production of type I IFNs which in turn will bind to their receptor. This should result in the phosphorylation and translocation of STAT molecules into the nucleus to promote the transcription of IFN-stimulated antiviral genes (ISGs). We herein tested the capacity of CHIKV clinical isolate to infect two different human fibroblast cell lines HS 633T and HT-1080 and we analyzed the resulting type I IFN innate immune response. Methods Indirect immunofluorescence and quantitative RT-PCR were used to test for the susceptibility of both fibroblast cell lines to CHIKV. Results Interestingly, the two fibroblast cell lines HS 633T and HT-1080 were differently susceptible to CHIKV infection and the former producing at least 30-fold higher viral load at 48 h post-infection (PI). We found that the expression of antiviral genes (RIG-I, IFN-β, ISG54 and ISG56) was more robust in the more susceptible cell line HS 633T at 48 h PI. Moreover, CHIKV was shown to similarly interfere with the nuclear translocation of pSTAT1 in both cell lines. Conclusion Critically, CHIKV can control the IFN response by preventing the nuclear translocation of pSTAT1 in both fibroblast cell lines. Counter-intuitively, the relative resistance of HT-1080 cells to CHIKV infection could not be attributed to more robust innate IFN- and ISG-dependent antiviral responses. These cell lines may prove to be valuable models to screen for novel mechanisms mobilized differentially by fibroblasts to control

  7. Establishment of the first humpback whale fibroblast cell lines and their application in chemical risk assessment.

    PubMed

    Burkard, Michael; Whitworth, Deanne; Schirmer, Kristin; Nash, Susan Bengtson

    2015-10-01

    This paper reports the first successful derivation and characterization of humpback whale fibroblast cell lines. Primary fibroblasts were isolated from the dermal connective tissue of skin biopsies, cultured at 37 °C and 5% CO2 in the standard mammalian medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS). Of nine initial biopsies, two cell lines were established from two different animals and designated HuWa1 and HuWa2. The cells have a stable karyotype with 2n=44, which has commonly been observed in other baleen whale species. Cells were verified as being fibroblasts based on their spindle-shaped morphology, adherence to plastic and positive immunoreaction to vimentin. Population doubling time was determined to be ∼41 h and cells were successfully cryopreserved and thawed. To date, HuWa1 cells have been propagated 30 times. Cells proliferate at the tested temperatures, 30, 33.5 and 37 °C, but show the highest rate of proliferation at 37 °C. Short-term exposure to para,para'-dichlorodiphenyldichloroethylene (p,p'-DDE), a priority compound accumulating in southern hemisphere humpback whales, resulted in a concentration-dependent loss of cell viability. The effective concentration which caused a 50% reduction in HuWa1 cell viability (EC50 value) was approximately six times greater than the EC50 value for the same chemical measured with human dermal fibroblasts. HuWa1 exposed to a natural, p,p'-DDE-containing, chemical mixture extracted from whale blubber showed distinctively higher sensitivity than to p,p'-DDE alone. Thus, we provide the first cytotoxicological data for humpback whales and with establishment of the HuWa cell lines, a unique in vitro model for the study of the whales' sensitivity and cellular response to chemicals and other environmental stressors. PMID:26363275

  8. Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

    NASA Astrophysics Data System (ADS)

    Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo; Mohamed, Azman Seeni; Saifuddin, Siti Nazmin; Masudi, Sam'an Malik; Mohamad, Dasmawati

    2015-04-01

    ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

  9. Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

    SciTech Connect

    Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo; Mohamed, Azman Seeni; Saifuddin, Siti Nazmin; Masudi, Sam’an Malik; Mohamad, Dasmawati

    2015-04-24

    ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

  10. Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

    PubMed Central

    2011-01-01

    Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

  11. Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami (Cypriniformes: Cyprinidae).

    PubMed

    Wang, Xiao-Ai; Yang, Jun-Xing; Chen, Xiao-Yong; Pan, Xiao-Fu

    2012-12-01

    Though Yunnan province contains some 562 known species of fish, no cell lines from any of these have been made available to date. To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami, a fish endemic to Fuxian Lake, Yunnan, China, we established and characterized the major features of a continuous cell line (AGF II) from the caudal fin tissue of A. grahami. This AGF II cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year. The cell line was maintained in DMEM/F12 supplemented with 10% FBS, with a cellular doubling time of 51.1 h. We continued with more experiments to optimize the culture and storage conditions, and found a variety of interesting results: cells could grow at temperature between 24 Degrees Celsius and 28 Degrees Celsius, with the optimal temperature of 28 Degrees Celsius. Likewise, the growth rate of A. grahami fin cells increased when the FBS proportion increased from 5% to 20%, with the optimal growth at the concentrations of 20% FBS; cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15; DMSO is a better cryoprotectant than Glycerol, EG and MeOH for AGFII cells with optimal concentration of 5% DMSO. Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52, with a modal peak at 48 chromosomes, accounting for 39.8% of all cells. Using the same primer pairs specific to mtDNA, the AGF II cell sequences obtained by PCR were identical to those from muscle tissues of A. grahami. Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A. grahami, also indicating that that current long-term artificial propagation of A. grahami has been successful. Finally, we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid, bright fluorescent signals were observed, suggesting that this cell line may be suitable for use in transfection

  12. Selective suicide gene therapy of colon cancer cell lines exploiting fibroblast growth factor 18 promoter.

    PubMed

    Teimoori-Toolabi, Ladan; Azadmanesh, Kayhan; Zeinali, Sirous

    2010-02-01

    Fibroblast growth factor 18 (FGF18) is one of the genes downstream of Wnt, one of the most important signaling pathways activated in colon cancer. An FGF18 promoter containing a single T-cell factor/lymphocyte enhancing factor 1 (TCF/LEF1) binding site was inserted upstream of a thymidine kinase (TK) suicide gene module, while a bacterial beta-Gal (LacZ) element served as the reporter gene. Following transient transfection with pUCFGF18LacZ, beta-Gal staining showed that 5% of SW480, 10% of HCT116, 0% of human umbilical vein endothelial cells (HUVECs) and 0% of normal colon cells (NCCs) had expressed LacZ. beta-Gal enzyme-linked immunosorbent assay revealed that the ratio of pUCFGF18LacZ activity to that of positive control was 0.09 and 0.25 in SW480 and HCT116, respectively (significantly higher than mock plasmid), while there were no significant changes in the beta-Gal expression in HUVEC and NCC cells transfected with pUCFGF18LacZ or mock plasmid. Following transfection with pUCFGF18TK and pUCCMVTK (positive control), cytotoxicity analysis of transfected cells showed that treatment with ganciclovir (GCV) significantly decreased SW480 and HCT116 cell survival at GCV concentrations above 20 microg/mL. An inverse correlation between GCV concentration and cell viability was evident in both colon cancer cell lines following transfection with these suicide plasmids. pUCFGF18TK and pUCCMVTK induced apoptosis after the administration of GCV in HCT116, but not in SW480, as demonstrated by M30 cytodeath antibody. This discrepancy may stem from differences in the mechanisms of TK/GCV-induced apoptosis in p53-proficient (HCT116) and -deficient (SW480) cells. The specific activity of the FGF18 promoter in HCT116 and SW480 may reflect the advantage of this promoter over artificial promoters containing artificial TCF/LEF binding sites. PMID:20187803

  13. Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

    PubMed

    Bao, Kai; Akguel, Baki; Bostanci, Nagihan

    2014-01-01

    In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. PMID:25471635

  14. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

  15. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    SciTech Connect

    Levy, J.R.; Olefsky, J.M.

    1988-05-05

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4/sup 0/C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37/sup 0/C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

  16. Calcium antagonists and low density lipoproteins metabolism by human fibroblasts and by human hepatoma cell line HEP G2.

    PubMed

    Corsini, A; Granata, A; Fumagalli, R; Paoletti, R

    1986-01-01

    The effect of Ca2+ antagonists (CA) on the receptor-mediated low density lipoprotein pathway has been investigated "in vitro" in human skin fibroblasts (HSF) and in human hepatoma cell line Hep G2. The specific binding and internalization of human 125I-labeled LDL are dose-dependently increased in HSF by CA of the verapamil series (verapamil, anipamil, gallopamil, ronipamil, and diltiazem), but neither by CA of the dihydropyridine series (nifedipine, nitrendipine) nor by flunarizine. BAY K 8644, a Ca2+ agonist, elicited an opposite effect. In the presence of the tested CA, LDL degradation is either unaffected (lower concentrations) or inhibited (higher concentrations). 125I-LDL uptake is stimulated also in fibroblasts from type IIa hypercholesterolemic patients, heterozygous for defective expression of LDL receptor. The enhanced cellular uptake of 125I-LDL was prevented by cycloheximide and by alpha-amanitin. CA of the verapamil series including diltiazem retained their effect in human hepatoma cell line Hep G2, a model proposed for hepatic metabolism of LDL. Our studies show that a) CA stimulate the high affinity binding and internalization of LDL in HSF and in human hepatoma cell line Hep G2; b) this stimulation involves DNA transcription and new protein synthesis; c) this effect is specific to one subgroup of Ca2+ antagonists (the verapamil class only). PMID:3006091

  17. A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison of fibroblasts and diverse cancer cell lines.

    PubMed

    Zoellner, Hans; Paknejad, Navid; Manova, Katia; Moore, Malcolm A S

    2015-12-01

    Differing stimuli affect cell stiffness while cancer metastasis is associated with reduced cell stiffness. Cell stiffness determined by atomic force microscopy has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. Ninety μm square fields were recorded from ten separate sites of cultured human dermal fibroblasts (HDF) and three sites each for melanoma (MM39, WM175, and MeIRMu), osteosarcoma (SAOS-2 and U2OS), and ovarian carcinoma (COLO316 and PEO4) cell lines, each site providing 1024 measurements as 32 × 32 square grids. Stiffness recorded below 0.8 μm height was occasionally influenced by substratum, so only stiffness recorded above 0.8 μm was analysed, but all sites were included for height and volume analysis. COLO316 had the lowest cell height and volume, followed by HDF (p < 0.0001) and then PEO4, SAOS-2, MeIRMu, WM175, U2OS, and MM39. HDF were more stiff than all other cells (p < 0.0001), while in descending order of stiffness were PEO4, COLO316, WM175, SAOS-2, U2OS, MM39, and MeIRMu (p < 0.02). Stiffness fingerprints comprised scattergrams of stiffness values plotted against the height at which each stiffness value was recorded and appeared unique for each cell type studied, although in most cases the overall form of fingerprints was similar, with maximum stiffness at low height measurements and a second lower peak occurring at high-height levels. We suggest that our stiffness-fingerprint analytical method provides a more nuanced description than previously reported and will facilitate study of the stiffness response to cell stimulation. PMID:26357955

  18. Genome-Wide Expression Analysis in Fibroblast Cell Lines from Probands with Pallister Killian Syndrome

    PubMed Central

    Wilkens, Alisha B.; Chatfield, Kathryn C.; Spinner, Nancy B.; Conlin, Laura K.; Zhang, Zhe; Krantz, Ian D.

    2014-01-01

    Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2. PMID:25329894

  19. Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) against UV irradiation on keratinocytes and fibroblasts cell lines.

    PubMed

    Zanatta, C F; Mitjans, M; Urgatondo, V; Rocha-Filho, P A; Vinardell, M P

    2010-01-01

    Considering the belief that natural lipids are safer for topical applications and that carotenoids are able to protect cells against photooxidative damage, we have investigated whether topical creams and lotions, produced with Buriti oil and commercial surfactants, can exert photoprotective effect against UVA and UVB irradiation on keratinocytes and fibroblasts. Cell treatment was divided into two steps, prior and after exposition to 30 min of UVA plus UVB radiation or to 60 min of UVA radiation. Emulsions prepared with ethoxylated fatty alcohols as surfactants and containing alpha-tocopherol caused phototoxic damage to the cells, especially when applied prior to UV exposure. Damage reported was due to prooxidant activity and phototoxic effect of the surfactant. Emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil and containing panthenol as active ingredient, were able to reduce the damages caused by radiation when compared to non-treated cells. When the two cell lines used in the study were compared, keratinocytes showed an increase in cell viability higher than fibroblasts. The Buriti oil emulsions could be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection, especially in after sun formulations. PMID:19766688

  20. Murine embryonic fibroblast cell lines differentiate into three mesenchymal lineages to different extents: new models to investigate differentiation processes.

    PubMed

    Dastagir, Khaled; Reimers, Kerstin; Lazaridis, Andrea; Jahn, Sabrina; Maurer, Viktor; Strauß, Sarah; Dastagir, Nadjib; Radtke, Christine; Kampmann, Andreas; Bucan, Vesna; Vogt, Peter M

    2014-08-01

    Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation. PMID:25068630

  1. Murine Embryonic Fibroblast Cell Lines Differentiate into Three Mesenchymal Lineages to Different Extents: New Models to Investigate Differentiation Processes

    PubMed Central

    Dastagir, Khaled; Lazaridis, Andrea; Jahn, Sabrina; Maurer, Viktor; Strauß, Sarah; Dastagir, Nadjib; Radtke, Christine; Kampmann, Andreas; Bucan, Vesna; Vogt, Peter M.

    2014-01-01

    Abstract Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as “feeder cells.” Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation. PMID:25068630

  2. A new fibroblast growth stimulating activity from the human megakaryoblastic leukaemia cell line ELF-153: in vitro and in vivo findings.

    PubMed Central

    Hassan, H. T.; Hanauske, A. R.; Lux, E.; Kleine, H. D.; Freund, M.

    1995-01-01

    Although the exact mechanism for the progression of myelofibrosis in acute megakaryoblastic leukaemia is unclear, certain humoral factors released from the proliferating megakaryoblasts that are unable to store these factors in their defective alpha-granules, including platelet derived growth factor (PDGF), fibroblast growth factors (FGF), platelet factor-4 (PF-4), transforming growth factor-beta (TGF-beta) and beta-thromboglobulin, could result in increased collagen synthesis by bone marrow fibroblasts. Recently, the human megakaryoblastic leukaemia cell line MEG-01 has been shown to produce both TGF-beta and PF-4 which have enhanced the growth of bone marrow fibroblasts. Therefore, we have examined the presence of a fibroblast growth stimulating activity and the humoral factors that might be responsible for it in the supernatant of the human megakaryoblastic leukaemia cell line ELF-153 recently established in our laboratory from a patient with acute myelofibrosis. A new fibroblast growth stimulating activity has been identified in the supernatant of the ELF-153 human megakaryoblastic leukaemia cell line that is independent of the percentage of fetal calf serum in NRK-49F fibroblast agar clonogenic assays and is not due to any of the known fibroblast growth stimulating humoral factors including PDGF, epithelial growth factor, TGF-alpha or beta, tumour necrosis factor-alpha, interleukin-1, 2, 4 or 6, FGF, fibronectin, PF-4 and factor VIII AG. Also, in vivo, subcutaneous injection of ELF-153 megakaryoblastic leukaemia cells into nude mice formed, in three out of the five mice after 6 weeks, subcutaneous tumours with a very rigid texture whose histological examination revealed dense infiltration by blast cells and pronounced reticular fibrosis. Immunohistochemistry demonstrated exclusive deposition of collagen III in the extracellular matrix whereas laminin and collagen IV were absent.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7488550

  3. METHOTREXATE AND MYOTREXATE INDUCE APOPTOSIS IN HUMAN MYOMA FIBROBLASTS (T hES CELL LINE) VIA MITOCHONDRIAL PATHWAY.

    PubMed

    Kastratović, Tatjana; Arsenijević, Slobodan; Matović, Zoran; Mitrović, Marina; Nikolić, Ivana; Milosavljević, Zoran; Protrka, Zoran; Šorak, Marija; Đurić, Janko

    2015-01-01

    Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. Although the local application of low doses of methotrexate (MTX) is used as an effective treatment of the myomas, myotrexate could be a promising new drug. This study investigated the cytotoxic and apoptotic effects of both MTX and myotrexate in human fibroblasts derived from the uterine fibroids (T hES cell line). The myotrexate adduct is an aqueous solution of MTX and L-arginine. Cells were treated with a graded concentrations of both MTX and myothrexate (0.1-16 µM) for 24 h. The cytotoxicity was assayed by MTT test, apoptosis was evaluated by Annexin V-FITC assay and their possible role in apoptosis was determined by immnu- flourescence. Both MTX and myotrexate induced apoptosis in T hES cells in a dose dependent manner (p < 0.001). Myotrexate significantly increased the percentage of AnnexinV positive cells, BAX/Bcl-2 ratio and subsequent caspase-3 activation compared to the MTX treated cells (p < 0.05). Both MTX or myotrexate treatment showed a diffuse staining of cytochrome c indicating its release from mitochondria to the cytosol, suggesting that their mechanisms of action most likely involves the mitochondrial apoptotic pathway. PMID:26642654

  4. Ochratoxin A and T-2 Toxin Induce Clonogenicity and Cell Migration in Human Colon Carcinoma and Fetal Lung Fibroblast Cell Lines.

    PubMed

    Abassi, Haila; Ayed-Boussema, Imen; Shirley, Sarah; Abid, Salwa; Bacha, Hassen

    2016-03-01

    T-2 toxin and Ochratoxin A (OTA) are toxic secondary metabolites produced by various fungi, and together they contaminate feedstuffs worldwide. T-2 toxin and OTA may exert carcinogenic action in rodent. Despite the various in vivo experiments, carcinogenicity of these two mycotoxins has not yet been proven for human. In this current study, we proposed to investigate, in Human colon carcinoma cells and fetal lung fibroblast-like cells transfected with MYC, the effect of T-2 toxin and OTA on cell clonogenicity and cell migration. Results of the present investigation showed that T2-toxin as well as OTA has an important clonogenic effect in all cell lines, suggesting that these mycotoxins could promote the transcription of c-myc gene. Furthermore, T-2 toxin and OTA enhanced the migration effect of HCT116 cells at very low concentrations, proposing that these mycotoxins may exhibit carcinogenesis-like properties in the studied cells. PMID:26849850

  5. Inhibitory Effects of Terminalia catappa on UVB-Induced Photodamage in Fibroblast Cell Line

    PubMed Central

    Wen, Kuo-Ching; Shih, I-Chen; Hu, Jhe-Cyuan; Liao, Sue-Tsai; Su, Tsung-Wei; Chiang, Hsiu-Mei

    2011-01-01

    This study investigated whether Terminalia catappa L. hydrophilic extract (TCLW) prevents photoaging in human dermal fibroblasts after exposure to UVB radiation. TCLW exhibited DPPH free radical scavenging activity and protected erythrocytes against AAPH-induced hemolysis. In the gelatin digestion assay, the rates of collagenase inhibition by TCL methanol extract, TCLW, and its hydrolysates were greater than 100% at the concentration of 1 mg/mL. We found that serial dilutions of TCLW (10–500 μg/mL) inhibited collagenase activity in a dose-dependent manner (82.3% to 101.0%). However, TCLW did not significantly inhibit elastase activity. In addition, TCLW inhibited MMP-1 and MMP-9 protein expression at a concentration of 25 μg/mL and inhibited MMP-3 protein expression at a concentration of 50 μg/mL. TCLW also promoted the protein expression of type I procollagen. We also found that TCLW attenuated the expression of MMP-1, -3, and -9 by inhibiting the phosphorylation of ERK, JNK, and p38. These findings suggest that TCLW increases the production of type I procollagen by inhibiting the activity of MMP-1, -3 and -9, and, therefore, has potential use in anti-aging cosmetics. PMID:20981325

  6. Inhibitory Effects of Terminalia catappa on UVB-Induced Photodamage in Fibroblast Cell Line.

    PubMed

    Wen, Kuo-Ching; Shih, I-Chen; Hu, Jhe-Cyuan; Liao, Sue-Tsai; Su, Tsung-Wei; Chiang, Hsiu-Mei

    2011-01-01

    This study investigated whether Terminalia catappa L. hydrophilic extract (TCLW) prevents photoaging in human dermal fibroblasts after exposure to UVB radiation. TCLW exhibited DPPH free radical scavenging activity and protected erythrocytes against AAPH-induced hemolysis. In the gelatin digestion assay, the rates of collagenase inhibition by TCL methanol extract, TCLW, and its hydrolysates were greater than 100% at the concentration of 1 mg/mL. We found that serial dilutions of TCLW (10-500 μg/mL) inhibited collagenase activity in a dose-dependent manner (82.3% to 101.0%). However, TCLW did not significantly inhibit elastase activity. In addition, TCLW inhibited MMP-1 and MMP-9 protein expression at a concentration of 25 μg/mL and inhibited MMP-3 protein expression at a concentration of 50 μg/mL. TCLW also promoted the protein expression of type I procollagen. We also found that TCLW attenuated the expression of MMP-1, -3, and -9 by inhibiting the phosphorylation of ERK, JNK, and p38. These findings suggest that TCLW increases the production of type I procollagen by inhibiting the activity of MMP-1, -3 and -9, and, therefore, has potential use in anti-aging cosmetics. PMID:20981325

  7. Radiation induced DNA damage and damage repair in human tumor and fibroblast cell lines assessed by histone H2AX phosphorylation

    SciTech Connect

    Mahrhofer, Hartmut; Buerger, Susann; Oppitz, Ulrich; Flentje, Michael; Djuzenova, Cholpon S. . E-mail: djuzenova_t@klinik.uni-wuerzburg.de

    2006-02-01

    Purpose: To analyze the radiation-induced levels of {gamma}H2AX and its decay kinetics in 10 human cell lines covering a wide range of cellular radiosensitivity (SF2, 0.06-0.63). Methods and Materials: Five tumor cell lines included Colo-800 melanoma, two glioblastoma (MO59J and MO59K), fibrosarcoma HT 1080, and breast carcinoma MCF7. Five primary skin fibroblasts lines included two normal strains, an ataxia telangiectasia strain, and two fibroblast strains from breast cancer patients with an adverse early skin reaction to radiotherapy. Cellular radiosensitivity was assessed by colony-forming test. Deoxyribonucleic acid damage and repair were analyzed according to nuclear {gamma}H2AX foci intensity, with digital image analysis. Results: The cell lines tested showed a wide degree of variation in the background intensity of immunostained nuclear histone {gamma}H2AX, which was higher for the tumor cell lines compared with the fibroblast strains. It was not possible to predict clonogenic cell survival (SF2) for the 10 cell lines studied from the radiation-induced {gamma}H2AX intensity. In addition, the slopes of the dose-response (0-4 Gy) curves, the rates of {gamma}H2AX disappearance, and its residual expression ({<=}18 h after irradiation) did not correlate with SF2 values. Conclusions: The results from 10 cell lines showed that measurements of immunofluorescence intensity by digital image analysis of phosphorylated histone H2AX as a surrogate marker of DNA double-strand breaks did not allow reliable ranking of cell strains according to their clonogenic survival after irradiation.

  8. Transgene-free human induced pluripotent stem cell line (HS5-SV.hiPS) generated from cesarean scar-derived fibroblasts.

    PubMed

    Rungsiwiwut, Ruttachuk; Pavarajarn, Wipawee; Numchaisrika, Pranee; Virutamasen, Pramuan; Pruksananonda, Kamthorn

    2016-01-01

    Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts. PMID:27345776

  9. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    SciTech Connect

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-04-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with /sup 125/I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

  10. Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction

    PubMed Central

    Guo, Yijie; Fukuda, Tomokazu; Nakamura, Shuichi; Bai, Lanlan; Xu, Jun; Kuroda, Kengo; Tomioka, Rintaro; Yoneyama, Hiroshi; Isogai, Emiko

    2015-01-01

    Leptospiral lipopolysaccharide (L-LPS) has shown potency in activating toll-like receptor 2 (TLR2) in pig fibroblasts (PEFs_NCC1), and causes the expression of proinflammatory cytokines. However, the stimulation by L-LPS was weak eliciting the function of TLR2 sufficiently in pig innate immunity responses during Leptospira infection. In this study, the immune response of pig embryonic fibroblast cell line (PEFs_SV40) was investigated and was found to be the high immune response, thus TLR2 is the predominate receptor of L-LPS in pig cells. Further, we found a strategy using the antibody against L-LPS, to prevent L-LPS interaction with TLR2 in pig cells which could impact on immune activation. PMID:25557825

  11. Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization.

    PubMed

    Kamada, Mizuna; Kumazaki, Tsutomu; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

    2012-06-01

    To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents. PMID:22273270

  12. Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

    PubMed Central

    Senevirathne, Mahinda; Kim, Soo-Hyun

    2010-01-01

    Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro. PMID:20607062

  13. Cadherin-11 Induces Rheumatoid Arthritis Fibroblast-Like Synoviocytes to Form Lining Layers in Vitro

    PubMed Central

    Kiener, Hans P.; Lee, David M.; Agarwal, Sandeep K.; Brenner, Michael B.

