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Sample records for human fibroblast cells

  1. Influence of three laser wavelengths on human fibroblasts cell culture.

    PubMed

    Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Campian, Radu; Crisan, Liana; Baciut, Grigore

    2013-02-01

    Although experimental studies in vitro and vivo have been numerous, the effect of laser wavelength irradiation on human fibroblast cell culture is poorly understood. This emphasizes the need of additional cellular and molecular research into laser influence with low energy and power. The aim of this study was to assess the influence of three different laser wavelengths on the human skin fibroblasts cell culture. We wanted to evaluate if near infrared lasers had any influence in healing of wounds by stimulating mitochondrial activity of fibroblasts. The cells were irradiated using 830-, 980- and 2,940-nm laser wavelengths. The irradiated cells were incubated and their mitochondrial activity was assessed by the MTT assay at 24, 48 and 72 h. Simultaneously, an apoptosis assay was assessed on the irradiated fibroblasts. It can be concluded that laser light of the near-infrared region (830 and 980 nm) influences fibroblasts mitochondrial activity compared to the 2,940-nm wavelength which produces apoptosis.

  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. Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

    PubMed

    Vartio, T; Kaelin, H; Vaheri, A

    1978-10-23

    The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.

  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. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells.

    PubMed

    Miyoshi, Keiko; Horiguchi, Taigo; Tanimura, Ayako; Hagita, Hiroko; Noma, Takafumi

    2015-01-01

    Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  6. Characterization of Mesenchymal Stem Cells from Human Vocal Fold Fibroblasts

    PubMed Central

    Hanson, Summer; Kim, Jaehyup; Quinchia Johnson, Beatriz H.; Bradley, Bridget; Breunig, Melissa; Hematti, Peiman; Thibeault, Susan L.

    2009-01-01

    Objective/Hypothesis Mesenchymal stem cells (MSCs) originally isolated from bone marrow, are fibroblast-looking cells that are now assumed to be present in the stromal component of many tissues. MSCs are characterized by a certain set of criteria including their growth culture characteristics, a combination of cell surface markers, and the ability to differentiate along multiple mesenchymal tissue lineages. We hypothesized that human vocal fold fibroblasts (hVFF) isolated from the lamina propria meet the criteria established to define MSCs and are functionally similar to MSCs derived from BM and adipose tissue. Study Design In vitro study Methods HVFF were previously derived from human vocal fold tissues. MSCs were derived from adipose tissue (AT), and BM of healthy donors, based on their attachment to culture dishes and their morphology, and expanded in culture. Cells were analyzed for standard cell surface markers identified on BM-derived MSCs as well as the ability to differentiate into cells of mesenchymal lineage, i.e. fat, bone and cartilage. We investigated the immunophenotype of these cells before and after interferon-γ (INF- γ) stimulation. Results HVFF displayed cell surface markers and multipotent differentiation capacity characteristic of MSCs. Furthermore, these cells exhibited similar patterns of expression of HLA and co-stimulatory molecules, after stimulation with INF- γ compared to MSCs derived from BM and AT. Conclusions Based on our findings hVFF derived from lamina propria have the same cell surface markers, immunophenotypic characteristics, and differentiation potential as BM- and AT-derived MSCs. We propose VF fibroblasts are MSCs resident in the vocal fold lamina propria. PMID:20131365

  7. Altered pattern of replication of human chromosomes in a human fibroblast-mouse cell hybrid.

    PubMed Central

    Farber, R A; Davidson, R L

    1978-01-01

    The pattern of terminal replication of the human chromosomes in a clone of hybrids between diploid human fibroblasts and mouse cells was analyzed by autoradiography. An average of 10 human chromosomes was present in the hybrid cells. Several of these chromosomes were found to terminate replication in a different order from the same chromosomes in the parental human fibroblasts. Chromosomes 4 and 5 completed replication later in the hybrid than in the fibroblasts (relative to the other human chromosomes). In contrast, chromosomes 7, 12, and 15 completed replication earlier in the hybrid than in the fibroblasts. These results suggest that the sequence of terminal chromosome replication in human fibroblasts is not irreversibly programmed into each chromosome. Images PMID:274734

  8. 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

  9. Genomic instability of gold nanoparticle treated human lung fibroblast cells.

    PubMed

    Li, Jasmine J; Lo, Soo-Ling; Ng, Cheng-Teng; Gurung, Resham Lal; Hartono, Deny; Hande, Manoor Prakash; Ong, Choon-Nam; Bay, Boon-Huat; Yung, Lin-Yue Lanry

    2011-08-01

    Gold nanoparticles (AuNPs) are one of the most versatile and widely researched materials for novel biomedical applications. However, the current knowledge in their toxicological profile is still incomplete and many on-going investigations aim to understand the potential adverse effects in human body. Here, we employed two dimensional gel electrophoresis to perform a comparative proteomic analysis of AuNP treated MRC-5 lung fibroblast cells. In our findings, we identified 16 proteins that were differentially expressed in MRC-5 lung fibroblasts following exposure to AuNPs. Their expression levels were also verified by western blotting and real time RT-PCR analysis. Of interest was the difference in the oxidative stress related proteins (NADH ubiquinone oxidoreductase (NDUFS1), protein disulfide isomerase associate 3 (PDIA3), heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and thioredoxin-like protein 1 (TXNL1)) as well as proteins associated with cell cycle regulation, cytoskeleton and DNA repair (heterogeneous nuclear ribonucleus protein C1/C2 (hnRNP C1/C2) and Secernin-1 (SCN1)). This finding is consistent with the genotoxicity observed in the AuNP treated lung fibroblasts. These results suggest that AuNP treatment can induce oxidative stress-mediated genomic instability.

  10. 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.

  11. 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

  12. 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

  13. Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

    PubMed

    Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

    2013-12-01

    Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture. PMID:23832306

  14. Basic fibroblast growth factor support of human embryonic stem cell self-renewal.

    PubMed

    Levenstein, Mark E; Ludwig, Tenneille E; Xu, Ren-He; Llanas, Rachel A; VanDenHeuvel-Kramer, Kaitlyn; Manning, Daisy; Thomson, James A

    2006-03-01

    Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.

  15. In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells.

    PubMed

    Seker, Sükran; Elçin, A Eser; Yumak, Tuğrul; Sınağ, Ali; Elçin, Y Murat

    2014-12-01

    The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7-8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun-Emmet-Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 μg/mL for 6, 24 and 48h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50-100 μg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.

  16. Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Improves Senescent Human Dermal Fibroblasts.

    PubMed

    Jung, Ji-Yong; Shim, Joong Hyun; Choi, Hyun; Lee, Tae Ryong; Shin, Dong Wook

    2015-08-13

    Adult skin stem cells are recognized as potential therapeutics to rejuvenate aged skin. We previously demonstrated that human dermal stem/progenitor cells (hDSPCs) with multipotent capacity could be enriched from human dermal fibroblasts using collagen type IV. However, the effects of hDSPCs on cellular senescence remain to be elucidated. In the present study, we investigated whether conditioned medium (CM) collected from hDSPC cultures (hDSPC-CM) exhibits beneficial effects on senescent fibroblasts. We found that hDSPC-CM promoted proliferation and decreased the expression level of senescence-associated β-galactosidase in senescent fibroblasts. In addition, p53 phosphorylation and p21 expression were significantly reduced in senescent fibroblasts treated with hDSPC-CM. hDSPC-CM restored the expression levels of collagen type I, collagen type III, and tissue inhibitor of metalloproteinase, and antagonized the increase of matrix metalloproteinase 1 expression. Finally, we demonstrated that hDSPC-CM significantly reduced reactive oxygen species levels by specifically up-regulating the expression level of superoxide dismutase 2. Taken together, these data suggest that hDSPC-CM can be applied as a potential therapeutic agent for improving human aged skin.

  17. 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

  18. 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

  19. Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts

    PubMed Central

    Maucksch, C; Firmin, E; Butler-Munro, C; Montgomery, JM; Dottori, M; Connor, B

    2012-01-01

    Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP) colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP)-expressing astrocytes. This study represents a novel virusfree approach for direct reprogramming of human fibroblasts to a neural precursor fate. PMID:24693194

  20. Human lung fibroblasts express interleukin-6 in response to signaling after mast cell contact.

    PubMed

    Fitzgerald, S Matthew; Lee, Steven A; Hall, H Kenton; Chi, David S; Krishnaswamy, Guha

    2004-04-01

    Asthma is a chronic inflammatory disease of the airways. Mast cell-derived cytokines may mediate both airway inflammation and remodeling. It has also been shown that fibroblasts can be the source of proinflammatory cytokines. In the human airways, mast cell-fibroblast interactions may have pivotal effects on modulating inflammation. To study this further, we cocultured normal human lung fibroblasts (NHLF) with a human mast cell line (HMC-1) and assayed for production of interleukin (IL)-6, an important proinflammatory cytokine. When cultured together, NHLF/HMC-1 contact induced IL-6 secretion. Separation of HMC-1 and NHLF cells by a porous membrane inhibited this induction. HMC-1-derived cellular membranes caused an increase in IL-6 production in NHLF. Activation of p38 MAPK was also seen in cocultures by Western blot, whereas IL-6 production in cocultures was significantly inhibited by the p38 inhibitor SB203580. IL-6 production in cocultures was minimally inhibited by a chemical inhibitor of nuclear factor-kappaB (Bay11), indicating that nuclear factor-kappaB may have a minimal role in signaling IL-6 production in mast cell/fibroblasts cocultures. Blockade of inter-cellular adhesion molecule-1, tumor necrosis factor-RI, and surface IL-1beta with neutralizing antibodies failed to significantly decrease IL-6 production in our coculture, indicating that other receptor-ligand associations may be responsible for this activation. These novel studies reveal the importance of cell-cell interactions in the complex milieu of airway inflammation.

  1. Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells

    SciTech Connect

    Wilhelm, S.M.; Collier, I.E.; Kronberger, A.; Eisen, A.Z.; Marmer, B.L.; Grant, G.A.; Bauer, E.A.; Goldberg, G.I.

    1987-10-01

    The authors have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO/sub 4//PAGE of 60 and 57 kDa. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively. Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. The data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.

  2. 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

  3. Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

    PubMed

    Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

  4. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

  5. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

  6. Implanted neonatal human dermal fibroblasts influence the recruitment of endothelial cells in mice

    PubMed Central

    Guerreiro, Susana G.; Brochhausen, Christoph; Negrão, Rita; Barbosa, Mário A.; Unger, Ronald E.; Kirkpatrick, C. James; Soares, Raquel; Granja, Pedro L.

    2012-01-01

    The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The vessels exhibited higher levels of hemoglobin, compared with the control matrix, implanted without fibroblasts, in which no vessel formation could be observed. No significant differences were detected in systemic inflammation. The presence of vessels originated from the host vasculature suggested that host vascular response was involved, which constitutes a fundamental aspect in the process of neovascularization. Fibroblasts implanted within matrigel increased the presence of endothelial cells with positive staining for CD31 and for CD34 and the production of collagen influencing the angiogenic process and promoting the formation of microvessels. New strategies in tissue engineering could be delineated with improved angiogenesis using neonatal fibroblasts. PMID:23507785

  7. Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

    PubMed Central

    Towler, James C.; Ebrahimi, Bahram; Lane, Brian; Davison, Andrew J.

    2012-01-01

    Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed. PMID:22258857

  8. 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.

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

    PubMed

    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

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

    PubMed

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

    2015-11-18

    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.

  11. Cu,Zn Superoxide Dismutase is a Peroxisomal Enzyme in Human Fibroblast and Hepatoma Cells

    NASA Astrophysics Data System (ADS)

    Keller, Gilbert-Andre; Warner, Thomas G.; Steimer, Kathelyn S.; Hallewell, Robert A.

    1991-08-01

    The intracellular localization of Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been examined by immunofluorescence using four monoclonal anti-Cu,Zn superoxide dismutase antibodies raised against a recombinant human Cu,Zn superoxide dismutase derivative produced and purified from Escherichia coli. Colocalization with catalase, a peroxisomal matrix enzyme, was used to demonstrate the peroxisomal localization of Cu,Zn superoxide dismutase in human fibroblasts and hepatoma cells. In the fibroblasts of Zellweger syndrome patients, the enzyme is not transported to the peroxisomal ghosts but, like catalase, remains in the cytoplasm. In addition, immunocryoelectron microscopy of yeast cells expressing human Cu,Zn superoxide dismutase showed that the enzyme is translocated to the peroxisomes.

  12. Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts.

    PubMed

    Ohshima, Susumu; Seyama, Atsushi

    2012-09-01

    Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells. PMID:22696268

  13. Induction of Stem Cell Gene Expression in Adult Human Fibroblasts without Transgenes

    PubMed Central

    Ambady, Sakthikumar; Holmes, William F.; Vilner, Lucy; Kole, Denis; Kashpur, Olga; Huntress, Victoria; Vojtic, Ina; Whitton, Holly; Dominko, Tanja

    2009-01-01

    Abstract Reprogramming of differentiated somatic cells into induced pluripotent stem (iPS) cells has potential for derivation of patient-specific cells for therapy as well as for development of models with which to study disease progression. Derivation of iPS cells from human somatic cells has been achieved by viral transduction of human fibroblasts with early developmental genes. Because forced expression of these genes by viral transduction results in transgene integration with unknown and unpredictable potential mutagenic effects, identification of cell culture conditions that can induce endogenous expression of these genes is desirable. Here we show that primary adult human fibroblasts have basal expression of mRNA for OCT4, SOX2, and NANOG. However, translation of these messages into detectable proteins and their subcellular localization depends on cell culture conditions. Manipulation of oxygen concentration and FGF2 supplementation can modulate expression of some pluripotency related genes at the transcriptional, translational, and cellular localization level. Changing cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2, and NANOG, and translocation of these transcription factors to the cell nucleus. We also show that culture conditions affect the in vitro lifespan of dermal fibroblasts, nearly doubling the number of population doublings before the cells reach replicative senescence. Our results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation, but this short-term induction may not be sufficient for acquisition of true pluripotency. Further investigation of the factors involved in inducing this response could lead to discovery of defined culture conditions capable of altering cell fate in vitro. This would alleviate the need for forced expression by transgenesis, thus eliminating the risk of

  14. Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts.

    PubMed

    Easley, Charles A; Miki, Toshio; Castro, Carlos A; Ozolek, John A; Minervini, Crescenzio F; Ben-Yehudah, Ahmi; Schatten, Gerald P

    2012-06-01

    Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. PMID:22686477

  15. Fibroblasts induce heparin synthesis in chondroitin sulfate E containing human bone marrow-derived mast cells

    SciTech Connect

    Gilead, L.; Bibi, O.; Razin, E. )

    1990-09-15

    Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above-mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers.

  16. Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

    SciTech Connect

    Bikfalvi, A.; Dupuy, E.; Inyang, A.L.; Tobelem, G. ); Fayein, N.; Courtois, Y. ); Leseche, G. )

    1989-03-01

    The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells. The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely {sup 125}I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound {sup 125}I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At this temperature, degradation of the internalized ligand was followed after 1 hour by the appearance of three major bands of 15,000 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

  17. Investigation of the phototoxic effect of ZnO nanorods on fibroblasts and melanoma human cells

    NASA Astrophysics Data System (ADS)

    Kishwar, S.; Siddique, M.; Israr-Qadir, M.; Nur, O.; Willander, M.; Öllinger, K.

    2014-11-01

    Photocytotoxic effects of as-grown and zinc oxide (ZnO) nanorods coated with 5-aminolevulinic acid (ALA) have been studied on human cells, i.e. melanoma and foreskin fibroblast, under dark and ultraviolet light exposures. Zinc oxide nanorods have been grown on the very sharp tip (diameter = 700 nm) of borosilicate glass pipettes and then were coated by the photosensitizer for targeted investigations inside human cells. The coated glass pipette’s tip with photosensitizer has been inserted inside the cells with the help of a micro-manipulator and irradiated through ultraviolet light (UVA), which reduces the membrane potential of the mitochondria leading to cell death. Cell viability loss has been detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay when exposed to the dissolved ZnO nanorods and the production of the reactive oxygen species (ROS) has been detected along with the enhanced cytotoxic effect under UVA irradiation. Additionally, the influence of the lipid soluble antioxidant vitamin E and water-soluble N-acetyl-cysteine toward the enhancement or reduction of the toxicity has been investigated. A comparative analysis of the toxic nature of ZnO nanorods has been drawn between normal human fibroblast and melanoma cells, which can be favorable for understanding the clinical setting for killing tumor cells.

  18. Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

    PubMed Central

    Bisson, Francis; Rochefort, Éloise; Lavoie, Amélie; Larouche, Danielle; Zaniolo, Karine; Simard-Bisson, Carolyne; Damour, Odile; Auger, François A.; Guérin, Sylvain L.; Germain, Lucie

    2013-01-01

    A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes. PMID:23443166

  19. In vitro generation of pancreatic endocrine cells from human adult fibroblast-like limbal stem cells.

    PubMed

    Criscimanna, Angela; Zito, Giovanni; Taddeo, Annalisa; Richiusa, Pierina; Pitrone, Maria; Morreale, Daniele; Lodato, Gaetano; Pizzolanti, Giuseppe; Citarrella, Roberto; Galluzzo, Aldo; Giordano, Carla

    2012-01-01

    Stem cells might provide unlimited supply of transplantable cells for β-cell replacement therapy in diabetes. The human limbus is a highly specialized region hosting a well-recognized population of epithelial stem cells, which sustain the continuous renewal of the cornea, and the recently identified stromal fibroblast-like stem cells (f-LSCs), with apparent broader plasticity. However, the lack of specific molecular markers for the identification of the multipotent limbal subpopulation has so far limited the investigation of their differentiation potential. In this study we show that the human limbus contains uncommitted cells that could be potentially harnessed for the treatment of diabetes. Fourteen limbal biopsies were obtained from patients undergoing surgery for ocular diseases not involving the conjunctiva or corneal surface. We identified a subpopulation of f-LSCs characterized by robust proliferative capacity, expressing several pluripotent stem cell markers and exhibiting self-renewal ability. We then demonstrated the potential of f-LSCs to differentiate in vitro into functional insulin-secreting cells by developing a four-step differentiation protocol that efficiently directed f-LSCs towards the pancreatic endocrine cell fate. The expression of specific endodermal, pancreatic, islet, and β-cell markers, as well as functional properties of f-LSC-derived insulin-producing cells, were evaluated during differentiation. With our stage-specific approach, up to 77% of f-LSCs eventually differentiated into cells expressing insulin (also assessed as C-peptide) and exhibited phenotypic features of mature β-cells, such as expression of critical transcription factors and presence of secretory granules. Although insulin content was about 160-fold lower than what observed in adult islets, differentiated cells processed ∼98% of their proinsulin content, similar to mature β-cells. Moreover, they responded in vitro in a regulated manner to multiple secretory stimuli

  20. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.

    PubMed

    Nakagawa, Masato; Koyanagi, Michiyo; Tanabe, Koji; Takahashi, Kazutoshi; Ichisaka, Tomoko; Aoi, Takashi; Okita, Keisuke; Mochiduki, Yuji; Takizawa, Nanako; Yamanaka, Shinya

    2008-01-01

    Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc(-) iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.

  1. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    PubMed

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

  2. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    PubMed

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated. PMID:26895068

  3. Functional melanocytes are readily reprogrammable from multilineage-differentiating stress-enduring (muse) cells, distinct stem cells in human fibroblasts.