    2006-01-01

    The synovial lining of diarthrodial joints is composed of a condensed network of synoviocytes that form an intact layer via cell-to-cell contacts with significant intercellular matrix spaces. However, the molecular basis for synovial lining formation and its structural integrity has not been previously defined. In this study, using a three-dimensional fibroblast-like synoviocyte in vitro organ culture system, we provide evidence that cadherin-11 expressed in fibroblast-like synoviocytes plays a determining role in establishing the synovial lining layer. Fibroblast-like synoviocytes that were grown in three-dimensional matrices demonstrated formation of a lining structure at the interface between the matrix and the fluid phase. Treatment of fibroblast-like synoviocyte organ cultures with a cadherin-11-Fc fusion protein efficiently abrogated lining layer organization. Moreover, because E-cadherin-expressing fibroblasts failed to organize a lining layer structure at the tissue boundary, this effect appears to be a distinct characteristic of fibroblasts expressing cadherin-11. We found that cadherin-11 mediated fibroblast-like synoviocyte cell-to-cell adhesion via formation of adherens junctions that were linked to and remodeled the actin cytoskeleton. Together, these studies implicate cadherin-11 in synovial tissue and lining layer formation and provide an in vitro system to model fibroblast-like synoviocyte behavior and function in organizing the synovial tissue. PMID:16651616

  14. In Vitro Study of Er:YAG and Er, Cr:YSGG Laser Irradiation on Human Gingival Fibroblast Cell Line.

    PubMed

    Talebi-Ardakani, Mohammad Reza; Torshabi, Maryam; Karami, Elahe; Arbabi, Elham; Rezaei Esfahrood, Zeinab

    2016-04-01

    The ultimate goal of the periodontal treatments is a regeneration of periodontium. Recently, laser irradiations are commonly used to improve wound repair. Because of many controversies about the effects of laser on soft tissue regeneration, more in vitro studies are still needed. The aim of the present in vitro study was to compare the effects of different doses of Er:YAG (erbium-doped:yttrium, aluminum, garnet) and Er, Cr:YSGG (erbium, chromium-doped: yttrium, scandium, gallium, garnet) laser treatment on human gingival fibroblasts (HGF) proliferation. In this randomized single-blind controlled in vitro trial, HGF cells were irradiated using Er:YAG and Er, Cr:YSGG laser for 10 and 30 seconds or remained unexposed as a control group. After a culture period of 24 and 48 hours, HGF cell proliferation was evaluated by MTT assay. The data were subjected to one-sided analysis of variance and Tukey multiple comparison tests. Our results showed Er:YAG application for 10 and 30 seconds as well as Er, Cr:YSGG irradiation for 10 and 30 seconds induced statistically significant (P<0.05) proliferation of HGF cells as compared with the control at 24 hours up to 18.39%, 26.22%, 21.21%, and 17.06% respectively. In 48 hour incubations, Er:YAG and Er, Cr:YSGG irradiation for 10 and 30 seconds significantly increased cellular proliferation up to 22.9%, 32.24%, 30.52% and 30.02% respectively (P<0.05). This study demonstrates that Er:YAG and Er, Cr:YSGG laser significantly increased HGF cell proliferation compared to the control specimens. This higher proliferation can lead to increased wound repair in clinical conditions. PMID:27309266

  15. A new approach to cancer therapy due to appropriate uptake and retention kinetics of meta-tetrahydroxy-phenylchlorin in a human fibroblast cell line.

    PubMed

    Wierrani, F; Fiedler, D; Schnitzhofer, G; Stewart, J C; Gharehbaghi, K; Henry, M; Grin, W; Grünberger, W; Krammer, B

    1996-04-01

    Studies have shown that meta-tetrahydroxy-phenylchlorin is an efficient tumor targeting agent for laser photodynamic therapy. The effectiveness of this approach for cancer treatment depends on drug concentration, incubation time and extracellular protein. We studied uptake and retention kinetics of mTHPC in a human fibroblast cell line. Our results clearly demonstrate a difference in the amount of extracellular mTHPC at an incubation temperature of 37 degrees C compared to 20 degrees C and 4 degrees C. pH-values were always constant and not responsible for the increase. Furthermore, both absorption and fluorescence of mTHPC increase when incubated at normal human body temperature. Incubation of human fibroblast cells with mTHPC (10 micg/mL) showed that intracellular mTHPC increases in a linear manner reaching saturation after 24 hours and declining until 48 hours with concommitant increase of supernatant mTHPC. Therefore, we believe that tumor cells can be treated optimally with PDT following a delay > 24 hours after drug administration with a minimum of damage to surrounding normal tissues. PMID:8937740

  16. Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum.

    PubMed

    Okamura, Kohji; Toyoda, Masashi; Hata, Kenichiro; Nakabayashi, Kazuhiko; Umezawa, Akihiro

    2015-12-01

    Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP), which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively. PMID:26697316

  17. Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

    PubMed Central

    Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

    2016-01-01

    Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540

  18. Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line.

    PubMed

    Kovacs, I; Yamamura, H I; Waite, S L; Varga, E V; Roeske, W R

    1998-02-01

    The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms. PMID:9454790

  19. Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

    PubMed

    Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

    2016-08-01

    Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc. PMID:27169394

  20. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

    PubMed Central

    Wiedłocha, A; Falnes, P O; Rapak, A; Muñoz, R; Klingenberg, O; Olsnes, S

    1996-01-01

    U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell. PMID:8524304

  1. FAP-α (Fibroblast activation protein-α) is involved in the control of human breast cancer cell line growth and motility via the FAK pathway

    PubMed Central

    2014-01-01

    Background Fibroblast Activation Protein alpha (FAP-α) or seprase is an integral membrane serine peptidase. Previous work has not satisfactorily explained both the suppression and promotion effects that have been observed in cancer. The purpose of this work was to investigate the role of FAP-α in human breast cancer. Expression of FAP-α was characterized in primary tumour samples and in cell lines, along with the effects of FAP-α expression on in vitro growth, invasion, attachment and migration. Furthermore the potential interaction of FAP-α with other signalling pathways was investigated. Results FAP-α was significantly increased in patients with poor outcome and survival. In vitro results showed that breast cancer cells over expressing FAP-α had increased growth ability and impaired migratory ability. The growth of MDA-MB-231 cells and the adhesion and invasion ability of both MCF-7 cells and MDA-MB-231 cells were not dramatically influenced by FAP-α expression. Over-expression of FAP-α resulted in a reduction of phosphorylated focal adhesion kinase (FAK) level in both cells cultured in normal media and serum-free media. An inhibitor to FAK restored the reduced motility ability of both MCF-7exp cells and MDA-MB-231exp cells and prevented the change in phosphorylated FAK levels. However, inhibitors to PI3K, ERK, PLCϒ, NWASP, ARP2/3, and ROCK had no influence this. Conclusions FAP-α in significantly associated with poor outcome in patients with breast cancer. In vitro, FAP-α promotes proliferation and inhibits migration of breast cancer cells, potentially by regulating the FAK pathway. These results suggest FAP-α could be a target for future therapies. PMID:24885257

  2. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

    We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

  3. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    PubMed Central

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

  4. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line.

    PubMed

    Śmieszek, Agnieszka; Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzińska, Aneta; Siudzińska, Anna; Marędziak, Monika; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  5. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    PubMed Central

    Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzińska, Aneta; Siudzińska, Anna; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  6. Modulatory Effects of Connexin-43 Expression on Gap Junction Intercellular Communications with Mast Cells and Fibroblasts

    PubMed Central

    Pistorio, Ashey L.; Ehrlich, H. Paul

    2011-01-01

    The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junctional intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin in RMC-1 cells. Fibroblasts express other connexins that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited α SMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis. PMID:21328609

  7. In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression.

    PubMed

    Rashnonejad, A; Gündüz, C; Süslüer, S Y; Onay, H; Durmaz, B; Bandehpour, M; Özkınay, F

    2016-01-01

    The reduced level of survival motor neuron (SMN) protein, caused by homozygous deletions in the SMN gene, led to a common neurodegenerative disorder known as spinal muscular atrophy (SMA). In spite of extensive efforts to find a cure for SMA, there is currently no effective treatment available for this devastating disease. In this study, restoration of SMN expression through 'gene-targeting' method in SMA fibroblast cells was attempted. We designed a 2697-bp gene-targeting cassette; it consisted of an SMN1 open reading frame expressing 38 kD SMN protein and the upstream and downstream regions of exon 1 of SMN1 gene at the ends as the homology arms. SMA fibroblast cells were transfected by gene-targeting cassette using Lipofectamine LTX-PLUS reagent. Occurrence of homologous recombination in selected cells was investigated by PCR analysis. Increased expression of SMN protein was shown by real-time PCR and western blotting analysis. The immunofluorescence analysis results demonstrated that the number of SMN nuclear structures, Gems, was the same as or greater than the number of Gems found in normal fibroblasts. The results of this study indicate that gene-targeting methods do, in fact, present as an alternative for restoration of SMN expression in SMA patients-derived cells in vitro. PMID:26331341

  8. The effects of acoustic vibration on fibroblast cell migration.

    PubMed

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. PMID:27612824

  9. Positive Correlation Between the Efficiency of Induced Pluripotent Stem Cells and the Development Rate of Nuclear Transfer Embryos When the Same Porcine Embryonic Fibroblast Lines Are Used As Donor Cells

    PubMed Central

    Xie, Bingteng; Wang, Jianyu; Liu, Shichao; Wang, Jiaqiang; Xue, Binghua; Li, Jingyu; Wei, Renyue; Zhao, Yanhua

    2014-01-01

    Abstract Induced pluripotent stem cells (iPSCs) and nuclear transfer (NT) are two of the primary routes to reprogram differentiated cells back to the pluripotent state. However, it is still unknown whether there is any correlation between the reprogramming efficiency of iPSCs and NT if the same donor cells are employed. In this study, six porcine embryonic fibroblast (PEF) lines from Landrace (L1, L6, L9) or Congjiang local pigs (C4, C5, C6) were used for iPSC induction and NT. Furthermore, the resultant iPSCs from four PEF lines (L1, L6, C4, and C5) were used for NT (iPSC-NT), and the expression of exogenous genes was detected in iPSC-NT embryos by real-time PCR. The results showed that the efficiency of iPSC lines established from different PEF lines were significantly different. When the same PEF lines were used as donor cells for NT, the blastocysts rates were also different among different PEF lines and positively related with iPSCs induction efficiency. When the iPSCs were used as donor cells for NT, compared with the source PEFs, the blastocysts rates were significantly decreased. Real-time PCR results indicated that exogenous genes (Oct4, c-Myc) continued to be expressed in iPSC-NT embryos. In summary, our results demonstrate that there was a positive correlation between iPSCs and NT reprogramming efficiency, although the mechanism of these two routes is different. This may provide a new method to select the appropriate donor cells for inducing iPSCs. PMID:24738969

  10. On the Thermus thermophilus HB8 potential pathogenicity triggered from rhamnolipids secretion: morphological alterations and cytotoxicity induced on fibroblastic cell line.

    PubMed

    Pantazaki, A A; Choli-Papadopoulou, T

    2012-05-01

    A limited number of bacterial strains usually grown under nutrient limitation secrete rhamnolipids (RLs), which are recorded as virulence factors that are implicated in the pathogenicity of a microorganism. The non-pathogenic T. thermophilus HB8 produces extracellular rhamnolipids (TthRLs) under defined cultivation conditions using sunflower seed oil and sodium gluconate as carbon sources. In particular, the secreted TthRLs have been isolated, purified and identified with ATR-FTIR. Their effects on the cells' viability were examined when they were supplemented in a culture of human skin fibroblasts. Purified TthRLs triggered a sequence of rapid and pronounced morphological alterations characterized by transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation, rounding up, distortion of nuclei and loss of lamellar processes, and finally disruption of membrane. The addition of TthRLs in the cultured fibroblasts caused cytotoxicity, in contrast to that of rhamnose that stimulated viability, as it was assessed by MTT test. These results revealed that among the constituents of RLs that are implicated in the cytotoxicity, it has to be attributed to the lipidic chain variation and not to the carbohydrate part. TthRLs cytotoxicity on fibroblasts is comparable, and provoked similar effects, to that caused by saponin white, a known surfactant. TthRLs secretion might be a crucial point for the transformation of a non-pathogenic bacterium to a pathogenic one under certain environmental conditions favoring their secretion. RLs secretion in the microorganism's world might be a general route for the passage in the pathogenicity to ensure their survival under nutrient limitation conditions. PMID:21611776

  11. Deguelin-induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down-regulation of fibroblast growth factor receptor 4 activity.

    PubMed

    Wu, Wei; Hai, Yang; Chen, Lu; Liu, Rui-Jin; Han, Yu-Xiang; Li, Wen-Hao; Li, Song; Lin, Shuo; Wu, Xin-Rong

    2016-04-01

    Deguelin, a natural component derived from leguminous plants, has been used as pesticide in some regions. Accumulating evidence show that deguelin has promising chemopreventive and therapeutic activities against cancer cells. This study shows that low concentrations of deguelin can lead to significant delay in zebrafish embryonic development through growth inhibition and induction of apoptosis. Furthermore, we identified fibroblast growth factor receptor 4 (FGFR4) as the putative target of deguelin. The candidate was initially identified by a microarray approach and then validated through in vitro experiments using hormone-responsive (MCF-7) and nonresponsive (MDA-MB-231) human breast cancer cell lines. The results show that deguelin suppressed cell proliferation and induced apoptosis in both cancer cell lines, but not in Hs 578Bst cells, by blocking PI3K/AKT and mitogen-activated protein kinases (MAPK) signaling. The FGFR4 mRNA and protein level also diminished in a dose-dependent manner. Interestingly, we found that forced FGFR4 overexpression attenuated deguelin-induced proliferative suppression and apoptotic cell death in both zebrafish and MCF-7 cell lines, p-AKT and p-ERK levels were restored upon FGFR4 overexpression. Taken together, our results strongly suggest that deguelin inhibition of PI3K/AKT and MAPK signaling in zebrafish and breast cancer cell lines is partially mediated through down-regulation of FGFR4 activity. PMID:27069628

  12. Effects of metal ions on fibroblasts and spiral ganglion cells.

    PubMed

    Paasche, G; Ceschi, P; Löbler, M; Rösl, C; Gomes, P; Hahn, A; Rohm, H W; Sternberg, K; Lenarz, T; Schmitz, K-P; Barcikowski, S; Stöver, T

    2011-04-01

    Degeneration of spiral ganglion cells (SGC) after deafness and fibrous tissue growth around the electrode carrier after cochlear implantation are two of the major challenges in current cochlear implant research. Metal ions are known to possess antimicrobial and antiproliferative potential. The use of metal ions could therefore provide a way to reduce tissue growth around the electrode array after cochlear implantation. Here, we report on in vitro experiments with different concentrations of metal salts with antiproliferative and toxic effects on fibroblasts, PC-12 cells, and freshly isolated spiral ganglion cells, the target cells for electrical stimulation by a cochlear implant. Standard cell lines (NIH/3T3 and L-929 fibroblasts and PC-12 cells) and freshly isolated SGC were incubated with concentrations of metal ions between 0.3 μmol/liter and 10 mmol/liter for 48 hr. Cell survival was investigated by neutral red uptake, CellQuantiBlue assay, or counting of stained surviving neurons. Silver ions exhibited distinct thresholds for proliferating and confluent cells. For zinc ions, the effective concentration was lower for fibroblasts than for PC-12 cells. SGC showed comparable thresholds for reduced cell survival not only for silver and zinc ions but also for copper(II) ions, indicating that these ions might be promising for reducing tissue growth on the surface of CI electrode arrays. These effects were also observed when combinations of two of these ions were investigated. PMID:21312225

  13. Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

    SciTech Connect

    Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei; Wang, Na-Na; Nesa, Effat un; Wang, Jian-Bo; Wang, Cong; Jia, Yi-Bin; Wang, Kai; Tian, Hui; Cheng, Yu-Feng

    2015-03-06

    Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiated fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.

  14. Differential expression of the human ST5 gene in HeLa-fibroblast hybrid cell lines mediated by YY1: evidence that YY1 plays a part in tumor suppression.

    PubMed Central

    Lichy, J H; Majidi, M; Elbaum, J; Tsai, M M

    1996-01-01

    Through a mutational analysis of a differentially regulated enhancer, we present evidence that supports a role for the transcription factor YY1 in tumor suppression in HeLa/fibroblast somatic cell hybrids. The human ST5 gene was previously shown to be expressed as three RNA species, 4.6, 3.1 and 2.8 kb in length. Whereas the two larger species are expressed at similar levels in all cell lines examined, the 2.8 kb mRNA is expressed specifically in non-tumorigenic hybrids. In this study, the basis for the differential expression of this mRNA species was investigated. The message was shown to originate from a promoter located within an intron of the ST5 gene. An enhancer located approximaely 1500 nt upstream of the start site was required for cell type specific expression. Mutational analysis of this enhancer revealed an AP1 site and five YY1 sites which were necessary for full enhancer activity. Levels of YY1 DNA binding activity were found to be as much as 6-fold higher in the non-tumorigenic cells relative to the tumorigenic cells, while AP1 activity was similar in both cell types. These results suggest that a signaling pathway targeting YY1 may play an important role in tumor suppression in HeLa-fibroblast hybrids. PMID:8972856

  15. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF) Cell Lines.

    PubMed

    Santhanam, Ramesh Kumar; Ahmad, Syahida; Abas, Faridah; Safinar Ismail, Intan; Rukayadi, Yaya; Tayyab Akhtar, Muhammad; Shaari, Khozirah

    2016-01-01

    Zanthoxylum rhetsa is an aromatic tree, known vernacularly as "Indian Prickly Ash". It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells. PMID:27231889

  16. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells.

    PubMed

    Salmenperä, Pertteli; Karhemo, Piia-Riitta; Räsänen, Kati; Laakkonen, Pirjo; Vaheri, Antti

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. PMID:27177832

  17. Exposure of human lung fibroblasts to ozone: cell mortality and hyaluronan metabolism

    SciTech Connect

    Mayer, D.; Branscheid, D. )

    1992-04-01

    Exposure of cultures of human lung fibroblasts to 0.5 ppm ozone for 20 h resulted in a significant increase in cellular mortality by 29%; after exposure to 2.5 ppm ozone for 4 h, the increase amounted to 74%. A marked difference in sensitivity to ozone was observed between fibroblast lines from different individuals. This variability in resistance to ozone was more evident after exposure to 0.5 ppm ozone for 20 h, when compared with 2.5 ppm ozone for 4 h. In one fibroblast line, synthesis of hyaluronan was enhanced by exposure to 0.5 ppm ozone for 20 h. The concentrations of hyaluronan in culture media increased in experiments using different fibroblast cell lines, a phenomenon that was obvious both if cell numbers and combined protein concentrations of cells and media are selected as references for hyaluronan concentrations.

  18. Induced pluripotent stem cells from goat fibroblasts.

    PubMed

    Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Gu, Chenghao; Wang, Ziyu; Dong, Fulu; Wang, Feng

    2013-12-01

    Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo. PMID:24123501

  19. Differential responses of scirrhous and well-differentiated gastric cancer cells to orthotopic fibroblasts.

    PubMed Central

    Yashiro, M.; Chung, Y. S.; Kubo, T.; Hato, F.; Sowa, M.

    1996-01-01

    Scirrhous gastric cancer cells proliferate rapidly with fibrosis, when the cancer cells invade into the submucosa of the stomach. To investigate the mechanisms responsible for the rapid proliferation, the growth interaction between gastric cancer cells and fibroblasts was examined. Human gastric cancer cell lines established from scirrhous carcinoma or well-differentiated adenocarcinoma were used. Human fibroblast cell lines were obtained from various organs. The growth interaction between gastric cancer cells and fibroblasts was examined by calculating the number of cancer cells or by measuring [3H]thymidine incorporation of cancer cells. Gastric fibroblasts specifically stimulated the growth of scirrhous gastric cancer cells, but not that of well-differentiated adenocarcinoma cells. The growth factor(s) produced from gastric fibroblasts were then partially purified and characterised. The growth-promoting factor(s) had apparent molecular weights of 10000 dalton and was sensitive both to heat and proteinase treatment. No inhibition for the factor(s) was achieved with defined anti-growth factor antibodies. In this study, differential responses of scirrhous and well-differentiated gastric cancer cells to orthotopic fibroblasts were shown. Rapid proliferation of scirrhous gastric carcinoma should be partly controlled by orthotopic fibroblasts. The growth factor(s) from gastric fibroblasts, which was distinct from various defined growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), transforming growth factor-alpha (TGF-alpha), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), insulin-like growth factor I (IGF-I), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and transforming growth factor beta 1 (TGF-beta 1) may play an important role in the progression of scirrhous gastric cancer cells. PMID:8855981

  20. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

    SciTech Connect

    Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

    2014-09-05

    Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

  1. Neoplastic transformation of human diploid fibroblast cells by chemical carcinogens

    PubMed Central

    Kakunaga, Takeo

    1978-01-01

    Cultured fibroblast cells derived from a skin biopsy sample taken from normal human adult were exposed to a potent carcinogen, 4-nitroquinoline 1-oxide. Alterations of cell growth pattern such as higher density and piling up of cells were noticed in some fractions of cultures that were successively subcultured after nitroquinoline oxide treatment. Morphologically altered cells retained this growth pattern and became established lines of transformed cells without showing the limited life-span characteristic of normal cells in culture. The transformed cells showed a higher saturation density and the ability to grow in soft agar, properties that are usually correlated with neoplastic transformation of cells in culture. Selection of preexisting transformed human cells as a mechanism of this observed transformation seemed unlikely because clones of these normal cells could also be used to assess the transforming effect of nitroquinoline oxide. Preliminary results suggest that numerous cell divisions were required for the development of the transformation after nitroquinoline oxide treatment of these human cells. When the transformed cell lines were injected subcutaneously into nude (athymic) mice, solid tumors were produced at the site of inoculation. Treatment with N-methyl-N′-nitro-N-nitrosoguanidine also induced cell transformation, in a manner similar to treatment with nitroquinoline oxide. However, transformation was not induced with (i) 4-aminoquinoline 1-oxide (a noncarcinogenic derivative of 4-nitroquinoline 1-oxide), (ii) 3-methylcholanthrene (a carcinogen that cannot be metabolically activated by the target cells employed), or (iii) the solvent dimethyl sulfoxide. Images PMID:418410

  2. Mesenchymal stem cells induce dermal fibroblast responses to injury

    SciTech Connect

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  3. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    PubMed Central

    Čížková, Alena; Stránecký, Viktor; Ivánek, Robert; Hartmannová, Hana; Nosková, Lenka; Piherová, Lenka; Tesařová, Markéta; Hansíková, Hana; Honzík, Tomáš; Zeman, Jiří; Divina, Petr; Potocká, Andrea; Paul, Jan; Sperl, Wolfgang; Mayr, Johannes A; Seneca, Sara; Houštĕk, Josef; Kmoch, Stanislav

    2008-01-01

    Background To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. Results Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for

  4. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.

    PubMed

    Questa, María; Romorini, Leonardo; Blüguermann, Carolina; Solari, Claudia María; Neiman, Gabriel; Luzzani, Carlos; Scassa, María Élida; Sevlever, Gustavo Emilio; Guberman, Alejandra Sonia; Miriuka, Santiago Gabriel

    2016-03-01

    Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls. PMID:27345989

  5. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System

    PubMed Central

    Ueno, Takayuki; Utsumi, Jun; Toi, Masakazu; Shimizu, Kazuharu

    2015-01-01

    The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment. PMID:26171396

  6. Protective and restorative effects of a Commiphora mukul gum resin and triheptanoin preparation on the CCL-110 skin fibroblast cell line.

    PubMed

    Ramachandran, C; Quirin, K-W; Resek, A; Melnick, S J

    2012-04-01

    Coenzyme Q10 (CoQ10) is a major ingredient in skin care products because of its anti-wrinkle effects, although it has some side effects especially at higher amounts. In this study, we compare the anti-wrinkle related properties of CoQ10 and a proprietary Commiphora mukul gum resin (guggul) and triheptanoin preparation (GU-TC7). GU-TC7 is prepared with a supercritical CO₂-co-solvent extraction with ethanol, standardized to 2% guggulsterones and triheptanoin, a triglyceride composed of three 7-carbon fatty acids. Treatment of CCL-110 skin fibroblasts with GU-TC7 demonstrates a mild proliferative effect compared to CoQ10 and increased type I collagen synthesis. Additionally, GU-TC7 inhibited matrix metalloproteinase-1 (MMP-1) expression in a dose-dependent manner at 20-100 μg mL⁻¹ and inhibited human elastase expression by more than 50% as compared to no elastase inhibition with CoQ10 treatment. These results suggest that GU-TC7 possesses properties that are applicable to the treatment of wrinkles and may be considered for its further evaluation in skin care products. PMID:22084831

  7. Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

    PubMed Central

    2014-01-01

    Introduction We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined. Methods Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed. Results Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation. Conclusions These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA. PMID:24467809

  8. Generation of Induced Pluripotent Stem Cells from Diabetic Foot Ulcer Fibroblasts Using a Nonintegrative Sendai Virus.

    PubMed

    Gerami-Naini, Behzad; Smith, Avi; Maione, Anna G; Kashpur, Olga; Carpinito, Gianpaolo; Veves, Aristides; Mooney, David J; Garlick, Jonathan A

    2016-08-01

    Diabetic foot ulcers (DFUs) are nonhealing chronic wounds that are a serious complication of diabetes. Since induced pluripotent stem cells (iPSCs) may offer a potent source of autologous cells to heal these wounds, we studied if repair-deficient fibroblasts, derived from DFU patients and age- and site-matched control fibroblasts, could be reprogrammed to iPSCs. To establish this, we used Sendai virus to successfully reprogram six primary fibroblast cell lines derived from ulcerated skin of two DFU patients (DFU8, DFU25), nonulcerated foot skin from two diabetic patients (DFF24, DFF9), and healthy foot skin from two nondiabetic patients (NFF12, NFF14). We confirmed reprogramming to a pluripotent state through three independent criteria: immunofluorescent staining for SSEA-4 and TRA-1-81, formation of embryoid bodies with differentiation potential to all three embryonic germ layers in vitro, and formation of teratomas in vivo. All iPSC lines showed normal karyotypes and typical, nonmethylated CpG sites for OCT4 and NANOG. iPSCs derived from DFUs were similar to those derived from site-matched nonulcerated skin from both diabetic and nondiabetic patients. These results have established for the first time that multiple, DFU-derived fibroblast cell lines can be reprogrammed with efficiencies similar to control fibroblasts, thus demonstrating their utility for future regenerative therapy of DFUs. PMID:27328415

  9. Cell cycle is disturbed in mucopolysaccharidosis type II fibroblasts, and can be improved by genistein.

    PubMed

    Moskot, Marta; Gabig-Cimińska, Magdalena; Jakóbkiewicz-Banecka, Joanna; Węsierska, Magdalena; Bocheńska, Katarzyna; Węgrzyn, Grzegorz

    2016-07-01

    Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by mutations resulting in deficiency of one of enzymes involved in degradation of glycosaminoglycans (GAGs). These compounds accumulate in cells causing their dysfunctions. Genistein is a molecule previously found to both modify GAG metabolism and modulate cell cycle. Therefore, we investigated whether the cell cycle is affected in MPS cells and if genistein can influence this process. Fibroblasts derived from patients suffering from MPS types I, II, IIIA and IIIB, as well as normal human fibroblasts (the HDFa cell line) were investigated. MTT assay was used for determination of cell proliferation, and the cell cycle was analyzed by using the MUSE® Cell Analyzer. While effects of genistein on cell proliferation were similar in both normal and MPS fibroblasts, fractions of cells in the G0/G1 phase were higher, and number of cells entering the S and G2/M phases was considerably lower in MPS II fibroblasts relative to control cells. Somewhat similar tendency, though significantly less pronounced, could be noted in MPS I, but only at longer times of incubation. However, this was not observed in MPS IIIA and MPS IIIB fibroblasts. Genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) was found to be able to partially correct the disturbances in the MPS II cell cycle, and to some extent in MPS I, at higher concentrations of this compound. The tendency to increase the fractions of cells entering the S and G2/M phases was also observed in MPS IIIA and IIIB fibroblasts treated with genistein. In conclusion, this is the first report indicating that the cell cycle can be impaired in MPS cells. The finding that genistein can improve the MPS II (and to some extent also MPS I) cell cycle provides an input to our knowledge on the molecular mechanisms of action of this compound. PMID:27016302

  10. A rice-derived recombinant human lactoferrin stimulates fibroblast proliferation, migration, and sustains cell survival.