    PubMed

    Tsuchiyama, Kenichiro; Wakao, Shohei; Kuroda, Yasumasa; Ogura, Fumitaka; Nojima, Makoto; Sawaya, Natsue; Yamasaki, Kenshi; Aiba, Setsuya; Dezawa, Mari

    2013-10-01

    The induction of melanocytes from easily accessible stem cells has attracted attention for the treatment of melanocyte dysfunctions. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily reprogrammed into functional melanocytes, whereas the remainder of the fibroblasts do not contribute to melanocyte differentiation. Muse cells can be isolated as cells positive for stage-specific embryonic antigen-3, a marker for undifferentiated human embryonic stem cells, and differentiate into cells representative of all three germ layers from a single cell, while also being nontumorigenic. The use of certain combinations of factors induces Muse cells to express melanocyte markers such as tyrosinase and microphthalmia-associated transcription factor and to show positivity for the 3,4-dihydroxy-L-phenylalanine reaction. When Muse cell-derived melanocytes were incorporated into three-dimensional (3D) cultured skin models, they localized themselves in the basal layer of the epidermis and produced melanin in the same manner as authentic melanocytes. They also maintained their melanin production even after the 3D cultured skin was transplanted to immunodeficient mice. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by using Muse cells, thereby contributing to autologous transplantation for melanocyte dysfunctions, such as vitiligo.

  4. Alterations in cell migration and cell viability of wounded human skin fibroblasts following visible red light exposure

    NASA Astrophysics Data System (ADS)

    Prabhu, Vijendra; Rao, Bola Sadashiva S.; Mahato, Krishna Kishore

    2014-02-01

    The present study intended to examine the effect of visible red light on structural and cellular parameters on wounded skin fibroblast cells. To achieve the stated objective, uniform scratch was created on confluent monolayered human skin fibroblast cells, and were exposed to single dose of He-Ne laser (15 mm spot, 6.6808 mWcm-2) at 1, 2, 3, 4, 5, 6 and 7 Jcm-2 in the presence and absence of 10% fetal bovine serum (FBS). Beam profile measurements of the expanded laser beam were conducted to ensure the beam uniformity. The influence of laser dose on the change in temperature was recorded using sensitive temperature probe. Additionally, following laser exposure cell migration and cell survival were documented at different time intervals on wounded human skin fibroblast cells grown in vitro. Beam profile measurements indicated more or less uniform power distribution over the whole beam area. Temperature monitoring of sham irradiated control and laser treatment groups displayed negligible temperature change indicating the absence of thermal effect at the tested laser doses. In the absence of 10% FBS, single exposure of different laser doses failed to produce any significant effects on cell migration or cell survival. However, in the presence of serum single exposure of 5 J/cm2 on wounded skin fibroblasts significantly enhanced the cell migration (P<0.05) compared to the other tested doses (1, 2, 3, 4, 6 and 7 J/cm2) and sham irradiated controls. In conclusion, the LLLT acts by improving cell migration and cell proliferation to produce measurable changes in wounded fibroblast cells.

  5. 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

  6. Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

    PubMed

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

    2012-01-01

    Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

  7. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  8. Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

    PubMed

    Moruzzi, Noah; Del Sole, Marianna; Fato, Romana; Gerdes, Jantje M; Berggren, Per-Olof; Bergamini, Christian; Brismar, Kerstin

    2014-08-01

    High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.

  9. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in space

    NASA Astrophysics Data System (ADS)

    Wu, Honglu; Story, Michael; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao

    2016-07-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NFkB and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for αa-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  10. Transient Gene and MicroRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-01-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NF(kappa)B and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for alpha-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  11. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in spaceflight.

    PubMed

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-06-01

    Microgravity, or an altered gravity environment different from the 1 g of the Earth, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies that have been conducted in space or by using simulated microgravity on the ground have focused on the growth or differentiation of these cells. It has not been specifically addressed whether nonproliferating cultured cells will sense the presence of microgravity in space. In an experiment conducted onboard the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 d, respectively, to investigate changes in gene and microRNA (miRNA) expression profiles in these cells. Results of the experiment showed that on d 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67(+) cells. Gene and miRNA expression data indicated activation of NF-κB and other growth-related pathways that involve hepatocyte growth factor and VEGF as well as the down-regulation of the Let-7 miRNA family. On d 14, when the cells were mostly nonproliferating, the gene and miRNA expression profile of the flight sample was indistinguishable from that of the ground sample. Comparison of gene and miRNA expressions in the d 3 samples, with respect to d 14, revealed that most of the changes observed on d 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for α-tubulin and fibronectin showed no difference between the flown and ground samples. Taken together, our study suggests that in true nondividing human fibroblast cells in culture, microgravity experienced in space has little effect on gene and miRNA expression profiles.-Zhang, Y., Lu, T., Wong, M., Wang, X., Stodieck, L., Karouia, F., Story, M., Wu, H. Transient gene and microRNA expression profile changes of

  12. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in spaceflight.

    PubMed

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-06-01

    Microgravity, or an altered gravity environment different from the 1 g of the Earth, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies that have been conducted in space or by using simulated microgravity on the ground have focused on the growth or differentiation of these cells. It has not been specifically addressed whether nonproliferating cultured cells will sense the presence of microgravity in space. In an experiment conducted onboard the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 d, respectively, to investigate changes in gene and microRNA (miRNA) expression profiles in these cells. Results of the experiment showed that on d 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67(+) cells. Gene and miRNA expression data indicated activation of NF-κB and other growth-related pathways that involve hepatocyte growth factor and VEGF as well as the down-regulation of the Let-7 miRNA family. On d 14, when the cells were mostly nonproliferating, the gene and miRNA expression profile of the flight sample was indistinguishable from that of the ground sample. Comparison of gene and miRNA expressions in the d 3 samples, with respect to d 14, revealed that most of the changes observed on d 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for α-tubulin and fibronectin showed no difference between the flown and ground samples. Taken together, our study suggests that in true nondividing human fibroblast cells in culture, microgravity experienced in space has little effect on gene and miRNA expression profiles.-Zhang, Y., Lu, T., Wong, M., Wang, X., Stodieck, L., Karouia, F., Story, M., Wu, H. Transient gene and microRNA expression profile changes of

  13. 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.

  14. Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

    PubMed Central

    Zhang, Ju; Zhang, Can-Wei; Du, Li-Qun; Wu, Xin-Yi

    2016-01-01

    AIM To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4′, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo. PMID:26949602

  15. Immunomodulatory Effects of Bee Venom in Human Synovial Fibroblast Cell Line

    PubMed Central

    Mohammadi, Ebrahim; Vatanpour, Hossein; H Shirazi, Farshad

    2015-01-01

    As in Iranian traditional medicine, bee venom (BV) is a promising treatment for the rheumatoid arthritis (RA) which is considered as a problematic human chronic inflammatory disease in the present time. Smoking is considered to be a major risk factor in RA onset and severity. The main aim of this study is to investigate the effects of BV on cigarette smoke-induced inflammatory response in fibroblast-like synoviocytes (FLS). Cytotoxicity of cigarette smoke condensate (CSC) and bee venom were determined by the tetrazolium (MTT) method in cultured synovial fibroblastes. The expression of interleukin-1β and sirtuin1 mRNA were analyzed by SYBR green real-time quantitative PCR. Differences between the mean values of treated and untreated groups were assessed by student t-test. Based on MTT assay, CSC and BV did not exert any significant cytotoxic effects up to 40 µg/mL and 10 µg/mL, respectively. Our results showed that interleukin-1β mRNA level was significantly up-regulated by CSC treatments in LPS-stimulated synoviocytes in a dose-dependent manner. Conversely, the expressions of IL-1β and Sirt1 were up-regulated even in lower concentrations of BV and attenuated at higher concentrations. Also, BV attenuated the CSC-induced and LPS-induced inflammatory responses in synovial fibroblasts. Our results support the epidemiological studies indicating pro-inflammatory effects of CSC and anti-inflammatory effects of BV on FLS cell line. PMID:25561937

  16. Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells.

    PubMed

    Liu, Xinjian; Li, Fang; Stubblefield, Elizabeth A; Blanchard, Barbara; Richards, Toni L; Larson, Gaynor A; He, Yujun; Huang, Qian; Tan, Aik-Choon; Zhang, Dabing; Benke, Timothy A; Sladek, John R; Zahniser, Nancy R; Li, Chuan-Yuan

    2012-02-01

    Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD. PMID:22105488

  17. Locomotory invasion of human cervical epithelium and avian fibroblasts by HeLa cells in vitro.

    PubMed

    Stephenson, E M

    1982-10-01

    The locomotory invasive ability of HeLa cells was tested against: (a) embryonic chick heart fibroblasts (CHF); and (b) normal epithelial cells from human cervix (HCE) in explant confrontations. Data for analyses were obtained from replicate cultures fixed 24 h after junction and from 24-h time-lapse films. The mean invasion index for HeLa versus CHF did not indicate significant obstruction but analyses of hourly radial advance and orientation frequencies showed that obstruction eventually developed as postjunctional incubation time increased. Early contacts between HeLa and CHF demonstrated non-reciprocity of type I contact inhibition of locomotion by the tumour cells, which continued moving in their original direction to underlap contact-inhibited fibroblasts and eventually to occupy spaces vacated by them. When CHF population density increased and free space diminished, HeLa cells displayed directional and probably substrate-dependent contact inhibition. The high invasion index of HeLa versus HCE was largely due to occupation of previous HCE territory by tumour cells and only occasionally to actual infiltration of the epithelial sheet. After contact with HeLa, ruffling substrate-adherent marginal epithelial cells displayed contractile, type I contact inhibition of locomotion. After orientation changes, they gradually retreated. Against HCE, HeLa cells exhibited non-reciprocity of type I contact inhibition and continued radially forward, following the retreating epithelial margin. They did not move onto exposed upper surfaces of epithelial cells and did not underlap marginal cells firmly adherent to the substratum. Invasion of the epithelial sheet was seen only when initial access beneath a cell with a non-adherent margin was available. The contact relationships of isolated invading HeLa cells with their epithelial neighbours suggested successive non-reciprocal contact inhibition reactions.

  18. Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells.

    PubMed Central

    Anisowicz, A; Zajchowski, D; Stenman, G; Sager, R

    1988-01-01

    Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokine-induced responses in gro gene expression are described. Human foreskin fibroblasts are shown to express approximately 10-fold elevated gro, myc, and fos mRNAs in response to serum and to phorbol 12-myristate 13-acetate stimulation, with early response kinetics indicative of growth regulation. In response to interleukin 1, however, in growing cells gro mRNA is elevated at least 100-fold but myc remains constant and fos is not expressed, suggesting a second regulatory pathway. In normal cultured mammary epithelial cells, gro is constitutively expressed, and elevated mRNA levels are induced by phorbol 12-myristate 13-acetate, but not by interleukin 1. However, most carcinoma cell lines examined do not express gro mRNA, suggesting a third function of gro as a negative growth regulator in epithelial cells. Images PMID:3264403

  19. Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons.

    PubMed

    Jiang, Houbo; Xu, Zhimin; Zhong, Ping; Ren, Yong; Liang, Gaoyang; Schilling, Haley A; Hu, Zihua; Zhang, Yi; Wang, Xiaomin; Chen, Shengdi; Yan, Zhen; Feng, Jian

    2015-01-01

    The direct conversion of fibroblasts to induced dopaminergic (iDA) neurons and other cell types demonstrates the plasticity of cell fate. The low efficiency of these relatively fast conversions suggests that kinetic barriers exist to safeguard cell-type identity. Here we show that suppression of p53, in conjunction with cell cycle arrest at G1 and appropriate extracellular environment, markedly increase the efficiency in the transdifferentiation of human fibroblasts to iDA neurons by Ascl1, Nurr1, Lmx1a and miR124. The conversion is dependent on Tet1, as G1 arrest, p53 knockdown or expression of the reprogramming factors induces Tet1 synergistically. Tet1 knockdown abolishes the transdifferentiation while its overexpression enhances the conversion. The iDA neurons express markers for midbrain DA neurons and have active dopaminergic transmission. Our results suggest that overcoming these kinetic barriers may enable highly efficient epigenetic reprogramming in general and will generate patient-specific midbrain DA neurons for Parkinson's disease research and therapy. PMID:26639555

  20. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    PubMed

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems. PMID:25063497

  1. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    PubMed

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems.

  2. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  3. Cd2+-Induced Alteration of the Global Proteome of Human Skin Fibroblast Cells

    PubMed Central

    2015-01-01

    Cadmium (Cd2+) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd2+ on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd2+, few have been targeted toward investigating the ability of Cd2+ to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd2+ exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC–MS/MS, to assess the Cd2+-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd2+ exposure. Our results unveiled that Cd2+ treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd2+ treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd2+ exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd2+-induced toxicity in human cells. PMID:24527689

  4. 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

  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. 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

  7. 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

  8. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

  9. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells.

    PubMed

    Smith, Leah J; Holmes, Amie L; Kandpal, Sanjeev Kumar; Mason, Michael D; Zheng, Tongzhang; Wise, John Pierce

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity.

  10. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells.

    PubMed

    Smith, Leah J; Holmes, Amie L; Kandpal, Sanjeev Kumar; Mason, Michael D; Zheng, Tongzhang; Wise, John Pierce

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. PMID:24823294

  11. Magnetite nanoparticles induced adaptive mechanisms counteract cell death in human pulmonary fibroblasts.

    PubMed

    Radu, Mihaela; Dinu, Diana; Sima, Cornelia; Burlacu, Radu; Hermenean, Anca; Ardelean, Aurel; Dinischiotu, Anca

    2015-10-01

    Magnetite nanoparticles (MNP) have attracted great interest for biomedical applications due to their unique chemical and physical properties, but the MNP impact on human health is not fully known. Consequently, our study proposes to highlight the biochemical mechanisms that underline the toxic effects of MNP on a human lung fibroblast cell line (MRC-5). The cytotoxicity generated by MNP in MRC-5 cells was dose and time-dependent. MNP-treated MRC-5 cells accumulated large amount of iron and reactive oxygen species (ROS) and exhibited elevated antioxidant scavenger enzymes. Reduced glutathione (GSH) depletion and enhanced lipid peroxidation (LPO) processes were also observed. The cellular capacity to counteract the oxidative damage was sustained by high levels of heat shock protein 60 (Hsp60), a protein that confers resistance against ROS attack and inhibition of cell death. While significant augmentations in nitric oxide (NO) and prostaglandine E2 (PGE2) levels were detected after 72 h of MNP-exposure only, caspase-1 was activated earlier starting with 24h post-treatment. Taken together, our results suggest that MRC-5 cells have the capacity to develop cell protection mechanisms against MNP. Detailed knowledge of the mechanisms induced by MNP in cell culture could be essential for their prospective use in various in vivo biochemical applications. PMID:26065626

  12. 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

  13. Therapeutic transdifferentiation of human fibroblasts into endothelial cells using forced expression of lineage-specific transcription factors

    PubMed Central

    Wong, Wing Tak; Cooke, John P

    2016-01-01

    Transdifferentiation is the direct conversion from one somatic cell type into another desired somatic cell type. This reprogramming method offers an attractive approach for regenerative medicine. Here, we demonstrate that neonatal fibroblasts can be transdifferentiated into endothelial cells using only four endothelial transcription factors, namely, ETV2, FLI1, GATA2, and KLF4. We observed a significant up-regulation of endothelial genes including KDR, CD31, CD144, and vWF in human neonatal foreskin (BJ) fibroblasts infected with the lentiviral construct encoding the open reading frame of the four transcription factors. We observed morphological changes in BJ fibroblasts from the fibroblastic spindle shape into a more endothelial-like cobblestone structures. Fluorescence-activated cell sorting analysis revealed that ~16% of the infected cells with the lentiviral constructs encoding 4F expressed CD31. The sorted cells were allowed to expand for 2 weeks and these cells were immunostained and found to express endothelial markers CD31. The induced endothelial cells also incorporated fluorescence-labeled acetylated low-density lipoprotein and efficiently formed capillary-like networks when seeded on Matrigel. These results suggested that the induced endothelial cells were functional in vitro. Taken together, we successfully demonstrated the direct conversion of human neonatal fibroblasts into endothelial cells by transduction of lentiviral constructs encoding endothelial lineage-specific transcription factors ETV2, FLI1, GATA2, and KLF4. The directed differentiation of fibroblasts into endothelial cells may have significant utility in diseases characterized by fibrosis and loss of microvasculature. PMID:27081470

  14. Uranyl Acetate Induces Oxidative Stress and Mitochondrial Membrane Potential Collapse in the Human Dermal Fibroblast Primary Cells

    PubMed Central

    Daraie, Bahram; Pourahmad, Jalal; Hamidi-Pour, Neda; Hosseini, Mir-Jamal; Shaki, Fatemeh; Soleimani, Masoud

    2012-01-01

    Cytotoxicity of depleted uranium, as a byproduct of military has been came to spotlight in recent decades. DU is known as a chemical rather than radioactive hazard and efforts to illustrating its mechanism is undergo, but the precise complete molecular mechanisms are still unclear. Recent studies showed that uranium induces biological changes in many different target tissues, such as the kidney, brain and skin. The aim of this study was to assess the impact of depleted uranium exposure at the cellular level in the human dermal fibroblast primary cells. The human dermal fibroblast primary cells incubated with different concentration (250-750 μM) of depleted uranium. Cytotoxicity and mitochondrial function in this cell lines were determined with the LDH leakage assay and the MTT test respectively. MDA levels were measured for determination of Lipid peroxidation in DU treated cells. Besides glutathione depletion and apoptosis phenotype detection were also assessed to complete the mechanistic screening. Results showed that the cell viability ameliorates in concentration and time dependent manners following in 24, 48 and 72 h incubation with DU. Moreover the significant increase in lipid peroxidation and significant decrease in cellular GSH recorded in DU treated human dermal fibroblast primary cells suggesting the preoxidant effect of uranyl ions. Cytoprotective effects of N-acetylcysteine (NAC) and dramatic decrease of cell viability in buthionin sulfoxamid (BSO) pretreated cells indicated the possibility of a critical role for glutathione system in DU detoxification. Death pattern, in fibroblast cells following DU treatment was varied from apoptosis to necrosis while the time and concentration increased. Since ROS formation is the initiation step for cell apoptosis, the present studies suggest Uranyl-induced toxicity in the human dermal fibroblast primary cells originated from oxidative stress and lead to occurrence of programmed cell death. PMID:24250472

  15. Enhancing the efficiency of direct reprogramming of human primary fibroblasts into dopaminergic neuron-like cells through p53 suppression.

    PubMed

    Liu, XinJian; Huang, Qian; Li, Fang; Li, Chuan-Yuan

    2014-09-01

    Dopaminergic (DA) neuron-like cells obtained through direct reprogramming of primary human fibroblasts offer exciting opportunities for treatment of Parkinson's disease. A significant obstacle is the low efficiency of conversion during the reprogramming process. Here, we demonstrate that the suppression of p53 significantly enhances the efficiency of transcription factor-mediated conversion of human fibroblasts into functional dopaminergic neurons. In particular, blocking p53 activity using a dominant-negative p53 (p53-DN) in IMR90 cells increases the conversion efficiency by 5-20 fold. The induced DA neuron-like cells exhibit dopamine neuron-specific gene expression, significant dopamine uptake and production capacities, and enables symptomatic relief in a rat Parkinson's disease model. Taken together, our findings suggest that p53 is a critical barrier in direct reprogramming of fibroblast into dopaminergic neurons. PMID:25129808

  16. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

    SciTech Connect

    Smith, Leah J.; Holmes, Amie L.; Kandpal, Sanjeev Kumar; Mason, Michael D.; Zheng, Tongzhang; Wise, John Pierce

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. - Highlights: • Particulate and soluble cobalt are cytotoxic and genotoxic to human lung cells. • Soluble cobalt induces more cytotoxicity compared to particulate cobalt. • Soluble and particulate cobalt induce similar levels of genotoxicity. • Particle-cell contact is required for particulate cobalt-induced toxicity.