    PubMed

    Tang, Ling; Cui, Tengjiao; Wu, James J; Liu-Mares, Wen; Huang, Ning; Li, Jie

    2010-01-01

    Human lactoferrin (hLF), a glycoprotein of the transferrin family, has recently been shown to stimulate wound repair through its antimicrobial effect and inflammation modulation. A recent study with several non-skin cell lines indicated that hLF may also have a stimulatory effect on cell proliferation. To explore the role of hLF in wound healing, we used recombinant human lactoferrin (holo-rhLF), derived from transgenic rice, to examine the effects of holo-rhLF on cell proliferation, migration, attachment, and survival in a human primary skin fibroblast culture system. This study revealed that holo-rhLF not only significantly stimulates fibroblast proliferation but also has synergistic effects with fibroblast growth factor-2 and antagonistic effects with transforming growth factor-beta1 on cell proliferation. Furthermore, using a chamber migration assay, our results demonstrate that holo-rhLF promotes fibroblast migration in a dosage-dependent manner. More importantly, holo-rhLF significantly increased cell viability and protected cells from death when they were stressed by either serum depletion or 12-O-tetradecanoylphorbol-13-acetate exposure. No significant effect was observed on cell attachment. In conclusion, these findings reveal the multiple functions of holo-rhLF in human skin fibroblasts and indicate its potential application in wound therapy by enhancing cell proliferation and migration as well as protecting cells from apoptosis. PMID:20082685

  11. Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts.

    PubMed

    Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W

    2016-02-19

    The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4(+) but not CD8(+) cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. PMID:26699196

  12. Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

    PubMed

    He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2016-08-01

    Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene. PMID:26341227

  13. Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

    PubMed

    Pickard, Adam; Cichon, Ann-Christin; Barry, Anna; Kieran, Declan; Patel, Daksha; Hamilton, Peter; Salto-Tellez, Manuel; James, Jacqueline; McCance, Dennis J

    2012-07-18

    Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. PMID:22643222

  14. Estradiol regulation of nucleotidases in female reproductive tract epithelial cells and fibroblasts.

    PubMed

    Shen, Zheng; Fahey, John V; Bodwell, Jack E; Rodriguez-Garcia, Marta; Rossoll, Richard M; Crist, Sarah G; Patel, Mickey V; Wira, Charles R

    2013-01-01

    The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5'-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5'-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT. PMID:23936114

  15. Fibroblast Cell-Based Therapy for Experimental Autoimmune Diabetes

    PubMed Central

    Jalili, Reza B.; Zhang, Yun; Hosseini-Tabatabaei, Azadeh; Kilani, Ruhangiz T.; Khosravi Maharlooei, Mohsen; Li, Yunyuan; Salimi Elizei, Sanam; Warnock, Garth L.; Ghahary, Aziz

    2016-01-01

    Type 1 diabetes (T1D) results from autoimmune destruction of insulin producing β cells of the pancreatic islets. Curbing autoimmunity at the initiation of T1D can result in recovery of residual β cells and consequently remission of diabetes. Here we report a cell-based therapy for autoimmune diabetes in non-obese diabetic (NOD) mice using dermal fibroblasts. This was achieved by a single injection of fibroblasts, expressing the immunoregulatory molecule indoleamine 2,3 dioxygenase (IDO), into peritoneal cavity of NOD mice shortly after the onset of overt hyperglycemia. Mice were then monitored for reversal of hyperglycemia and changes in inflammatory / regulatory T cell profiles. Blood glucose levels dropped into the normal range in 82% of NOD mice after receiving IDO-expressing fibroblasts while all control mice remained diabetic. We found significantly reduced islet inflammation, increased regulatory T cells, and decreased T helper 17 cells and β cell specific autoreactive CD8+ T cells following IDO cell therapy. We further showed that some of intraperitoneal injected fibroblasts migrated to local lymph nodes and expressed co-inhibitory molecules. These findings suggest that IDO fibroblasts therapy can reinstate self-tolerance and alleviate β cell autoreactivity in NOD mice, resulting in remission of autoimmune diabetes. PMID:26765526

  16. Fibroblast Cell-Based Therapy for Experimental Autoimmune Diabetes.

    PubMed

    Jalili, Reza B; Zhang, Yun; Hosseini-Tabatabaei, Azadeh; Kilani, Ruhangiz T; Khosravi Maharlooei, Mohsen; Li, Yunyuan; Salimi Elizei, Sanam; Warnock, Garth L; Ghahary, Aziz

    2016-01-01

    Type 1 diabetes (T1D) results from autoimmune destruction of insulin producing β cells of the pancreatic islets. Curbing autoimmunity at the initiation of T1D can result in recovery of residual β cells and consequently remission of diabetes. Here we report a cell-based therapy for autoimmune diabetes in non-obese diabetic (NOD) mice using dermal fibroblasts. This was achieved by a single injection of fibroblasts, expressing the immunoregulatory molecule indoleamine 2,3 dioxygenase (IDO), into peritoneal cavity of NOD mice shortly after the onset of overt hyperglycemia. Mice were then monitored for reversal of hyperglycemia and changes in inflammatory/regulatory T cell profiles. Blood glucose levels dropped into the normal range in 82% of NOD mice after receiving IDO-expressing fibroblasts while all control mice remained diabetic. We found significantly reduced islet inflammation, increased regulatory T cells, and decreased T helper 17 cells and β cell specific autoreactive CD8+ T cells following IDO cell therapy. We further showed that some of intraperitoneal injected fibroblasts migrated to local lymph nodes and expressed co-inhibitory molecules. These findings suggest that IDO fibroblasts therapy can reinstate self-tolerance and alleviate β cell autoreactivity in NOD mice, resulting in remission of autoimmune diabetes. PMID:26765526

  17. Mesenchymal stromal cells and fibroblasts: a case of mistaken identity?

    PubMed

    Hematti, Peiman

    2012-05-01

    The plastic-adherent fibroblast-looking cells that can be isolated and culture-expanded from bone marrow and many other tissues are widely known as mesenchymal stromal cells (MSC). In addition to their fibroblast-like morphology, they are characterized by a panel of cell-surface markers and their potential to differentiate into bone, fat and cartilage. Based on their intriguing immunomodulatory and regenerative properties, MSC are being investigated as cellular therapeutics for a variety of clinical indications. However, many questions regarding the true identity and functionality of these cells in vivo remain unanswered. Fibroblasts, known for a much longer time but still poorly characterized, are also considered to be a ubiquitous stromal element of almost all tissues and are believed to play a role in tissue homeostasis. Despite the presence of MSC and fibroblasts in almost all tissues, similar morphology and other shared characteristics, the exact relationship between MSC and fibroblasts has remained undetermined. In this review, based on recent and old, but often neglected, literature it is suggested that ex vivo culture-expanded MSC and fibroblasts are indistinguishable by morphology, cell-surface markers, differentiation potential and immunologic properties. PMID:22458957

  18. Fibroblast nemosis induces angiogenic responses of endothelial cells

    SciTech Connect

    Enzerink, Anna; Rantanen, Ville; Vaheri, Antti

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  19. Potentiation of fibroblast growth by nodular sclerosing Hodgkin's disease cell cultures.

    PubMed

    Newcom, S R; O'Rourke, L

    1982-07-01

    Cell cultures were established from 8 lymph nodes replaced by nodular sclerosing Hodgkin's disease. Serum-containing and serum-free conditioned media from these cultures potentiated fibroblast growth and were found to be consistently more potent than fibroblast growth factor, 100 ng/ml, every other day. Both a proliferative response and transformation-like growth were observed using BALB/c 3T3 cells, human diploid fibroblasts, and human embryonic fibroblasts as target cells. The Hodgkin's disease growth factor(s) was not produced by fibroblasts or lymphocytes in the Hodgkin's cultures and was most potent when the Hodgkin's cultures had been enriched with Hodgkin's giant cells. Removal of normal macrophages decreased the proliferative activity but did not eliminate it or nonadherent growth of 3T3 cells in agar. Control cultures of 6 nonmalignant lymph nodes, a Lennert's lymphoma, a mixed cellularity Hodgkin's disease lymph node, and a malignant histiocytosis cell line suggested that among lymph node disorders, this feature may be relatively specific for nodular sclerosing Hodgkin's disease. PMID:6211204

  20. Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase.

    PubMed

    Green, H; Chan, T

    1973-11-23

    In the presence of 10(-4) to 10(-5) molar adenosine, established cell lines of fibroblastic or lymphoid origin die of pyrimidine starvation. Less than lethal concentrations inhibit cell growth. Over a broad concentration range, the effects of adenosine are prevented by providing a suitable pyrimidine source. We suggest that the recently described immune deficiency disease associated with absence of adenosine deaminase may be the result of pyrimidine starvation induced by adenosine nucleotides in cells of the lymphoid system. PMID:4795749

  1. Altered response of fibroblasts from human tympanosclerotic membrane to interacting mast cells: implication for tissue remodeling.

    PubMed

    Pawelczyk, Tadeusz; Sakowicz-Burkiewicz, Monika; Wesserling, Martyna; Grden, Marzena; Kuczkowski, Jerzy

    2014-12-01

    Several lines of evidence suggest that a tympanosclerotic (TMS) lesion often develops secondary to acute and chronic otitis media. Histological findings indicate that fibroblasts and inflammatory cells, including mast cells, play a key role in the tympanosclerotic plaque formation. However, details on the functional characteristics of tympanosclerotic fibroblasts (Fs(TMS)) are scanty. Therefore the aim of our study was to examine the activity of human fibroblasts from tympanosclerotic lesions and to evaluate the influence of stimulated by crosslinking of IgE receptor mast cells (HMC-1(FcɛRI)) on fibroblast functional behavior. We observed that fibroblasts from normal tympanic membrane (Fs(TM)) released less TNF-α, TGF-β1 and IL-6 compared to Fs(TMS). Fs(TMS) but not Fs(TM) upon interaction with HMC-1(FcɛRI) released increased quantities of TNF-α and TGF-β1. Exposing the fibroblast to HMC-1(FcɛRI) cells resulted in an increased synthesis of proteins including collagen. We noted that the COL2A1 transcript level increased ∼5- and ∼12-fold in Fs(TM) and Fs(TMS) co-cultured with HMC-1(FcɛRI), respectively. Both Fs(TM) and Fs(TMS) upon maintenance in the primary culture released significant quantities of matrix metalloproteinase 9 (MMP-9). However, Fs(TMS) released ∼5-fold more MMP-9 activity compared to the Fs(TM) cultures. The mast cell-induced release of TNF-α, TGF-β1 and MMP-9 sustained for a longer time in Fs(TMS) cultures compared to Fs(TM). Concluding, our data strongly indicate that increased fibroblast sensitivity to mast cell stimulation greatly contributes to the excessive fibrosis and pathological remodeling of the tympanic membrane. We postulate that the persistency of the Fs(TMS) activated state could be an important factor in the pathogenesis of tympanosclerosis. PMID:25310903

  2. Cell proliferation in vitro modulates fibroblast collagenase activity

    SciTech Connect

    Lindblad, W.J.; Flood, L.

    1986-05-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a /sup 14/C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/..mu..g DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of /sup 3/H-thymidine and /sup 3/H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion.

  3. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity.

    PubMed

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography-Mass Spectrometry (GC-MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC-MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  4. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

    PubMed Central

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  5. Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.

    PubMed

    Ohshima, Susumu; Seyama, Atsushi

    2016-06-01

    Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc. PMID:26917432

  6. Investigation of the optical properties of normal fibroblasts and fibroblasts cultured with cancer cells in terahertz frequency range

    NASA Astrophysics Data System (ADS)

    Strepitov, E. A.; Liakhov, E. P.; Balbekin, N. S.; Khodzitsky, M. K.; Smolyanskaya, O. A.; Trulyov, A. S.; Serebryakova, M. K.

    2015-07-01

    The optical properties of normal fibroblasts and fibroblasts cultured with cancer cells were studied in the frequency range of 0.2 - 1.0 THz. The results show the possibility to distinguish healthy cells from corrupted ones using their optical parameters.

  7. Biphasic Response of Ciprofloxacin in Human Fibroblast Cell Cultures

    PubMed Central

    Hincal, Filiz; Gürbay, Aylin; Favier, Alain

    2003-01-01

    To investigate the possibility of the involvement of an oxidative stress induction in the mechanism of the cytotoxic effect of quinolone antibiotics, we examined the viability of human fibroblast cells exposed to ciprofloxacin (CPFX), and measured the levels of lipid peroxidation (LP), glutathione (GSH), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). The data showed that the effect of CPFX on the viability of cells, as determined by neutral red uptake assay, was time-dependent, and the dose-response relation was biphasic. Cytotoxicity was not observed in the concentration range 5–150 mg/l CPFX when the cells were incubated for 24 h. In contrast, lower concentrations (5 and 12.5 mg/l) of CPFX increased the cell growth in all incubation periods tested. Marked decreases in the viability of fibroblasts were observed at concentrations 50 and 75 mg/l, and ≥50 mg/l, following 48 and 72 h exposure, respectively (p < 0.05). However, when the cells were exposed to > 75 mg/l CPFX for 48 h, no cytotoxicity was observed. By exposing fibroblast cultures to 75 mg/l CPFX for 48 h, an induction of LP enhancement and a marked decrease in intracellular GSH were observed. Vitamin E pretreatment of the cells lowered the level of LP, increased the total GSH content, and provided significant protection against CPFX-induced cytotoxicity. The biphasic effect of CPFX possibly resulted from the complex dose-dependent relationships between reactive oxygen species (ROS), cell proliferation, and cell viability. It was previously reported, in fact, for several cell models that ROS exert a biphasic effect on cell growth. Furthermore, cultured fibroblasts release their own free radicals, and the inhibition of endogenous ROS inhibits the fibroblast cell proliferation, whereas the effect of exogenous ROS is biphasic. PMID:19330132

  8. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    SciTech Connect

    Nobe, Koji; Nobe, Hiromi; Yoshida, Hiroko; Kolodney, Michael S.; Paul, Richard J.; Honda, Kazuo

    2010-08-20

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.

  9. Effects of dexamethasone on human synovial fibroblast-like cells, from osteoarthritic joints, in culture

    SciTech Connect

    Vento, R.; Torregrossa, M.V.; Giuliano, M.; Grecomoro, G.; Piccione, F. )

    1990-01-01

    The effect of Dexamethasone (DEX) on cell division and macromolecular synthesis was investigated in a line (Mc Coy cells, A 9) of synovial fibroblast-like cells derived from human osteoarthritic joints. DEX markedly reduced the proliferation of Mc Coy cells in a time and dose-dependent manner. The maximal inhibition was found at 500 nM DEX 24 h after incubation and was accompanied by the appearance of giant macrophage-like cells. After DEX treatment cells showed increased content of DNA, proteins and RNA together with the reduction of ({sup 3}H)-thymidine incorporation into the TCA-precipitable fraction.

  10. Long-Term Quiescent Fibroblast Cells Transit into Senescence

    PubMed Central

    Marthandan, Shiva; Priebe, Steffen; Hemmerich, Peter; Klement, Karolin; Diekmann, Stephan

    2014-01-01

    Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development. PMID:25531649

  11. Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells

    SciTech Connect

    Powers, T.P.; Davidson, R.L.; Shows, T.B.

    1994-02-01

    Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells. 46 refs., 5 figs., 2 tabs.

  12. ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

    PubMed Central

    Skolucka, Nina; Daczewska, Malgorzata; Saczko, Jolanta; Chwilkowska, Agnieszka; Choromanska, Anna; Kotulska, Malgorzata; Kaminska, Iwona; Kulbacka, Julita

    2011-01-01

    Objective To estimate electroporation (EP) influence on malignant and normal cells. Methods Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following: 250, 1 000, 1 750, 2 500 V/cm; 50 µs by 5 impulses for every case. The viability of cells after EP was estimated by MTT assay. The ultrastructural analysis was observed by transmission electron microscope (Zeiss EM 900). Results In the current study we observed the intracellular effect following EP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP. Conversely, we showed that EP in some conditions can stimulate cells to proliferation. Some changes induced by EP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters of EP (250 and 1 000 V/cm). After applying higher electric field intensities (2 500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications after EP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP. Conclusions We can claim that EP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells. PMID:23569735

  13. Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells

    SciTech Connect

    Riabowol, K.T.; Vosatka, R.J.; Ziff, E.B.; Lamb, N.J.; Feramisco, J.R.

    1988-04-01

    Transcription of the protooncogene c-fos is increased >10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G/sub o/) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, the authors microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, they found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G/sub o/ to G/sub 1/ of the cell cycle, its function is also required during the early G/sub 1/ portion of the cell cycle to allow subsequent DNA synthesis.

  14. Conversion of monkey fibroblasts to transplantable telencephalic neuroepithelial stem cells.

    PubMed

    Ai, Zongyong; Xiang, Zheng; Li, Yuemin; Liu, Guoku; Wang, Hong; Zheng, Yun; Qiu, Xiaoyan; Zhao, Shumei; Zhu, Xiaoqing; Li, Yanhua; Ji, Weizhi; Li, Tianqing

    2016-01-01

    Non-human primates provide optimal models for the development of stem cell therapies. Although somatic cells have been converted into neural stem/progenitor cells, it is unclear whether telencephalic neuroepithelial stem cells (NESCs) with stable properties can be generated from fibroblasts in primate. Here we report that a combination of transcription factors (Oct4, Sox2, Klf4) with a new culture medium induces rhesus monkey fibroblasts into NESCs, which can develop into miniature neural tube (NT)-like structures at a cell level. Furthermore, single induced NESCs (iNESCs) can generate later-stage 3D-NTs after grown on matrigel in suspension culture. iNESCs express NT cell markers, have a unique gene expression pattern biasing towards telencephalic patterning, and give rise to cortical neurons. Via transplantation, single iNESCs can extensively survive, regenerate myelinated neuron axons and synapse structures in adult monkey striatum and cortex, and differentiate into cortical neurons. Successful transplantation is closely associated with graft regions and grafted cell identities. The ability to generate defined and transplantable iNESCs from primate fibroblasts under a defined condition with predictable fate choices will facilitate disease modeling and cell therapy. PMID:26584346

  15. The growth regulatory fibroblast IK channel is the prominent electrophysiological feature of rat prostatic cancer cells.

    PubMed

    Rane, S G

    2000-03-16

    Physiological effectors for mitogenic cell growth control remain to be determined for mammalian tumor cells, particularly those derived from prostatic tissue. One such effector for mitogenic Ras/MAPK signaling in fibroblasts is an intermediate-conductance, calcium-activated potassium channel (FIK). In this study patch-clamp electrophysiology was used to show that both AT2.1 and MatLyLu rat prostate cancer cell lines express high levels of a current identified as FIK, based on the following criteria: activation by elevation of intracellular calcium, voltage independence, potassium selectivity, and block by charybdotoxin (ChTX) and the Stichodactyla helianthus potassium channel neurotoxin (StK). FIK current densities in AT2.1 and MatLyLu cells were comparable to the high levels seen in fibroblasts transfected with oncogenic Ras or Raf, suggesting hyperactivity of the Ras/MAPK pathway in prostatic cancer cells. Voltage-gated sodium current was present in most MatLyLu cells but absent from AT2.1 cells, and all AT2.1 cells had voltage-gated potassium currents. Thus, FIK is the main electrophysiological feature of rat prostatic cancer cells as it is for mitogenically active fibroblasts, suggesting it may play a similar growth regulatory role in both. PMID:10708575

  16. Responses of fibroblasts and glial cells to nanostructured platinum surfaces

    NASA Astrophysics Data System (ADS)

    Pennisi, C. P.; Sevcencu, C.; Dolatshahi-Pirouz, A.; Foss, M.; Lundsgaard Hansen, J.; Nylandsted Larsen, A.; Zachar, V.; Besenbacher, F.; Yoshida, K.

    2009-09-01

    The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

  17. Cell culture-derived HCV cannot infect synovial fibroblasts

    PubMed Central

    Nadeem, Abd-Elshafy D.; Thomas, Pietschmann; Ulf, Müller-Ladner; Elena, Neumann; Anggakusuma, A; Mohamed, Bahgat M.; Frank, Pessler; Patrick, Behrendt

    2015-01-01

    Worldwide 170 million individuals are infected with hepatitis C virus (HCV), up to 45 million of whom are affected by arthropathy. It is unclear whether this is due to viral infection of synovial cells or immune-mediated mechanisms. We tested the capacity of primary synovial fibroblasts to support HCV propagation. Out of the four critical HCV receptors, only CD81 was expressed to any significant extent in OASF and RASF. Consistent with this, pseudotyped HCV particles were unable to infect these cells. Permissiveness for HCV replication was investigated by transfecting cells with a subgenomic replicon of HCV encoding a luciferase reporter. OASF and RASF did not support replication of HCV, possibly due to low expression levels of miR-122. In conclusion, primary human synovial fibroblasts are unable to support propagation of HCV in vitro. HCV-related arthropathy is unlikely due to direct infection of these cells. PMID:26643193

  18. Cells of the synovium in rheumatoid arthritis. Synovial fibroblasts

    PubMed Central

    Müller-Ladner, Ulf; Ospelt, Caroline; Gay, Steffen; Distler, Oliver; Pap, Thomas

    2007-01-01

    For some time synovial fibroblasts have been regarded simply as innocent synovial cells, mainly responsible for synovial homeostasis. During the past decade, however, a body of evidence has accumulated illustrating that rheumatoid arthritis synovial fibroblasts (RASFs) are active drivers of joint destruction in rheumatoid arthritis. Details regarding the intracellular signalling cascades that result in long-term activation and synthesis of proinflammatory molecules and matrix-degrading enzymes by RASFs have been analyzed. Molecular, cellular and animal studies have identified various interactions with other synovial and inflammatory cells. This expanded knowledge of the distinct role played by RASFs in the pathophysiology of rheumatoid arthritis has moved these fascinating cells to the fore, and work to identify targeted therapies to inhibit their joint destructive potential is underway. PMID:18177509

  19. Spontaneous establishment of an Epstein-Barr virus-infected fibroblast line from the synovial tissue of a rheumatoid arthritis patient.

    PubMed Central

    Koide, J; Takada, K; Sugiura, M; Sekine, H; Ito, T; Saito, K; Mori, S; Takeuchi, T; Uchida, S; Abe, T

    1997-01-01

    An Epstein-Barr virus (EBV)-infected fibroblast line, designated DSEK, was spontaneously established from synovial tissue of a patient with rheumatoid arthritis (RA). DSEK cells expressed EBV nuclear antigens EBNA-1 and EBNA-2 and latent membrane protein LMP-1. Cell surface markers of DSEK cells were similar to those of EBV-negative fibroblast clones derived from synoviocytes and were negative for lymphocyte and macrophage markers. DSEK cells expressed CD44, CD58, and HLA-DR antigens and spontaneously produced interleukin-10 basic fibroblast growth factor and transforming growth factor beta1. These results indicate that rheumatoid synoviocytes can be a target for EBV infection and suggest that EBV may play a role in the pathogenesis of RA. PMID:9032386

  20. Rat embryo fibroblast cells expressing human papillomavirus 1a genes exhibit altered growth properties and tumorigenicity.

    PubMed Central

    Green, M; Brackmann, K H; Loewenstein, P M

    1986-01-01

    Human papillomavirus 1a (HPV1a) induces benign tumors (papillomas or warts) in humans under natural conditions of infection but has not been found to replicate significantly in cell culture or in experimental animals. To establish model systems to study the oncogenic properties and expression of HPV genes, we established cell lines by cotransfecting the 3Y1 rat fibroblast cell line with HPV1a DNA constructs containing an intact early gene region and the Tn5 neomycin resistance gene. Most cell lines selected for expression of the neomycin resistance gene by treatment with the antibiotic G-418 contained viral DNA in a high-molecular-weight form. The growth characteristics of several cell lines containing high copy numbers of HPV1a DNA were studied further. They were shown to differ from the parental cell line and from G-418-resistant cell lines that did not incorporate viral DNA in the following properties: morphological alteration, increased cell density at confluence, growth in 0.5% serum, efficient anchorage-independent growth in soft agar, and rapid formation of tumors in nude mice. Those cell lines that possessed altered growth properties and tumorigenicity were found to express abundant quantities of polyadenylated virus-specific RNA species in the cytoplasm. Images PMID:3023676

  1. Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts.

    PubMed Central

    Moe, O W; Miller, R T; Horie, S; Cano, A; Preisig, P A; Alpern, R J

    1991-01-01

    Increased Na/H antiporter activity has been demonstrated after in vivo chronic metabolic acidosis as well as in vitro acid preincubation of cultured rabbit renal tubule cells. To study the underlying molecular mechanisms of this adaptive increase in Na/H antiporter activity, the present studies examined the effect of low pH media on Na/H antiporter activity and mRNA abundance in cultured renal tubule cells. Na/H antiporter activity was increased by 60% in a mouse renal cortical tubule cell line (MCT), and by 90% in an opossum kidney cell line (OKP) after 24 h of preincubation in acid (low [HCO3]) media. The ethylisopropylamiloride sensitivity of the Na/H antiporters were different in these two cell lines (MCT IC50 = 65 nM; OKP IC50 = 4.5 microM). In MCT cells, Na/H antiporter mRNA abundance measured by RNA blots increased by two- to fivefold after 24 h in low [HCO3] media. Na/H antiporter mRNA abundance was also increased in MCT cells with high CO2 preincubation as well as in rat renal cortex with in vivo chronic acid feeding. In contrast to renal epithelia, acid preincubation of NIH 3T3 fibroblasts led to suppression of Na/H antiporter activity. RNA blots of 3T3 fibroblasts revealed the same size Na/H antiporter transcript as in MCT cells. However, Na/H antiporter mRNA levels were suppressed by acid preincubation. These studies demonstrate differential regulation of Na/H antiporter activity and mRNA abundance in renal epithelial cells and fibroblasts in response to an acidotic environment. Images PMID:1658050

  2. Tumor-associated fibroblasts promote the proliferation and decrease the doxorubicin sensitivity of liposarcoma cells

    PubMed Central

    HARATI, KAMRAN; DAIGELER, ADRIEN; HIRSCH, TOBIAS; JACOBSEN, FRANK; BEHR, BJÖRN; WALLNER, CHRISTOPH; LEHNHARDT, MARCUS; BECERIKLI, MUSTAFA

    2016-01-01

    The reasons for the distinct chemoresistance of liposarcomas and their high risk of local recurrence still remain unclear. Depending on the histological subtype of liposarcoma, first-line therapy with the cytostatic agent, doxorubicin, only achieves response rates of approximately 36%. Approximatley 70% of all local recurrences develop in spite of complete surgical resection of the primary tumor with microscopically negative margins. In this study, we aimed to assess the influence of tumor-associated fibroblasts (TAFs) obtained from surgically removed liposarcomas on the well-established human liposarcoma SW872 cell line. Intratumoral TAFs were isolated from intermediate- and high-grade liposarcoma samples. The human liposarcoma cell line, SW872, was co-cultured with the corresponding TAFs or with dermal fibroblasts as a control. The proliferation (by BrdU assay), cell viability (by MTT assay) and sensitivity to doxorubicin (using the iCELLigence system) of the co-cultured SW872 cells were examined. The SW872 cells exhibited a significant increase in proliferation and viability when co-cultured with the TAFs. As detected by real-time cell analysis, the SW872 cells co-cultured with the TAFs exhibited a diminished response towards doxorubicin. Notably, co-culture with TAFs obtained from high-grade liposarcoma samples resulted in higher proliferation and increased chemoresistance than co-culture with TAFs obtained from intermediate-grade liposarcoma samples. The findings of the present study thus indicate that TAFs from liposarcomas enhance the proliferation and decrease the chemosensitivity of SW872 liposarcoma cells significantly compared with normal fibroblasts from the dermis. TAFs from more malignant liposarcomas promoted tumor cell proliferation and chemoresistance more strikingly than TAFs from less malignant liposarcomas. These data provide evidence for the influence of the tumor microenvironment on liposarcoma and support for further investigations in

  3. Skin Biopsy and Patient-Specific Stem Cell Lines

    PubMed Central

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  4. Fibroblast activation protein predicts prognosis in clear cell renal cell carcinoma.

    PubMed

    López, José I; Errarte, Peio; Erramuzpe, Asier; Guarch, Rosa; Cortés, Jesús M; Angulo, Javier C; Pulido, Rafael; Irazusta, Jon; Llarena, Roberto; Larrinaga, Gorka

    2016-08-01

    Clear cell renal cell carcinoma is a complex disease with only partial response to therapy and scarce reliable clinical parameters indicative of progression and survival. Fibroblast activation protein expression has been correlated with prognosis in several malignancies but never in renal cancer. We aim to analyze the immunohistochemical expression of fibroblast activation protein in 208 clear cell renal cell carcinomas and to evaluate its impact on the prognosis and survival. A positive cytoplasmic immunostaining of this protein in the stromal fibroblasts associated to cancer cells is associated with large tumor diameter (≥4cm), high-grade (G3/4) tumors, and high-stage (≥pT3) tumors. Fibroblast activation protein-positive cases had significantly shorter survivals after 5 (P=.00015), 10 (P=.0000042), and 15 (P=.000043) years of follow-up, with a hazard ratio of 0.31. Multivariate analysis showed that fibroblast activation protein (P=.00117) was stronger than grade and stage in predicting clinical aggressiveness in clear cell renal cell carcinoma. This study confirms the usefulness of fibroblast activation protein detection in the stromal fibroblast associated to cancer in clear cell renal cell carcinoma and adds a new immunohistochemical marker to predict clinical behavior in these patients. PMID:27063470

  5. Establishment, Culture, and Characterization of Guinea Pig Fetal Fibroblast Cell

    PubMed Central

    Mahboobi, Reza; Dianatpour, Mehdi; Zare, Shahrokh; Hosseini, Seyed Ebrahim

    2014-01-01

    Establishment of Guinea pig fetal fibroblast cells and their biological evaluation before and after cryopreservation were the main purposes of this study. After determination of the proper age of pregnancy by ultrasonography, 30 days old fetuses of Guinea pigs were recovered. Their skins were cut into small pieces (1 mm2) and were cultured. When reaching 80–90% confluence, the cells were passaged. Cells of the second and eighth passages were cultured in 24-well plates (4 × 104 cells/well) for 6 days and three wells per day were counted. The average cell counts at each time point were then plotted against time and the population doubling time (PDT) was determined. Then, vials of cells (2 × 106 cells/mL) were cryopreserved for 1 month and after thawing, the cell viability was evaluated. The PDT of the second passage was about 23 h and for the eighth passage was about 30 h. The viability of the cultures was 95% in the second passage and 74.5% in the eighth passage. It was shown that the Guinea pig fetal fibroblast cell culture can be established using the adherent culture method while, after freezing, the viability indices of these cells were favorable. PMID:24790770

  6. Blasted Cell Line Names

    PubMed Central

    Wang, Jing; Byers, Lauren A.; Yordy, John S.; Liu, Wenbin; Shen, Li; Baggerly, Keith A.; Giri, Uma; Myers, Jeffrey N.; Ang, K. Kian; Story, Michael D.; Girard, Luc; Minna, John D.; Heymach, John V.; Coombes, Kevin R.

    2010-01-01

    Background: While trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. Most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name. Results: Reasoning that the variant names could be described in terms of mutations or deletions of character strings, we implemented a simple version of the Needleman-Wunsch global sequence alignment algorithm and applied it to the cell line names. All correct matches were found by this procedure. Incorrect matches only occured when a cell line was present in one data set but not in the other. The raw match scores tended to be substantially worse for the incorrect matches. Conclusions: A simple application of the Needleman-Wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets. PMID:21082038

  7. Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.

    PubMed

    Chao, A C; Dix, J A; Sellers, M C; Verkman, A S

    1989-12-01

    The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells. PMID:2482083

  8. Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties

    PubMed Central

    Scherzer, Michael T.; Waigel, Sabine; Donninger, Howard; Arumugam, Vennila; Zacharias, Wolfgang; Clark, Geoffrey; Siskind, Leah J.; Soucy, Patricia; Beverly, Levi

    2015-01-01

    Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME. PMID:26371754

  9. Intracellular collagen fibrils: evidence of an intracellular source from experiments with tendon fibroblasts and fibroblastic tumour cells.

    PubMed Central

    Michna, H

    1988-01-01

    This study was designed to substantiate one or both of the two hypotheses for the explanation of intracellular collagen fibrils in collagen-producing cells. The more obvious is the phagocytosis of extracellular collagen fibrils by the cell and the other is a form of autophagocytosis of newly synthesised collagenous products. Information was collected on fibroblasts from murine tendons after exercise and simultaneously stimulating collagen synthesis by treatment with an anabolic steroid hormone. Moreover, in vivo and in vitro fibroblastic tumour cells which demonstrate enhanced protein synthesis were also treated with the anabolic steroid. The findings of intracellular collagen fibrils in tendon fibroblasts and the sarcoma cells after experimentally stimulating collagen synthesis are discussed in the light of the hypothesis that the findings may represent steps of autophagocytosis of newly synthesised collagenous products in the absence of a control mechanism to remove collagenous products which cannot be secreted. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:3225213

  10. Irradiated fibroblast-induced bystander effects on invasive growth of squamous cell carcinoma under cancer-stromal cell interaction.

    PubMed

    Kamochi, Noriyuki; Nakashima, Masahiro; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Sugihara, Hajime; Toda, Shuji; Kudo, Sho

    2008-12-01

    The irradiated fibroblast-induced response of non-irradiated neighboring cells is called 'radiation-induced bystander effect', but it is unclear in non-irradiated human squamous cell carcinoma (SCC) cells. The present study shows that irradiated fibroblasts promoted the invasive growth of T3M-1 SCC cells, but not their apoptosis, more greatly than non-irradiated fibroblasts, using collagen gel invasion assay, immunohistochemistry and Western blot. The number of irradiated fibroblasts decreased to about 30% of that of non-irradiated fibroblasts, but irradiated fibroblasts increased the growth marker ki-67 display of SCC cells more greatly than non-irradiated fibroblasts. Irradiated fibroblasts did not affect the apoptosis marker ss-DNA expression of SCC cells. Irradiated fibroblasts enhanced the display of the following growth-, invasion- and motility-related molecules in SCC cells more greatly than non-irradiated fibroblasts: c-Met, Ras, mitogen-activated protein kinase (MAPK) cascade (Raf-1, MEK-1 and ERK-1/2), matrix metalloproteinase-1 and -9, laminin 5 and filamin A. Irradiated fibroblasts, but not non-irradiated ones, formed irradiation-induced foci (IRIF) of the genomic instability marker p53-binding protein 1 (53BP1) and expressed transforming growth factor-beta1 (TGF- beta1). Irradiated fibroblasts in turn enabled SCC cells to enhance 53BP1 IRIF formation more extensively than non-irradiated fibroblasts. Finally, effects of irradiated fibroblasts on growth and apoptosis of another HEp-2 SCC cell type were similar to those of T3M-1. These results suggest that irradiated fibroblasts promotes invasion and growth of SCC cells by enhancement of invasive growth-related molecules above through TGF- beta1-mediated bystander mechanism, in which irradiated fibroblast-induced genomic instability of SCC cells may be involved. PMID:19018771

  11. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    NASA Astrophysics Data System (ADS)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-02-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  12. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    PubMed Central

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-01-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis. PMID:25660754

  13. Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

    PubMed Central

    Ohta, Kunimasa; Kawano, Rie; Ito, Naofumi

    2012-01-01

    The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy. PMID:23300571

  14. Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

    PubMed

    Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

    2009-02-01

    Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity. PMID:19154348

  15. Overexpression of the IGF-II/M6P Receptor in Mouse Fibroblast Cell Lines Differentially Alters Expression Profiles of Genes Involved in Alzheimer’s Disease-Related Pathology

    PubMed Central

    Wang, Yanlin; Thinakaran, Gopal; Kar, Satyabrata

    2014-01-01

    Alzheimer’s disease (AD) is the most common type of senile dementia affecting elderly people. The processing of amyloid precursor protein (APP) leading to the generation of β-amyloid (Aβ) peptide contributes to neurodegeneration and development of AD pathology. The endocytic trafficking pathway, which comprises of the endosomes and lysosomes, acts as an important site for Aβ generation, and endocytic dysfunction has been linked to increased Aβ production and loss of neurons in AD brains. Since insulin-like growth factor-II (IGF-II) receptor plays a critical role in the transport of lysosomal enzymes from the trans-Golgi network to endosomes, it is likely that the receptor may have a role in regulating Aβ metabolism in AD pathology. However, very little is known on how altered levels of the IGF-II receptor can influence the expression/function of various molecules involved in AD pathology. To address this issue, we evaluated the expression profiles of 87 selected genes related to AD pathology in mouse fibroblast MS cells that are deficient in murine IGF-II receptor and corresponding MS9II cells overexpressing ∼500 times the human IGF-II receptors. Our results reveal that an elevation in IGF-II receptor levels alters the expression profiles of a number of genes including APP as well as enzymes regulating Aβ production, degradation and clearance mechanisms. Additionally, it influences the expression of various lysosomal enzymes and protein kinases that are involved in Aβ toxicity. IGF-II receptor overexpression also alters expression of several genes involved in intracellular signalling as well as cholesterol metabolism, which play a critical role in AD pathology. The altered gene profiles observed in this study closely match with the corresponding protein levels, with a few exceptions. These results, taken together, suggest that an elevation in IGF-II receptor levels can influence the expression profiles of transcripts as well as proteins that are involved

  16. Form and Function in Cell Motility: From Fibroblasts to Keratocytes

    PubMed Central

    Herant, Marc; Dembo, Micah

    2010-01-01

    Abstract It is plain enough that a horse is made for running, but similar statements about motile cells are not so obvious. Here the basis for structure-function relations in cell motility is explored by application of a new computational technique that allows realistic three-dimensional simulations of cells migrating on flat substrata. With this approach, some cyber cells spontaneously display the classic irregular protrusion cycles and handmirror morphology of a crawling fibroblast, and others the steady gliding motility and crescent morphology of a fish keratocyte. The keratocyte motif is caused by optimal recycling of the cytoskeleton from the back to the front so that more of the periphery can be devoted to protrusion. These calculations are a step toward bridging the gap between the integrated mechanics and biophysics of whole cells and the microscopic molecular biology of cytoskeletal components. PMID:20409459

  17. Human pancreatic beta-like cells converted from fibroblasts

    PubMed Central

    Zhu, Saiyong; Russ, Holger A.; Wang, Xiaojing; Zhang, Mingliang; Ma, Tianhua; Xu, Tao; Tang, Shibing; Hebrok, Matthias; Ding, Sheng

    2016-01-01

    Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes. PMID:26733021

  18. Human pancreatic beta-like cells converted from fibroblasts.

    PubMed

    Zhu, Saiyong; Russ, Holger A; Wang, Xiaojing; Zhang, Mingliang; Ma, Tianhua; Xu, Tao; Tang, Shibing; Hebrok, Matthias; Ding, Sheng

    2016-01-01

    Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes. PMID:26733021

  19. Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis

    PubMed Central

    Salmanzadeh, Alireza; Kittur, Harsha; Sano, Michael B.; C. Roberts, Paul; Schmelz, Eva M.; Davalos, Rafael V.

    2012-01-01

    Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool. PMID:22536308

  20. GRP78 is required for cell proliferation and protection from apoptosis in chicken embryo fibroblast cells.

    PubMed

    Jeon, M; Choi, H; Lee, S I; Kim, J S; Park, M; Kim, K; Lee, S; Byun, S J

    2016-05-01

    Chicken serum has been suggested as a supplement to promote chicken cell proliferation and development. However, the molecular mechanisms by which chicken serum stimulates chicken cell proliferation remain unknown. Here, we evaluated the effects of chicken serum supplementation on chicken embryo fibroblast (CEF) and DF-1 cell proliferation. We also sought to elucidate the molecular pathways involved in mediating the effects of chicken serum on fibroblasts and DF-1 cells by overexpression of chicken 78 kDa glucose-regulated protein (chGRP78), which is important for cell growth and the prevention of apoptosis. Our data demonstrated that the addition of 5% chicken serum significantly enhanced fibroblast proliferation. Moreover, knockdown of chGRP78 using siRNA decreased fibroblast proliferation and increased apoptosis. Based on these results, we suggest that the chGRP78-mediated signaling pathway plays a critical role in chicken serum-stimulated fibroblast survival and anti-apoptosis. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells through the inhibition of apoptosis and may lead to the development of new treatments for avian disease. PMID:26944959

  1. Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.

    PubMed

    Brunetti, Dario; Perota, Andrea; Lagutina, Irina; Colleoni, Silvia; Duchi, Roberto; Calabrese, Fiorella; Seveso, Michela; Cozzi, Emanuele; Lazzari, Giovanna; Lucchini, Franco; Galli, Cesare

    2008-12-01

    The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained. PMID:18823265

  2. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    SciTech Connect

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. )

    1991-03-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

  3. CLO: The cell line ontology

    PubMed Central

    2014-01-01

    Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

  4. Effect of microemulsions on cell viability of human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  5. Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2

    PubMed Central

    MIYAHARA, Daichi; OISHI, Isao; MAKINO, Ryuichi; KURUMISAWA, Nozomi; NAKAYA, Ryuma; ONO, Tamao; KAGAMI, Hiroshi; TAGAMI, Takahiro

    2015-01-01

    An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2. PMID:26727404

  6. Differentiated fibroblastic progenies of human embryonic stem cells for toxicology screening.

    PubMed

    Cao, Tong; Lu, Kai; Fu, Xin; Heng, Boon Chin

    2008-03-01

    Immortalized cell lines and live animal models are commonly used for cytotoxicity screening of biomedical devices and materials. However, these assays poorly reflect human physiology and have numerous other disadvantages. An alternative may be to utilize differentiated fibroblastic progenies of human embryonic stem cells (hESC) for in vitro toxicology screening. These were generated through random spontaneous differentiation within standard culture media, over several passages. The cytotoxic response of the differentiated hESC fibroblastic progenies (pH9) to mitomycin C was observed to be not only very similar to the L929 cell line, but was, in fact, more sensitive. At an initial seeding density of 1000 cells/well (0.33 cm(2)), the proliferation index was observed to decrease 19.0% from 1.638 to 1.326 for the L929 cell line, as the dosage of mitomycin C was gradually increased from 0 to 1.54 microg/mL. By contrast, pH9 displayed a corresponding 40.5% drop in proliferation index from 3.713 to 2.209. At a higher seeding density of 2000 cells/well (0.33 cm(2)), the proliferation index was observed to decrease 27.0% from 1.213 to 0.885 for the L929 cell line, whereas pH9 displayed a corresponding 43.7% drop in proliferation index from 3.711 to 2.091. Hence, it is apparent that pH9 exhibited a more sensitive dose-response to mitomycin C compared to L929, which could be advantageous for cytotoxicity screening assays. Additionally, this study also demonstrated that a highly purified and well-defined phenotypic population of differentiated hESC progenies is not necessary for high reproducibility and accuracy in cytotoxic response. PMID:18241121

  7. Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.

    PubMed

    Kubaczka, Caroline; Senner, Claire E; Cierlitza, Monika; Araúzo-Bravo, Marcos J; Kuckenberg, Peter; Peitz, Michael; Hemberger, Myriam; Schorle, Hubert

    2015-11-01

    Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation. PMID:26412560

  8. ASF-4-1 fibroblast-rich culture increases chemoresistance and mTOR expression of pancreatic cancer BxPC-3 cells at the invasive front in vitro, and promotes tumor growth and invasion in vivo

    PubMed Central

    FUJIWARA, MASAYA; KANAYAMA, KAZUKI; HIROKAWA, YOSHIFUMI S.; SHIRAISHI, TAIZO

    2016-01-01

    Pancreatic cancer develops dense stromal tissue through the desmoplastic reaction. The aim of the present study was to assess the effects of a fibroblast-rich environment on the malignant potential of pancreatic cancer. Cells from the human pancreatic cancer cell line BxPC-3 were mixed at a ratio of 1:3 (fibroblast-rich) or 1:1 (fibroblast-poor) with cells from the human skin fibroblast line ASF-4-1. In the fibroblast-rich co-culture, tumor budding was observed and BxPC-3 cells were found to be more resistant to gemcitabine than those in the fibroblast-poor co-culture. Immunohistochemistry revealed that the expression of mammalian target of rapamycin was increased at the invasive front of fibroblast-rich co-cultures. In addition, in mouse xenografts of fibroblast-rich co-cultures, tumors were larger and had a higher Ki-67 index compared with that of the fibroblast-poor co-culture xenografts. These results indicate that fibroblast-rich co-cultures may promote the malignant potential of the pancreatic cancer cell line BxPC-3, both in vitro and in vivo. PMID:27073551

  9. Recombinogenic Telomeres in Diploid Sorex granarius (Soricidae, Eulipotyphla) Fibroblast Cells

    PubMed Central

    Draskovic, I.; Minina, J. M.; Karamysheva, T. V.; Novo, C. L.; Liu, W.-Y.; Porreca, R. M.; Gibaud, A.; Zvereva, M. E.; Skvortsov, D. A.; Rubtsov, N. B.

    2014-01-01

    The telomere structure in the Iberian shrew Sorex granarius is characterized by unique, striking features, with short arms of acrocentric chromosomes carrying extremely long telomeres (up to 300 kb) with interspersed ribosomal DNA (rDNA) repeat blocks. In this work, we investigated the telomere physiology of S. granarius fibroblast cells and found that telomere repeats are transcribed on both strands and that there is no telomere-dependent senescence mechanism. Although telomerase activity is detectable throughout cell culture and appears to act on both short and long telomeres, we also discovered that signatures of a recombinogenic activity are omnipresent, including telomere-sister chromatid exchanges, formation of alternative lengthening of telomeres (ALT)-associated PML-like bodies, production of telomere circles, and a high frequency of telomeres carrying marks of a DNA damage response. Our results suggest that recombination participates in the maintenance of the very long telomeres in normal S. granarius fibroblasts. We discuss the possible interplay between the interspersed telomere and rDNA repeats in the stabilization of the very long telomeres in this organism. PMID:24842907

  10. Placenta Mesenchymal Stem Cell Derived Exosomes Confer Plasticity on Fibroblasts.

    PubMed

    Tooi, Masayuki; Komaki, Motohiro; Morioka, Chikako; Honda, Izumi; Iwasaki, Kengo; Yokoyama, Naoki; Ayame, Hirohito; Izumi, Yuichi; Morita, Ikuo

    2016-07-01

    Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc. PMID:26640165

  11. Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

    PubMed

    Yu, G; Okawa, H; Okita, K; Kamano, Y; Wang, F; Saeki, M; Yatani, H; Egusa, H

    2016-01-01

    Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of

  12. Glial cell line-derived neurotrophic factor in combination with insulin-like growth factor 1 and basic fibroblast growth factor promote in vitro culture of goat spermatogonial stem cells.

    PubMed

    Bahadorani, M; Hosseini, S M; Abedi, P; Abbasi, H; Nasr-Esfahani, M H

    2015-01-01

    Growth factors are increasingly considered as important regulators of spermatogonial stem cells (SSCs). This study investigated the effects of various growth factors (GDNF, IGF1, bFGF, EGF and GFRalpha-1) on purification and colonization of undifferentiated goat SSCs under in vitro and in vivo conditions. Irrespective of the culture condition used, the first signs of developing colonies were observed from day 4 of culture onwards. The number of colonies developed in GDNF + IGF1 + bFGF culture condition was significantly higher than the other groups (p < 0.05). In contrast, the size of colonies developed in GDNF + EGF + LIF culture condition was significantly higher than the other groups (p < 0.05). Immunocytochemical stationing for specific biomarkers of somatic cells (vimentin, alpha-inhibin and α-SMA) and spermatogonial cells (PLZF, THY 1, VASA, alpha-1 integrin, bet-1 integrin and DBA) revealed that both cell types existed in developing colonies, irrespective of the culture condition used. Even though, the relative abundance of VASA, FGFR3, OCT4, PLZF, BCL6B and THY1 transcription factors in GDNF + IGF1 + bFGF treatment group was significantly higher than the other groups (p < 0.05). Additionally, goat SSCs developed in the latter culture condition could colonize within the seminiferous tubules of the germ-cell depleted recipient mice following xenotransplantation. Obtained results demonstrated that combination of GDNF with IGF1 and bFGF promote in vitro culture of goat SSCs while precludes uncontrolled proliferation of somatic cells. PMID:26154310

  13. Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells.

    PubMed

    Xie, Fei; Gong, Kerui; Li, Ke; Zhang, Mingliang; Chang, Judy C; Jiang, Shizhong; Ye, Lin; Wang, Jiaming; Tan, Yuting; Kan, Yuet Wai

    2016-08-15

    Fibroblasts can be transdifferentiated directly into other somatic cells such as cardiomyocytes, hematopoietic cells, and neurons. An advantage of somatic cell differentiation without first generating induced pluripotent stem cells (iPSCs) is that it avoids contamination of the differentiated cells with residual iPSCs, which may cause teratoma. However, since primary fibroblasts from biopsy undergo senescence during repeated culture, it may be difficult to grow transdifferentiated cells in sufficient numbers for future therapeutic purposes. To circumvent this problem, we reversibly immortalized primary fibroblasts by using the piggyBac transposon to deliver the human telomerase reverse transcriptase (hTERT) gene hTERT plus SV40 Large T. Both approaches enabled fibroblasts to grow continuously without senescence, and neither caused teratoma formation in immunodeficient mice. However, fibroblasts immortalized with hTERT plus SV40 large T antigen accumulated chromosomal rearrangements, whereas fibroblasts immortalized with hTERT retained the normal karyotype. To transdifferentiate hTERT-immortalized fibroblasts into other somatic lineage cells, we transiently transfected them with episomal OCT4 and cultured them under neural cell growth condition with transposase to remove the transposon. Tripotent neural progenitor cells were seamlessly and efficiently generated. Thus, reversible immortalization of primary fibroblasts with hTERT will allow potential autologous cell-based therapeutics that bypass and simulate iPSC generation. PMID:27328768

  14. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration

    PubMed Central

    Murphy, Malea M.; Lawson, Jennifer A.; Mathew, Sam J.; Hutcheson, David A.; Kardon, Gabrielle

    2011-01-01

    Muscle regeneration requires the coordinated interaction of multiple cell types. Satellite cells have been implicated as the primary stem cell responsible for regenerating muscle, yet the necessity of these cells for regeneration has not been tested. Connective tissue fibroblasts also are likely to play a role in regeneration, as connective tissue fibrosis is a hallmark of regenerating muscle. However, the lack of molecular markers for these fibroblasts has precluded an investigation of their role. Using Tcf4, a newly identified fibroblast marker, and Pax7, a satellite cell marker, we found that after injury satellite cells and fibroblasts rapidly proliferate in close proximity to one another. To test the role of satellite cells and fibroblasts in muscle regeneration in vivo, we created Pax7CreERT2 and Tcf4CreERT2 mice and crossed these to R26RDTA mice to genetically ablate satellite cells and fibroblasts. Ablation of satellite cells resulted in a complete loss of regenerated muscle, as well as misregulation of fibroblasts and a dramatic increase in connective tissue. Ablation of fibroblasts altered the dynamics of satellite cells, leading to premature satellite cell differentiation, depletion of the early pool of satellite cells, and smaller regenerated myofibers. Thus, we provide direct, genetic evidence that satellite cells are required for muscle regeneration and also identify resident fibroblasts as a novel and vital component of the niche regulating satellite cell expansion during regeneration. Furthermore, we demonstrate that reciprocal interactions between fibroblasts and satellite cells contribute significantly to efficient, effective muscle regeneration. PMID:21828091

  15. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  16. Indirect longitudinal cytotoxicity of root canal sealers on L929 cells and human periodontal ligament fibroblasts.