  17. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

    PubMed

    Wang, Xiao; Peden, Keith; Murata, Haruhiko

    2015-12-16

    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination

  18. Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

    PubMed

    Levenstein, Mark E; Berggren, W Travis; Lee, Ji Eun; Conard, Kevin R; Llanas, Rachel A; Wagner, Ryan J; Smith, Lloyd M; Thomson, James A

    2008-12-01

    Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

  19. Characterization of Endoneurial Fibroblast-like Cells from Human and Rat Peripheral Nerves

    PubMed Central

    Richard, Laurence; Védrenne, Nicolas; Vallat, Jean-Michel

    2014-01-01

    Endoneurial fibroblast-like cells (EFLCs) are one of the cell populations present in the peripheral nervous system. The role and immunophenotypic characteristics of EFLCs are not well known and led us to perform a histological and cytological study of EFLCs in normal human and rat peripheral nerves. We found that all EFLCs express CD34, neural/glial antigen 2 (NG2), and prolyl-4-hydrolase-beta. In addition, half of the EFLCs in normal peripheral nerves express platelet-derived growth factor receptor-β (PDGFR-β) and some also express the intermediate filament nestin in vivo (at a lower level than Schwann cells, which express high levels of nestin). Using cell cultures of purified EFLCs, we characterized subpopulations of EFLCs expressing PDGFR-β alone or PDGFR-β and nestin. Experimental nerve lesions in rat resulted in an increase in nestin-positive EFLCs, which returned to normal levels after 8 days. This suggests that some EFLCs could have a different proliferative and/or regenerative potential than others, and these EFLCs may play a role in the initial phase of nerve repair. These “activated” EFLCs share some immunophenotypic similarities with pericytes and Interstitial cells of Cajal, which have progenitor cell potentials. This raises the questions as to whether a proportion of EFLCs have a possible role as endoneurial progenitor cells. PMID:24670794

  20. Malvidin Protects WI-38 Human Fibroblast Cells Against Stress-induced Premature Senescence

    PubMed Central

    Seo, Hye Rin; Choi, Mi Jin; Choi, Ji Myung; Ko, Jong Cheol; Ko, Jee Yeon; Cho, Eun Ju

    2016-01-01

    Background: Malvidin is one of the most abundant components in red wines and black rice. The effects of malvidin on aging and lifespan under oxidative stress have not been fully understood. This study focused on the anti-aging effect of malvidin on stress-induced premature senescence (SIPS) in WI-38 human lung-derived diploid fibroblasts. Methods: In order to determine the viability of WI-38 cells, MTT assay was conducted, and malondialdehyde level was determined using thiobarbituric acid-reactive substance assay. Protein expression of inflammation-related factors was also evaluated by Western blot analysis. Results: Acute and chronic oxidative stress via hydrogen peroxide (H2O2) treatment led to SIPS in WI-38 cells, which showed decreased cell viability, increased lipid peroxidation, and a shortened lifespan in comparison with non-H2O2-treated WI-38 cells. However, malvidin treatment significantly attenuated H2O2-induced oxidative stress by inhibiting lipid peroxidation and increasing cell viability. Furthermore, the lifespan of WI-38 cells was prolonged by malvidin treatment. In addition, malvidin downregulated the expression of oxidative stress-related proteins, including NF-κB, COX-2, and inducible nitric oxide synthase. Furthermore, protein expression levels of p53, p21, and Bax were also regulated by malvidin treatment in WI-38 cells undergoing SIPS. Conclusions: Malvidin may potentially inhibit the aging process by controlling oxidative stress. PMID:27051647

  1. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    SciTech Connect

    Kakudo, Natsuko . E-mail: kakudon@takii.kmu.ac.jp; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-07-27

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration.

  2. Inhibition of the Differentiation of Monocyte-Derived Dendritic Cells by Human Gingival Fibroblasts

    PubMed Central

    Séguier, Sylvie; Tartour, Eric; Guérin, Coralie; Couty, Ludovic; Lemitre, Mathilde; Lallement, Laetitia; Folliguet, Marysette; Naderi, Samah El; Terme, Magali; Badoual, Cécile; Lafont, Antoine; Coulomb, Bernard

    2013-01-01

    We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism. PMID:23936476

  3. Distinct Cell Stress Responses Induced by ATP Restriction in Quiescent Human Fibroblasts

    PubMed Central

    Yalamanchili, Nirupama; Kriete, Andres; Alfego, David; Danowski, Kelli M.; Kari, Csaba; Rodeck, Ulrich

    2016-01-01

    Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor (TF) landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated TFs and altered TF subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases. PMID:27757122

  4. Cooling rate dependent biophysical and viability response shift with attachment state in human dermal fibroblast cells.

    PubMed

    Choi, Jeunghwan; Bischof, John C

    2011-12-01

    While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.

  5. Fibroblast Activation Protein Expression by Stromal Cells and Tumor-Associated Macrophages in Human Breast Cancer

    PubMed Central

    Julia, Tchou; Zhang Paul, J; Yingtao, Bi; Celine, Satija; Rajrupa, Marjumdar; Stephen, TL; Lo, A; Haiying, Chen; Carolyn, Mies; June, Carl H; Jose, Conejo-Garcia; Ellen, Puré

    2013-01-01

    Summary Fibroblast activation protein (FAP) has long been known to be expressed in the stroma of breast cancer. However, very little is known if the magnitude of FAP expression within the stroma may have prognostic value and reflect the heterogeneous biology of the tumor cell. An earlier study had suggested that stromal FAP expression in breast cancer was inversely proportional to prognosis. We, therefore, hypothesized that stromal FAP expression may correlate with clinicopathologic variables and may serve as an adjunct prognostic factor in breast cancer. We evaluated the expression of FAP in a panel of breast cancer tissues (n=52) using a combination of immunostain analyses at the tissue and single cell level using freshly frozen or freshly digested human breast tumor samples respectively. Our results showed that FAP expression was abundantly expressed in the stroma across all breast cancer subtypes without significant correlation with clinicopathologic factors. We further identified a subset of FAP positive or FAP+ stromal cells that also expressed CD45, a pan-leukocyte marker. Using freshly dissociated human breast tumor specimens (n=5), we demonstrated that some of these FAP+ CD45+ cells were CD11b+CD14+MHC-II+ indicating that they were likely tumor associated macrophages (TAMs). Although FAP+CD45+ cells have been demonstrated in the mouse tumor stroma, our results demonstrating that human breast TAMs expressed FAP was novel and suggested that existing and future FAP directed therapy may have dual therapeutic benefits targeting both stromal mesenchymal cells and immune cells such as TAMs. More work is needed to explore the role of FAP as a potential targetable molecule in breast cancer treatment. PMID:24074532

  6. Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach

    PubMed Central

    Mirakhori, Fahimeh; Zeynali, Bahman; Rassouli, Hassan; Shahbazi, Ebrahim; Hashemizadeh, Shiva; Kiani, Sahar; Salekdeh, Ghasem Hosseini; Baharvand, Hossein

    2015-01-01

    Background A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach. Methodology/Principal Findings Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo. Conclusions/Significance These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture. PMID:26266943

  7. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    SciTech Connect

    Yakubov, Eduard; Rechavi, Gidi; Rozenblatt, Shmuel; Givol, David

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  8. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  9. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on the cellular response to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

  10. 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

  11. Radiation induced bystander effect by GAP junction channels in human fibroblast cell

    NASA Astrophysics Data System (ADS)

    Furusawa, Y.; Shao, C.; Aoki, M.; Kobayashi, Y.; Funayama, T.; Ando, K.

    The chemical factor involved in bystander effect and its transfer pathway were investigated in a confluent human fibroblast cell (AG1522) population. Micronuclei (MN) and G1-phase arrest were detected in cells irradiated by carbon (~100 keV/μm) ions at HIMAC. A very low dose irradiation showed a high effectiveness in producing MN, suggesting a bystander effect. This effectiveness was enhanced by 8-Br-cAMP treatment that increases gap junctional intercellular communication (GJIC). On the other hand, the effect was reduced by 5% DMSO treatment, which reduce the reactive oxygen species (ROS), and suppressed by 100 μM lindane treatment, an inhibitor of GJIC. In addition, the radiation-induced G1-phase arrest was also enhanced by cAMP, and reduced or suppressed by DMSO or lindane. A microbeam device (JAERI) was also used for these studies. It was found that exposing one single cell in a confluent cell population to exactly one argon (~1260 keV/μm) or neon (~430 keV/ μm) ion, additional MN could be detected in many other unirradiated cells. The yield of MN increased with the number of irradiated cells. However, there was no significant difference in the MN induction when the cells were irradiated by increasing number of particles. MN induction by bystander effect was partly reduced by DMSO, and effectively suppressed by lindane. Our results obtained from both random irradiation and precise numbered irradiation indicate that both GJIC and ROS contributed to the radiation-induced bystander effect, but the cell gap junction channels likely play an essential role in the release and transfer of radiation-induced chemical factors.

  12. High content analysis of human fibroblast cell cultures after exposure to space radiation.

    PubMed

    Dieriks, Birger; De Vos, Winnok; Meesen, Geert; Van Oostveldt, Kaat; De Meyer, Tim; Ghardi, Myriam; Baatout, Sarah; Van Oostveldt, Patrick

    2009-10-01

    Space travel imposes risks to human health, in large part by the increased radiation levels compared to those on Earth. To understand the effects of space radiation on humans, it is important to determine the underlying cellular mechanisms. While general dosimetry describes average radiation levels accurately, it says little about the actual physiological impact and does not provide biological information about individual cellular events. In addition, there is no information about the nature and magnitude of a systemic response through extra- and intercellular communication. To assess the stress response in human fibroblasts that were sent into space with the Foton-M3 mission, we have developed a pluralistic setup to measure DNA damage and inflammation response by combining global and local dosimetry, image cytometry and multiplex array technology, thereby maximizing the scientific output. We were able to demonstrate a significant increase in DNA double-strand breaks, determined by a twofold increase of the gamma-H2AX signal at the level of the single cell and a threefold up-regulation of the soluble signal proteins CCL5, IL-6, IL-8, beta-2 microglobulin and EN-RAGE, which are key players in the process of inflammation, in the growth medium.

  13. High content analysis of human fibroblast cell cultures after exposure to space radiation.

    PubMed

    Dieriks, Birger; De Vos, Winnok; Meesen, Geert; Van Oostveldt, Kaat; De Meyer, Tim; Ghardi, Myriam; Baatout, Sarah; Van Oostveldt, Patrick

    2009-10-01

    Space travel imposes risks to human health, in large part by the increased radiation levels compared to those on Earth. To understand the effects of space radiation on humans, it is important to determine the underlying cellular mechanisms. While general dosimetry describes average radiation levels accurately, it says little about the actual physiological impact and does not provide biological information about individual cellular events. In addition, there is no information about the nature and magnitude of a systemic response through extra- and intercellular communication. To assess the stress response in human fibroblasts that were sent into space with the Foton-M3 mission, we have developed a pluralistic setup to measure DNA damage and inflammation response by combining global and local dosimetry, image cytometry and multiplex array technology, thereby maximizing the scientific output. We were able to demonstrate a significant increase in DNA double-strand breaks, determined by a twofold increase of the gamma-H2AX signal at the level of the single cell and a threefold up-regulation of the soluble signal proteins CCL5, IL-6, IL-8, beta-2 microglobulin and EN-RAGE, which are key players in the process of inflammation, in the growth medium. PMID:19772463

  14. Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation*

    PubMed Central

    Pinheiro, Ana Rita; Paramos-de-Carvalho, Diogo; Certal, Mariana; Costa, Maria Adelina; Costa, Cristina; Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Sévigny, Jean; Correia-de-Sá, Paulo

    2013-01-01

    Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors. PMID:23918924

  15. Wound healing potential of Spirulina platensis extracts on human dermal fibroblast cells.

    PubMed

    Syarina, Pauzi Nur Aimi; Karthivashan, Govindarajan; Abas, Faridah; Arulselvan, Palanisamy; Fakurazi, Sharida

    2015-01-01

    Blue-green alga (Spirulina platensis) is a well renowned nutri-supplement due to its high nutritional and medicinal properties. The aim of this study was to examine the wound healing efficiency of Spirulina platensis at various solvent extracts using in vitro scratch assay on human dermal fibroblast cells (HDF). Various gradient solvent extracts (50 μg/ml of methanolic, ethanolic and aqueous extracts) from Spirulina platensis were treated on HDF cells to acquire its wound healing properties through scratch assay and in this investigation we have used allantoin, as a positive control to compare efficacy among the phytoextracts. Interestingly, aqueous extract were found to stimulate proliferation and migration of HDF cells at given concentrations and enhanced closure rate of wound area within 24 hours after treatment. Methanolic and ethanolic extracts have shown proliferative effect, however these extracts did not aid in the migration and closure of wound area when compared to aqueous extract. Based on phytochemical profile of the plant extracts analyzed by LC-MS/MS, it was shown that compounds supposedly involved in accelerating wound healing are cinnamic acid, narigenin, kaempferol, temsirolimus, phosphatidylserine isomeric derivatives and sulphoquinovosyl diacylglycerol. Our findings concluded that blue-green algae may pose potential biomedical application to treat various chronic wounds especially in diabetes mellitus patients.

  16. Cell fusion in space: plasma membrane fusion in human fibroblasts during short term microgravity

    NASA Astrophysics Data System (ADS)

    Jongkind, J. F.; Visser, P.; Verkerk, A.

    During short-term microgravity in sounding rocket experiments (6 min.) the cytoskeleton undergoes changes and therefore it is possible that cell processes which are dependent on the structure and function of the cytoskeleton are influenced. A cell fusion experiment, initiated by a short electric pulse, was chosen as a model experiment for this sounding rocket experiment. Confluent monolayers of primary human skin fibroblasts, grown on coverslips, were mounted between two electrodes (distance 0.5 cm) and fused by discharging a capacitor (68 muF 250 V; 10 msec) in a low conductive medium. During a microgravity experiment in which nearly all the requirements for an optimal result were met (only the recovery of the payload was delayed) results were found that indicated that microgravity during 6 minutes did not influence cell fusion since the percentage of fused products did not change during microgravity. Within the limits of discrimination using morphological assays microgravity has no influence on the actin/cortical cytoskeleton just after electrofusion.

  17. Reprogramming of Mouse, Rat, Pig, and Human Fibroblasts into iPS Cells

    PubMed Central

    Wu, Sean M.

    2012-01-01

    The induction of pluripotency in somatic cells by transcription factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically-modified large animal models or potentially transplantable donor tissues or organs. In this unit, we describe the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts. We focus on lenti- and retroviral infection as the main platform for pluripotent transcription factor overexpression since these reagents are widely-available and remain the most efficient way to generate iPSC colonies. We hope to illustrate the basic process for iPSC generation in these four species in such a way that would enable the lowering of the entry barrier into iPSC biology by new investigators. PMID:22237859

  18. Wound healing potential of Spirulina platensis extracts on human dermal fibroblast cells.

    PubMed

    Syarina, Pauzi Nur Aimi; Karthivashan, Govindarajan; Abas, Faridah; Arulselvan, Palanisamy; Fakurazi, Sharida

    2015-01-01

    Blue-green alga (Spirulina platensis) is a well renowned nutri-supplement due to its high nutritional and medicinal properties. The aim of this study was to examine the wound healing efficiency of Spirulina platensis at various solvent extracts using in vitro scratch assay on human dermal fibroblast cells (HDF). Various gradient solvent extracts (50 μg/ml of methanolic, ethanolic and aqueous extracts) from Spirulina platensis were treated on HDF cells to acquire its wound healing properties through scratch assay and in this investigation we have used allantoin, as a positive control to compare efficacy among the phytoextracts. Interestingly, aqueous extract were found to stimulate proliferation and migration of HDF cells at given concentrations and enhanced closure rate of wound area within 24 hours after treatment. Methanolic and ethanolic extracts have shown proliferative effect, however these extracts did not aid in the migration and closure of wound area when compared to aqueous extract. Based on phytochemical profile of the plant extracts analyzed by LC-MS/MS, it was shown that compounds supposedly involved in accelerating wound healing are cinnamic acid, narigenin, kaempferol, temsirolimus, phosphatidylserine isomeric derivatives and sulphoquinovosyl diacylglycerol. Our findings concluded that blue-green algae may pose potential biomedical application to treat various chronic wounds especially in diabetes mellitus patients. PMID:27004048

  19. Wound healing potential of Spirulina platensis extracts on human dermal fibroblast cells

    PubMed Central

    Syarina, Pauzi Nur Aimi; Karthivashan, Govindarajan; Abas, Faridah; Arulselvan, Palanisamy; Fakurazi, Sharida

    2015-01-01

    Blue-green alga (Spirulina platensis) is a well renowned nutri-supplement due to its high nutritional and medicinal properties. The aim of this study was to examine the wound healing efficiency of Spirulina platensis at various solvent extracts using in vitro scratch assay on human dermal fibroblast cells (HDF). Various gradient solvent extracts (50 μg/ml of methanolic, ethanolic and aqueous extracts) from Spirulina platensis were treated on HDF cells to acquire its wound healing properties through scratch assay and in this investigation we have used allantoin, as a positive control to compare efficacy among the phytoextracts. Interestingly, aqueous extract were found to stimulate proliferation and migration of HDF cells at given concentrations and enhanced closure rate of wound area within 24 hours after treatment. Methanolic and ethanolic extracts have shown proliferative effect, however these extracts did not aid in the migration and closure of wound area when compared to aqueous extract. Based on phytochemical profile of the plant extracts analyzed by LC-MS/MS, it was shown that compounds supposedly involved in accelerating wound healing are cinnamic acid, narigenin, kaempferol, temsirolimus, phosphatidylserine isomeric derivatives and sulphoquinovosyl diacylglycerol. Our findings concluded that blue-green algae may pose potential biomedical application to treat various chronic wounds especially in diabetes mellitus patients. PMID:27004048

  20. Ultraviolet-B Protective Effect of Flavonoids from Eugenia caryophylata on Human Dermal Fibroblast Cells

    PubMed Central

    Patwardhan, Juilee; Bhatt, Purvi

    2015-01-01

    Background: The exposure of skin to ultraviolet-B (UV-B) radiations leads to deoxyribonucleic acid (DNA) damage and can induce production of free radicals which imbalance the redox status of the cell and lead to increased oxidative stress. Clove has been traditionally used for its analgesic, anti-inflammatory, anti-microbial, anti-viral, and antiseptic effects. Objective: To evaluate the UV-B protective activity of flavonoids from Eugenia caryophylata (clove) buds on human dermal fibroblast cells. Materials and Methods: Protective ability of flavonoid-enriched (FE) fraction of clove was studied against UV-B induced cytotoxicity, anti-oxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2 antioxidant response element (Nrf2 ARE) pathway. Results: FE fraction showed a significant antioxidant potential. Pretreatment of cells with FE fraction (10–40 μg/ml) reversed the effects of UV-B induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: The present study demonstrated for the first time that the FE fraction from clove could confer UV-B protection probably through the Nrf2-ARE pathway, which included the down-regulation of Nrf2 and HO-1. These findings suggested that the flavonoids from clove could potentially be considered as UV-B protectants and can be explored further for its topical application to the area of the skin requiring protection. SUMMARY Pretreatment of human dermal fibroblast with flavonoid-enriched fraction of Eugenia caryophylata attenuated effects of ultraviolet-B radiationsIt also conferred protection through nuclear factor E2-related factor 2-antioxidant response pathway and increased tolerance of cells against oxidative stress

  1. Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair.

    PubMed

    Tseng, Ting-Chen; Hsieh, Fu-Yu; Dai, Niann-Tzyy; Hsu, Shan-Hui

    2016-09-01

    Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, β-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future. PMID:27341268

  2. Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors

    SciTech Connect

    Schweigerer, L.; Neufeld, G.; Mergia, A.; Abraham, J.A.; Fiddes, J.C.; Gospodarowicz, D.

    1987-02-01

    Cultured human embryonal rhabdomyosarcoma cells express the basic fibroblast growth factor (bFGF) gene and they produced bFGF, which is apparently composed of two microheterogenous forms with M/sub r/s of 16,500 and 17,200, respectively. bFGF was purified by radioreceptor binding assays or cross-linking experiments. bFGF derived from the rhabdomyosarcoma cells stimulates their own proliferation and that of human or bovine vascular endothelial cells. It is conceivable that the rhabdomyosarcoma-derived bFGF stimulates the growth and neovascularization of human rhabdomyosarcomas and that it may thereby contribute to the development of these tumors.

  3. Quercitrin for periodontal regeneration: effects on human gingival fibroblasts and mesenchymal stem cells

    PubMed Central

    Gómez-Florit, Manuel; Monjo, Marta; Ramis, Joana M.

    2015-01-01

    Periodontal disease (PD) is the result of an infection and chronic inflammation of the gingiva that may lead to its destruction and, in severe cases, alveolar bone and tooth loss. The ultimate goal of periodontal treatment is to achieve periodontal soft and hard tissues regeneration. We previously selected quercitrin, a catechol-containing flavonoid, as a potential agent for periodontal applications. In this study, we tested the ability of quercitrin to alter biomarker production involved in periodontal regeneration on primary human gingival fibroblasts (hGF) and primary human mesenchymal stem cells (hMSC) cultured under basal and inflammatory conditions. To mimic PD inflammatory status, interleukin-1 beta (IL-1β) was used. The expression of different genes related to inflammation and extracellular matrix were evaluated and prostaglandin E2 (PGE2) production was quantified in hGFs; alkaline phosphatase (ALP) activity and calcium content were analysed in hMSCs. Quercitrin decreased the release of the inflammatory mediator PGE2 and partially re-established the impaired collagen metabolism induced by IL-1β treatment in hGFs. Quercitrin also increased ALP activity and mineralization in hMSCs, thus, it increased hMSCs differentiation towards the osteoblastic lineage. These findings suggest quercitrin as a novel bioactive molecule with application to enhance both soft and hard tissue regeneration of the periodontium. PMID:26558438

  4. The effect of ursolic and oleanolic acids on human skin fibroblast cells.

    PubMed

    Wójciak-Kosior, Magdalena; Paduch, Roman; Matysik-Woźniak, Anna; Niedziela, Piotr; Donica, Helena

    2011-01-01

    In this article, we look at how ursolic and oleanolic acids can be used for the purpose of quality control of natural products used in dermatocosmetology as well as of various other therapeutic preparations. Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenes and they are constituents of many medicinal herbs. In this study, we analyzed the cytotoxic and anti-proliferative activity of OA and UA against normal human skin fibroblasts (HSF). Additionally, the scavenging activity of free radicals of both acids was analyzed. The sensitivity of cells to OA and UA activity was determined using a standard spectrophotometric (MTT) assay. The free radical scavenging activity of OA and UA was measured using the DPPH• test. The F-actin cytoskeletal proteins organization was analyzed using TRITC-phalloidine fluorescent staining. The cytotoxic activity of the analyzed acids was determined using Neutral Red (NR) uptake assay. Of the two isomeric compounds, UA showed a higher cytotoxic activity against HSF cells than did OA. Our investigations showed that OA, in view of its non-toxic nature, may be used as a supplementary factor for dermal preparations.

  5. Antioxidant and anti-lipid peroxidation activities of Tamarindus indica seed coat in human fibroblast cells.

    PubMed

    Nakchat, Oranuch; Meksuriyen, Duangdeun; Pongsamart, Sunanta

    2014-02-01

    Antioxidant activity and total phenolic content of tamarind seed coat extracts (TSCEs) were compared between the two extracts using boiling-water (TSCE-W) and 70% ethanol (TSCE-E) for extraction. TSCE-W, consisting of the highest phenolic content, possessed 2,2-diphenyl-1 -picrylhydrazyl (DPPH) radical scavenging and anti-lipid peroxidation activities much higher than TSCE-E and Trolox. Additionally, both TSCEs also exhibited superoxide anion and hydrogen peroxide scavenging activities higher than Trolox and BHA. Anti-lipid peroxidation and cytotoxicity of TSCE-W were also studied in human foreskin fibroblast CCD-1064Sk cells. Cytotoxic effect was not observed when exposed to TSCE-W up to 1 mg/mL for 12-48 h. However, TSCE-W significantly attenuated lipid peroxidation in H202-damaged cells. HPLC analysis showed the presence of (+)-catechin, (-)-epicatechin, and procyanidin B2 in TSCE-W, which could be responsible for antioxidant and anti-lipid peroxidation activities. The results suggest that an inexpensive and simple boiling-water extraction of TSCE-W may provide a valuable natural antioxidant source having anti-lipid peroxidation for health food additives, nutraceuticals as well as cosmeceuticals.

  6. Antioxidant and anti-lipid peroxidation activities of Tamarindus indica seed coat in human fibroblast cells.

    PubMed

    Nakchat, Oranuch; Meksuriyen, Duangdeun; Pongsamart, Sunanta

    2014-02-01

    Antioxidant activity and total phenolic content of tamarind seed coat extracts (TSCEs) were compared between the two extracts using boiling-water (TSCE-W) and 70% ethanol (TSCE-E) for extraction. TSCE-W, consisting of the highest phenolic content, possessed 2,2-diphenyl-1 -picrylhydrazyl (DPPH) radical scavenging and anti-lipid peroxidation activities much higher than TSCE-E and Trolox. Additionally, both TSCEs also exhibited superoxide anion and hydrogen peroxide scavenging activities higher than Trolox and BHA. Anti-lipid peroxidation and cytotoxicity of TSCE-W were also studied in human foreskin fibroblast CCD-1064Sk cells. Cytotoxic effect was not observed when exposed to TSCE-W up to 1 mg/mL for 12-48 h. However, TSCE-W significantly attenuated lipid peroxidation in H202-damaged cells. HPLC analysis showed the presence of (+)-catechin, (-)-epicatechin, and procyanidin B2 in TSCE-W, which could be responsible for antioxidant and anti-lipid peroxidation activities. The results suggest that an inexpensive and simple boiling-water extraction of TSCE-W may provide a valuable natural antioxidant source having anti-lipid peroxidation for health food additives, nutraceuticals as well as cosmeceuticals. PMID:24597144

  7. Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

    PubMed

    Stevens, Roy H; de Moura Martins Lobo Dos Santos, Caroline; Zuanazzi, David; de Accioly Mattos, Marcelo Barbosas; Ferreira, Davis Fernandes; Kachlany, Scott C; Tinoco, Eduardo M B

    2013-01-01

    Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens. PMID:23022667

  8. Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells, follicular dendritic cells and a third superficial stromal cell type

    PubMed Central

    Steiniger, Birte S; Wilhelmi, Verena; Seiler, Anja; Lampp, Katrin; Stachniss, Vitus

    2014-01-01

    At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD271− and CD271+/− stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27+ and switched B lymphocytes, but also scattered IgD++ B lymphocytes and variable numbers of CD4+ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far. PMID:24890772

  9. Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion.

    PubMed

    Du, Shou-Hui; Tay, Johan Chin-Kang; Chen, Can; Tay, Felix-Chang; Tan, Wee-Kiat; Li, Zhen-Dong; Wang, Shu

    2015-08-01

    Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However, medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers, primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4, Sox2 Klf4, and c-Myc (OSKM) transcription factor genes, with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore, the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion, especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment.

  10. 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

  11. Analysis of Heavy Ion-Induced Chromosome Aberrations in Human Fibroblast Cells Using In Situ Hybridization

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Durante, Marco; Furusawa, Yoshiya; George, Kerry; Kawata, Tetsuya; Cucinotta, Francis A.

    2003-01-01

    Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon Si, or with Fe ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 0 C for 24 hours after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Unrejoined chromosomal breaks and complex exchanges were analyzed in the irradiated samples. In order to verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after high-LET radiation, and consequently, the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/micron, the highest LET value in the present study. For samples exposed to 200 MeV/nucleon Fe ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique that allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy dose of the Fe ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges, values for which were higher than those obtained after a 6 Gy gamma exposure. After 0.7 Gy of Fe ions, most complex aberrations were found to involve three or four chromosomes, indicating the maximum number of chromosome domains traversed by a single Fe ion track. 2

  12. DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low laser irradiation

    NASA Astrophysics Data System (ADS)

    Hawkins Evans, D.; Mbene, A.; Zungu, I.; Houreld, N.; Abrahamse, H.

    2009-02-01

    Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2

  13. 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

  14. Data on cell viability of human lung fibroblasts treated with polyphenols-rich extract from Plinia trunciflora (O. Berg) Kausel).

    PubMed

    Calloni, Caroline; Silva Santos, Luciana Fernandes; Martínez, Luana Soares; Salvador, Mirian

    2016-03-01

    Jaboticaba (Plinia trunciflora (O. Berg) Kausel) is a Brazilian native berry, which presents high levels of polyphenols. Here we provide data related to the effects of the polyphenols-rich extract from jaboticaba on the cell viability, mitochondrial complex I (nicotinamide adenine dinucleotide/CoQ oxidoreductase) activity and ATP biosynthesis of human lung fibroblast cells (MRC-5) treated with amiodarone. The data presented in this article demonstrate that the polyphenols-rich extract from jaboticaba was able to reduce cell death as well as the decrease in complex I activity and ATP biosynthesis caused by amiodarone in MRC-5 cells.

  15. Data on cell viability of human lung fibroblasts treated with polyphenols-rich extract from Plinia trunciflora (O. Berg) Kausel)

    PubMed Central

    Calloni, Caroline; Silva Santos, Luciana Fernandes; Martínez, Luana Soares; Salvador, Mirian

    2016-01-01

    Jaboticaba (Plinia trunciflora (O. Berg) Kausel) is a Brazilian native berry, which presents high levels of polyphenols. Here we provide data related to the effects of the polyphenols-rich extract from jaboticaba on the cell viability, mitochondrial complex I (nicotinamide adenine dinucleotide/CoQ oxidoreductase) activity and ATP biosynthesis of human lung fibroblast cells (MRC-5) treated with amiodarone. The data presented in this article demonstrate that the polyphenols-rich extract from jaboticaba was able to reduce cell death as well as the decrease in complex I activity and ATP biosynthesis caused by amiodarone in MRC-5 cells. PMID:26870757

  16. Interactions of human cytomegalovirus with human fibroblasts.

    PubMed

    Vonka, V; Benyesh-Melnick, M

    1966-01-01

    Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Interactions of human cytomegalovirus with human fibroblasts. J. Bacteriol. 91:213-220. 1966.-Virus attachment of human cytomegalovirus to human embryo lung fibroblasts was found to be temperature-independent, from 4 to 37 C. Prolonged incubation at 4 C, however, resulted in inactivation of a high proportion of attached virus. Virus penetration seemed to be temperature-dependent, occurring at 37 C but not at 4 C. Detailed studies of the growth curve of the virus were made. Cell-associated virus preceded the appearance of virus in the fluid phase by 2 to 5 days. Complement-fixing antigen could be detected, but only when the cytopathic effect was advanced, and it was demonstrable only in the cell-associated fraction. Under methyl cellulose, decreasing the bicarbonate concentration in the overlay from 0.225 to 0.15% resulted in marked increase in plating efficiency with all strains tested. However, varying the concentration of bicarbonate from 0.3 to 0.15% in fluid medium did not influence the growth of virus.

  17. Metabolism of benzo(a)pyrene by human epithelial and fibroblastic cells: metabolite patterns and DNA adduct formation

    SciTech Connect

    Bartley, J.; Bartholomew, J.C.; Stampfer, M.R.

    1982-01-01

    It was demonstrated in cell culture that mammary epithelial cells from normal human breast specimens metabolize benzo(a)pyrene (BaP) and form adducts with the bases of their DNA more readily and at lower concentrations of BaP than do fibroblasts from the same specimens. BaP metabolism and adduct formation was determined in the same incubations with epithelial cells grown out in early passage from each of three specimens and with fibroblasts from one of these specimens. The metabolite pattern of the epithelial cells was indicative of preferential formation of 7, 8-dihydrodiol-9, 10-dihydroepoxybenzo(a)pyrene the ultimate carcinogen. In contrast, fibroblasts formed mainly mono- and dihydroxide derivatives of BaP. The metabolite pattern from epithelial cells was compatible with the ease in which adducts between DNA and the diolepoxide of benzo(a)pyrene were formed. These results provide evidence that chemical carcinogens should be considered as possible factors in the induction of breast cancer in women.

  18. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    PubMed

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  19. Stiffening of Human Skin Fibroblasts with Age

    PubMed Central

    Schulze, Christian; Wetzel, Franziska; Kueper, Thomas; Malsen, Anke; Muhr, Gesa; Jaspers, Soeren; Blatt, Thomas; Wittern, Klaus-Peter; Wenck, Horst; Käs, Josef A.

    2010-01-01

    Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix. PMID:20959083

  20. In vitro cytotoxic and anti-inflammatory effects of myrrh oil on human gingival fibroblasts and epithelial cells.

    PubMed

    Tipton, D A; Lyle, B; Babich, H; Dabbous, M Kh

    2003-06-01

    Limited scientific studies suggest that myrrh (Commiphora molmol) has antibacterial and anti-inflammatory activities. This study determined myrrh oil (MO) cytotoxicity to human gingival fibroblasts and epithelial cells and its effect, measured by ELISA, on interleukin (IL)-1beta-stimulated IL-6 and IL-8 production. Cell viability and cytotoxicity were determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay) and by release of lactate dehydrogenase (LDH) from membrane damaged (LDH release assay) cells, respectively. Based on the MTT assay, 24- and 48-h exposures to fibroblast and epithelial cell (24-h only) viability. At 48 h, 0.0005-0.001% MO decreased epithelial cell viability 30-50%. After 24 and 48 h, MO, at >/=0.005%, maximally decreased viability of all cell lines. In the LDH release assay, exposure to cells. At 24 h, >/=0.0025% MO caused maximal cytotoxicity; cells were more susceptible to cytotoxic effects of MO. There was little or no detectable IL-1beta-stimulated production of IL-6 or IL-8 by cells exposed to >/=0.0025% MO, probably reflective of loss of viability. At subtoxic MO levels (0.00001-0.001%), there was a significant reduction of IL-1beta-stimulated IL-6 and IL-8 production by fibroblasts, but not by epithelial cells. PMID:12781209

  1. Cell death, chromosome damage and mitotic delay in normal human, ataxia telangiectasia and retinoblastoma fibroblasts after x-irradiation.

    PubMed

    Zampetti-Bosseler, F; Scott, D

    1981-05-01

    We recently showed (Scott and Zampetti-Bosseler 1980) that X-ray sensitive mouse lymphoma cells sustain more chromosome damage, mitotic delay and spindle defects than X-ray resistant cells. We proposed that (a) chromosome aberrations contribute much more to lethality than spindle defects, and (b) that DNA lesions are less effectively repaired in the sensitive cells and give rise to more G2 mitotic delay and chromosome aberrations. Our present results on human fibroblasts with reported differential sensitivity to ionizing radiation (i.e. normal donors and patients with ataxia telangiectasia and retinoblastoma) support the first hypothesis since we observed a positive correlation between chromosome aberration frequencies and cell killing and no induced spindle defects. Our second hypothesis is however not substantiated since X-ray sensitive fibroblasts from the ataxia patient suffered less mitotic delay than cells from normal donors. A common lesion for mitotic delay and chromosome aberrations can still be assumed by adopting the hypothesis of Painter and Young (1981) that the defect in ataxia cells is not in repair but in a failure of DNA damage to initiate mitotic delay. In contrast to other reports, we found the retinoblastoma cells to be of normal radiation sensitivity (cell killing and aberration).

  2. Human Dental Pulp Stem Cells and Gingival Fibroblasts Seeded into Silk Fibroin Scaffolds Have the Same Ability in Attracting Vessels

    PubMed Central

    Woloszyk, Anna; Buschmann, Johanna; Waschkies, Conny; Stadlinger, Bernd; Mitsiadis, Thimios A.

    2016-01-01

    Neovascularization is one of the most important processes during tissue repair and regeneration. Current healing approaches based on the use of biomaterials combined with stem cells in critical-size bone defects fail due to the insufficient implant vascularization and integration into the host tissues. Therefore, here we studied the attraction, ingrowth, and distribution of blood vessels from the chicken embryo chorioallantoic membrane into implanted silk fibroin scaffolds seeded with either human dental pulp stem cells or human gingival fibroblasts. Perfusion capacity was evaluated by non-invasive in vivo Magnetic Resonance Imaging while the number and density of blood vessels were measured by histomorphometry. Our results demonstrate that human dental pulp stem cells and gingival fibroblasts possess equal abilities in attracting vessels within silk fibroin scaffolds. Additionally, the prolonged in vitro pre-incubation period of these two cell populations favors the homogeneous distribution of vessels within silk fibroin scaffolds, which further improves implant survival and guarantees successful healing and regeneration. PMID:27148078

  3. Human Dental Pulp Stem Cells and Gingival Fibroblasts Seeded into Silk Fibroin Scaffolds Have the Same Ability in Attracting Vessels.

    PubMed

    Woloszyk, Anna; Buschmann, Johanna; Waschkies, Conny; Stadlinger, Bernd; Mitsiadis, Thimios A

    2016-01-01

    Neovascularization is one of the most important processes during tissue repair and regeneration. Current healing approaches based on the use of biomaterials combined with stem cells in critical-size bone defects fail due to the insufficient implant vascularization and integration into the host tissues. Therefore, here we studied the attraction, ingrowth, and distribution of blood vessels from the chicken embryo chorioallantoic membrane into implanted silk fibroin scaffolds seeded with either human dental pulp stem cells or human gingival fibroblasts. Perfusion capacity was evaluated by non-invasive in vivo Magnetic Resonance Imaging while the number and density of blood vessels were measured by histomorphometry. Our results demonstrate that human dental pulp stem cells and gingival fibroblasts possess equal abilities in attracting vessels within silk fibroin scaffolds. Additionally, the prolonged in vitro pre-incubation period of these two cell populations favors the homogeneous distribution of vessels within silk fibroin scaffolds, which further improves implant survival and guarantees successful healing and regeneration. PMID:27148078

  4. Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation.