    PubMed

    Araki, K; Suda, H; Spångberg, L S

    1994-02-01

    The cytotoxicity of two root canal sealers was evaluated in vitro. The powder components of both sealers, mainly zinc, were the same. The liquid for one sealer, Canals, was clove oil (included eugenol in more than 80%) and other materials. For the other, Canals-N, the liquid was composed of higher fatty acids and glycol. The experiments included two cell lines, heteroploid L929 mouse fibroblasts and diploid human periodontal ligament fibroblasts. Cytotoxicity was assessed using the radiochromium release method with 4-h exposure time. The assay involved using insert chambers in multiwell arrays to produce indirect contact of materials with the cell monolayer at a controlled distance of approximately 1 mm. This model also allowed for the longitudinal study of the same material sample to assess time-dependent changes in toxicity. Freshly mixed Canals was highly toxic (p < 0.01) to both cell lines. On and after 24 h of setting no toxicity was detected. At no time could cytotoxicity be observed when experimenting with Canals-N. These results indicate that both materials have a low content of water diffusible toxic components. Substituting eugenol can further decrease the toxicity of the sealer. PMID:8006567

  17. Collagen matrix as a tool in studying fibroblastic cell behavior.

    PubMed

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486

  18. Collagen matrix as a tool in studying fibroblastic cell behavior

    PubMed Central

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486

  19. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  20. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts – the ability to alter epithelial differentiation – that might also explain the loss of tissue function and organization that is a hallmark of aging. PMID:15657080

  1. Lysine hydroxylation of collagen in a fibroblast cell culture system

    NASA Technical Reports Server (NTRS)

    Uzawa, Katsuhiro; Yeowell, Heather N.; Yamamoto, Kazushi; Mochida, Yoshiyuki; Tanzawa, Hideki; Yamauchi, Mitsuo

    2003-01-01

    The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.

  2. Tumor-associated fibroblasts promote the proliferation and decrease the doxorubicin sensitivity of liposarcoma cells.

    PubMed

    Harati, Kamran; Daigeler, Adrien; Hirsch, Tobias; Jacobsen, Frank; Behr, Björn; Wallner, Christoph; Lehnhardt, Marcus; Becerikli, Mustafa

    2016-06-01

    The reasons for the distinct chemoresistance of liposarcomas and their high risk of local recurrence still remain unclear. Depending on the histological subtype of liposarcoma, first-line therapy with the cytostatic agent, doxorubicin, only achieves response rates of approximately 36%. Approximatley 70% of all local recurrences develop in spite of complete surgical resection of the primary tumor with microscopically negative margins. In this study, we aimed to assess the influence of tumor-associated fibroblasts (TAFs) obtained from surgically removed liposarcomas on the well-established human liposarcoma SW872 cell line. Intratumoral TAFs were isolated from intermediate- and high-grade liposarcoma samples. The human liposarcoma cell line, SW872, was co-cultured with the corresponding TAFs or with dermal fibroblasts as a control. The proliferation (by BrdU assay), cell viability (by MTT assay) and sensitivity to doxorubicin (using the iCELLigence system) of the co-cultured SW872 cells were examined. The SW872 cells exhibited a significant increase in proliferation and viability when co-cultured with the TAFs. As detected by real-time cell analysis, the SW872 cells co-cultured with the TAFs exhibited a diminished response towards doxorubicin. Notably, co-culture with TAFs obtained from high-grade liposarcoma samples resulted in higher proliferation and increased chemoresistance than co-culture with TAFs obtained from intermediate-grade liposarcoma samples. The findings of the present study thus indicate that TAFs from liposarcomas enhance the proliferation and decrease the chemosensitivity of SW872 liposarcoma cells significantly compared with normal fibroblasts from the dermis. TAFs from more malignant liposarcomas promoted tumor cell proliferation and chemoresistance more strikingly than TAFs from less malignant liposarcomas. These data provide evidence for the influence of the tumor microenvironment on liposarcoma and support for further investigations in

  3. Fibroblasts induce epithelial to mesenchymal transition in breast tumor cells which is prevented by fibroblasts treatment with histamine in high concentration.

    PubMed

    Porretti, Juliana C; Mohamad, Nora A; Martín, Gabriela A; Cricco, Graciela P

    2014-06-01

    Epithelial to mesenchymal transition (EMT) of cancer cells is an essential process in cancer progression. Cancer cells that undergone EMT loose cell-cell contacts, acquire mesenchymal properties and develop migratory and invasive abilities. In previous studies we have demonstrated that histamine may modify the invasive phenotype of pancreatic and mammary tumor cells. In this work we proposed to investigate whether histamine may also influence the interaction between tumor cells and normal fibroblasts. The potential activation of normal CCD-1059Sk fibroblasts by histamine and EMT phenotypic changes induced in MCF-7 and MDA-MB-231 breast tumor cells by the conditioned media (CM) derived from fibroblasts were evaluated. Initially, we determined the presence of H1, H2 and H4 histamine receptors and matrix metalloproteinase 2 (MMP2) mRNA in CCD-1059Sk fibroblasts. MMP2 gelatinolytic activity, cell migration and alpha-smooth muscle actin expression were increased in fibroblasts by low doses (<1μM) and decreased by high doses (20μM) of histamine. MCF-7 cells cultured with CM from fibroblasts exhibited spindle-shaped morphology, cell spreading and cytoplasmic expression of β-catenin but there was no change in MMP2 activity and cell migration. MDA-MB-231 cells cultured with CM from fibroblasts showed a more elongated phenotype, cell spreading, cytoplasmic β-catenin, increased MMP2 activity and endogenous TGF-β1 expression, and enhanced cell migration and invasion. Notably, all these features were reversed when mammary tumor cells were cultured with CM from fibroblasts treated with 20μM histamine. In conclusion, high doses of histamine may prevent the activation of fibroblasts and also avert the EMT related changes induced in epithelial tumor cells by fibroblasts CM. PMID:24685678

  4. Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibroblasts. Regulation by inflammatory mediators.

    PubMed Central

    Schrey, M. P.; Patel, K. V.

    1995-01-01

    Malignant human breast tumours contain high levels of prostaglandin E2 (PGE2). However, the mechanisms controlling PGE2 production in breast cancer are unknown. This in vitro study investigates the capacity for PGE2 synthesis and metabolism in several human breast cancer cell lines and early passage human breast fibroblasts and seeks to identify potential regulatory factors which may control these pathways. Basal PGE2 production rose up to 30-fold in breast fibroblast lines on addition of exogenous arachidonic acid (10 microM), whereas no such changes were observed in six out of seven cancer cell lines, with the exception of modest increases in MDA-MB-231 cells. Interleukin 1 beta (IL-1 beta) also induced PGE2 production in breast fibroblasts in the presence of excess substrate, consistent with cyclo-oxygenase induction by the cytokine. Under these conditions only Hs578T cells and MDA-MB-231 cells demonstrated large increases in PGE2 in response to IL-1 beta or phorbol ester; no such responses were seen in MCF-7, T47-D, ZR-75-1, BT-20 or CLF-90-1 cells. In the absence of added arachidonate, bradykinin (BK) and endothelin-1 (ET-1), potentiated PGE2 production in IL-1 beta-treated fibroblasts, possibly by mobilising endogenous substrate. PGE2 also stimulated ET-1 production by breast cancer cells. In co-cultures with T47-D cells both basal and stimulated PGE2 production by breast fibroblasts was greatly reduced. This appeared to be due to metabolic inactivation by the cancer cell since T47-D cells readily converted PGE2 to 15-keto-PGE2. This apparent 15-hydroxy-PG dehydrogenase activity was stimulated by TPA and inhibited by cycloheximide. In conclusion, breast fibroblasts, particularly under the influence of inflammatory mediators, provide a potentially rich source for PGE2 production in breast tumours, whereas significant contributions from the epithelial tumour component may be restricted to cancer cells exhibiting an invasive phenotype. Metabolic inactivation by

  5. Mammary fibroblasts regulate morphogenesis of normal and tumorigenic breast epithelial cells by mechanical and paracrine signals

    PubMed Central

    Lühr, Inke; Friedl, Andreas; Overath, Thorsten; Tholey, Andreas; Kunze, Thomas; Hilpert, Felix; Sebens, Susanne; Arnold, Norbert; Rösel, Frank; Oberg, Hans-Heinrich; Maass, Nicolai; Mundhenke, Christoph; Jonat, Walter; Bauer, Maret

    2013-01-01

    Stromal factors play a critical role in the development of the mammary gland. Using a three dimensional-coculture model we demonstrate a significant role for stromal fibroblasts in the regulation of normal mammary epithelial morphogenesis and the control of tumor growth. Both soluble factors secreted by fibroblasts and fibroblast-derived modifications of the matrix compliance contribute to the regulation of epithelial cell morphogenesis. Readjustment of matrix tension by fibroblasts can even induce a phenotypic reversion of breast carcinoma cells. These data offer a basis to develop new strategies for the normalization of the tumor stroma as an innovative target in cancer therapy. PMID:22776560

  6. Skin telocytes versus fibroblasts: two distinct dermal cell populations.

    PubMed

    Kang, Yuli; Zhu, Zaihua; Zheng, Yonghua; Wan, Weiguo; Manole, Catalin G; Zhang, Qiangqiang

    2015-11-01

    It is already accepted that telocytes (TCs) represent a new type of interstitial cells in human dermis. In normal skin, TCs have particular spatial relations with different dermal structures such as blood vessels, hair follicles, arrector pili muscles or segments of sebaceous and/or eccrine sweat glands. The distribution and the density of TCs is affected in various skin pathological conditions. Previous studies mentioned the particular (ultra)structure of TCs and also their immunophenotype, miR imprint or proteome, genome or secretome features. As fibroblast is the most common intersitital cell (also in human dermis), a dedicated comparison between human skin TCs and fibroblasts (Fbs) was required to be performed. In this study, using different techniques, we document several points of difference between human dermis TCs and Fbs. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM), we demonstrated TCs with their hallmark cellular prolongations - telopodes. Thus, we showed their ultrastructural distinctiveness from Fbs. By RayBio Human Cytokine Antibody Array V analyses performed on the supernatant from separately cultured TCs and Fbs, we detected the cytokine profile of both cell types, individually. Two of 79 detected cytokines - epithelial-derived neutrophil-activating peptide 78 and granulocyte chemotactic protein-2 - were 1.5 times higher in the supernatant of TCs (comparing with Fbs). On the other hand, 37 cytokines were at least 1.5 higher in Fbs supernatant (comparing with TCs), and among them six cytokines - interleukin 5, monocyte chemotactic protein-3 (MCP-3), MCP-4, macrophage inflammatory protein-3, angiogenin, thrombopoietin - being 9.5 times higher (results also confirmed by ELISA testing). In summary, using different techniques, we showed that human dermal TCs and Fbs are different in terms of ultrastructure and cytokine profile. PMID:26414534

  7. Skin telocytes versus fibroblasts: two distinct dermal cell populations

    PubMed Central

    Kang, Yuli; Zhu, Zaihua; Zheng, Yonghua; Wan, Weiguo; Manole, Catalin G; Zhang, Qiangqiang

    2015-01-01

    It is already accepted that telocytes (TCs) represent a new type of interstitial cells in human dermis. In normal skin, TCs have particular spatial relations with different dermal structures such as blood vessels, hair follicles, arrector pili muscles or segments of sebaceous and/or eccrine sweat glands. The distribution and the density of TCs is affected in various skin pathological conditions. Previous studies mentioned the particular (ultra)structure of TCs and also their immunophenotype, miR imprint or proteome, genome or secretome features. As fibroblast is the most common intersitital cell (also in human dermis), a dedicated comparison between human skin TCs and fibroblasts (Fbs) was required to be performed. In this study, using different techniques, we document several points of difference between human dermis TCs and Fbs. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM), we demonstrated TCs with their hallmark cellular prolongations – telopodes. Thus, we showed their ultrastructural distinctiveness from Fbs. By RayBio Human Cytokine Antibody Array V analyses performed on the supernatant from separately cultured TCs and Fbs, we detected the cytokine profile of both cell types, individually. Two of 79 detected cytokines – epithelial-derived neutrophil-activating peptide 78 and granulocyte chemotactic protein-2 – were 1.5 times higher in the supernatant of TCs (comparing with Fbs). On the other hand, 37 cytokines were at least 1.5 higher in Fbs supernatant (comparing with TCs), and among them six cytokines – interleukin 5, monocyte chemotactic protein-3 (MCP-3), MCP-4, macrophage inflammatory protein-3, angiogenin, thrombopoietin – being 9.5 times higher (results also confirmed by ELISA testing). In summary, using different techniques, we showed that human dermal TCs and Fbs are different in terms of ultrastructure and cytokine profile. PMID:26414534

  8. cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator.

    PubMed Central

    Rommens, J M; Dho, S; Bear, C E; Kartner, N; Kennedy, D; Riordan, J R; Tsui, L C; Foskett, J K

    1991-01-01

    A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl(-)-indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell current-voltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl- flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (delta F508), the most common mutation causing cystic fibrosis. These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl- channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basic mechanisms underlying this CFTR-associated ion permeability and to study the structure and function of the CFTR. Images PMID:1715567

  9. High-LET Radiation Induced Chromosome Aberrations in Normal and Ataxia Telangiectasia Fibroblast Cells

    NASA Astrophysics Data System (ADS)

    Kawata, Tetsuya; George, Ms Kerry; Cucinotta, Francis A.; Shigematsu, Naoyuki; Ito, Hisao; Furusawa, Yoshiya; Uno, Takashi

    We investigated the effects of heavy ions beams on chromosomal aberrations in normal and AT cells. Normal and AT fibroblast cells arrested at G0/G1 phase were irradiated with 2 Gy of X-rays, 490 MeV/u Silicon (LET 55 keV/micron), 500 MeV/u Iron (LET 185 keV/micron) and 200 MeV/u Iron (LET 440 keV/micron) particles, and then cells were allowed to repair for 24 hours at 37 degrees before subculture. Calyculin-A induced PCC method was employed to collect G2/M chromosomes and whole DNA probes 1 and 3 were used to analyze chromosomal aberrations such as color-junctions, deletions, simple exchanges (incomplete and reciprocal exchanges) and complex-type exchanges. The percentages of aberrant cells were higher when normal and AT cells were exposed to heavy ions compared to X-rays, and had a tendency to increase with increasing LET up to 185 keV/micron and then decreased at 440 keV/micron. When the frequency of color-junctions per cell was compared after X-ray exposure, AT cells had around three times higher frequency of color-junctions (mis-rejoining) than normal cells. However, at 185 keV/micron there was no difference in the frequency of color-junctions between two cell lines. It was also found that the frequency of simple exchanges per cell was almost constant in AT cells regardless LET levels, but it was LET dependent for normal cells. Interestingly, the frequency of simple exchanges was higher for normal fibroblast cells when it was compared at 185 keV/micron, but AT cells had more complex-type exchanges at the same LET levels. Heavy ions are more efficient in inducing chromosome aberrations in normal and AT cells compared to X-rays, and the aberration types between normal and AT fibroblast appeared different probably due to difference in the ATM gene function.

  10. Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells

    PubMed Central

    Gunji, Yoko; Iwasawa, Shunichiro; Nelson, Amy; Farid, Maha; Ikari, Jun; Liu, Xiangde; Wang, Xingqi; Michalski, Joel; Smith, Lynette; Iqbal, Javeed; Behery, Radwa El; West, William; Yelamanchili, Sowmya; Rennard, Deborah; Holz, Olaf; Mueller, Kai-Christian; Magnussen, Helgo; Rabe, Klaus; Castaldi, Peter J; Rennard, Stephen I.

    2014-01-01

    Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs. PMID

  11. Microbubble-Enhanced Ultrasound Gene Transfer into Fibroblast Cells

    NASA Astrophysics Data System (ADS)

    Hirayama, Kota; Kaneko, Yukio; Tei, Yuichi; Matsumoto, Yoichiro

    2007-05-01

    Ultrasound finds many applications in the medical field, including ultrasound imaging, non-invasive treatment of tumors and lithotripsy. Ultrasound also has a potential to deliver some therapeutic materials, such as genes, drugs or proteins into cells. It is known that microbubbles can improve the delivery efficiency. It is believed that therapeutic materials can pass through the cell membrane whose permeability is increased by microbubble destruction or the ultrasound pressure. In this study, we investigated the delivery of GFP plasmid gene into the fibroblast cells. Ultrasound (frequency = 2.1 MHz, duty cycle = 10%) was used to irradiate the cultured cells through a medium that contains microbubbles and GFP plasmid. GFP plasmid transfection could be easily observed by fluorescence microscopy. Ultrasound irradiation under a variety of conditions resulted in successful GFP plasmid delivery. Microbubbles enhanced GFP transfection, and conclusions were drawn as to the relationship between gene transfection and various ultrasound exposure parameters. We also investigated the effect of ultrasound intensity on cell viability.

  12. Modelling Action Potential Generation and Propagation in Fibroblastic Cells

    NASA Astrophysics Data System (ADS)

    Torres, J. J.; Cornelisse, L. N.; Harks, E. G. A.; Theuvenet, A. P. R.; Ypey, D. L.

    2003-04-01

    Using a standard Hodgkin-Huxley (HH) formalism, we present a mathematical model for action potential (AP) generation and intercellular AP propagation in quiescent (serum-deprived) normal rat kidney (NRK) fibroblasts [1], based on the recent experimental identification of the ion channels involved [2]. The principal ion channels described are those of an inwardly rectifying K+ conductance (GKIR), an L-type calcium conductance (GCaL), an intracellular calcium activated Cl- conductance (GCl(Ca)), a residual leak conductance Gleak, and gap junctional channels between the cells (Ggj). The role of each one of these components in the particular shape of the AP wave-form has been analyzed and compared with experimental observations. In addition, we have studied the role of subcellular processes like intracellular calcium dynamics and calcium buffering in AP generation. AP propagation between cells was reconstructed in a hexagonal model of cells coupled by Ggj with physiological conductance values. The model revealed an excitability mechanism of quiescent NRK cells with a particular role of intracellular calcium dynamics. It allows further explorations of the mechanism of signal generation and transmission in NRK cell cultures and its dependence on growth conditions.

  13. Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells

    PubMed Central

    Lamb, Acacia; Golemis, Erica A.; Cukierman, Edna

    2008-01-01

    Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest significant differences in proliferation, differentiation, response to drugs, and other parameters. However, these substrates do not closely resemble the mesenchymal microenvironment that is typically associated with advanced carcinomas in vivo, which is produced to a large extent by fibroblasts. In this study, we have evaluated the ability of a fibroblast-derived three-dimensional matrix to regulate the growth of a panel of 11 human tumor epithelial cell lines. Although proliferative and morphological responses to three-dimensional cues segregated independently, general responsiveness to the matrix correlated with the ability of matrix to influence drug responses. Fibroblast-derived three-dimensional matrix increased β1-integrin-dependent survival of a subset of human cancer cell lines during taxol treatment, while it sensitized or minimally influenced survival of other cells. β1-integrin-dependent changes in cell resistance to taxol did not correlate with degree of modulation of FAK and Akt, implying additional signaling factors are involved. Based on these results, we propose these matrices potentially have value as in vitro drug screening platforms. PMID:18411046

  14. Downregulation of caveolin-1 upregulates the expression of growth factors and regulators in co-culture of fibroblasts with cancer cells

    PubMed Central

    SHI, XIAO-YU; XIONG, LI-XIA; XIAO, LIANG; MENG, CHUANG; QI, GUAN-YUN; LI, WEN-LIN

    2016-01-01

    Reduced expression levels of caveolin-1 (Cav-1) in tumor stromal fibroblasts influences the occurrence and progression of tumors, particularly in breast cancer, but the relevant molecular mechanism is unclear. The present study aimed to clarify the potential mechanism underlying the promotion of tumor growth by reduced Cav-1 expression levels, by investigating Cav-1-targeted molecules in fibroblasts and breast cancer cells. The expression of growth factors in the ESF fibroblast cell line transfected with Cav-1 small interfering RNA (siRNA) was examined. The expression of apoptotic regulators in the BT474 breast cancer cell line that was co-cultured with the fibroblasts, was also investigated. The transfection of Cav-1-targeting siRNA in ESF cells resulted in efficient and specific inhibition of Cav-1 expression. The downregulation of Cav-1 increased the expression and secretion of stromal cell-derived factor-1 (SDF-1), epidermal growth factor (EGF) and fibroblast-specific protein-1 (FSP-1) in ESF cells. This resulted in the accelerated proliferation of the breast cancer cells. Tumor protein 53-induced glycolysis and apoptosis regulator (TIGAR) was upregulated in the BT474 cells under the condition of co-culture with Cav-1 siRNA fibroblasts, while levels of reactive oxygen species (ROS) were decreased, resulting in apoptosis inhibition in the breast cancer cells. These results demonstrated that the downregulation of Cav-1 promoted the growth of breast cancer cells through increasing SDF-1, EGF and FSP-1 in tumor stromal fibroblasts, and TIGAR levels in breast cancer cells. To the best of our knowledge, the present study supports the hypothesis that Cav-1 possesses tumor-suppressor properties, with the mechanism of Cav-1-dependent signaling involving the regulation of SDF-1, EGF, FSP-1 and TIGAR. PMID:26647977

  15. Dual role of sphingosine kinase-1 in promoting the differentiation of dermal fibroblasts and the dissemination of melanoma cells.

    PubMed

    Albinet, V; Bats, M-L; Huwiler, A; Rochaix, P; Chevreau, C; Ségui, B; Levade, T; Andrieu-Abadie, N

    2014-06-26

    Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression. PMID:23893239

  16. Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

    PubMed Central

    Latif, Najma; Quillon, Alfred; Sarathchandra, Padmini; McCormack, Ann; Lozanoski, Alec; Yacoub, Magdi H.; Chester, Adrian H.

    2015-01-01

    Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the

  17. Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

    PubMed

    Molcanyi, Marek; Mehrjardi, Narges Zare; Schäfer, Ute; Haj-Yasein, Nadia Nabil; Brockmann, Michael; Penner, Marina; Riess, Peter; Reinshagen, Clemens; Rieger, Bernhard; Hannes, Tobias; Hescheler, Jürgen; Bosche, Bert

    2014-01-01

    Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed. PMID:25249934

  18. Investigation of native fluorescence spectral difference among prostate cancer cell lines with different risk levels

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Xue, Jianpeng; Xu, Baogang; Wang, Wubao; Gu, Yueqing; Tang, Rui; Achilefu, S.; Ackerstaff, Ellen; Koutcher, Jason A.; Alfano, R. R.

    2013-03-01

    The alteration of native fluorophores among different types of cancer cell lines was investigated by the fluorescence spectroscopy. Different types of cancer cell lines with different risk levels, such as moderate metastatic (DU-145) and advanced metastatic (PC-3) cell lines as well as normal cell line (Fibroblast), were excited by the selective excitation wavelength of 300 nm to explore changes of the relative contents of tryptophan and NADH using principal component analysis (PCA). The higher relative content of tryptophan was observed in the advanced metastatic cancer cell lines in comparison with the moderate metastatic and non aggressive cell lines.

  19. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

    PubMed Central

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-01-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double-stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome-scale lentiviral single-guide RNA library, could be applied to a loss-of-function genetic screen, although the loss-of-function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription-PCR and western blot analysis indicated that the PFFs were Cas9-positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  20. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer.

    PubMed

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-09-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double‑stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome‑scale lentiviral single‑guide RNA library, could be applied to a loss‑of‑function genetic screen, although the loss‑of‑function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline‑inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription‑PCR and western blot analysis indicated that the PFFs were Cas9‑positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  1. Paracrine Effects of Adipose-Derived Stem Cells on Keratinocytes and Dermal Fibroblasts

    PubMed Central

    Lee, Seung Ho; Jin, Sang Yun; Song, Jin Seok; Seo, Kyle K.

    2012-01-01

    Background Adipose-derived stem cells (ASCs) are mesenchymal stem cells that have recently been applied to tissue repair and regeneration. Keratinocytes and dermal fibroblasts play key roles in cutaneous wound healing. Objective We investigated the paracrine effects of ASCs on HaCaT cells (i.e., immortalized human keratinocytes) and human dermal fibroblasts to explore the mechanism of the effects of ASCs on cutaneous wound healing. Methods HaCaT cells and primary cultured human dermal fibroblasts were treated with 50% conditioned medium of ASCs (ASC-CM). Viability, in vitro wound healing, and fibroblast-populated collagen lattice contraction assays were conducted, and reverse transcription-polymerase chain reaction (RT-PCR) for the type I procollagen α1 chain gene was performed. Results The proliferation of HaCaT cells and fibroblasts was increased by ASC-CM in the viability assay. ASC-CM promoted in vitro wound healing of HaCaT cells and increased the contraction of the fibroblast-populated collagen lattice. RT-PCR showed that the transcription of the type I procollagen α1 chain gene in fibroblasts was upregulated by ASC-CM. Conclusion The stimulatory effect of ASC on cutaneous wound healing may be partially mediated by paracrine effects of ASCs on other skin cells. Application of ASCs or ASC-derived molecules could be an innovative therapeutic approach in the treatment of chronic wounds and other conditions. PMID:22577262

  2. Electric Cell-Substrate Impedance Sensing (ECIS) with Microelectrode Arrays for Investigation of Cancer CellFibroblasts Interaction

    PubMed Central

    Tran, Trong Binh; Baek, Changyoon; Min, Junhong

    2016-01-01

    The tumor microenvironment, including stromal cells, surrounding blood vessels and extracellular matrix components, has been defined as a crucial factor that influences the proliferation, drug-resistance, invasion and metastasis of malignant epithelial cells. Among other factors, the communications and interaction between cancer cells and stromal cells have been reported to play pivotal roles in cancer promotion and progression. To investigate these relationships, an on-chip co-culture model was developed to study the cellular interaction between A549—human lung carcinoma cells and MRC-5—human lung epithelial cells in both normal proliferation and treatment conditions. In brief, a co-culture device consisting of 2 individual fluidic chambers in parallel, which were separated by a 100 μm fence was utilized for cell patterning. Microelectrodes arrays were installed within each chamber including electrodes at various distances away from the confrontation line for the electrochemical impedimetric sensing assessment of cell-to-cell influence. After the fence was removed and cell-to-cell contact occurred, by evaluating the impedance signal responses representing cell condition and behavior, both direct and indirect cell-to-cell interactions through conditioned media were investigated. The impact of specific distances that lead to different influences of fibroblast cells on cancer cells in the co-culture environment was also defined. PMID:27088611

  3. Paracrine anti-fibrotic effects of neonatal cells and living cell constructs on young and senescent human dermal fibroblasts.