    PubMed

    Ueno, Nobuhiro; Shimizu, Akio; Kanai, Michiyuki; Iwaya, Yugo; Ueda, Shugo; Nakayama, Jun; Seo, Misuzu Kurokawa

    2016-01-01

    Deregulated expression of fibroblast growth factor receptors (FGFRs) and their ligands plays critical roles in tumorigenesis. The gene expression of an alternatively spliced isoforms of FGFR3, FGFR3IIIc, was analyzed by RT-PCR in samples from patients with esophageal carcinoma (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). The incidence of FGFR3IIIc was higher in EC [12/16 (75%); p=0.073] than in non-cancerous mucosa (NCM) [6/16 (38%)]. Indeed, an immunohistochemical analysis of early-stage ESCC showed that carcinoma cells expressing FGFR3IIIc stained positively with SCC-112, a tumor marker, and Ki67, a cell proliferation marker, suggesting that the expression of FGFR3IIIc promotes cell proliferation. We used EC-GI-10 cells endogenously expressing FGFR3IIIc as a model of ESCC to provide mechanistic insight into the role of FGFR3IIIc in ESCC. The knockdown of endogenous FGFR3 using siRNA treatment significantly abrogated cell proliferation and the overexpression of FGFR3IIIc in cells with enhanced cell proliferation. EC-GI-10 cells and ESCC from patients with EC showed endogenous expression of FGF2, a specific ligand for FGFR3IIIc, suggesting that the upregulated expression of FGFR3IIIc may create autocrine FGF signaling in ESCC. Taken together, FGFR3IIIc may have the potential to be an early-stage tumor marker and a molecular target for ESCC therapy.

  5. Detection of DNA Damage by Space Radiation in Human Fibroblast Cells Flown on the International Space Station

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Lu, Tao; Wong, Michael; Beno, Jonathan; Countryman, Stefanie; Stodieck, Louis; Karouia, Fathi; Zhang, Ye

    2015-01-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the early discovery of the Van Allen Belt, reports on effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a small number of changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells fixed on Days 3 and 14 after reaching orbit were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used marker for DNA double strand breaks. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground control. Human fibroblast cells were also exposed to low dose rate gamma rays, as well as to protons and Fe ions. Comparison of the pattern and distribution of the foci after gamma ray and charged particle exposure to our flight results confirmed that the foci found in the flown cells were indeed induced by space radiation.

  6. Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent.

    PubMed Central

    Ogryzko, V V; Hirai, T H; Russanova, V R; Barbie, D A; Howard, B H

    1996-01-01

    Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component. PMID:8756678

  7. CD10 expressed by fibroblasts and melanoma cells degrades endothelin-1 secreted by human keratinocytes.

    PubMed

    Xie, Lining; Moroi, Yoichi; Takahara, Masakazu; Tsuji, Gaku; Oba, Junna; Hayashida, Sayaka; Takeuchi, Satoshi; Shan, Baoen; Uchi, Hiroshi; Furue, Masutaka

    2011-01-01

    Endothelin-1 (ET-1) is a potent multifunctional peptide linked to wound healing, pigmentation, carcinogenesis, and fibrosclerotic processes in the skin. Whereas ET-1 was thought to be digested by receptor-mediated endocytosis, it is also reported to be biochemically degraded by the neutral endopeptidase CD10 using kidney homogenates. Although keratinocytes (KC) and fibroblasts (Fb) are sources of both ET-1 and CD10, respectively, there is no report investigating the direct association between CD10 expression and its function in relation to ET-1 degradation in the skin. CD10 expression in melanoma cells is associated with clinical prognosis, suggesting an important role in the invasive and metastatic potential of melanoma cells. Here, cultured KC produced much higher amounts of ET-1 than did cultured Fb or melanoma cells. In contrast, KC and A375 melanoma cells did not express CD10, while Fb, SK-MEL-28 and G361 melanoma cells constitutively expressed CD10. KC-derived ET-1 was down-modulated by both CD10-positive Fb and CD10-positive melanoma cells, and the inhibition was partially reversed under substitution conditions using CD10-knockdown Fb or CD10-knockdown melanoma cells. This indicates that CD10 on cultured Fb and melanoma cells is biochemically active in the degradation or down-modulation of ET-1 secreted from KC. These findings may lead to better understanding of skin homeostasis and of the malignant potential of melanoma.

  8. Determine the yield of micronucleated cells in primary human fibroblasts exposed to focused soft X-rays.

    SciTech Connect

    Kevin M. Prise

    2007-01-02

    This project was a small part of a larger collaborative study headed by Dr Aloke Chatterjee, (Lawrence Berkeley National Laboratory) and including Drs Les Braby, John Ford (Texas A&M) and Kathy Held (MGH Boston), which was developing an integrated theoretical and experimental model of the radiation-induced bystander response. Our part of the study has been to determine the effectiveness of soft X-rays at inducing chromosomal damage under conditions of direct and bystander exposure. The aim was to compare this with the effectiveness of the low energy 60 kV electron microbeam available at Texas A&M. Previous studies have been performed with primary human fibroblasts measuring micronuclei formation to determine the relative yields of direct versus bystander mediated micronuclei formation after cells were individually irradiated utilizing our novel focused soft X-ray microprobe, which is capable of producing localized submicron beams of carbon-K (278 eV) X-rays. Only a brief overview is given here as the study has been published in several papers. Our original hypothesis was to study yields of bystander-induced micronucleated cells in both wild-type and mutant fibroblast from mouse embryo fibroblasts. Difficulties with the level of background micronuclei in the MEFs prevented systematic studies of bystander responses in the laboratories involved in the collaboration. We then performed these studies with AG1522 primary human fibroblast cells using a siRNA approach developed by John Ford at Texas A&M to knock down DNA PKcs in the first instance. Our soft X-ray source has been in routine use for carbon-K X-rays and is now available with Aluminium-K (1.49 keV) and titanium-K (4.5 keV), although the dose-rate from titanium is still too low at present for most experiments, where large numbers of cells need to be exposed. A separately funded project developed a new soft X-ray microprobe which will give much greater flexibility for changing energies and giving high dose

  9. Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

    PubMed Central

    Shahbazi, Ebrahim; Moradi, Sharif; Nemati, Shiva; Satarian, Leila; Basiri, Mohsen; Gourabi, Hamid; Zare Mehrjardi, Narges; Günther, Patrick; Lampert, Angelika; Händler, Kristian; Hatay, Firuze Fulya; Schmidt, Diana; Molcanyi, Marek; Hescheler, Jürgen; Schultze, Joachim L.; Saric, Tomo; Baharvand, Hossein

    2016-01-01

    Summary Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts. PMID:27052315

  10. Oxidative stress impairs the repair of oxidative DNA base modifications in human skin fibroblasts and melanoma cells.

    PubMed

    Eiberger, Wolfgang; Volkmer, Beate; Amouroux, Rachel; Dhérin, Claudine; Radicella, J Pablo; Epe, Bernd

    2008-06-01

    Irradiation of mammalian cells with solar light is associated with the generation of reactive oxygen species (ROS) and oxidative stress, which is mediated in part by endogenous photosensitizers absorbing in the visible range of the solar spectrum. Accordingly, oxidative DNA base modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are the predominant types of DNA damage in cells irradiated at wavelengths >400 nm. We have analysed the repair of oxidative purine modifications in human skin fibroblasts and melanoma cells using an alkaline elution technique, both under normal conditions and after depletion of glutathione. Similar repair rates were observed in fibroblasts and melanoma cells from three different patients (t1/2 approximately 4h). In both cell types, glutathione depletion (increased oxidative stress) caused a pronounced repair retardation even under non-toxic irradiation conditions. Furthermore, the cleavage activity at 8-oxoG residues measured in protein extracts of the cells dropped transiently after irradiation. An addition of dithiothreitol restored normal repair rates. Interestingly, the repair rates of cyclobutane pyrimidine dimers (t1/2 approximately 18 h), AP sites (t1/2 approximately 1h) and single-strand breaks (t1/2 <30 min) were not affected by the light-induced oxidative stress. We conclude that the base excision repair of oxidative purine modifications is surprisingly vulnerable to oxidative stress, while the nucleotide excision repair of pyrimidine dimers is not. PMID:18436486

  11. Induction of chromatin damage and distribution of isochromatid breaks in human fibroblast cells exposed to heavy ions

    NASA Technical Reports Server (NTRS)

    Kawata, Tetsuya; Ito, Hisao; Motoori, Ken; Ueda, Takuya; Shigematsu, Naoyuki; Furusawa, Yoshiya; Durante, Marco; George, Kerry; Wu, Honglu; Cucinotta, Francis A.

    2002-01-01

    The frequency of chromatid breaks and the distribution of isochromatid breaks were measured in G2-phase normal human fibroblasts prematurely condensed a short time after exposure to low- or high-LET radiations. The average number of isochromatid breaks from a single particle traversal increased with increasing LET values, while the average number of chromatid-type breaks appeared to reach a plateau. The distribution of isochromatid breaks after high-LET iron particles exposure was overdispersed compared to gamma-rays, indicating that a single iron particle traversal through a cell nucleus can produce multiple isochromatid breaks.

  12. Enhancing structural support of the dermal microenvironment activates fibroblasts, endothelial cells, and keratinocytes in aged human skin in vivo.

    PubMed

    Quan, Taihao; Wang, Frank; Shao, Yuan; Rittié, Laure; Xia, Wei; Orringer, Jeffrey S; Voorhees, John J; Fisher, Gary J

    2013-03-01

    The dermal extracellular matrix (ECM) provides strength and resiliency to skin. The ECM consists mostly of type I collagen fibrils, which are produced by fibroblasts. Binding of fibroblasts to collagen fibrils generates mechanical forces, which regulate cellular morphology and function. With aging, collagen fragmentation reduces fibroblast-ECM binding and mechanical forces, resulting in fibroblast shrinkage and reduced function, including collagen production. Here, we report that these age-related alterations are largely reversed by enhancing the structural support of the ECM. Injection of dermal filler, cross-linked hyaluronic acid, into the skin of individuals over 70 years of age stimulates fibroblasts to produce type I collagen. This stimulation is associated with localized increase in mechanical forces, indicated by fibroblast elongation/spreading, and mediated by upregulation of type II TGF-β receptor and connective tissue growth factor. Interestingly, enhanced mechanical support of the ECM also stimulates fibroblast proliferation, expands vasculature, and increases epidermal thickness. Consistent with our observations in human skin, injection of filler into dermal equivalent cultures causes elongation of fibroblasts, coupled with type I collagen synthesis, which is dependent on the TGF-β signaling pathway. Thus, fibroblasts in aged human skin retain their capacity for functional activation, which is restored by enhancing structural support of the ECM.

  13. Epigenetic Induction of Definitive and Pancreatic Endoderm Cell Fate in Human Fibroblasts

    PubMed Central

    Faas, Marijke M.; de Vos, Paul; Verfaillie, Catherine M.

    2016-01-01

    Reprogramming can occur by the introduction of key transcription factors (TFs) as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) combined with a chromatin remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (5AZA) CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions. PMID:27403168

  14. Enhancement of mucus accumulation in a human gastric scirrhous carcinoma cell line (KATO-III) by fibroblast-tumor cell interaction.

    PubMed

    Yamamoto, R; Iishi, H; Tatsuta, M; Nakamura, H; Terada, N; Komatsu, K; Matsusaka, T

    1990-01-01

    Human fibroblasts (WI-38 cells) were found to enhance mucus accumulation by human scirrhous carcinoma cells (KATO-III cells). Coculture of KATO-III with WI-38 cells resulted in enlargement of the KATO-III cells and increases in the proportions of PAS- and colloidal iron-positive KATO-III cells. These morphological alterations were reversed when the KATO-III cells were again cultured without WI-38 cells. Conditioned media from cultures of WI-38 cells or cocultures of KATO-III and WI-38 cells induced the same morphological alterations in KATO-III cells, suggesting that WI-38 cells produce a factor or factors that enhance mucus accumulation in KATO-III cells. This factor seemed to be a protein with a molecular weight of more than 10,000 daltons. PMID:1974095

  15. 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

  16. UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts.

    PubMed

    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

  17. Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression.

    PubMed

    Sciannamblo, Mariateresa; Chigorno, Vanna; Passi, Alberto; Valaperta, Rea; Zucchi, Ileana; Sonnino, Sandro

    2002-03-01

    In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density. GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation. Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase.

  18. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

    PubMed Central

    Seneviratne, Chaminda Jayampath

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  19. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens.

    PubMed

    Sriram, Gopu; Natu, Vaishali Prakash; Islam, Intekhab; Fu, Xin; Seneviratne, Chaminda Jayampath; Tan, Kai Soo; Cao, Tong

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  20. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

    PubMed Central

    Seneviratne, Chaminda Jayampath

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  1. JNK/SAPK Signaling Is Essential for Efficient Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells.

    PubMed

    Neganova, Irina; Shmeleva, Evgenija; Munkley, Jennifer; Chichagova, Valeria; Anyfantis, George; Anderson, Rhys; Passos, Joao; Elliott, David J; Armstrong, Lyle; Lako, Majlinda

    2016-05-01

    Reprogramming of somatic cells to the phenotypic state termed "induced pluripotency" is thought to occur through three consecutive stages: initiation, maturation, and stabilisation. The initiation phase is stochastic but nevertheless very important as it sets the gene expression pattern that permits completion of reprogramming; hence a better understanding of this phase and how this is regulated may provide the molecular cues for improving the reprogramming process. c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPKs) are stress activated MAPK kinases that play an essential role in several processes known to be important for successful completion of the initiation phase such as cellular proliferation, mesenchymal to epithelial transition (MET) and cell cycle regulation. In view of this, we postulated that manipulation of this pathway would have significant impacts on reprogramming of human fibroblasts to induced pluripotent stem cells. Accordingly, we found that key components of the JNK/SAPK signaling pathway increase expression as early as day 3 of the reprogramming process and continue to rise in reprogrammed cells throughout the initiation and maturation stages. Using both chemical inhibitors and RNA interference of MKK4, MKK7 and JNK1, we tested the role of JNK/SAPK signaling during the initiation stage of neonatal and adult fibroblast reprogramming. These resulted in complete abrogation of fully reprogrammed colonies and the emergence of partially reprogrammed colonies which disaggregated and were lost from culture during the maturation stage. Inhibition of JNK/SAPK signaling resulted in reduced cell proliferation, disruption of MET and loss of the pluripotent phenotype, which either singly or in combination prevented establishment of pluripotent colonies. Together these data provide new evidence for an indispensable role for JNK/SAPK signaling to overcome the well-established molecular barriers in human somatic cell induced

  2. Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

    PubMed

    Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

    2009-01-01

    Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells. PMID:19402346

  3. Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts.

    PubMed

    Khazaie, Niusha; Massumi, Mohammad; Wee, Ping; Salimi, Mahdieh; Mohammadnia, Abdulshakour; Yaqubi, Moein

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs.

  4. Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts

    PubMed Central

    Khazaie, Niusha; Massumi, Mohammad; Wee, Ping; Salimi, Mahdieh; Mohammadnia, Abdulshakour; Yaqubi, Moein

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs. PMID:26938987

  5. Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

    PubMed

    Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

    2016-08-01

    Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future.

  6. Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

    PubMed

    Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

    2016-08-01

    Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. PMID:27252227

  7. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin.

    PubMed

    Gospodarowicz, D; Brown, K D; Birdwell, C R; Zetter, B R

    1978-06-01

    Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of

  8. Detection of DNA Damage by Space Radiation in Human Fibroblast Cells Flown on the International Space Station

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Zhang, Ye

    2016-01-01

    Although charged particles in space have been detected with radiation detectors on board the spacecraft since the early discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells were kept at 370 C in space and fixed on Days 3 and 14 after reaching orbit. After returning to the ground, the fixed cells were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used biomarker for DNA double strand breaks. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground controls. To confirm that the foci data from the flight study was actually induced from space radiation exposure, human fibroblast cells were exposed to low- and high-LET protons and high-LET Fe ions on the ground. High-LET protons and Fe ions were found to induce foci of the pattern that were observed in the flown cells.

  9. Detection of DNA damage by space radiation in human fibroblast cells flown on the International Space Station

    NASA Astrophysics Data System (ADS)

    Wu, Honglu; Feiveson, Alan; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao; Wong, Michael

    2016-07-01

    Although charged particles in space have been detected with radiation detectors on board the spacecraft since the early discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells were kept at 370C in space and fixed on Days 3 and 14 after reaching orbit. After returning to the ground, the fixed cells were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used biomarker for DNA double strand breaks. The 3-dimensional γg-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground controls. To confirm that the foci data from the flight study was actually induced from space radiation exposure, human fibroblast cells were exposed to low- and high-LET protons and high-LET Fe ions on the ground. High-LET protons and Fe ions were found to induce foci of the pattern that were observed in the flown cells.

  10. Cytotoxic effects of amitriptyline in human fibroblasts.

    PubMed

    Moreno-Fernández, A M; Cordero, M D; de Miguel, M; Delgado-Rufino, M D; Sánchez-Alcázar, J A; Navas, P

    2008-01-14

    Amitriptyline is a tricyclic antidepressant widely used in the treatment of chronic pain. The objective of the present study was to investigate the potential cytotoxic effects of amitriptyline in human fibroblasts primary culture. Human fibroblast cells were cultured from healthy subjects and incubated with 50 microM and 100 microM amitriptyline. Cell counting was performed to study dose-dependency of toxicity. Lipid peroxidation analysis and western blotting for antioxidants catalase and mitochondrial superoxide dismutase (MnSOD) were carried out in order to evaluate oxidative stress. To investigate mitochondria damage the following determinations were made: cytochrome c, citrate synthase, and mitochondrial membrane potential (DeltaPsi(m)). Amitriptyline reduced significantly the number of cultured cells, resulting in a decrease of 45.2%, 65.0% and 94.9% when treated with 20 microM, 50 microM and 100 microM amitriptyline, respectively. This drug enhanced the production of oxidized products during lipid peroxidation, inverting the reduced/oxidized ratio to 25% reduction and 75% oxidation after 24h of amitriptyline administration. A decreased in catalase protein levels has been also observed. Moreover, amitriptyline treatment induced a significant decrease of cytochrome c, DeltaPsi(m), and citrate synthase activity; revealing mitochondrial damage. These findings suggest that amitriptyline has a strong cytotoxic effect in human fibroblasts, decreasing growth rate and mitochondrial activity, and increasing oxidative stress.

  11. Increase in VEGF secretion from human fibroblast cells by bioactive glass S53P4 to stimulate angiogenesis in bone.

    PubMed

    Detsch, Rainer; Stoor, Patricia; Grünewald, Alina; Roether, Judith A; Lindfors, Nina C; Boccaccini, Aldo R

    2014-11-01

    Bioactive glasses (BAGs) are being investigated for the repair and reconstruction of bone defects, as they exhibit osteoconductive and osteostimulatory potential. However, successful bone regeneration requires also the neovascularization of the construct which is, among other factors, guided by vascular endothelial growth factor (VEGF). In this study, BAG S53P4 (53% SiO2 , 23% Na2 O, 20% CaO, 4% P2 O5 ) is investigated in relation to VEGF-release and response of fibroblast cells. Human CD-18CO fibroblasts were cultivated in contact with different granules of different sizes (0.5-0.8 mm, 1.0-2.0 mm, and 2.0-3.15 mm) and at different concentrations (0-1 wt/vol % of BAG) for 72 h. The analysis of morphology revealed no toxic effect for all granule sizes and concentrations. Compared with the reference, lactate dehydrogenase-activity of CCD-18CO cells increased in contact with BAG samples. The VEGF release from CCD-18CO fibroblasts cultured on different granule sizes and at different concentrations after 72 h of incubation was quantified. It was found that particles of 0.5-0.8 mm and 1.0-2.0 mm in size enhanced VEGF release, whereas BAG particle sizes of 2.0-3.15 mm led to inhibition of VEGF release. The results are relevant to understand the influence of the particle size and concentration of BAG S53P4 on VEGF expression and neovascularization. PMID:24357515

  12. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan.

    PubMed Central

    Pfefferkorn, E R

    1984-01-01

    Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The kinetics of [3H]tryptophan uptake into the acid-soluble pools of control and gamma interferon-treated cultures were identical during the first 48 sec. Thereafter uptake of [3H]tryptophan into the acid-soluble pool of control fibroblasts reached the expected plateau after 96 sec. In contrast, uptake of [3H]tryptophan continued for at least 12 min in the gamma interferon-treated cultures. At that time, the acid-soluble pool of the gamma interferon-treated cultures contained 8 times the radioactivity of the control cultures. This continued accumulation was the result of rapid intracellular degradation of [3H]tryptophan into kynurenine and N-formylkynurenine that leaked slowly from the cells. These two metabolites were also recovered from the medium of cultures treated for 1 or 2 days with gamma interferon. Human recombinant alpha and beta interferons, which have no antitoxoplasma activity, did not induce any detectable degradation of tryptophan. Several hypotheses are presented to explain how the intracellular degradation of tryptophan induced by gamma interferon could restrict the growth of an obligate intracellular parasite. Images PMID:6422465

  13. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    SciTech Connect

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  14. Investigation of Adaptive Responses in Bystander Cells in 3D Cultures Containing Tritium-Labeled and Unlabeled Normal Human Fibroblasts

    PubMed Central

    Pinto, Massimo; Azzam, Edouard I.; Howell, Roger W.

    2010-01-01

    The study of radiation-induced bystander effects in normal human cells maintained in three-dimensional (3D) architecture provides more in vivo-like conditions and is relevant to human risk assessment. Linear energy transfer, dose and dose rate have been considered as critical factors in propagating radiation-induced effects. This investigation uses an in vitro 3D tissue culture model in which normal AG1522 human fibroblasts are grown in a carbon scaffold to investigate induction of a G1 arrest in bystander cells that neighbor radiolabeled cells. Cell cultures were co-pulse-labeled with [3H]deoxycytidine (3HdC) to selectively irradiate a minor fraction of cells with 1–5 keV/μm β particles and bromodeoxyuridine (BrdU) to identify the radiolabeled cells using immunofluorescence. The induction of a G1 arrest was measured specifically in unlabeled cells (i.e. bystander cells) using a flow cytometry-based version of the cumulative labeling index assay. To investigate the relationship between bystander effects and adaptive responses, cells were challenged with an acute 4 Gy γ-radiation dose after they had been kept under the bystander conditions described above for several hours, and the regulation of the radiation-induced G1 arrest was measured selectively in bystander cells. When the average dose rate in 3HdC-labeled cells (<16% of population) was 0.04–0.37 Gy/h (average accumulated dose 0.14–10 Gy), no statistically significant stressful bystander effects or adaptive bystander effects were observed as measured by magnitude of the G1 arrest, micronucleus formation, or changes in mitochondrial membrane potential. Higher dose rates and/or higher LET may be required to observe stressful bystander effects in this experimental system, whereas lower dose rates and challenge doses may be required to detect adaptive bystander responses. PMID:20681788

  15. Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-β and elongated cell shape.

    PubMed

    Wang, Wenbo; Li, Jie; Wang, Keyun; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Ye, Mingliang; Zou, Hanfa; Liu, Wei

    2016-03-01

    Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling. PMID:26632599

  16. Cytotoxic effect of silorane and methacrylate based composites on the human dental pulp stem cells and fibroblasts

    PubMed Central

    Shafiei, Fereshteh; Razmkhah, Mahboobeh; Attar, Armin; Alavi, Ali A.

    2014-01-01

    Objectives: The aim of this study was to compare the cytotoxic effect of a methacrylate-based and a silorane-based composite on the human dental pulp stem cells (DPSCs) versus human dental pulp fibroblasts (DPFs). Study Design: Samples of the Filtek Z250 and P90 were polymerized and immersed in the culture medium to obtain extracts after incubation for one, seven and 14 days. Magnetic cell sorting based on the CD146 expression was performed to purify DPSCs and DPFs. After incubation of both cells with the extracts, cytotoxicity was determined using the MTT test. Results: For the extracts of first and seventh day, both composites showed significantly lower cytotoxicity on DPSCs than DPFs (p=0.003). In addition, there was a significant difference in the time-group interaction of both materials indicating different cytotoxic behaviours (p=0.014). In contrast to Z250, exposure to the 14th day extract of P90 resulted in higher cell viability compared to that of day seven. Conclusions: DPSCs are less susceptible to the cytotoxic effect of the composites than DPFs. Compared to Z250, the cytotoxic effect of silorane-based composite decreases as the time passes on. This difference should be considered, particularly in deep cavities, in order to preserve the regenerative capacity of the pulp. Key words:Composite resins, Dental pulp, Mesenchymal Stromal Cells, Silorane, Toxicology. PMID:24608214

  17. The messenger RNA sequences in human fibroblast cells induced with poly rI.rC to produce interferon.

    PubMed Central

    Raj, N B; Pitha, P M

    1980-01-01

    Induction of human fibroblast cells with poly rI.rC induces interferon mRNA which can be translated into interferon precursor in wheat germ cell free system or in Xenopus oocytes into biologically active interferon. The extent of gene expression in the poly rI.rC induced cells was compared to that of the uninduced cells by hybridization of the mRNA to complementary DNA. Homologous template driven hybridization of cDNA revealed the presence of two clearly defined transitions in the total poly A RNA from the induced cells; abundant class and a scarce class comprising approximately 37,000 diverse species of RNA. Heterologus hybridization of the cDNA with total uninduced mRNA showed that the majority of the mRNA sequences are the same in both the induced and uninduced cells. The results of the hybridization using cDNA prepared to the fraction enriched for interferon mRNA, however, showed that about 4% of the sequences present in the interferon enriched fraction are not present in the uninduced cells. These differences may result from the poly rI.rC induced alterations in gene expression. PMID:6160473

  18. Reversible regulation of cell cycle-related genes by epigallocatechin gallate for hibernation of neonatal human tarsal fibroblasts.

    PubMed

    Bae, Jung Yoon; Kanamune, Jun; Han, Dong-Wook; Matsumura, Kazuaki; Hyon, Suong-Hyu

    2009-01-01

    We investigated the hibernation effect of epigallocatechin-3-O-gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G(2)/M phases of cell cycle with concomitant increase in that of the G(0)/G(1) phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions.

  19. Influence of Fe3O4 Nanoparticles in Hydroxyapatite Scaffolds on Proliferation of Primary Human Fibroblast Cells

    NASA Astrophysics Data System (ADS)

    Maleki-Ghaleh, H.; Aghaie, E.; Nadernezhad, A.; Zargarzadeh, M.; Khakzad, A.; Shakeri, M. S.; Beygi Khosrowshahi, Y.; Siadati, M. H.

    2016-06-01

    Modern techniques for expanding stem cells play a substantial role in tissue engineering: the raw material that facilitates regeneration of damaged tissues and treats diseases. The environmental conditions and bioprocessing methods are the primary determinants of the rate of cultured stem cell proliferation. Bioceramic scaffolds made of calcium phosphate are effective substrates for optimal cell proliferation. The present study investigates the effects of two bioceramic scaffolds on proliferating cells in culture media. One scaffold was made of hydroxyapatite and the other was a mixture of hydroxyapatite and ferromagnetic material (Fe3O4 nanoparticles). Disk-shaped (10 mm × 2 mm) samples of the two scaffolds were prepared. Primary human fibroblast proliferation was 1.8- and 2.5-fold faster, respectively, when cultured in the presence of hydroxyapatite or ferrous nanoparticle/hydroxyapatite mixtures. Optical microscopy images revealed that the increased proliferation was due to enhanced cell-cell contact. The presence of magnetic Fe3O4 nanoparticles in the ceramic scaffolds significantly increased cell proliferation compared to hydroxyapatite scaffolds and tissue culture polystyrene.

  20. Gene Expression Profile Changes and Cellular Responses to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Rohde, Larry; Wu, Honglu

    2016-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. In addition, DNA in space can be damaged by toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage affects the accuracy of the radiation risk assessment for astronauts and the mutation rate in microorganisms. Although possible synergistic effects of space radiation and microgravity have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on cellular responses to DNA damage, confluent human fibroblast cells (AG1522) flown on the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB). Damages in the DNA were quantified by immunofluorescence staining for ?-H2AX, which showed similar percentages of different types of stained cells between flight and ground. However, there was a slight shift in the distribution of the ?-H2AX foci number in the flown cells with countable foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. A microarray analysis of gene expressions in response to bleomycin treatment was also performed. Comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. Similar responses at the RNA level between different gravity conditions were also observed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly

  1. Abietic acid inhibits UVB-induced MMP-1 expression in human dermal fibroblast cells through PPARα/γ dual activation.

    PubMed

    Jeon, Youngsic; Jung, Yujung; Youm, Jong-Kyung; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2015-02-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and consist of three isotypes: PPARα, PPARβ/δ and PPARγ. PPARs are expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, these receptors are highly studied in dermato-endocrine research, and their ligands are targets for the treatment of various skin disorders, such as photoageing and chronological ageing of skin. Intensive studies have revealed that PPARα/γ functions in photoageing and age-related inflammation by regulating matrix metalloproteinases (MMPs) via nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). However, the detailed mechanism of PPARα/γ's role in photoageing has not yet been elucidated. In this study, we confirmed that abietic acid (AA) is a PPARα/γ dual ligand and significantly decreased UVB-induced MMP-1 expression by downregulating UVB-induced MAPK signalling and downstream transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in Hs68 human dermal fibroblast cells. Treatment of cells with AA and GW6471 or bisphenol A diglycidyl ether (BADGE), PPARα or PPARγ antagonists, respectively, reversed the effect on UVB-induced MMP-1 expression and inflammatory signalling pathway activation. Taken together, our data suggest that AA acts as a PPARα/γ dual activator to inhibit UVB-induced MMP-1 expression and age-related inflammation by suppressing NF-κB and the MAPK/AP-1 pathway and can be a useful agent for improving skin photoageing. PMID:25496486

  2. Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

    PubMed Central

    Ueba, T; Nosaka, T; Takahashi, J A; Shibata, F; Florkiewicz, R Z; Vogelstein, B; Oda, Y; Kikuchi, H; Hatanaka, M

    1994-01-01

    Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression. Images PMID:8090761

  3. The expression of pluripotency genes and neuronal markers after neurodifferentiation in fibroblasts co-cultured with human umbilical cord blood mononuclear cells.

    PubMed

    Marinowic, D R; Domingues, M F; Machado, D C; DaCosta, J C

    2015-01-01

    Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation. PMID:25134818

  4. Paclitaxel inhibits cell proliferation and collagen lattice contraction via TGF-β signaling pathway in human tenon's fibroblasts in vitro.

    PubMed

    Chen, Ninghong; Guo, Dadong; Guo, Yuanyuan; Sun, Yuanyuan; Bi, Hongsheng; Ma, Xiaohua

    2016-04-15

    As an anti-microtubule agent, paclitaxel has been widely applied clinically. However, the effects of paclitaxel on human tenon's fibroblast (HTF) proliferation and migration in vitro was still unclear. In the present study, we explored the influences of paclitaxel on HTF cell proliferation, cell viability, cell cycle phase distribution under various concentrations of paclitaxel (i.e., 0, 10(-8), 10(-7), 10(-6)mol/l) via real-time cell electronic system and flow cytometry, further determined the expression of TGF-β1 and connective tissue growth factor (CTGF) after treatment with different concentrations of paclitaxel. Moreover, extra cellular matrix production and collagen lattice contraction assay were also explored. The results indicate that paclitaxel could apparently inhibit the cell viability, induces the elevation of S and G2/M phases of HTFs, and downregulates the expression of both TGF-β1 and CTGF. Meanwhile, the levels of fibronectin extra domain A (EDA), collagen and collagen lattice contraction were apparently reduced after treatment with paclitaxel. Overall, paclitaxel could apparently inhibit the proliferation of HTFs and leads to cell cycle arrest at both S and G2/M phases, attenuates the generation of collagen and collagen lattice contraction, decreases the expressions of TGF-β1, CTGF and fibronectin EDA. The inhibitory mechanism of paclitaxel on HTFs is involved in TGF-β1 signaling pathway.

  5. Fibroblast growth factor-23 induces cellular senescence in human mesenchymal stem cells from skeletal muscle.

    PubMed

    Sato, Chisato; Iso, Yoshitaka; Mizukami, Takuya; Otabe, Koji; Sasai, Masahiro; Kurata, Masaaki; Sanbe, Takeyuki; Sekiya, Ichiro; Miyazaki, Akira; Suzuki, Hiroshi

    2016-02-12

    Although muscle wasting and/or degeneration are prevalent in patients with chronic kidney disease, it remains unknown whether FGF-23 influences muscle homeostasis and regeneration. Mesenchymal stem cells (MSCs) in skeletal muscle are distinct from satellite cells and have a known association with muscle degeneration. In this study we sought to investigate the effects of FGF-23 on MSCs isolated from human skeletal muscle in vitro. The MSCs expressed FGF receptors (1 through 4) and angiotensin-II type 1 receptor, but no traces of the Klotho gene were detected. MSCs and satellite cells were treated with FGF-23 and angiotensin-II for 48 h. Treatment with FGF-23 significantly decreased the number of MSCs compared to controls, while treatment with angiotensin-II did not. FGF-23 and angiotensin-II both left the cell counts of the satellite cells unchanged. The FGF-23-treated MSCs exhibited the senescent phenotype, as judged by senescence-associated β-galactosidase assay, cell morphology, and increased expression of p53 and p21 in western blot analysis. FGF-23 also significantly altered the gene expression of oxidative stress regulators in the cells. In conclusion, FGF-23 induced premature senescence in MSCs from skeletal muscle via the p53/p21/oxidative-stress pathway. The interaction between the MSCs and FGF-23 may play a key role in the impaired muscle reparative mechanisms of chronic kidney disease. PMID:26797283

  6. Fibroblast growth factor-23 induces cellular senescence in human mesenchymal stem cells from skeletal muscle.

    PubMed

    Sato, Chisato; Iso, Yoshitaka; Mizukami, Takuya; Otabe, Koji; Sasai, Masahiro; Kurata, Masaaki; Sanbe, Takeyuki; Sekiya, Ichiro; Miyazaki, Akira; Suzuki, Hiroshi

    2016-02-12

    Although muscle wasting and/or degeneration are prevalent in patients with chronic kidney disease, it remains unknown whether FGF-23 influences muscle homeostasis and regeneration. Mesenchymal stem cells (MSCs) in skeletal muscle are distinct from satellite cells and have a known association with muscle degeneration. In this study we sought to investigate the effects of FGF-23 on MSCs isolated from human skeletal muscle in vitro. The MSCs expressed FGF receptors (1 through 4) and angiotensin-II type 1 receptor, but no traces of the Klotho gene were detected. MSCs and satellite cells were treated with FGF-23 and angiotensin-II for 48 h. Treatment with FGF-23 significantly decreased the number of MSCs compared to controls, while treatment with angiotensin-II did not. FGF-23 and angiotensin-II both left the cell counts of the satellite cells unchanged. The FGF-23-treated MSCs exhibited the senescent phenotype, as judged by senescence-associated β-galactosidase assay, cell morphology, and increased expression of p53 and p21 in western blot analysis. FGF-23 also significantly altered the gene expression of oxidative stress regulators in the cells. In conclusion, FGF-23 induced premature senescence in MSCs from skeletal muscle via the p53/p21/oxidative-stress pathway. The interaction between the MSCs and FGF-23 may play a key role in the impaired muscle reparative mechanisms of chronic kidney disease.

  7. The effect of platelet-derived growth factor on cell division and glycosaminoglycan synthesis by human skin and scar fibroblasts.

    PubMed

    Savage, K; Siebert, E; Swann, D

    1987-07-01

    The effect of platelet-derived growth factor (PDGF) on cell division and glycosaminoglycan (GAG) synthesis by fibroblasts isolated from skin and scar was measured. We found that PDGF stimulates cell division more efficiently in normal skin fibroblasts than in scar fibroblasts and decreases GAG synthesis in skin and scar fibroblasts. Using a 4-h pulse label with [3H]thymidine ([3H]Thd) following a 20-h incubation of confluent monolayer cultures with 0-5 units PDGF/ml Dulbecco's modified Eagle's medium, we found a concentration-dependent increase in [3H]Thd incorporation. After incubation of fibroblasts with [3H]glucosamine and 35SO4 in the presence or absence of PDGF, labeled constituents were isolated from the extracellular, pericellular, and cellular fractions by pronase digestion and column chromatography on Sepharose CL4B or DEAE-cellulose and analyzed by cellulose acetate electrophoresis. The presence of PDGF decreased the total amount of 35S incorporated into macromolecules by skin and scar fibroblasts and resulted in an altered distribution of labeled GAGs. Dermal fibroblasts exposed to PDGF for 24 h incorporated a greater percentage of radiolabeled 35S into dermatan sulfate prime (DS') and less into dermatan sulfate (DS) in the extracellular fractions and a greater percentage of 35S into heparan sulfate (HS) in the pericellular fractions than did parallel cultures grown in the absence of PDGF. It is thought than PDGF may have an effect on scar formation by increasing the fibroblast population in the wound tissue and by affecting the total amount and types of matrix components synthesized.

  8. CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion.

    PubMed

    Liu, Ju-Fang; Hou, Sheng-Mou; Tsai, Chun-Hao; Huang, Chun-Yin; Hsu, Chin-Jung; Tang, Chih-Hsin

    2013-05-01

    CCN4 is a cysteine-rich protein that belongs to the Cyr61, CTGF, Nov family of matricellular proteins. Here, we investigated the intracellular signaling pathways involved in CCN4-induced vascular cell adhesion molecule-1 expression in human osteoarthritis synovial fibroblasts. Stimulation of OASFs with CCN4 induced VCAM-1 expression. CCN4-induced VCAM-1 expression was attenuated by αvβ5 or α6β1 integrin antibody, Syk inhibitor, PKCδ inhibitor (rottlerin), JNK inhibitor (SP600125), and AP-1 inhibitors (curcumin and tanshinone). Stimulation of cells with CCN4 increased Syk, PKCδ, and JNK activation. Treatment of OASFs with CCN4 also increased c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element in the VCAM-1 promoter. Moreover, up-regulation of VCAM-1 increased the adhesion of monocytes to OASF monolayers, and this adhesion was attenuated by transfection with a VCAM-1 siRNA. Our results suggest that CCN4 increases VCAM-1 expression in human OASFs via the Syk, PKCδ, JNK, c-Jun, and AP-1 signaling pathways. The CCN4-induced VCAM-1 expression promoted monocyte adhesion to human OASFs. PMID:23313051

  9. Arctiin induces an UVB protective effect in human dermal fibroblast cells through microRNA expression changes.

    PubMed

    Lee, Ghang Tai; Cha, Hwa Jun; Lee, Kwang Sik; Lee, Kun Kook; Hong, Jin Tae; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Bae, Seunghee

    2014-03-01

    Ultraviolet (UV) radiation induces severe alterations in the molecular and cellular components of normal human dermal fibroblast (NHDF) cells by disrupting many intracellular transduction cascades. Although UV responses have been well documented at the genome and proteome levels, UV protective effects have not been elucidated at these levels. The aim of the present study was to demonstrate that arctiin, a phytochemical isolated from the plant Arctium lappa, induced a protective effect against UVB radiation by changing microRNA (miRNA) expression profiles. Using flow cytometry, and water-soluble tetrazolium salt (WST-1)-based cell viability, wound healing, and DNA repair assays we showed that pretreatment with arctiin prior to UVB irradiation reduced UVB-induced apoptosis, cell migration defects, and DNA damage in NHDF cells. It was also found that arctiin‑induced UVB protection is associated with altered miRNA expression profiles. Bioinformatic analysis revealed that the deregulated miRNAs were functionally involved in mitogen-activated protein kinase (MAPK) signaling and cancer signaling pathways. The results suggest that arctiin acts as a UVB protective agent by altering specific miRNA expression in NHDF cells.