    PubMed

    Pratsinis, Harris; Armatas, Andreas; Dimozi, Anastasia; Lefaki, Maria; Vassiliu, Pantelis; Kletsas, Dimitris

    2013-01-01

    Senescent cells observed in the area of chronic wounds have been proposed to affect wound healing. Therapeutic approaches against chronic wounds include, among others, the local application of living cell constructs (LCCs), containing fibroblasts and/or keratinocytes. Accordingly, the aim of the present work was to examine the effects of factors secreted by early passage neonatal fibroblasts and LCCs--in the form of a conditioned medium (CM)--on senescent adult dermal fibroblasts regarding functions related to the healing process, i.e., cell proliferation, alpha-smooth muscle actin and metalloproteinase expression, and collagen synthesis. Target cells were fibroblasts senescent either due to subsequent divisions (replicative senescence) or due to an exogenous stress (stress-induced premature senescence). No effect on the proliferation of senescent fibroblasts was observed, as expected. All CMs were found to inhibit overall collagen synthesis both in early passage and in senescent fibroblasts. The LCC-derived CM was found to be more potent than fibroblast-derived CMs and, furthermore, to inhibit alpha-smooth muscle actin expression. In conclusion, these results may indicate anti-contractile and anti-fibrotic activities of factor(s) secreted by neonatal skin fibroblasts, and more intensely by LCCs on adult donor-derived fibroblasts. These activities seem to persist during senescence of the target cells. PMID:24581241

  4. Production of Pigs by Hand-Made Cloning Using Mesenchymal Stem Cells and Fibroblasts.

    PubMed

    Yang, Zhenzhen; Vajta, Gábor; Xu, Ying; Luan, Jing; Lin, Mufei; Liu, Cong; Tian, Jianing; Dou, Hongwei; Li, Yong; Liu, Tianbin; Zhang, Yijie; Li, Lin; Yang, Wenxian; Bolund, Lars; Yang, Huanming; Du, Yutao

    2016-08-01

    Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding. PMID:27459584

  5. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    SciTech Connect

    Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  6. Impedance analysis of fibroblastic cell layers measured by electric cell-substrate impedance sensing

    NASA Astrophysics Data System (ADS)

    Lo, Chun-Min; Ferrier, Jack

    1998-06-01

    Impedance measurements of cell layers cultured on gold electrode surfaces obtained by electric cell-substrate impedance sensing provide morphological information such as junctional resistance and cell-substrate separation. Previously, a model that assumes that cells have a disklike shape and that electric currents flow radially underneath the ventral cell surface and then through the paracellular space has been used to theoretically calculate the impedance of the cell-covered electrode. In this paper we propose an extended model of impedance analysis for cell layers where cellular shape is rectangular. This is especially appropriate for normal fibroblasts in culture. To verify the model, we analyze impedance data obtained from four different kinds of fibroblasts that display a long rectangular shape. In addition, we measure the average cell-substrate separation of human gingival fibroblasts at different temperatures. At temperatures of 37, 22, and 4 °C, the average separation between ventral cell surface and substratum are 46, 55, and 89 nm, respectively.

  7. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    PubMed

    Harris, David M; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

    2011-01-01

    Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy. PMID:21731684

  8. Voacamine modulates the sensitivity to doxorubicin of resistant osteosarcoma and melanoma cells and does not induce toxicity in normal fibroblasts.

    PubMed

    Condello, Maria; Cosentino, Dario; Corinti, Silvia; Di Felice, Gabriella; Multari, Giuseppina; Gallo, Francesca Romana; Arancia, Giuseppe; Meschini, Stefania

    2014-04-25

    In previous studies it has been demonstrated that the plant alkaloid voacamine (1), used at noncytotoxic concentrations, enhanced the cytotoxicity of doxorubicin and exerted a chemosensitizing effect on cultured multidrug-resistant (MDR) U-2 OS-DX osteosarcoma cells. The in vitro investigations reported herein gave the following results: (i) the chemosensitizing effect of 1, in terms of drug accumulation and cell survival, was confirmed using SAOS-2-DX cells, another MDR osteosarcoma cell line; (ii) compound 1 enhanced the cytotoxic effect of doxorubicin also on the melanoma cell line Me30966, intrinsically drug resistant and P-glycoprotein-negative; (iii) at the concentrations used to sensitize tumor cells, 1 was not cytotoxic to normal cells (human fibroblasts). These findings suggest possible applications of voacamine (1) in integrative oncologic therapies against resistant tumors. PMID:24720452

  9. Voacamine Modulates the Sensitivity to Doxorubicin of Resistant Osteosarcoma and Melanoma Cells and Does Not Induce Toxicity in Normal Fibroblasts

    PubMed Central

    2015-01-01

    In previous studies it has been demonstrated that the plant alkaloid voacamine (1), used at noncytotoxic concentrations, enhanced the cytotoxicity of doxorubicin and exerted a chemosensitizing effect on cultured multidrug-resistant (MDR) U-2 OS-DX osteosarcoma cells. The in vitro investigations reported herein gave the following results: (i) the chemosensitizing effect of 1, in terms of drug accumulation and cell survival, was confirmed using SAOS-2-DX cells, another MDR osteosarcoma cell line; (ii) compound 1 enhanced the cytotoxic effect of doxorubicin also on the melanoma cell line Me30966, intrinsically drug resistant and P-glycoprotein-negative; (iii) at the concentrations used to sensitize tumor cells, 1 was not cytotoxic to normal cells (human fibroblasts). These findings suggest possible applications of voacamine (1) in integrative oncologic therapies against resistant tumors. PMID:24720452

  10. FSP1+ fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells

    PubMed Central

    Sun, Lina; Sun, Chenming; Liang, Zhanfeng; Li, Hongran; Chen, Lin; Luo, Haiying; Zhang, Hongmei; Ding, Pengbo; Sun, Xiaoning; Qin, Zhihai; Zhao, Yong

    2015-01-01

    Thymic epithelial cells (TECs) form a 3-dimentional network supporting thymocyte development and maturation. Besides epithelium and thymocytes, heterogeneous fibroblasts are essential components in maintaining thymic microenvironments. However, thymic fibroblast characteristics, development and function remain to be determined. We herein found that thymic non-hematopoietic CD45-FSP1+ cells represent a unique Fibroblast specific protein 1 (FSP1)—fibroblast-derived cell subset. Deletion of these cells in FSP1-TK transgenic mice caused thymus atrophy due to the loss of TECs, especially mature medullary TECs (MHCIIhigh, CD80+ and Aire+). In a cyclophosphamide-induced thymus injury and regeneration model, lack of non-hematopoietic CD45-FSP1+ fibroblast subpopulation significantly delayed thymus regeneration. In fact, thymic FSP1+ fibroblasts released more IL-6, FGF7 and FSP1 in the culture medium than their FSP1- counterparts. Further experiments showed that the FSP1 protein could directly enhance the proliferation and maturation of TECs in the in vitro culture systems. FSP1 knockout mice had significantly smaller thymus size and less TECs than their control. Collectively, our studies reveal that thymic CD45-FSP1+ cells are a subpopulation of fibroblasts, which is crucial for the maintenance and regeneration of TECs especially medullary TECs through providing IL-6, FGF7 and FSP1. PMID:26445893

  11. Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    PubMed Central

    Fausther, Michel; Goree, Jessica R.; Lavoie, Élise G.; Graham, Alicia L.; Sévigny, Jean; Dranoff, Jonathan A.

    2015-01-01

    The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis. PMID:25822334

  12. Effect of Fe3O4 Nanoparticles on Skin Tumor Cells and Dermal Fibroblasts

    PubMed Central

    Alili, Lirija; Chapiro, Swetlana; Marten, Gernot U.; Schmidt, Annette M.; Zanger, Klaus; Brenneisen, Peter

    2015-01-01

    Iron oxide (Fe3O4) nanoparticles have been used in many biomedical approaches. The toxicity of Fe3O4 nanoparticles on mammalian cells was published recently. Though, little is known about the viability of human cells after treatment with Fe3O4 nanoparticles. Herein, we examined the toxicity, production of reactive oxygen species, and invasive capacity after treatment of human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) with Fe3O4 nanoparticles. These nanoparticles had an average size of 65 nm. Fe3O4 nanoparticles induced oxidative stress via generation of reactive oxygen species (ROS) and subsequent initiation of lipid peroxidation. Furthermore, the question was addressed of whether Fe3O4 nanoparticles affect myofibroblast formation, known to be involved in tumor invasion. Herein, Fe3O4 nanoparticles prevent the expression alpha-smooth muscle actin and therefore decrease the number of myofibroblastic cells. Moreover, our data show in vitro that concentrations of Fe3O4 nanoparticles, which are nontoxic for normal cells, partially reveal a ROS-triggered cytotoxic but also a pro-invasive effect on the fraction of squamous cancer cells surviving the treatment with Fe3O4 nanoparticles. The data herein show that the Fe3O4 nanoparticles appear not to be adequate for use in therapeutic approaches against cancer cells, in contrast to recently published data with cerium oxide nanoparticles. PMID:26090418

  13. Continual Cell Deformation Induced via Attachment to Oriented Fibers Enhances Fibroblast Cell Migration

    PubMed Central

    Qin, Sisi; Ricotta, Vincent; Simon, Marcia; Clark, Richard A. F.; Rafailovich, Miriam H.

    2015-01-01

    Fibroblast migration is critical to the wound healing process. In vivo, migration occurs on fibrillar substrates, and previous observations have shown that a significant time lag exists before the onset of granulation tissue. We therefore conducted a series of experiments to understand the impact of both fibrillar morphology and migration time. Substrate topography was first shown to have a profound influence. Fibroblasts preferentially attach to fibrillar surfaces, and orient their cytoplasm for maximal contact with the fiber edge. In the case of en-mass cell migration out of an agarose droplet, fibroblasts on flat surfaces emerged with an enhanced velocity, v = 52μm/h, that decreases to the single cell value, v = 28μm/h within 24 hours and remained constant for at least four days. Fibroblasts emerging on fibrillar surfaces emerged with the single cell velocity, which remained constant for the first 24 hours and then increased reaching a plateau with more than twice the initial velocity within the next three days. The focal adhesions were distributed uniformly in cells on flat surfaces, while on the fibrillar surface they were clustered along the cell periphery. Furthermore, the number of focal adhesions for the cells on the flat surfaces remained constant, while it decreased on the fibrillar surface during the next three days. The deformation of the cell nuclei was found to be 50% larger on the fiber surfaces for the first 24 hours. While the mean deformation remained constant on the flat surface, it increased for the next three days by 24% in cells on fibers. On the fourth day, large actin/myosin fibers formed in cells on fibrillar surfaces only and coincided with a change from the standard migration mechanism involving extension of lamellipodia, and retraction of the rear, to one involving strong contractions oriented along the fibers and centered about the nucleus. PMID:25774792

  14. Apoptosis-Like Cell Death Induction and Aberrant Fibroblast Properties in Human Incisional Hernia Fascia

    PubMed Central

    Diaz, Ramon; Quiles, Maria T.; Guillem-Marti, Jordi; Lopez-Cano, Manuel; Huguet, Pere; Ramon-y-Cajal, Santiago; Reventos, Jaume; Armengol, Manel; Arbos, Maria A.

    2011-01-01

    Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo. PMID:21641387

  15. Sialylation of vitronectin regulates stress fiber formation and cell spreading of dermal fibroblasts via a heparin-binding site.

    PubMed

    Miyamoto, Yasunori; Tanabe, Mio; Date, Kimie; Sakuda, Kanoko; Sano, Kotone; Ogawa, Haruko

    2016-04-01

    Vitronectin (VN) plays an important role in tissue regeneration. We previously reported that VN from partial hepatectomized (PH) rats results in a decrease of sialylation of VN and de-sialylation of VN decreases the cell spreading of hepatic stellate cells. In this study, we analyzed the mechanism how sialylation of VN regulates the properties of mouse primary cultured dermal fibroblasts (MDF) and a dermal fibroblast cell line, Swiss 3T3 cells. At first, we confirmed that VN from PH rats or de-sialylated VN also decreased cell spreading in MDF and Swiss 3T3 cells. The de-sialylation suppressed stress fiber formation in Swiss 3T3 cells. Next, we analyzed the effect of the de-sialylation of VN on stress fiber formation in Swiss 3T3 cells. RGD peptide, an inhibitor for a cell binding site of VN, did not affect the cell attachment of Swiss 3T3 cells on untreated VN but significantly decreased it on de-sialylated VN, suggesting that the de-sialylation attenuates the binding activity of an RGD-independent binding site in VN. To analyze a candidate RGD-independent binding site, an inhibition experiment of stress fiber formation for a heparin binding site was performed. The addition of heparin and treatment of cells with heparinase decreased stress fiber formation in Swiss 3T3 cells. Furthermore, de-sialylation increased the binding activity of VN to heparin, as detected by surface plasmon resonance (SPR). These results demonstrate that sialylation of VN glycans regulates stress fiber formation and cell spreading of dermal fibroblast cells via a heparin binding site. PMID:26979432

  16. Lineage Tracing Reveals Distinctive Fates for Mesothelial Cells and Submesothelial Fibroblasts during Peritoneal Injury

    PubMed Central

    Chen, Yi-Ting; Chang, Yu-Ting; Pan, Szu-Yu; Chou, Yu-Hsiang; Chang, Fan-Chi; Yeh, Pei-Ying; Liu, Yuan-Hung; Chiang, Wen-Chih; Chen, Yung-Ming; Wu, Kwan-Dun; Tsai, Tun-Jun; Duffield, Jeremy S.

    2014-01-01

    Fibrosis of the peritoneal cavity remains a serious, life-threatening problem in the treatment of kidney failure with peritoneal dialysis. The mechanism of fibrosis remains unclear partly because the fibrogenic cells have not been identified with certainty. Recent studies have proposed mesothelial cells to be an important source of myofibroblasts through the epithelial–mesenchymal transition; however, confirmatory studies in vivo are lacking. Here, we show by inducible genetic fate mapping that type I collagen–producing submesothelial fibroblasts are specific progenitors of α-smooth muscle actin–positive myofibroblasts that accumulate progressively in models of peritoneal fibrosis induced by sodium hypochlorite, hyperglycemic dialysis solutions, or TGF-β1. Similar genetic mapping of Wilms’ tumor-1–positive mesothelial cells indicated that peritoneal membrane disruption is repaired and replaced by surviving mesothelial cells in peritoneal injury, and not by submesothelial fibroblasts. Although primary cultures of mesothelial cells or submesothelial fibroblasts each expressed α-smooth muscle actin under the influence of TGF-β1, only submesothelial fibroblasts expressed α-smooth muscle actin after induction of peritoneal fibrosis in mice. Furthermore, pharmacologic inhibition of the PDGF receptor, which is expressed by submesothelial fibroblasts but not mesothelial cells, attenuated the peritoneal fibrosis but not the remesothelialization induced by hypochlorite. Thus, our data identify distinctive fates for injured mesothelial cells and submesothelial fibroblasts during peritoneal injury and fibrosis. PMID:24854266

  17. Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent

    PubMed Central

    Alkasalias, Twana; Flaberg, Emilie; Kashuba, Vladimir; Alexeyenko, Andrey; Pavlova, Tatiana; Savchenko, Andrii; Szekely, Laszlo; Klein, George; Guven, Hayrettin

    2014-01-01

    Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3. PMID:25404301

  18. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells.

    PubMed

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients' outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCMTGF, FCMPDGF) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCMB). FCMTGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCMTGF≫FCMPDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCMTGF>FCMPDGF) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. PMID:24394543

  19. Estrogen Receptor Alpha (ERα)-Associated Fibroblasts Promote Cell Growth in Prostate Cancer.

    PubMed

    Da, Jun; Lu, Mujun; Wang, Zhong

    2015-12-01

    Estrogen receptor (ER) is expressed in cancer-associated fibroblasts (CAFs) in the stromal compartment of cancerous prostate. However, the effect of ERα in CAF cells on prostate cancer (PCa) cell growth remains unclear. We used lentiviral transduction to stably express ERα in CAF cells isolated from transgenic adenocarcinoma of the mouse prostate model. MTT and 3D colony-formation assays demonstrated that conditioned medium from ERα-expressing CAF cells (CAF-ERα+) promoted cell proliferation and colony growth of various PCa cell lines, such as PC3, LNCaP, 22RV1, and C4-2. We further confirmed the in vitro data by orthotopically co-implanting 22RV1, transfected with firefly luciferase, and CAF-ERα+ cells in vivo using mouse model. Mice co-implanted with CAF-ERα+ exhibited stronger luciferase signals and bigger tumor size compared to animals co-implanted with CAF that do not express ER. Our results demonstrate that ER expressed in CAF might play a pro-proliferative role in PCa. PMID:27259327

  20. The prognostic significance of fibroblast growth factor receptor 4 in non-small-cell lung cancer

    PubMed Central

    Huang, Hong-ping; Feng, Hui; Qiao, Hong-bo; Ren, Ze-xiang; Zhu, Ge-dong

    2015-01-01

    Background Fibroblast growth factor receptor 4 (FGFR4) has been proved to be correlated with progression and prognosis in many cancers. However, the significance of FGFR4 in non-small-cell lung cancer (NSCLC) is still not well elucidated. Methods In our experiment, we detected FGFR4 expression in 237 samples of NSCLC with immunohistochemistry, and further analyzed the correlation between FGFR4 and clinicopathologic features of NSCLC with chi-square test. Moreover, we evaluated the prognostic value of FGFR4 by Kaplan–Meier survival curve and Cox regression model. By regulating the expression of FGFR4 by overexpression or knockdown, we assessed the role of FGFR4 on NSCLC cell proliferation. Results FGFR4 expression was high in NSCLC (46.8%, 111/237). FGFR4 expression was significantly associated with tumor diameter (P=0.039). With univariate (P=0.009) and multivariate (P=0.002) analysis, FGFR4 was identified as an independent prognostic factor in NSCLC (P=0.009). Moreover, FGFR4 can promote the proliferation of NSCLC cell lines. Conclusion FGFR4 is an independent prognostic biomarker in NSCLC. FGFR4 can accelerate the proliferation of NSCLC cell lines, indicating FGFR4 could be a potential drug target of NSCLC. PMID:26045670

  1. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    SciTech Connect

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  2. Cancer: An Oxidative Crosstalk between Solid Tumor Cells and Cancer Associated Fibroblasts.

    PubMed

    Arcucci, Alessandro; Ruocco, Maria Rosaria; Granato, Giuseppina; Sacco, Anna Maria; Montagnani, Stefania

    2016-01-01

    Redox balance is associated with the regulation of several cell signalling pathways and functions. In fact, under physiological conditions, cells maintain a balance between oxidant and antioxidant systems, and reactive oxygen species (ROS) can act as second messengers to regulate cell proliferation, cell death, and other physiological processes. Cancer tissues usually contain higher levels of ROS than normal tissues, and this ROS overproduction is associated with tumor development. Neoplastic tissues are very heterogeneous systems, composed of tumor cells and microenvironment that has a critical role in tumor progression. Cancer associated fibroblasts (CAFs) represent the main cell type of tumor microenvironment, and they contribute to tumor growth by undergoing an irreversible activation process. It is known that ROS can be transferred from cancer cells to fibroblasts. In particular, ROS affect the behaviour of CAFs by promoting the conversion of fibroblasts to myofibroblasts that support tumor progression and dissemination. Furthermore, the wrecking of redox homeostasis in cancer cells and tumor microenvironment induces a metabolic reprogramming in tumor cells and cancer associated fibroblasts, giving advantage to cancer growth. This review describes the role of ROS in tumor growth, by focusing on CAFs activation and metabolic interactions between cancer cells and stromal fibroblasts. PMID:27595103

  3. Cancer: An Oxidative Crosstalk between Solid Tumor Cells and Cancer Associated Fibroblasts

    PubMed Central

    Arcucci, Alessandro; Ruocco, Maria Rosaria; Granato, Giuseppina; Sacco, Anna Maria

    2016-01-01

    Redox balance is associated with the regulation of several cell signalling pathways and functions. In fact, under physiological conditions, cells maintain a balance between oxidant and antioxidant systems, and reactive oxygen species (ROS) can act as second messengers to regulate cell proliferation, cell death, and other physiological processes. Cancer tissues usually contain higher levels of ROS than normal tissues, and this ROS overproduction is associated with tumor development. Neoplastic tissues are very heterogeneous systems, composed of tumor cells and microenvironment that has a critical role in tumor progression. Cancer associated fibroblasts (CAFs) represent the main cell type of tumor microenvironment, and they contribute to tumor growth by undergoing an irreversible activation process. It is known that ROS can be transferred from cancer cells to fibroblasts. In particular, ROS affect the behaviour of CAFs by promoting the conversion of fibroblasts to myofibroblasts that support tumor progression and dissemination. Furthermore, the wrecking of redox homeostasis in cancer cells and tumor microenvironment induces a metabolic reprogramming in tumor cells and cancer associated fibroblasts, giving advantage to cancer growth. This review describes the role of ROS in tumor growth, by focusing on CAFs activation and metabolic interactions between cancer cells and stromal fibroblasts. PMID:27595103

  4. Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase.

    PubMed

    Xie, Ying; Zhao, Xiaoe; Jia, Hongxiang; Ma, Baohua

    2013-01-01

    Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened. PMID:23271363

  5. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene.

    PubMed

    Reznickova, Alena; Novotna, Zdenka; Kolska, Zdenka; Kasalkova, Nikola Slepickova; Rimpelova, Silvie; Svorcik, Vaclav

    2015-01-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells. PMID:25953566

  6. Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture.

    PubMed

    Mougneau, E; Lemieux, L; Rassoulzadegan, M; Cuzin, F

    1984-09-01

    Two distinct forms of the myc oncogene were assayed for their ability to induce, in cultured rat fibroblast cells, the alterations of cellular growth controls observed upon transfer of the gene of polyoma virus encoding only the large T protein (plt). Both of these rearranged myc genes and the plt gene had been previously shown to cooperate with ras oncogenes for transformation of rat embryo fibroblasts (REF) and were thought to induce the same early step ("immortalization") of the tumoral transformation pathway. We now report that these two different oncogenes elicite the same response in the following biological assays: (i) reduction of the requirements in serum factors for growth in culture of cells of the established FR3T3 line; (ii) expression of transformed properties in low serum medium after transfer into FR3T3 cells expressing only the middle T protein of polyoma virus (MTT lines); (iii) conferring on REF cells the ability to grow as clonal colonies after seeding at low cell density; (iv) conferring on REF cells the ability to grow continuously in cell culture. These congruent phenotypes suggest that the activities of the large T and myc proteins result in the induction of the same molecular events. These results also provide simple biological assays and selective systems for oncogenes of the myc class. PMID:6091107

  7. TSPAN12 is a critical factor for cancer-fibroblast cell contact-mediated cancer invasion.

    PubMed

    Otomo, Ryo; Otsubo, Chihiro; Matsushima-Hibiya, Yuko; Miyazaki, Makoto; Tashiro, Fumio; Ichikawa, Hitoshi; Kohno, Takashi; Ochiya, Takahiro; Yokota, Jun; Nakagama, Hitoshi; Taya, Yoichi; Enari, Masato

    2014-12-30

    Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non-cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the β-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced β-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy. PMID:25512506

  8. Hyperbaric oxygen attenuates cell growth in skin fibroblasts cultured in a high-glucose medium.

    PubMed

    Lin, Hen-I; Chu, Shi-Jye; Perng, Wann-Cherng; Wu, Chin-Pyng; Lin, Zuei-Yin; Huang, Kun-Lun

    2008-01-01

    Hyperglycemia and hypoxia synergistically retard diabetic wound healing. We investigated the direct effect of hyperbaric and normobaric hyperoxia on skin fibroblasts cultured in a high-glucose medium. Detroit 551 human dermal fibroblasts cultured in Dulbecco's modified Eagle's medium containing d-glucose had reduced cell survival compared with cells grown in normal glucose medium; survival was 27.5+/-3.8% lower in 25 mM glucose and 30.6+/-3.7% lower in 50 mM glucose. Cell survival decreased because of inhibition of cell proliferation and enhanced cell death. Daily hyperbaric oxygen therapy at 2.5 atmosphere absolute for 90 minutes on 3 consecutive days reduced cell proliferation and increased cell death in normal cultured fibroblasts. Hyperbaric oxygen therapy and high-glucose medium had a synergistic effect and reduced survival by 37.6+/-4.4% (25 mM glucose) and 39.6+/-5.1% (50 mM glucose). The effects of hyperbaric oxygen and high-glucose medium were associated with overproduction of reactive oxygen species. Our results suggest that direct exposure of skin fibroblasts to hyperbaric oxygen affects cell growth and superimposes the toxic effect of high glucose. This cytotoxicity may be related to the production of reactive oxygen species in the fibroblasts. PMID:18638270

  9. Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease

    PubMed Central

    Suganya, Rangaswamy; Chakraborty, Anirban; Miriyala, Sumitra; Hazra, Tapas K.; Izumi, Tadahide

    2015-01-01

    The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEFla (MEFlowAPE1), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEFla cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEFla was lower than that in the wild-type MEF (MEFwt), indicating the low DNA damage stress in MEFla under the normal growth condition. Oxidative phosphorylation activity in MEFla was lower than in MEFwt, while the glycolysis rates in MEFla were higher than in MEFwt. In addition, we observed decreased intracellular oxidative stress in MEFla. These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability. PMID:25645679

  10. The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

    PubMed Central

    Murase, Hiromi; Shimazawa, Masamitsu; Kakino, Mamoru; Ichihara, Kenji; Tsuruma, Kazuhiro; Hara, Hideaki

    2013-01-01

    Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation. PMID:24416064

  11. Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.