  10. Arctiin induces an UVB protective effect in human dermal fibroblast cells through microRNA expression changes.

    PubMed

    Lee, Ghang Tai; Cha, Hwa Jun; Lee, Kwang Sik; Lee, Kun Kook; Hong, Jin Tae; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Bae, Seunghee

    2014-03-01

    Ultraviolet (UV) radiation induces severe alterations in the molecular and cellular components of normal human dermal fibroblast (NHDF) cells by disrupting many intracellular transduction cascades. Although UV responses have been well documented at the genome and proteome levels, UV protective effects have not been elucidated at these levels. The aim of the present study was to demonstrate that arctiin, a phytochemical isolated from the plant Arctium lappa, induced a protective effect against UVB radiation by changing microRNA (miRNA) expression profiles. Using flow cytometry, and water-soluble tetrazolium salt (WST-1)-based cell viability, wound healing, and DNA repair assays we showed that pretreatment with arctiin prior to UVB irradiation reduced UVB-induced apoptosis, cell migration defects, and DNA damage in NHDF cells. It was also found that arctiin‑induced UVB protection is associated with altered miRNA expression profiles. Bioinformatic analysis revealed that the deregulated miRNAs were functionally involved in mitogen-activated protein kinase (MAPK) signaling and cancer signaling pathways. The results suggest that arctiin acts as a UVB protective agent by altering specific miRNA expression in NHDF cells. PMID:24398562

  11. 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

  12. The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

    PubMed

    Opländer, Christian; Cortese, Miriam M; Korth, Hans-Gert; Kirsch, Michael; Mahotka, Csaba; Wetzel, Wiebke; Pallua, Norbert; Suschek, Christoph V

    2007-09-01

    Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.

  13. In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

    PubMed

    Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

    2008-02-01

    Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p < 0.05), while in the case of HBF an equal or even higher number of cells adhered to PE was observed. The number of adhered osteoblast cells was almost equal and in some days even higher than the number of adhered cells on negative control sample, while in the case of fibroblast this difference was significantly higher than TPS (p < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were observed to be undergoing cell division. These findings indicate that beta-TCP/HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast

  14. Neoplastic transformation of human lung fibroblast MRC-5 SV2 cells induced by benzo[a]pyrene and confluence culture.

    PubMed

    Zhu, Huijun; Gooderham, Nigel

    2002-08-15

    Benzo[a]pyrene (BaP) is potent rodent carcinogen and a reputed human carcinogen. Although much is known about its metabolic activation leading to DNA damage, the mechanisms of its actions are not as well understood at a cellular level. In addressing this, we have established an in vitro model that follows the progression toward neoplastic transformation induced by BaP. The model uses immortal nontumorigenic human lung fibroblast MRC-5 SV2 cells as effectors, cocultured with a metabolically competent human lymphoblastoid line h1A1v2 (activator cells). Treatment of the coculture with BaP for 48 h induced a dose-dependent decrease in cloning efficiency of the MRC-5 SV2 cells; nevertheless, cultures continued to progress to confluence. At prolonged confluence culture (day 11), an elevation in the proportion of G2-M phase cells was detected by flow cytometry. By day 15, the G2-M phase peak disappeared, accordant with the appearance of a population with DNA content greater than the cells in G2-M phase. These changes in DNA ploidy were coincident with changes in morphology, specifically the appearance of enlarged and irregular-shaped nuclei. Confluence culture of BaP-treated MRC-5 SV2 cells for more than 2 weeks resulted in cell death; however, a few colonies survived the crisis to reach confluence again after an additional 10-14 days. The number of death-resistant colonies was proportional to the dose of BaP, with the majority of the cells exhibiting abnormal morphology. The degree of morphological change progressively increased with successive rounds of confluence. Cells that survived three rounds of confluence adopted a vastly different morphology, becoming polygonal, spindle, or other irregular-shaped, and acquired the ability to form large dense clumps that grew in an anchorage-independent manner. In parallel experiments, treatment with the vehicle alone (DMSO) resulted in substantially less death resistance and lower numbers of high-density clumps. Our studies

  15. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

    PubMed Central

    Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258

  16. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

    PubMed

    Mehalick, Leslie A; Poulsen, Christopher; Fischer, Carol L; Lanzel, Emily A; Bates, Amber M; Walters, Katherine S; Cavanaugh, Joseph E; Guthmiller, Janet M; Johnson, Georgia K; Wertz, Philip W; Brogden, Kim A

    2015-12-01

    Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2-10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

  17. UVA-induced ROS generation inhibition by Oenothera paradoxa defatted seeds extract and subsequent cell death in human dermal fibroblasts.

    PubMed

    Jaszewska, Edyta; Soin, Magdalena; Filipek, Agnieszka; Naruszewicz, Marek

    2013-09-01

    UVA radiation stimulates the production of reactive oxygen species (ROS), which react with lipids, proteins and other intracellular molecules leading to oxidative stress, cellular damage and ultimately cell death. There is, therefore, a growing need for substances exhibiting antioxidant activity, which may support repair mechanisms of the skin. This study evaluates the protective effect of the aqueous Oenothera paradoxa Hudziok defatted seeds extract, rich in polyphenolic compounds, against UVA (25 and 50J/cm(2))-induced changes in normal human dermal fibroblasts (NHDFs). The tested extract (0.1-10μg/ml) has decreased, in a concentration-dependent fashion, the UVA-induced release of lactate dehydrogenase (LDH) into the culture medium, the ROS production (with the use of 2',7'-dichlorodihydrofluorescein diacetate) and lipid peroxidation (utilizing redox reactions with ferrous ions) as compared to the control cells (incubated without the extract). Moreover, the extract increased the number of viable (calcein positive) cells decreasing the number of cells in late apoptosis (annexin V-FITC and propidium iodide positive). Thus our results show that O. paradoxa defatted seeds extract may be beneficial for the prevention of UVA skin damage.

  18. Cationic Glycopolymers for the Delivery of pDNA to Human Dermal Fibroblasts and Rat Mesenchymal Stem Cells

    PubMed Central

    Kizjakina, Karina; Bryson, Joshua M.; Grandinetti, Giovanna; Reineke, Theresa M.

    2014-01-01

    Progenitor and pluripotent cell types offer promise as regenerative therapies but transfecting these sensitive cells has proven difficult. Herein, a series of linear trehalose-oligoethyleneamine “click” copolymers were synthesized and examined for their ability to deliver plasmid DNA (pDNA) to two progenitor cell types, human dermal fibroblasts (HDFn) and rat mesenchymal stem cells (RMSC). Seven polymer vehicle analogs were synthesized in which three parameters were systematically varied: the number of secondary amines (4–6) within the polymer repeat unit (Tr433, Tr530, and Tr632), the end group functionalities [PEG (Tr4128PEG-a, Tr4118PEG-b), triphenyl (Tr4107-c), or azido (Tr499-d)], and the molecular weight (degree of polymerization of about 30 or about 100) and the biological efficacy of these vehicles was compared to three controls: Lipofectamine 2000, JetPEI, and Glycofect. The trehalose polymers were all able to bind and compact pDNA polyplexs, and promote pDNA uptake and gene expression [luciferase and enhanced green fluorescent protein (EGFP)] with these primary cell types and the results varied significantly depending on the polymer structure. Interestingly, in both cell types, Tr433 and Tr530 yielded the highest luciferase gene expression. However, when comparing the number of cells transfected with a reporter plasmid encoding enhanced green fluorescent protein, Tr433 and Tr4107-c yielded the highest number of HDFn cells positive for EGFP. Interestingly, with RMSC, all of the higher molecular weight analogs (Tr4128PEG-a, Tr4118PEG-b, Tr4107-c, Tr499-d) yielded high percentages of cells positive for EGFP (30–40%). PMID:22138032

  19. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    NASA Astrophysics Data System (ADS)

    Laranjeira, Marta S.; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-04-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol-gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.

  20. Human breast cancer cells contain a phosphoramidon-sensitive metalloproteinase which can process exogenous big endothelin-1 to endothelin-1: a proposed mitogen for human breast fibroblasts.

    PubMed Central

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

    1995-01-01

    Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen. PMID:7880721

  1. Fermented Acanthopanax koreanum Root Extract Reduces UVB- and H2O2-Induced Senescence in Human Skin Fibroblast Cells.

    PubMed

    Park, Min-Ja; Bae, Young-Seuk

    2016-07-28

    The present study assessed the effects of an aqueous extract of Acanthopanax koreanum root (AE) and of AE following fermentation by lactic acid bacteria (Lactobacillus plantarum and Bifidobacterium bifidum) (AEF) on human skin fibroblast HS68 cells exposed to ultraviolet B (UVB) irradiation and oxidative stress. AEF effectively antagonized the senescence-associated β-galactosidase staining and upregulation of p53 and p21(Cip1/WAF1) induced by UVB or H2O2 treatment in HS68 cells. It also exhibited excellent antioxidant activities in radical scavenging assays and reduced the intracellular level of reactive oxygen species induced by UVB or H2O2 treatment. The antioxidant and antisenescent activities of AEF were greater than those of nonfermented A. koreanum extract. AEF significantly repressed the UVB- or H2O2-induced activities of matrix metalloproteinase (MMP)-1 and -3, overexpression of MMP-1, and nuclear factor κB (NF-κB) activation. This repression of NF-κB activation and MMP-1 overexpression was attenuated by a mitogen-activated protein kinase activator, suggesting that this AEF activity was dependent on this signaling pathway. Taken together, these data indicated that AEF-mediated antioxidant and anti-photoaging activities may produce anti-wrinkle effects on human skin. PMID:27090187

  2. Effect of myrrh oil on IL-1beta stimulation of NF-kappaB activation and PGE(2) production in human gingival fibroblasts and epithelial cells.

    PubMed

    Tipton, D A; Hamman, N R; Dabbous, M Kh

    2006-03-01

    Anecdotal and scientific evidence suggest that myrrh oil (MO) has anti-inflammatory properties. Subtoxic MO levels decrease interleukin (IL)-1beta-stimulated production of the inflammatory cytokine IL-6 by human gingival fibroblasts, but not epithelial cells. IL-1beta upregulates IL-6 via PGE(2), and via NF-kappaB, a transcription factor for many inflammatory mediator genes. NF-kappaB is inhibited by sesquiterpene compounds (from plants other than myrrh). This study determined MO effect on IL-1beta-stimulated PGE(2) production and NF-kappaB activation in gingival fibroblasts and epithelial cells. Cells were preincubated with MO, exposed to IL-1beta, cytoplasmic and nuclear fractions were isolated, and activated NF-kappaB was measured using an ELISA-based assay. IL-1beta increased nuclear activated NF-kappaB levels in fibroblasts and epithelial cells [10- and 2.5-fold over controls, respectively (p=0.0001)], and these increases were not significantly affected by MO. PGE(2) was measured in cell supernatants by ELISA, after preincubation with MO and exposure to IL-1beta. MO inhibited IL-1beta-stimulated PGE(2) production by fibroblasts (p=0.001), but not epithelial cells. The data suggest that gingival epithelial cells and fibroblasts may differ in the magnitude of NF-kappaB activation after IL-1beta stimulation, and that MO inhibition of IL-1beta-stimulated IL-6 production in fibroblasts is due in part to inhibition of PGE(2), but not NF-kappaB activation. (Supported by NIDCR DE-0725.). PMID:16112536

  3. Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling

    PubMed Central

    Correll, Kelly; Schiel, John A.; Finigan, Jay H.; Prekeris, Rytis; Mason, Robert J.

    2014-01-01

    There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS. PMID:24748602

  4. Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

    SciTech Connect

    Marianne B. Sowa; Wilfried Goetz; Janet E. Baulch; Dinah N. Pyles; Jaroslaw Dziegielewski; Susannah Yovino; Andrew R. Snyder; Sonia M. de Toledo; Edouard I. Azzam; William F. Morgan

    2008-06-30

    Purpose: To investigate radiation induced bystander responses and to determine the role of gap junction intercellular communication and the radiation environment in propagating this response. Materials and Methods: We use medium transfer and targeted irradiation to examine radiation induced bystander effects in primary human fibroblast (AG1522) and human colon carcinoma (RKO36) cells. We examined the effect of variables such as gap junction intercellular communication, linear energy transfer (LET), and the role of the radiation environment in non-targeted responses. Endpoints included clonogenic survival, micronucleus formation and foci formation at histone 2AX over doses ranging from 10 to 100 cGy. Results: The results show no evidence of a low-LET radiation induced bystander response for the endpoints of clonogenic survival and induction of DNA damage. Nor do we see evidence of a high-LET, Fe ion radiation (1 GeV/n) induced bystander effect. However, direct comparison for 3.2 MeV α-particle exposures showed a statistically significant medium transfer bystander effect for this high-LET radiation. Conclusions: From our results, it is evident that there are many confounding factors influencing bystander responses as reported in the literature. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

  5. Accumulation of distinct prelamin A variants in human diploid fibroblasts differentially affects cell homeostasis

    SciTech Connect

    Candelario, Jose; Borrego, Stacey; Reddy, Sita; Comai, Lucio

    2011-02-01

    Lamin A is a component of the nuclear lamina that plays a major role in the structural organization and function of the nucleus. Lamin A is synthesized as a prelamin A precursor which undergoes four sequential post-translational modifications to generate mature lamin A. Significantly, a large number of point mutations in the LMNA gene cause a range of distinct human disorders collectively known as laminopathies. The mechanisms by which mutations in lamin A affect cell function and cause disease are unclear. Interestingly, recent studies have suggested that alterations in the normal lamin A pathway can contribute to cellular dysfunction. Specifically, we and others have shown, at the cellular level, that in the absence of mutations or altered splicing events, increased expression of wild-type prelamin A results in a growth defective phenotype that resembles that of cells expressing the mutant form of lamin A, termed progerin, associated with Hutchinson-Gilford Progeria syndrome (HGPS). Remarkably, the phenotypes of cells expressing elevated levels of wild-type prelamin A can be reversed by either treatment with farnesyltransferase inhibitors or overexpression of ZMPSTE24, a critical prelamin A processing enzyme, suggesting that minor increases in the steady-state levels of one or more prelamin A intermediates is sufficient to induce cellular toxicity. Here, to investigate the molecular basis of the lamin A pathway toxicity, we characterized the phenotypic changes occurring in cells expressing distinct prelamin A variants mimicking specific prelamin A processing intermediates. This analysis demonstrates that distinct prelamin A variants differentially affect cell growth, nuclear membrane morphology, nuclear distribution of lamin A and the fundamental process of transcription. Expression of prelamin A variants that are constitutively farnesylated induced the formation of lamin A aggregates and dramatic changes in nuclear membrane morphology, which led to reduced

  6. 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.

  7. Adipose-derived stem cells promote human dermal fibroblast function and increase senescence-associated β‑galactosidase mRNA expression through paracrine effects.

    PubMed

    Shen, Xiao; Du, Yunpeng; Shen, Weimin; Xue, Bin; Zhao, Yu

    2014-12-01

    Adipose‑derived stem cells (ADSCs) are known to secrete various cytokines, which affect fibroblast function through paracrine effects. In the present study, the paracrine effects of ADSCs on the function and senescence of young and aged human dermal fibroblasts (HDFs) were investigated in vitro. ADSCs and HDFs were isolated from healthy donors and flow cytometry was used for immunophenotype identification. ADSCs were co‑cultured with young or aged human dermal fibroblasts in Transwell plates, and control groups were established accordingly. Cellular proliferation was measured by an MTT assay. Type I collagen, matrix metalloproteinase‑1 (MMP‑1) and senescence-associated β‑galactosidase (SA‑β‑GAL) mRNA expression were measured by quantitative polymerase chain reaction. It was identified that ADSCs promoted proliferation of co‑cultured HDFs and induced increased expression of type I collagen and decreased expression of MMP‑1. The co‑cultured HDFs exhibited increased expression of SA‑β‑GAL. These results demonstrated that ADSCs improve fibroblast function through paracrine effects. The increased expression of SA‑β‑GAL indicated an accelerated aging process. It is proposed that ADSCs may improve fibroblast function, but not reverse the age status in vitro.

  8. Cholesteatoma Fibroblasts Promote Epithelial Cell Proliferation through Overexpression of Epiregulin

    PubMed Central

    Yoshikawa, Mamoru; Kojima, Hiromi; Yaguchi, Yuichiro; Okada, Naoko; Saito, Hirohisa; Moriyama, Hiroshi

    2013-01-01

    To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b). To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial–mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma. PMID:23826119

  9. Blockade of Ca2+-activated K+ channels inhibits proliferation of human endothelial cells induced by basic fibroblast growth factor.

    PubMed

    Wiecha, J; Münz, B; Wu, Y; Noll, T; Tillmanns, H; Waldecker, B

    1998-01-01

    Basic fibroblast growth factor (bFGF) exerts angiogenic and mitogenic properties in human tissue. Since changes in ion currents modulate essential Ca2+-dependent intracellular pathways in endothelial cells, we have investigated a possible contribution of Ca2+-activated K+ channels (BKCa) on bFGF-induced endothelial cell proliferation. The patch-clamp technique was used to identify BKCa and to study their modulation by bFGF in cultured endothelial cells of human umbilical cord veins (HUVEC). Cell counts of HUVEC were carried out on different days to analyze bFGF-induced cell proliferation and its influence by the specific BKCa blocker iberiotoxin (IBX). Using single-channel recordings, we found characteristic BKCa with a single-channel slope conductance of 170.3 +/- 2.1 pS (n = 7), half-maximal activation at internal pCa = 5.7 (n = 5; test potential: 80 mV), and dose-dependent block by IBX (25-100 nmol/l). In cell-attached patches bFGF (50 ng/ml) caused a significant increase in the open-state probability (NPo) after 6 min at test potentials of 80 and 100 mV (n = 28; p < 0.001), respectively, which lasted up to 30 min. After preincubation with pertussis toxin (100 ng/ml; 4 h) bFGF superfusion did not cause a significant increase in BKCa activity until 25 min had passed (n = 20; p < 0.01). Addition of 100 nmol/l IBX to the pipette solution caused a total block of BKCa within 2 min in cell-attached patches, whereas bFGF (50 ng/ml) was not able to activate BKCa. When incubated with IBX (25-100 nmol/l) every 2 days, bFGF-induced proliferation of HUVEC was significantly decreased by 50 (-41%) and 100 nmol/l (-50%) IBX (n = 5; p < 0.001) after 7 days. We conclude that activation of BKCa by bFGF may play an important role in bFGF-induced proliferation of human endothelial cells and thus might be important in the process of angiogenesis and vascular remodelling.