    PubMed Central

    van Giersbergen, P L; Shatzer, S A; Henderson, A K; Lai, J; Nakanishi, S; Yamamura, H I; Buck, S H

    1991-01-01

    Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors. PMID:1848006

  12. Integrin alpha 11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells.

    PubMed

    Zhu, Chang-Qi; Popova, Svetlana N; Brown, Ewan R S; Barsyte-Lovejoy, Dalia; Navab, Roya; Shih, Warren; Li, Ming; Lu, Ming; Jurisica, Igor; Penn, Linda Z; Gullberg, Donald; Tsao, Ming-Sound

    2007-07-10

    Integrin alpha11 (ITGA11/alpha11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal alpha11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human alpha11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WT(shIGF2)) fibroblasts resulted in a decreased growth rate of A549+WT(shIGF2), compared with A549+WT tumors. The results indicate that alpha11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma-stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth. PMID:17600088

  13. Guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata

    PubMed Central

    Bae, Won-Gyu; Kim, Jangho; Choung, Yun-Hoon; Chung, Yesol; Suh, Kahp Y.; Pang, Changhyun; Chung, Jong Hoon; Jeong, Hoon Eui

    2015-01-01

    Engineering complex extracellular matrix (ECM) is an important challenge for cell and tissue engineering applications as well as for understanding fundamental cell biology. We developed the methodology for fabrication of precisely controllable multiscale hierarchical structures using capillary force lithography in combination with original wrinkling technique for the generation of well-defined native ECM-like platforms by culturing fibroblast cells on the multiscale substrata [1]. This paper provides information on detailed characteristics of polyethylene glycol-diacrylate multiscale substrata. In addition, a possible model for guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata is proposed. PMID:26543882

  14. Identification and partial characterization of the fibroblast growth factor receptor of baby hamster kidney cells

    SciTech Connect

    Neufeld, G.; Gospodarowicz, D.

    1985-11-05

    The binding of biologically active, SVI-labeled basic fibroblast growth factor (FGF) to baby hamster kidney-derived cell line cells (BHK-21) was studied at 4 degrees C. Unlabeled FGF displaced cell surface bound SVI-FGF, but platelet-derived growth factor, epidermal growth factor, insulin, or transferrin did not. Binding was saturable both as a function of time and as a function of increasing SVI-FGF concentrations. Scatchard analysis of the binding data revealed the presence of about 1.2 X 10(5) binding sites/cell with an apparent KD of 270 pM. The number of the binding sites was down-regulated following preincubation of the cells with FGF. The density of binding sites/cell also decreased as an inverse function of cell density. When SVI-FGF binding was studied in a BHK-21 cell membrane preparation, it was found that the membranal binding site displayed a lower KD of 21 pM. SVI-FGF was covalently cross-linked to its cell surface receptor on intact BHK-21 cells using the homobifunctional agent disuccinimidyl suberate. Two macromolecular species with an apparent molecular weight of 145,000 and SV,000, respectively, were labeled under both reducing and nonreducing conditions. Unlabeled FGF competed with SVI-FGF for binding to both macromolecular species. The labeling of the macromolecules was also inhibited by heparin. No labeling was observed in the absence of the cross-linkers or when heat-inactivated SVI-FGF was used instead of radiolabeled, biologically active FGF.

  15. Brief Azacytidine Step Allows The Conversion of Suspension Human Fibroblasts into Neural Progenitor-Like Cells

    PubMed Central

    Mirakhori, Fahimeh; Zeynali, Bahman; Kiani, Sahar; Baharvand, Hossein

    2015-01-01

    In recent years transdifferentiation technology has enabled direct conversion of human fibroblasts to become a valuable, abundant and accessible cell source for patient-specific induced cell generation in biomedical research. The majority of transdifferentiation approaches rely upon viral gene delivery which due to random integration with the host genome can cause genome instability and tumorigenesis upon transplantation. Here, we provide a simple way to induce neural progenitor-like cells from human fibroblasts without genetic manipulation by changing physicochemical culture properties from monolayer culture into a suspension in the presence of a chemical DNA methyltransferase inhibitor agent, Azacytidine. We have demonstrated the expression of neural progenitor-like markers, morphology and the ability to spontaneously differentiate into neural-like cells. This approach is simple, inexpensive, lacks genetic manipulation and could be a foundation for future chemical neural transdifferentiation and a safe induction of neural progenitor cells from human fibroblasts for clinical applications. PMID:25870845

  16. Changes in the gene expression of co-cultured human fibroblast cells and osteosarcoma cells: the role of microenvironment

    PubMed Central

    Salvatore, Viviana; Focaroli, Stefano; Teti, Gabriella; Mazzotti, Antonio; Falconi, Mirella

    2015-01-01

    Background The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. Methods In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. Results In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. Conclusions These findings demonstrated that the tumor

  17. Thyroid cell lines in research on goitrogenesis.

    PubMed

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  18. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  19. Naïve adult stem cells isolation from primary human fibroblast cultures.

    PubMed

    Wenzel, Vera; Roedl, Daniela; Ring, Johannes; Djabali, Karima

    2013-01-01

    Over the last decade, several adult stem cell populations have been identified in human skin (1-4). The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory (5, 6). These early studies described a multipotent precursor cell population from adult mammalian dermis (5). These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons (5). Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers (6). Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies. Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies (7). The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable (7). These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures (7). We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are

  20. CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells.

    PubMed

    Zhang, Hui; Kho, Alvin T; Wu, Qing; Halayko, Andrew J; Limbert Rempel, Karen; Chase, Robert P; Sweezey, Neil B; Weiss, Scott T; Kaplan, Feige

    2016-09-01

    Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid-regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal-epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/- mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal-epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)-induced human fetal lung fibroblast line (MRC5). LPS-induced upregulation of the proinflammatory cytokines IL-8 and CCL2 was exacerbated in MRC5-CRISPLD2(knockdown) cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL-8, IL-6, CCL2. LPS-stimulated expression of proinflammatory mediators by human lung epithelial HAEo- cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF-CRISPLD2(knockdown) suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood. PMID:27597766

  1. Embryonic germ cell lines and their derivation from mouse primordial germ cells.

    PubMed

    Labosky, P A; Barlow, D P; Hogan, B L

    1994-01-01

    When primordial germ cells of the mouse are cultured on feeder layers with the addition of the polypeptide signalling molecules leukaemia inhibitory factor, Steel factor and basic fibroblast growth factor they give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells (EG cells) can be induced to differentiate extensively in culture and also form teratocarcinomas when injected into nude mice. Additionally, they contribute to chimeras when injected into host blastocysts. We have derived multiple EG cell lines from 8.5 days post coitum (dpc) embryos of C57BL/6 inbred mice. Four independent EG cell lines with normal male karyotypes have formed chimeras (up to 70% coat colour chimerism) when injected into BALB/c host blastocysts. Chimeric mice from all four cell lines are fertile, but only those from one line have transmitted coat colour markers through the germline. Studies have also been carried out to determine whether gonadal primordial germ cells can give rise to pluripotent EG cells. Germ cells from gonads of 15.5 dpc C57BL/6 embryos and newborn mice failed to produce EG cell lines. EG cell lines capable of forming teratocarcinomas and coat colour chimeras have been established from primordial germ cells of 12.5 dpc genital ridges. We are currently testing the genomic imprinting status of the insulin-like growth factor type 2 receptor gene (Igf2r) in our different EG cell lines. PMID:7835148

  2. Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

    PubMed Central

    Fuentes-Calvo, Isabel; Blázquez-Medela, Ana M.; Santos, Eugenio; López-Novoa, José M.; Martínez-Salgado, Carlos

    2010-01-01

    Background Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells. Methodology/Principal Findings Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras−/−,N-ras−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras−/−) embrionary fibroblasts by mating of K-ras+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras−/− fibroblasts. Conclusions/Significance We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations. PMID:20090846

  3. The Disintegrin-like and Cysteine-rich domains of ADAM-9 Mediate Interactions between Melanoma Cells and Fibroblasts*

    PubMed Central

    Zigrino, Paola; Nischt, Roswitha; Mauch, Cornelia

    2011-01-01

    A characteristic of malignant cells is their capacity to invade their surrounding and to metastasize to distant organs. During these processes, proteolytic activities of tumor and stromal cells modify the extracellular matrix to produce a microenvironment suitable for their growth and migration. In recent years the family of ADAM proteases has been ascribed important roles in these processes. ADAM-9 is expressed in human melanoma at the tumor-stroma border where direct or indirect interactions between tumor cells and fibroblasts occur. To analyze the role of ADAM-9 for the interaction between melanoma cells and stromal fibroblasts, we produced the recombinant disintegrin-like and cysteine-rich domain of ADAM-9 (DC-9). Melanoma cells and human fibroblasts adhered to immobilized DC-9 in a Mn2+-dependent fashion suggesting an integrin-mediated process. Inhibition studies showed that adhesion of fibroblasts was mediated by several β1 integrin receptors independent of the RGD and ECD recognition motif. Furthermore, interaction of fibroblasts and high invasive melanoma cells with soluble recombinant DC-9 resulted in enhanced expression of MMP-1 and MMP-2. Silencing of ADAM-9 in melanoma cells significantly reduced cell adhesion to fibroblasts. Ablation of ADAM-9 in fibroblasts almost completely abolished these cellular interactions and melanoma cell invasion in vitro. In summary, these results suggest that ADAM-9 expression plays an important role in mediating cell-cell contacts between fibroblasts and melanoma cells and that these interactions contribute to proteolytic activities required during invasion of melanoma cells. PMID:21135106

  4. Basal Cell Carcinoma in Gorlin’s Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

    PubMed Central

    Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

    2015-01-01

    Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings

  5. Cholesterol stimulation of HDL binding to human endothelial cells EAhy 926 and skin fibroblasts: evidence for a mechanism independent of cellular metabolism.

    PubMed

    Bernini, F; Bellosta, S; Corsini, A; Maggi, F M; Fumagalli, R; Catapano, A L

    1991-04-24

    The properties of the HDL binding site on the permanent human cell line EAhy 926 were studied. This cell line presents with highly differentiated functions of vascular endothelium. EAhy 926 cells possess HDL3 saturable binding sites with a Kd of about 20 micrograms/ml, which were up-regulated by cholesterol and were pronase- and EDTA-insensitive. Furthermore, HDL3 promoted cholesterol efflux from EAhy 926 cells in a dose-dependent manner. Thus, the HDL-binding site in EAhy 926 cells is similar to that present in fibroblasts, smooth muscle cells and endothelial cells. Up-regulation of HDL binding by cholesterol did not require de novo synthesis of HDL 'receptor' protein, as shown by the lack of effect of cycloheximide and alpha-amanitin and also occurred in fixed, non-living cells. Similar results were obtained using human skin fibroblasts. From these data we conclude that: (a) EAhy 926 cells are a good model for studying the HDL interaction with endothelial cells; (b) a mechanism independent of cellular metabolism is involved in the cholesterol-mediated up-regulation of HDL binding sites in EAhy 926 cells and human skin fibroblasts. PMID:1851638

  6. ANALYSIS OF A MARINE FISH CELL LINE FROM A MALE SHEEPSHEAD

    EPA Science Inventory

    Chromosomes from consecutive culture passages of a developing cell line from fin fibroblasts of a male sheepshead (Archosargus probatocephalus) were analyzed. It was demonstrated that the modal chromosome number is 48. The chromosome types found in these cells included 8 submetac...

  7. Proteome alteration induced by hTERT transfection of human fibroblast cells

    PubMed Central

    Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

    2008-01-01

    Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase

  8. Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion.

    PubMed

    Poleganov, Marco Alexander; Eminli, Sarah; Beissert, Tim; Herz, Stephanie; Moon, Jung-Il; Goldmann, Johanna; Beyer, Arianne; Heck, Rosario; Burkhart, Isabell; Barea Roldan, Diana; Türeci, Özlem; Yi, Kevin; Hamilton, Brad; Sahin, Ugur

    2015-11-01

    mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections. PMID:26381596

  9. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

    SciTech Connect

    Arora, S.; Jain, J.; Rajwade, J.M.; Paknikar, K.M.

    2009-05-01

    Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 {mu}g/mL and 100 {mu}g/mL SNP, respectively, although with minor decrease in confluence. IC{sub 50} values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 {mu}g/mL and 449 {mu}g/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC{sub 50} SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with {approx} 1/2 IC{sub 50} concentration of SNP (i.e. 30 {mu}g/mL and 225 {mu}g/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH ({approx} 1.2 fold) and depletion of lipid peroxidation ({approx} 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ({approx} 1.4 fold) and GSH ({approx} 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 {mu}g/mL in primary fibroblasts, 12.5 {mu}g/mL in primary liver cells) than the necrotic concentration (100 {mu}g/mL in primary fibroblasts, 500 {mu}g/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC{sub 50} SNP (resulting in apoptosis

  10. In-Vitro Biocompatibility Studies of Plasma-Nitrided Titanium Alloy β-21S Using Fibroblast Cells

    NASA Astrophysics Data System (ADS)

    Mohan, L.; Raja, M. D.; Uma, T. S.; Rajendran, N.; Anandan, C.

    2016-04-01

    In the present work, titanium alloy β-21S was nitrided in a low-pressure RF plasma with 100% nitrogen and 20% hydrogen-diluted nitrogen at 800 °C for 4 h and the samples were evaluated for in-vitro biocompatibility by using NIH 3T3 fibroblast cell line. Cellular behavior was evaluated in terms of cell morphology and its viability. FESEM was exploited to observe the morphology of the cells fixed over the surface of the implant. Fibroblasts were seemed to be well distributed over the surface with its characteristic spindle-like shape. Over all, the results indicate that nitriding provided a compatible surface for cell attachment and cell growth. Cell viability and proliferation was assessed by using standard MTT assay. Compared with substrate, the nitrided samples exhibited high-percentage cell viability demonstrating their increased biocompatibility. In addition, the nitrided samples facilitate bone-like apatite formation and exhibited a gradual increase of apatite formation after immersion in Hanks' solution.

  11. NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

    PubMed Central

    Kayamori, Kou; Katsube, Ken-ichi; Sakamoto, Kei; Ohyama, Yoshio; Hirai, Hideaki; Yukimori, Akane; Ohata, Yae; Akashi, Takumi; Saitoh, Masao; Harada, Kiyoshi; Harada, Hiroyuki; Yamaguchi, Akira

    2016-01-01

    Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs. PMID:27124156

  12. Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts

    PubMed Central

    Wu, Yuehong; Zhang, Yong; Mishra, Anuja; Tardif, Suzette D.; Hornsby, Peter J.

    2010-01-01

    Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, α-fetoprotein; ectoderm, βIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 × 105 newborn marmoset skin fibroblasts, we obtained ∼100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and of those 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy. PMID:20363201

  13. Distinct function of estrogen receptor α in smooth muscle and fibroblast cells in prostate development.

    PubMed

    Vitkus, Spencer; Yeh, Chiuan-Ren; Lin, Hsiu-Hsia; Hsu, Iawen; Yu, Jiangzhou; Chen, Ming; Yeh, Shuyuan

    2013-01-01

    Estrogen signaling, through estrogen receptor (ER)α, has been shown to cause hypertrophy in the prostate. Our recent report has shown that epithelial ERα knockout (KO) will not affect the normal prostate development or homeostasis. However, it remains unclear whether ERα in different types of stromal cells has distinct roles in prostate development. This study proposed to elucidate how KO of ERα in the stromal smooth muscle or fibroblast cells may interrupt cross talk between prostate stromal and epithelial cells. Smooth muscle ERαKO (smERαKO) mice showed decreased glandular infolding with the proximal area exhibiting a significant decrease. Fibroblast ERαKO mouse prostates did not exhibit this phenotype but showed a decrease in the number of ductal tips. Additionally, the amount of collagen observed in the basement membrane was reduced in smERαKO prostates. Interestingly, these phenotypes were found to be mutually exclusive among smERαKO or fibroblast ERαKO mice. Compound KO of ERα in both fibroblast and smooth muscle showed combined phenotypes from each of the single KO. Further mechanistic studies showed that IGF-I and epidermal growth factor were down-regulated in prostate smooth muscle PS-1 cells lacking ERα. Together, our results indicate the distinct functions of fibroblast vs. smERα in prostate development. PMID:23204329

  14. UVB-Induced Cell Death Signaling Is Associated with G1-S Progression and Transcription Inhibition in Primary Human Fibroblasts

    PubMed Central

    Ortolan, Tatiana Grohmann; Menck, Carlos Frederico M.

    2013-01-01

    DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions. PMID:24155908

  15. Cyclin A is redundant in fibroblasts but essential in hematopoietic and embryonic stem cells.

    PubMed

    Kalaszczynska, Ilona; Geng, Yan; Iino, Tadafumi; Mizuno, Shin-ichi; Choi, Yoon; Kondratiuk, Ilona; Silver, Daniel P; Wolgemuth, Debra J; Akashi, Koichi; Sicinski, Piotr

    2009-07-23

    Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation. PMID:19592082

  16. Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts.

    PubMed

    Ueno, Koji; Takeuchi, Yuriko; Samura, Makoto; Tanaka, Yuya; Nakamura, Tamami; Nishimoto, Arata; Murata, Tomoaki; Hosoyama, Tohru; Hamano, Kimikazu

    2016-01-01

    The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers. PMID:27329845

  17. Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts

    PubMed Central

    Ueno, Koji; Takeuchi, Yuriko; Samura, Makoto; Tanaka, Yuya; Nakamura, Tamami; Nishimoto, Arata; Murata, Tomoaki; Hosoyama, Tohru; Hamano, Kimikazu

    2016-01-01

    The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers. PMID:27329845

  18. Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis

    SciTech Connect

    Pellett, O.L.; Smith, M.L.; Greene, A.A.; Schneider, J.A. )

    1988-05-01

    Cystinosis is an autosomal recessive disease in which three clinical forms are recognized: infantile nephropathic, with renal tubular damage by 1 year of age and progressive glomerular insufficiency; intermediate, with tubular and glomerular insufficiency beginning at a later age; benign, with no kidney damage. Skin fibroblasts cultured from patients with all types of cystinosis show increased intralysosomal free (nonprotein) cystine; however, fibroblasts from heterozygotes have normal free-cystine values. To determine whether genetic complementation occurs between the different forms, somatic cell hybrids were constructed between cells from a patient with infantile nephropathic cystinosis and cells from patients with other types of cystinosis. If complementation occurred, the hybrids would be expected to have normal cystine levels. To construct hybrid cells, a universal parent cell type (TG1-neo), which was hypoxanthine/aminopterin/thymidine (HAT) sensitive and G418 resistant was constructed from an infantile nephropathic cystinosis fibroblast strain. Polyethylene glycol fusion of TG1-neo with other cells that are not HAT sensitive or G418 resistant allowed for selection of hybrid cells in a medium containing HAT and the aminoglycoside G418. As indicated by elevated cystine levels, complementation did not occur between TG1-neo and two different benign cystinosis strains, an intermediate cystinosis strain, or another nephropathic cystinosis cell strain. When a normal fibroblast strain was fused with TG1-neo, all 15 hybrid clones studied contained normal amounts of intracellular free cystine.

  19. Isolation and characterization of SSEA3(+) stem cells derived from goat skin fibroblasts.

    PubMed

    Yang, Zhongcai; Liu, Jun; Liu, Hongliang; Qiu, Mingning; Liu, Qingqing; Zheng, Liming; Pang, Meijun; Quan, Fusheng; Zhang, Yong

    2013-06-01

    Novel stem cells expressing stage-specific embryonic antigen 3 (SSEA-3) reside among human dermal fibroblasts and are known as multilineage-differentiating stress-enduring (Muse) cells. They enhance the generation efficiency of induced pluripotent stem cells. However, Muse cells have only been found in humans. We aimed to isolate SSEA3-positive cells from terminally differentiated skin fibroblasts of adult goat and determine their pluripotency. Cell clusters from SSEA3(+) populations possessed stem cell-like morphological features and normal karyotypes, were consistently positive for alkaline phosphatase, and expressed stem cell pluripotency markers. These SSEA3(+) cells remained undifferentiated over eight passages in suspension culture and were able to differentiate into cells of all three germ layers in vitro and in vivo. Our combined findings suggest that a subset of adult stem cells expressing SSEA3 also exist among adult goat skin fibroblasts. We are the first to report that multipotent adult goat cells exist among terminally differentiated goat skin in suspension culture. Our results also provide a promising platform for generation of a transgenic goat, because the undifferentiated state of stem cells was thought to be more efficient as donor cells for somatic cell nuclear transfer. PMID:23668861

  20. Intravitreous transplantation of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delays photoreceptor cell degeneration in Royal College of Surgeons rats

    PubMed Central

    Uteza, Yves; Rouillot, Jean-Sébastien; Kobetz, Alexandra; Marchant, Dominique; Pecqueur, Sèverine; Arnaud, Emmanuelle; Prats, Hervé; Honiger, Jiri; Dufier, Jean-Louis; Abitbol, Marc; Neuner-Jehle, Martin

    1999-01-01

    We developed an experimental approach with genetically engineered and encapsulated mouse NIH 3T3 fibroblasts to delay the progressive degeneration of photoreceptor cells in dark-eyed Royal College of Surgeons rats. These xenogeneic fibroblasts can survive in 1.5-mm-long microcapsules made of the biocompatible polymer AN69 for at least 90 days under in vitro and in vivo conditions because of their stable transfection with the gene for the 18-kDa form of the human basic fibroblast growth factor (hFGF-2). Furthermore, when transferred surgically into the vitreous cavity of 21-day-old Royal College of Surgeons rats, the microencapsulated hFGF-2-secreting fibroblasts provoked a local delay of photoreceptor cell degeneration, as seen at 45 days and 90 days after transplantation. This effect was limited to 2.08 mm2 (45 days) and 0.95 mm2 (90 days) of the retinal surface. In both untreated eyes and control globes with encapsulated hFGF-2-deficient fibroblasts, the rescued area (of at most 0.08 mm2) was significantly smaller at both time points. Although, in a few ocular globes, surgical trauma induced a reorganization of the retinal cytoarchitecture, neither microcapsule rejection nor hFGF-2-mediated tumor formation were detected in any treated eyes. These findings indicate that encapsulated fibroblasts secreting hFGF-2 or perhaps other agents can be applied as potential therapeutic tools to treat retinal dystrophies. PMID:10077648

  1. Transcriptomic study of high‑glucose effects on human skin fibroblast cells.

    PubMed

    Pang, Lingxia; Wang, Youpei; Zheng, Meiqin; Wang, Qing; Lin, Hong; Zhang, Liqing; Wu, Lingjian

    2016-03-01

    Skin ulcers are a common complication of diabetes mellitus (DM). Fibroblasts are located within the dermis of skin tissue and can be damaged by diabetes. However, the underlying mechanism of how DM affects fibroblasts remains elusive. To understand the effects of DM on fibroblasts, the current study mimicked DM by high‑glucose (HG) supplementation in the culture medium of human foreskin primary fibroblast cells, and the analysis of transcriptomic changes was conducted. RNA sequencing‑based transcriptome analysis identified that, upon HG stress, 463 genes were upregulated and 351 genes downregulated (>1.5‑fold changes; P<0.05). These altered genes were distributed into 20 different pathways. In addition, gene ontology (GO) analysis indicated that 31 GO terms were enriched. Among the pathways identified, nuclear factor κB (NF‑κB) pathway genes were highly expressed, and the addition of Bay11‑7082, a typical NF‑κB signaling inhibitor, blocked the previously observed alterations in plasminogen activator inhibitor 1 (PAI1), an inflammation marker and frizzled class receptor 8 (FZD8), a Wnt signaling gene, expression that resulted from HG stress. Furthermore, an inhibitor of Wnt signaling diminished the role of Bay11‑7082 in the regulation of PAI1 expression under HG conditions, suggesting that Wnt signaling may function downstream of the NF‑κB pathway to protect fibroblast cells from HG stress. To the best of our knowledge, the current study is the first analysis of transcriptomic responses under HG stress in human fibroblasts. The data provided here may aid the understanding of the molecular mechanisms by which fibroblast cells are damaged in the skin of patients with DM. PMID:26820167

  2. Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors.

    PubMed

    Deng, Yanfei; Liu, Qingyou; Luo, Chan; Chen, Shibei; Li, Xiangping; Wang, Caizhu; Liu, Zhenzhen; Lei, Xiaocan; Zhang, Huina; Sun, Hongliang; Lu, Fenghua; Jiang, Jianrong; Shi, Deshun

    2012-09-01

    Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future. PMID:22420535

  3. Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

    PubMed Central

    Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

    2012-01-01

    Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

  4. mir-1-mediated paracrine effect of cancer-associated fibroblasts on lung cancer cell proliferation and chemoresistance.

    PubMed

    Li, Jianmin; Guan, Jing; Long, Xiaoping; Wang, Yang; Xiang, Xudong

    2016-06-01

    Lung cancer is the leading cause of cancer-related mortality in humans worldwide. Moreover, the overall 5-year survival rate is only 15%. Pathologically almost 80% of all lung cancer cases are non-small cell lung cancer (NSCLC). Cancer-associated fibroblasts (CAFs) have been found to exist in a large number of NSCLCs. CAFs have been proven to promote tumor progression, metastasis and resistance to therapy through paracrine effects in most solid tumors. In the present study, firstly we isolated CAFs from patient tissues and demonstrated that they promoted cell proliferation and chemoresistance to cisplatin in the lung cancer cell lines A549 and 95D in a paracrine manner. Secondly, using ELISA and quantative PCR, we found that a higher amount of stromal cell-derived factor 1 (SDF-1) existed in the CAFs rather than that observed in the normal fibroblasts (NFs). Thirdly, we detected that SDF-1 facilitated lung cancer cell proliferation and drug resistance via the CXCR4-mediated signaling pathway which involved NF-κB and Bcl-xL. Moreover, we also confirmed that the expression level of SDF-1 in the CAFs was negatively regulated by microRNA mir-1 through microRNA overexpression and quantitative PCR. Overall, our data provide one explanation for the effects of CAFs on lung cancer cells. Meanwhile, our results also suggest CAFs as a potential therapeutic target in tumor treatment. PMID:27035564

  5. Prevention of asbestos-induced cell death in rat lung fibroblasts and alveolar macrophages by scavengers of active oxygen species

    SciTech Connect

    Shatos, M.A.; Doherty, J.M.; Marsh, J.P.; Mossman, B.T.

    1987-10-01

    The possible modulation of asbestos-related cell death using antioxidants in both target and effector cells of asbestosis was investigated. After exposure to crocidolite asbestos at a range of concentrations (2.5-25 ..mu..gcm/sup 2/ dish), the viability of a normal rat lung fibroblast line and freshly isolated alveolar macrophages (AM) was determined. In comparison to fibroblasts, AM were more resistant to the cytotoxic effects of asbestos. Cytotoxic concentrations of asbestos then were added to both cell types in combination with the antioxidants, superoxide dismutase (SOD), a scavenger of superoxide (O/sub 2//sup -./), and catalase, an enzyme scavenging H/sub 2/O/sub 2/. Dimethylthiourea (DMTU), a scavenger of the hydroxyl radical (OH/sup ./) and deferoxamine, an iron chelator, also were evaluated in similar studies. Results showed significant dosage-dependent reduction of asbestos-associated cell death with all agents. In contrast, asbestos-induced toxicity was not ameliorated after addition of chemically inactivated SOD and catalase or bovine serum albumin. Results above suggest asbestos-induced cell damage is mediated by active oxygen species. In this regard, the iron associated with the fiber andor its interaction with cell membranes might be critical in deriving a modified Haber-Weiss (Fenton-type) reaction resulting in production of OH/sup ./.

  6. Antitumor cell-complex vaccines employing genetically modified tumor cells and fibroblasts.

    PubMed

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Diaz, Ana; Algás, Rosa; Aliño, Salvador F

    2014-02-01

    The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok) and a low producer (p2F). Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP) IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01). When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05). Significant survival (40%) was only observed in the groups vaccinated with free transfected B16 cells. PMID:24556729

  7. Muse Cells, a New Type of Pluripotent Stem Cell Derived from Human Fibroblasts.

    PubMed

    Liu, Qi; Zhang, Ru-zhi; Li, Di; Cheng, Sai; Yang, Yu-hua; Tian, Ting; Pan, Xiao-ru

    2016-04-01

    A new type of mesenchymal stem cells (MSCs) that expresses stage-specific embryonic antigen 3 (SSEA-3) and the mesenchymal cell marker CD105 are known as multilineage-differentiating stress-enduring (Muse) cells. Studies have shown that stem cells in suspension cultures are more likely to generate embryoid body-like stem cell spheres and maintain an undifferentiated phenotype and pluripotency. We separated Muse cells derived from human dermal fibroblasts by long-term trypsin incubation (LTT) through suspension cultures in methylcellulose. The Muse cells obtained expressed several pluripotency markers, including Nanog, Oct4, Sox2, and SSEA-3, and could differentiate in vitro into cells of the three germ layers, such as hepatocytes (endodermal), neural cells (ectodermal) and adipocytes, and osteocytes (mesodermal cells). These cells showed a low level of DNA methylation and a high nucleo-cytoplasmic ratio. Our study provides an innovative and exciting platform for exploring the potential cell-based therapy of various human diseases using Muse cells as well as their great possibility for regenerative medicine. PMID:27055628

  8. Antitumor Cell-Complex Vaccines Employing Genetically Modified Tumor Cells and Fibroblasts

    PubMed Central

    Miguel, Antonio; Herrero, María José; Sendra, Luis; Botella, Rafael; Diaz, Ana; Algás, Rosa; Aliño, Salvador F.

    2014-01-01

    The present study evaluates the immune response mediated by vaccination with cell complexes composed of irradiated B16 tumor cells and mouse fibroblasts genetically modified to produce GM-CSF. The animals were vaccinated with free B16 cells or cell complexes. We employed two gene plasmid constructions: one high producer (pMok) and a low producer (p2F). Tumor transplant was performed by injection of B16 tumor cells. Plasma levels of total IgG and its subtypes were measured by ELISA. Tumor volumes were measured and survival curves were obtained. The study resulted in a cell complex vaccine able to stimulate the immune system to produce specific anti-tumor membrane proteins (TMP) IgG. In the groups vaccinated with cells transfected with the low producer plasmid, IgG production was higher when we used free B16 cell rather than cell complexes. Nonspecific autoimmune response caused by cell complex was not greater than that induced by the tumor cells alone. Groups vaccinated with B16 transfected with low producer plasmid reached a tumor growth delay of 92% (p ≤ 0.01). When vaccinated with cell complex, the best group was that transfected with high producer plasmid, reaching a tumor growth inhibition of 56% (p ≤ 0.05). Significant survival (40%) was only observed in the groups vaccinated with free transfected B16 cells. PMID:24556729

  9. Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast

    PubMed Central

    Qu, Ling; Zhu, Rui; Li, Hongguo; Xue, Yingsen; Liu, Xincheng

    2016-01-01

    Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration. PMID:27525012

  10. Celecoxib suppresses fibroblast growth factor-2 expression in pancreatic ductal adenocarcinoma PANC-1 cells.

    PubMed

    Li, Jing; Luo, Miaosha; Wang, Yan; Shang, Boxin; Dong, Lei

    2016-09-01

    The inhibition of cyclooxygenase (COX)-2 has been reported to suppress growth and induce apoptosis in human pancreatic cancer cells. Nevertheless, the precise biological mechanism of how celecoxib, a selective COX-2 inhibitor, regulates the growth and invasion of pancreatic tumors is not completely understood. It has been shown that fibroblast growth factor-2 (FGF-2) and its receptor levels correlate with the inhibition of cancer cell proliferation, migration and invasion in pancreatic ductal adenocarcinoma (PDAC). Therefore, the aim of the present study was to examine the hypothesis that the antitumor activity of celecoxib in PDAC may be exerted through modulation of FGF-2 function. In the present study, we evaluated the effects of celecoxib on the proliferation, migration, invasion and apoptosis of the PANC-1 cell line. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to examine the expression of FGF-2, FGFR-2, ERK1/2 and MMPs. In the present study, FGF-2 and FGFR-2 were expressed in PANC-1 cells and FGF-2 exerted a stimulatory effect on phosphorylated extracellular signal regulated kinase (p-ERK) expression. Celecoxib treatment suppressed FGF-2 and FGFR-2 expression and decreased MMP-2, MMP-9 and p-ERK expression in the PANC-1 cells. Furthermore, celecoxib treatment caused the resistance of PANC-1 cells to FGF-2 induced proliferation, migration and invasion ability, as well as the increase in their apoptotic rate. Our data provide evidence that targeting FGF-2 with celecoxib may be used as an effective treatment in PDAC. PMID:27430377

  11. Direct reprogramming of human fibroblasts into sweat gland-like cells.

    PubMed

    Zhao, Zhiliang; Xu, Mengyao; Wu, Meng; Ma, Kui; Sun, Mengli; Tian, Xiaocheng; Zhang, Cuiping; Fu, Xiaobing

    2015-01-01

    The skin of patients with an extensive deep burn injury is repaired by a process that leaves a hypertrophic scar without sweat glands and therefore loses the function of perspiration. The aim of this study was to identify whether the key factors related to sweat gland development could directly reprogram fibroblasts into sweat gland-like cells. After introducing the NF-κB and Lef-1 genes into fibroblasts, we found that stably transfected fibroblasts expressed specific markers of sweat glands, including CEA, CK7, CK14 and CK19, both at the protein and mRNA levels. The immunofluorescence staining also showed positive expression of CEA, CK7, CK14 and CK19 in induced fibroblasts, but there were no positive cells in the control groups. The expression of Shh and Cyclin D1, downstream genes of NF-κB and Lef-1, were also significantly increased during regeneration. The induced fibroblasts were implanted into an animal model. Twenty days later, iodine-starch perspiration tests showed that 7 out of the 10 cell-treated paws were positive for perspiration, with a distinctive black point-like area appearing in the center of the paw. Contralateral paws tested negative. Histological examination of skin biopsies from experimental and control paws revealed that sweat glands were fully reconstructed in the test paws, with integral, secretory and ductal portions, but were not present in the control paws. This is the first report of successful reprogramming of fibroblasts into sweat gland-like cells, which will provide a new cell source for sweat gland regeneration in patients with extensive deep burns. PMID:26566868

  12. Fibroblast contraction of collagen lattices in vitro: inhibition by chronic inflammatory cell mediators.

    PubMed

    Ehrlich, H P; Wyler, D J

    1983-09-01

    Fibroblast-populated collagen lattices (FPCL), prepared in petri dishes with serum-containing culture medium and incubated at 37 degrees C, undergo progressive and symmetric contraction (reduction in size) over a period of days. The in vitro contraction process requires viable cells with intact cytoskeletal elements, is associated with cell elongation, and is believed to represent a fibroblast function which also occurs in vivo during wound healing and tissue fibrosis. We report that soluble mediators elaborated by chronic inflammatory cells cultured in vitro, when added to FPCL, inhibit lattice contraction. Granulomas, isolated from the liver of Schistosoma mansoni-infected mice, secrete a factor(s) with an estimated molecular weight between 13,700 and 43,000 daltons (gel filtration: Sephadex G-200) and pI = 6 (preparative isoelectrofocusing in granular gel) which inhibits lattice contraction but is not toxic to fibroblasts. Supernatants (cell-free conditioned culture medium) of cultured macrophages isolated from these granulomas also contain this activity. The contraction inhibitory activity present in granuloma culture supernatants is abrogated by the addition of indomethacin to the lattices, while the addition of prostaglandin E2 (PGE2) alone to lattices inhibits contraction. Furthermore, culture supernatants interfere with fibroblast elongation in lattices. We propose that the ability of fibroblasts to contract collagen lattices in vitro and a fibrotic mass in vivo may be regulated by soluble products of chronic inflammatory cells, including macrophages. This process may be mediated by fibroblast-derived prostaglandins which alter cytoskeletal functions and has implications for understanding regulation of tissue fibrogenesis in a variety of diseases. PMID:6885932

  13. Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts

    PubMed Central

    Chen, Joseph C.; Roan, Nadia R.

    2016-01-01

    Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency. PMID:27347495

  14. Human mast cell basic fibroblast growth factor in pulmonary fibrotic disorders.

    PubMed Central

    Inoue, Y.; King, T. E.; Tinkle, S. S.; Dockstader, K.; Newman, L. S.

    1996-01-01

    Mast cells (MCs) are abundant in fibrotic tissue, although their role in fibrogenesis remains obscure. Recent studies suggest MCs may produce basic fibroblast growth factor (bFGF). To evaluate the hypothesis that MC bFGF contributes to the fibrotic response in human interstitial lung disease, we studied lung tissue, bronchoalveolar lavage fluid and serum in 1) idiopathic pulmonary fibrosis, 2) chronic beryllium disease and sarcoidosis, 3) control subjects with no disease or who were beryllium sensitized with normal lung histology. Diseased subjects underwent clinical assessments to stage disease severity. We determined that most bFGF+ cells in lung interstitium are MCs and are most abundant in idiopathic pulmonary fibrosis. Distribution of bFGF+ MCs matched that of extracellular matrix deposition and correlated with the extent of fibrosis morphometrically. Only one bFGF isoform (17.8 kd) was found in idiopathic pulmonary fibrosis and chronic beryllium disease lung tissues and interacted with heparin-like molecules in the lung. Using a human MC line, we verified that MCs express bFGF mRNA and protein that localizes to cytoplasmic granules. Clinically, bFGF concentrations in bronchoalveolar lavage fluid and serum were highest in disease states and correlated with bronchoalveolar lavage cellularity and severity of gas exchange abnormalities, supporting a role for MC bFGF in the pulmonary fibrotic response and its clinical consequence. Images Figure 1 Figure 2 Figure 7 Figure 10 Figure 11 Figure 12 PMID:8952537

  15. Establishment and characterization of a cell line from the Chinese soft-shelled turtle Pelodiscus sinensis.

    PubMed

    Guo, Haijie; Xia, Zhaonan; Tang, Wei; Mao, Zhijuan; Qian, Guoying; Wang, Caisheng

    2016-06-01

    The establishment and partial characterization of Pelodiscus sinensis continuous cell line is described here. A novel P. sinensis fibroblast cell line, designated PSF, was established from heart tissue by the semi-digestion explant culture technique. Since its initiation in July 2013, the cell line has been subcultured at 30°C in minimal essential medium (MEM) containing 15% (v/v) fetal bovine serum for more than 50 passages. The growth curve of the cell line revealed the population doubling time was 51.1 h. Karyotyping analysis indicated the modal chromosome number was 66, and no microbial contamination was detected. The PSF cell line produced significant fluorescent signals after transfection with plasmid pEGFP-C3. Analysis of mitochondrial cytochrome D-loop sequences revealed 96% identity among other Chinese turtle subspecies. Several cell line characterizations included morphological analysis and immunocytochemistry, which revealed the origin of the PSF cell line was fibroblast-like cells. Measurement of the isoenzymes lactic dehydrogenase and malic dehydrogenase showed no cross-contamination of this cell line with other species. This newly established cell line will be a valuable tool for transgenic and genetic manipulation studies and will act as an efficient instrument for studies of the viral diseases of the soft-shelled turtle. PMID:27059326

  16. Molecular Communication between Tumor-Associated Fibroblasts and Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Leef, George; Thomas, Sufi Mary

    2013-01-01

    Over the past few decades, it has become increasingly clear that the lethality of cancers depends on more than the malignant cells themselves. The environment those malignant cells are exposed to is just as important a determinant of their behavior. Head and neck squamous cell carcinoma (HNSCC) is both common and deadly. It is the 6th most frequently occurring cancers, and prognosis is still generally poor. Recent evidence indicates that activated fibroblasts residing within the tumor stroma play a significant role in promoting the aggressive spread often seen in head and neck cancer. Tumor associated fibroblasts (TAFs) have also been implicated in facilitating angiogenesis and suppressing the normal anti-tumor function of immune cells. Studying the signaling molecules involved in these processes will facilitate the development of promising targets and inhibitors to prevent tumor-associated fibroblasts from exerting their reinforcing effects on the tumor. In this article, we review the recent literature on the signals used in tumor associated fibroblast communication, with a focus on potential therapeutic targets. Further, we highlight the lead candidates for TAF-targeted therapeutic interventions. Future anticancer strategies may achieve better results than current approaches by targeting the support cells in tumor stroma in addition to the cancerous cells. PMID:23357526

  17. Fibroblastic reticular cell-derived lysophosphatidic acid regulates confined intranodal T-cell motility

    PubMed Central

    Takeda, Akira; Kobayashi, Daichi; Aoi, Keita; Sasaki, Naoko; Sugiura, Yuki; Igarashi, Hidemitsu; Tohya, Kazuo; Inoue, Asuka; Hata, Erina; Akahoshi, Noriyuki; Hayasaka, Haruko; Kikuta, Junichi; Scandella, Elke; Ludewig, Burkhard; Ishii, Satoshi; Aoki, Junken; Suematsu, Makoto; Ishii, Masaru; Takeda, Kiyoshi; Jalkanen, Sirpa; Miyasaka, Masayuki; Umemoto, Eiji

    2016-01-01

    Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network. DOI: http://dx.doi.org/10.7554/eLife.10561.001 PMID:26830463

  18. Direct Conversion of Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules.

    PubMed

    Hu, Wenxiang; Qiu, Binlong; Guan, Wuqiang; Wang, Qinying; Wang, Min; Li, Wei; Gao, Longfei; Shen, Lu; Huang, Yin; Xie, Gangcai; Zhao, Hanzhi; Jin, Ying; Tang, Beisha; Yu, Yongchun; Zhao, Jian; Pei, Gang

    2015-08-01

    Neuronal conversion from human fibroblasts can be induced by lineage-specific transcription factors; however, the introduction of ectopic genes limits the therapeutic applications of such induced neurons (iNs). Here, we report that human fibroblasts can be directly converted into neuronal cells by a chemical cocktail of seven small molecules, bypassing a neural progenitor stage. These human chemical-induced neuronal cells (hciNs) resembled hiPSC-derived neurons and human iNs (hiNs) with respect to morphology, gene expression profiles, and electrophysiological properties. This approach was further applied to generate hciNs from familial Alzheimer's disease patients. Taken together, our transgene-free and chemical-only approach for direct reprogramming of human fibroblasts into neurons provides an alternative strategy for modeling neurological diseases and for regenerative medicine. PMID:26253202

  19. Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts

    PubMed Central

    Kim, Jonghun; Kim, Kee-Pyo; Lim, Kyung Tae; Lee, Seung Chan; Yoon, Juyong; Song, Guangqi; Hwang, Seon In; Schöler, Hans R.; Cantz, Tobias; Han, Dong Wook

    2015-01-01

    The ability to generate integration-free induced hepatocyte-like cells (iHeps) from somatic fibroblasts has the potential to advance their clinical application. Here, we have generated integration-free, functional, and expandable iHeps from mouse somatic fibroblasts. To elicit this direct conversion, we took advantage of an oriP/EBNA1-based episomal system to deliver a set of transcription factors, Gata4, Hnf1a, and Foxa3, to the fibroblasts. The established iHeps exhibit similar morphology, marker expression, and functional properties to primary hepatocytes. Furthermore, integration-free iHeps prolong the survival of fumarylacetoacetate-hydrolase-deficient (Fah−/−) mice after cell transplantation. Our study provides a novel concept for generating functional and expandable iHeps using a non-viral, non-integrating, plasmid-based system that could facilitate their pharmaceutical and biomedical application. PMID:26503743

  20. Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.

    PubMed

    Kim, Jonghun; Kim, Kee-Pyo; Lim, Kyung Tae; Lee, Seung Chan; Yoon, Juyong; Song, Guangqi; Hwang, Seon In; Schöler, Hans R; Cantz, Tobias; Han, Dong Wook

    2015-01-01

    The ability to generate integration-free induced hepatocyte-like cells (iHeps) from somatic fibroblasts has the potential to advance their clinical application. Here, we have generated integration-free, functional, and expandable iHeps from mouse somatic fibroblasts. To elicit this direct conversion, we took advantage of an oriP/EBNA1-based episomal system to deliver a set of transcription factors, Gata4, Hnf1a, and Foxa3, to the fibroblasts. The established iHeps exhibit similar morphology, marker expression, and functional properties to primary hepatocytes. Furthermore, integration-free iHeps prolong the survival of fumarylacetoacetate-hydrolase-deficient (Fah(-/-)) mice after cell transplantation. Our study provides a novel concept for generating functional and expandable iHeps using a non-viral, non-integrating, plasmid-based system that could facilitate their pharmaceutical and biomedical application. PMID:26503743

  1. Activation of fibroblast and papilla cells by glycolipid biosurfactants, mannosylerythritol lipids.

    PubMed

    Morita, Tomotake; Kitagawa, Masaru; Yamamoto, Shuhei; Suzuki, Michiko; Sogabe, Atsushi; Imura, Tomohiro; Fukuoka, Tokuma; Kitamoto, Dai

    2010-01-01

    Mannosylerythritol lipids (MELs), the extracellular glycolipids produced from feedstock by yeasts belonging to the genus Pseudozyma, are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics, the cell activating property of MELs was investigated using cultured fibroblast and papilla cells, and a three-dimensional cultured human skin model. The di-acetylated MEL (MEL-A) produced from soybean oil significantly increased the viability of the fibroblast cells over 150% compared with that of control cells. On the other hand, no cell activation was observed by the treatment with MEL-A produced from olive oil. The mono-acetylated MEL (MEL-B) hardly increased the cell viability. The viability of the fibroblast cells decreased with the addition of more than 1 microg/L of MELs, whereas the cultured human skin cells showed high viability with 5 microg/L of MELs. Interestingly, the papilla cells were dramatically activated with 0.001 microg/L of MEL-A produced from soybean oil: the cell viability reached at 150% compared with that of control cells. Consequently, the present MEL-A produced from soybean oil should have a potential as a new hair growth agent stimulating the papilla cells. PMID:20625237

  2. Interferon-dependent induction of mRNA for the major histocompatibility antigens in human fibroblasts and lymphoblastoid cells.

    PubMed Central

    Fellous, M; Nir, U; Wallach, D; Merlin, G; Rubinstein, M; Revel, M

    1982-01-01

    In human cells treated with interferons, there is an increase in the amount of HLA-A,B,C and beta 2-microglobulin exposed on the cell surface. We have used a cloned HLA-A,B,C cDNA probe to demonstrate by molecular hybridization that this effect of interferon is preceded by a large increase in the amount of HLA mRNA in the cell. This effect was found in five different human cell lines, with purified leukocyte and fibroblast interferons. The increase in HLA mRNA is comparable in its kinetics and dose-response to the induction of (2'-5') oligo(A) synthetase mRNA by interferons. Therefore, interferons seem to activate at least two cellular genes which have different biochemical functions. Images PMID:6179076

  3. Lineage Reprogramming of Fibroblasts into Proliferative Induced Cardiac Progenitor Cells by Defined Factors.

    PubMed

    Lalit, Pratik A; Salick, Max R; Nelson, Daryl O; Squirrell, Jayne M; Shafer, Christina M; Patel, Neel G; Saeed, Imaan; Schmuck, Eric G; Markandeya, Yogananda S; Wong, Rachel; Lea, Martin R; Eliceiri, Kevin W; Hacker, Timothy A; Crone, Wendy C; Kyba, Michael; Garry, Daniel J; Stewart, Ron; Thomson, James A; Downs, Karen M; Lyons, Gary E; Kamp, Timothy J

    2016-03-01

    Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy. PMID:26877223

  4. Cancer-associated fibroblasts up-regulate CCL2, CCL26, IL6 and LOXL2 genes related to promotion of cancer progression in hepatocellular carcinoma cells.

    PubMed

    Lin, Zu-Yau; Chuang, Yen-Hwang; Chuang, Wan-Long

    2012-10-01

    Impact of different cancer-associated fibroblast (CAF) cell lines on proliferation, migration, invasion and differential expressions of genes in different hepatocellular carcinoma (HCC) cell lines was investigated. Two human CAF cell lines (F26/KMUH, F28/KMUH) and two human HCC cell lines (HCC24/KMUH, HCC38/KMUH) were studied. Influence of F28/KMUH cells on expressions of genes in HCC38/KMUH cells was detected by microarray to select genes for further analysis. Both CAF cell lines promoted proliferation (all P<0.05), migration (all P<0.05) and Matrigel invasion (all P<0.0001) of both HCC cell lines. F26/KMUH cells showed stronger promoted effects on, firstly, proliferation of HCC24/KMUH cells (P=0.0064) and, secondly, migration of both HCC cell lines than F28/KMUH cells did (all P<0.002). Ten up-regulated genes (APLN, CCL2, CCL26, CXCR4, IL6, MUC1, LOXL2, PDGFA, PGK1, VEGFA) related to proliferation, migration, invasion and angiogenesis of HCC detected by microarray were selected for quantitative reverse transcriptase-polymerase chain reaction analysis. Both CAF cell lines had same tendency of effects on differential expressions of genes in same HCC cell line, but expressions of genes between different HCC cell lines were not consistent. Only CCL2, CCL26, IL6 and LOXL2 genes were consistently up-regulated in both HCC cell lines. In conclusion, the effects of CAFs to promote proliferation, migration and invasion of HCC cells are influenced by the characteristics of both CAFs and HCC cells. Up-regulations of CCL2, CCL26, IL6 and LOXL2 genes in cancer cells are part of the common effects of CAFs on HCC cells. PMID:22739041

  5. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    NASA Astrophysics Data System (ADS)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  6. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis

    PubMed Central

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-01-01

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment. PMID:26576741

  7. Evaluation of Glucose Uptake in Normal and Cancer Cell Lines by Positron Emission Tomography.

    PubMed

    Maddalena, Francesca; Lettini, Giacomo; Gallicchio, Rosj; Sisinni, Lorenza; Simeon, Vittorio; Nardelli, Anna; Venetucci, Angela Assunta; Storto, Giovanni; Landriscina, Matteo

    2015-01-01

    To date, there is no definitive demonstration of the utility of positron emission tomography (PET) in studying glucose metabolism in cultured cell lines. Thus, this study was designed to compare PET to more standardized methods for the quantitative assessment of glucose uptake in nontransformed and transformed living cells and to validate PET for metabolic studies in vitro. Human colon and breast carcinoma cell lines and mouse embryo fibroblasts were evaluated for [(18)F]fluorodeoxyglucose ([(18)F]FDG) uptake by PET and autoradiography and 2-deoxyglucose (2-DG) incorporation by colorimetric assay and analyzed for the radiotoxic effects of [(18)F]FDG and the expression levels of glucose transporters. Indeed, [(18)F]FDG incorporation on PET was comparable to [(18)F]FDG uptake by autoradiography and 2-DG incorporation by colorimetric assay, although radiotracer-based methods exhibited more pronounced differences between individual cell lines. As expected, these data correlated with glucose transporters 1 to 4 and hexokinase II expression in tumor cell lines and mouse fibroblasts. Notably, [(18)F]FDG incorporation resulted in low apoptotic rates, with fibroblasts being slightly more sensitive to radiotracer-induced cell death. The quantitative analysis of [(18)F]FDG uptake in living cells by PET represents a valuable and reproducible method to study tumor cell metabolism in vitro, being representative of the differences in the molecular profile of normal and tumor cell lines. PMID:26461458

  8. MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

    PubMed

    Liu, Haijun; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Wang, Xingang; Zhang, Wei; Liao, Hong; Yao, Honghong; Chao, Jie

    2016-01-15

    Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis. PMID:26608530

  9. Manufactured silver nanoparticles of different sizes induced DNA strand breaks and oxidative DNA damage in hepatoma and leukaemia cells and in dermal and pulmonary fibroblasts.

    PubMed

    Ávalos, A; Haza, A I; Morales, P

    2015-01-01

    Many classes of silver nanoparticles (AgNPs) have been synthesized and widely applied, but no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. Therefore, the purpose of this study was to compare the potential genotoxic effects (DNA strand breaks and oxidative DNA damage) of 4.7 nm coated and 42 nm uncoated AgNPs, using the comet assay, in four relevant human cell lines (hepatoma, leukaemia, and dermal and pulmonary fibroblasts) in order to understand the impact of such nanomaterials on cellular DNA. The results indicated that in all cell lines tested, 4.7 nm coated (0.1-1.6 μg ml⁻¹) and 42 nm uncoated (0.1-6.7 μg ml⁻¹) AgNPs increased DNA strand breaks in a dose- and size-dependent manner following 24 h treatment, the smaller AgNPs being more genotoxic. Human pulmonary fibroblasts showed the highest sensitivity to the AgNPs. A modified comet assay using endonuclease III and formamidopyrimidine- DNA glycosylase restriction enzymes showed that in tumoral and normal human dermal fibroblasts, pyrimidines and purines were oxidatively damaged by both AgNPs, but the damage was not size-dependent. However, in human pulmonary fibroblasts, no oxidative damage was observed after treatment with 42 nm AgNPs. In conclusion, both AgNP sizes induced DNA damage in human cells, and this damage could be related to oxidative stress. PMID:25958309

  10. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

    PubMed Central

    Kemény, Lajos V.; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

    2016-01-01

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells’ nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma–stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments. PMID:27271591