  10. BAY 11-7085 induces glucocorticoid receptor activation and autophagy that collaborate with apoptosis to induce human synovial fibroblast cell death

    PubMed Central

    Relic, Biserka; Charlier, Edith; Deroyer, Celine; Malaise, Olivier; Neuville, Sophie; Desoroux, Aline; Gillet, Philippe; de Seny, Dominique; Malaise, Michel G.

    2016-01-01

    Inhibition of proapoptotic pathways in synovial fibroblasts is one of the major causes of synovial proliferation and hyperplasia in rheumatic diseases. We have shown previously that NF-κB inhibitor BAY 11-7085, through inactivation of PPAR-γ, induces apoptosis in human synovial fibroblasts. In this work we showed that BAY 11-7085 induced autophagy that preceded BAY 11-7085-induced apoptosis. Of interest, BAY 11-7085 induced Serine 211 phosphorylation and degradation of glucocorticoid receptor (GR). Glucocorticoid prednisolone induced both activation and degradation of GR, as well as autophagy in synovial fibroblasts. BAY 11-7085-induced cell death was significantly decreased with glucocorticoid inhibitor mifepristone and with inhibitors of autophagy. Both BAY 11-7085-induced autophagy and GR activation were down regulated with PPAR-γ agonist, 15d-PGJ2 and MEK/ERK inhibitor UO126. Inhibition of autophagy markedly decreased endogenous and BAY 11-7085-induced ERK phosphorylation, suggesting a positive feed back loop between ERK activation and autophagy in synovial fibroblasts. Co-transfection of MEK1 with PPAR-γ1 in HEK293 cells caused known inhibitory phosphorylation of PPAR-γ1 (Serine 112) and enhanced GR degradation, in the absence or presence of prednisolone. Furthermore, GR was both phosphorylated on Serine 211 and down regulated in synovial fibroblasts during serum starvation induced autophagy. These results showed that GR activation and PPAR-γ inactivation mediated BAY 11-7085-induced autophagy. PMID:26993765

  11. Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

    PubMed Central

    Sando, G N; Ma, G P; Lindsley, K A; Wei, Y P

    1990-01-01

    We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing. PMID:2150334

  12. Analysis of unrejoined chromosomal breakage in human fibroblast cells exposed to low- and high-LET radiation

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Furusawa, Yoshiya; George, Kerry; Kawata, Tetsuya; Cucinotta, Francis A.

    2002-01-01

    Reported studies of DNA breakage induced by radiation of various qualities have generally shown a higher fraction of unrejoined residual breaks after high-LET exposure. This observation is supported by the argument that high-LET radiation induced DNA breaks that are more complex in nature and, thus, less likely to be repaired. In most cases the doses used in these studies were very high. We have studied unrejoined chromosome breaks by analyzing chromosome aberrations using a fluorescence in situ hybridization (FISH) technique with a combination of whole chromosome specific probes and probes specific for the telomere region of the chromosomes. Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon Si, or with Fe ions at either 200 and 500 MeV/nucleon, and were allowed to repair at 37 degrees C for 24 hours after exposure. A chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Results showed that the frequency of unrejoined chromosome breaks was higher after high-LET radiation, and the ratio of unrejoined to misrejoined chromosome breaks increased steadily with LET up a peak value at 440 keV/microm.

  13. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

    PubMed Central

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice. PMID:26091287

  14. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

    PubMed

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

  15. 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

  16. 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.

  17. Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans.

    PubMed

    Helgeland, K; Nordby, O

    1993-05-01

    A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired. PMID:8496779

  18. Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage.

    PubMed

    Varani, James; Perone, Patricia; Spahlinger, Diana M; Singer, Lisa M; Diegel, Kelly L; Bobrowski, Walter F; Dunstan, Robert

    2007-08-01

    Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin.

  19. Autophagy in human skin fibroblasts: Comparison between young and aged cells and evaluation of its cellular rhythm and response to Ultraviolet A radiation.

    PubMed

    Pernodet, Nadine; Dong, Kelly; Pelle, Edward

    2016-01-01

    Autophagic mechanisms play critical roles in cell maintenance. Damaged organelles that are not removed by autophagosomes, which act by engulfing and degrading these cellular components, have been linked to various pathologies. Recently, the progression of aging has also been correlated to a compromised autophagic response. Here, we report for the first time a significant reduction in autophagic levels in synchronized aged normal human skin fibroblasts as compared to young fibroblasts. We measured a 77.9% reduction in autophagy as determined by reverse transcription-polymerase chain reaction for LC3B expression, a microtubule-associated protein correlated to late stage autophagosome formation. In addition, we visualized these same changes by immunocytofluorescence with antibodies directed against LC3B. By harvesting synchronized, as well as unsynchronized cells over time, we were also able to measure for the first time a nighttime peak in autophagy that was present in young but absent in aged fibroblasts. Finally, since human skin is constantly subjected to environmentally induced oxidative stress from sunlight, we exposed fibroblasts to 10 J/cm2 ultraviolet A and found, in good agreement with current literature, not only that irradiation could partially reactivate autophagy in the aged cells, but also that this increase was phase shifted earlier from its endogenous temporal pattern because of its loss of synchronization with circadian rhythm.

  20. [Lysosomal glycosidase activity in cultured human fibroblasts].

    PubMed

    Beliaeva, I D; Ivleva, T S; Vidershaĭn, G Ia

    1984-11-01

    A study was made of the activity of 3 lysosomal glycosidases -beta-D-galactosidase (K. P. 3.2.1.23), alpha-L-fucosidase (K. P. 3.2.1.51), N-acetyl-beta-D-hexosoaminidase (K. P. 3.2.1.52) depending on the time after subcultivation and duration of the passage of human skin embryonal and postembryonal fibroblasts. It was established that changes in the specific activity of the enzymes should be calculated with reference to the cell rather than to protein whose amount might vary considerably. It was also found that for measuring the specific activity of enzymes, of great importance are the procedures of cell removal from the base layer (by mechanical scraping off or by trypsin solution) and the regimen of the homogenization of cell preparations.

  1. 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

  2. Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

    PubMed

    Schafer, I A; Jamieson, A M; Petrelli, M; Price, B J; Salzman, G C

    1979-01-01

    Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

  3. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

    PubMed Central

    Poulsen, Christopher; Mehalick, Leslie A.; Fischer, Carol L.; Lanzel, Emily A.; Bates, Amber M.; Walters, Katherine S.; Cavanaugh, Joseph E.; Guthmiller, Janet M.; Johnson, Georgia K.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 µM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propridium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2 to 10.0 µM long-chain bases and GML were not cytotoxic; 40.0 to 80.0 µM long-chain bases, but not GML, were cytotoxic; and 80.0 µM long-chain bases induced cellular damage and death in less than 20 minutes. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections. PMID:26005054

  4. 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

  5. The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts

    SciTech Connect

    Paulsen, Michelle T.; Ljungman, Mats . E-mail: ljungman@umich.edu

    2005-11-15

    Juglone (5-hydroxy-1,4-naphtoquinone) is a natural toxin produced by walnut trees. In this study we show that juglone differentially reduces viability of human cells in culture. Normal fibroblast were found to be especially sensitive to juglone and lost viability primarily through a rapid apoptotic and necrotic response. This response may have been triggered by DNA damage since juglone induced a rapid and strong phosphorylation of H2AX in all phases of the cell cycle. Furthermore, juglone inhibits mRNA synthesis in human fibroblasts in a dose-dependent manner. Surprisingly, juglone caused a drastic reduction of the basal level of p53 in human fibroblasts and this loss could not be fully rescued by proteasome and calpain I inhibitors. However, when cells were pretreated with UV light or ionizing radiation, juglone was not able to reduce the cellular levels of activated p53. Our results show that juglone has multiple effects on cells such as the induction of DNA damage, inhibition of transcription, reduction of p53 protein levels and the induction of cell death.

  6. Preclinical safety studies on autologous cultured human skin fibroblast transplantation.

    PubMed

    Zeng, Wei; Zhang, Shuying; Liu, Dai; Chai, Mi; Wang, Jiaqi; Zhao, Yuming

    2014-01-01

    Recently, FDA approved the clinical use of autologous fibroblasts (LAVIV™) for the improvement of nasolabial fold wrinkles in adults. The use of autologous fibroblasts for the augmentation of dermal and subcutaneous defects represents a potentially exciting natural alternative to the use of other filler materials for its long-term corrective ability and absence of allergic adverse effects proved by clinical application. However, compared to the clinical evidence, preclinical studies are far from enough. In this study, human skin-derived fibroblasts were cultured and expanded for both in vitro and in vivo observations. In vitro, the subcultured fibroblasts were divided into two groups. One set of cells underwent cell cycle and karyotype analysis at passages 5 and 10. The second group of cells was cocultured in medium with different concentrations of human skin extract D for the measurement of collagen concentration and cell count. In vivo, the subcultured fibroblasts were injected into nude mice subcutaneously. Biopsies were taken for morphology observation and specific collagen staining at 1, 2, and 3 months after injection. The results in vitro showed no significant differences in cell cycle distribution between passages 5 and 10. Cell proliferation and secretion were inhibited as the concentration of extract D increased. In vivo, the fibroblasts were remarkably denser on the experimental side with no dysplastic cells. Mitotic cells were easily observed at the end of the first month but were rare at the end of the third month. Type III collagen was detected at the end of the first month, while collagen type I was positive at the end of the second month. The content of both collagens increased as time passed. The above results indicated that the use of the autologous fibroblasts was safe, providing a basic support for clinical use of fibroblasts.

  7. In Vitro Study of Surface Modified Poly(ethylene glycol)-Impregnated Sintered Bovine Bone Scaffolds on Human Fibroblast Cells

    PubMed Central

    Pramanik, Sumit; Ataollahi, Forough; Pingguan-Murphy, Belinda; Oshkour, Azim Ataollahi; Osman, Noor Azuan Abu

    2015-01-01

    Scaffold design from xenogeneic bone has the potential for tissue engineering (TE). However, major difficulties impede this potential, such as the wide range of properties in natural bone. In this study, sintered cortical bones from different parts of a bovine-femur impregnated with biodegradable poly(ethylene glycol) (PEG) binder by liquid phase adsorption were investigated. Flexural mechanical properties of the PEG-treated scaffolds showed that the scaffold is stiffer and stronger at a sintering condition of 1000°C compared with 900°C. In vitro cytotoxicity of the scaffolds evaluated by Alamar Blue assay and microscopic tests on human fibroblast cells is better at 1000°C compared with that at 900°C. Furthermore, in vitro biocompatibility and flexural property of scaffolds derived from different parts of a femur depend on morphology and heat-treatment condition. Therefore, the fabricated scaffolds from the distal and proximal parts at 1000°C are potential candidates for hard and soft TE applications, respectively. PMID:25950377

  8. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    PubMed

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  9. Truly Incomplete and Complex Chromosomal Exchanges in Human Fibroblast Cells Exposed In Situ to Energetic Heavy Ions

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Durante, marco; Furusawa, Yoshiya; George, Kerry; Kawata, Tetsuya; Cucinotta, Francis A.

    2003-01-01

    Confluent human fibroblast cells (AG 1522) were irradiated with gamma rays, 490 MeV/nucleon Si, or with Fe ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 C for 24 hours after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. In order to verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after high-LET radiation, and consequently, the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/micron, the highest LET value in the present study. For samples exposed to 200 MeV/nucleon Fe ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique that allow identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy dose of the Fe ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges, values for which were higher than those obtained after a 6 Gy gamma exposure. After 0.7 Gy of Fe ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single Fe ion track.

  10. Effects of mechanical strain on human mesenchymal stem cells and ligament fibroblasts in a textured poly(L-lactide) scaffold for ligament tissue engineering.

    PubMed

    Kreja, Ludwika; Liedert, Astrid; Schlenker, Heiter; Brenner, Rolf E; Fiedler, Jörg; Friemert, Benedikt; Dürselen, Lutz; Ignatius, Anita

    2012-10-01

    The purpose of this study was to prove the effect of cyclic uniaxial intermittent strain on the mRNA expression of ligament-specific marker genes in human mesenchymal stem cells (MSC) and anterior cruciate ligament-derived fibroblasts (ACL-fibroblasts) seeded onto a novel textured poly(L-lactide) scaffold (PLA scaffold). Cell-seeded scaffolds were mechanically stimulated by cyclic uniaxial stretching. The expression of ligament matrix gene markers: collagen types I and III, fibronectin, tenascin C and decorin, as well as the proteolytic enzymes matrix metalloproteinase MMP-1 and MMP-2 and their tissue specific inhibitors TIMP-1 and TIMP-2 was investigated by analysing the mRNA expression using reverse transcriptase polymerase chain reaction and related to the static control. In ACL-fibroblasts seeded on PLA, mechanical load induced up-regulation of collagen types I and III, fibronectin and tenascin C. No effect of mechanical stimulation on the expression of ligament marker genes was found in undifferentiated MSC seeded on PLA. The results indicated that the new textured PLA scaffold could transfer the mechanical load to the ACL-fibroblasts and improved their ligament phenotype. This scaffold might be suitable as a cell-carrying component of ACL prostheses.

  11. Exploring long-term protection of normal human fibroblasts and epithelial cells from chemotherapy in cell culture

    PubMed Central

    Apontes, Pasha; Leontieva, Olga V.; Demidenko, Zoya N.; Li, Fengzhi; Blagosklonny, Mikhail V.

    2011-01-01

    Killing of proliferating normal cells limits chemotherapy of cancer. Several strategies to selectively protect normal cells were previously suggested. Here we further explored the protection of normal cells from cell cycle-specific chemotherapeutic agents such as mitotic inhibitors (MI). We focused on a long-term cell recovery (rather than on a short-term cell survival) after a 3-day exposure to MI (paclitaxel and nocodazole). In three normal human cell types (RPE, NKE, WI-38t cells) but not in cancer cells with mutant p53, pre-treatment with nutlin-3a, a non-genotoxic inducer of wt p53, caused G1 and/or G2 arrest, thus preventing lethal mitotic arrest caused by MI and allowing normal cells to recover after removal of MI. Rapamycin, an inhibitor of the nutrient-sensing mTOR pathway, potentiated the protective effect of nutlin-3a in normal cells. Also, a combination of rapamycin and metformin, an anti-diabetic drug, induced G1 and G2 arrest selectively in normal cells and thereby protected them from MI. A combination of metformin and rapamycin also protected normal cells in low glucose conditions, whereas in contrast it was cytotoxic for cancer cells. Based on these data and the analysis of the literature, we suggest that a rational combination of metformin and rapamycin can potentiate chemotherapy with mitotic inhibitors against cancer, while protecting normal cells, thus further increasing the therapeutic window. PMID:21447859

  12. Interactions of 1D- and 2D-layered inorganic nanoparticles with fibroblasts and human mesenchymal stem cells

    PubMed Central

    Rashkow, Jason Thomas; Talukdar, Yahfi; Lalwani, Gaurav; Sitharaman, Balaji

    2015-01-01

    Aim This study investigates the effects of tungsten disulfide nanotubes (WSNTs) and molybdenum disulfide nanoplatelets (MSNPs) on fibroblasts (NIH-3T3) and mesenchymal stem cells (MSCs) to determine safe dosages for potential biomedical applications. Materials & methods Cytotoxicity of MSNPs and WSNTs (5–300 µg/ml) on NIH-3T3 and MSCs was assessed at 6, 12 or 24 h. MSC differentiation to adipocytes and osteoblasts was assessed following treatment for 24 h. Results Only NIH-3T3 cells treated with MSNPs showed dose or time dependent increase in cytotoxicity. Differentiation markers of MSCs in treated groups were unaffected compared with untreated controls. Conclusion MSNPs and WSNTs at concentrations less than 50 µg/ml are potentially safe for treatment of fibroblasts or MSCs for up to 24 h. PMID:26080694

  13. Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsy

    SciTech Connect

    Tyrrell, R.M.; Pidoux, M.

    1986-06-01

    Repair efficiency of cultured cells may be estimated by measuring the ability of a particular cell type to support virus damaged by an appropriate agent. In this study we have compared the inactivation of ultraviolet (254 nm)-damaged herpes simplex virus in human fibroblast and epidermal keratinocyte cell lines derived from the same foreskin biopsy and found the epithelial cells to be a factor of 3 times less efficient in supporting the damaged virus. The two different cell types show comparable ultraviolet inactivation of clone-forming ability, indicating that the difference is specific to viral host cell reactivation. This study required the development of a quantitative infectious centers assay for the measurement of viral titer in human epithelial cells, a system which may be of more general application in studies of potential human carcinogens.

  14. Cytokine-mediated PGE2 expression in human colonic fibroblasts.

    PubMed

    Kim, E C; Zhu, Y; Andersen, V; Sciaky, D; Cao, H J; Meekins, H; Smith, T J; Lance, P

    1998-10-01

    We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2 production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1beta (IL-1beta; 10 ng/ml) or tumor necrosis factor-alpha (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1beta in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1beta caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 micromol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo. PMID:9755052

  15. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    PubMed

    Kirker, Kelly R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  16. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    PubMed

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes.

  17. Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes.

    PubMed Central

    Imokawa, G; Yada, Y; Morisaki, N; Kimura, M

    1998-01-01

    To clarify the paracrine linkage between human fibroblasts and melanocytes in cutaneous pigmentation, we studied the effects of human fibroblast-derived factors on the proliferation of human melanocytes. In medium conditioned for 4 days with human fibroblast culture, factors were produced that markedly stimulated DNA synthesis of human melanocytes. The stimulatory effect was higher in medium conditioned with fibroblasts from aged skin than in medium conditioned with fibroblasts from young skin, and was interrupted by inhibitors of tyrosine kinase, such as tyrphostin, genistein and herbimycin, but not by inhibitors of protein kinases C and A, such as H-7 and phloretin. The conditioned medium was also capable of activating mitogen-activated protein kinase of human melanocytes, with old fibroblasts being more effective than young ones. Analysis of factors released into the conditioned medium revealed that levels of hepatocyte growth factor (HGF) and stem cell factor (SCF) were increased in old-fibroblast-conditioned medium compared with young-fibroblast-conditioned medium. In contrast, levels of basic fibroblast growth factor (bFGF) were similar in both media. When the conditioned medium was treated with HGF antibody with or without SCF antibody, the increase in DNA synthesis by human melanocytes was decreased to 20% of the elevated level, whereas antibodies to bFGF had no effect. Analysis of the medium conditioned for 4 days after cytokine application demonstrated that, of the cytokines tested, interleukin 1alpha and tumour necrosis factor alpha are highly effective in stimulating HGF secretion by old fibroblasts. HGF and SCF, but not bFGF, were markedly increased in culture medium in the presence of IL-1alpha, and this stimulatory effect was confined to young human fibroblasts. These findings suggest that SCF and HGF derived from human fibroblasts may play a part in regulating cutaneous pigmentation during inflammation and aging. PMID:9494091

  18. Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

    PubMed

    Palma, C S; Tannous, M A; Malta, T M; Russo, E M S; Covas, D T; Picanço-Castro, V

    2013-04-02

    Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and βCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

  19. Engineering human neo-tendon tissue in vitro with human dermal fibroblasts under static mechanical strain.

    PubMed

    Deng, Dan; Liu, Wei; Xu, Feng; Yang, Yang; Zhou, Guangdong; Zhang, Wen Jie; Cui, Lei; Cao, Yilin

    2009-12-01

    Proper cell source is one of the key issues for tendon engineering. Our previous study showed that dermal fibroblasts could be used to successfully engineer tendon in vivo and tenocytes could engineer neo-tendon in vitro with static strain. This study further investigated the possibility of engineering human neo-tendon tissue in vitro using dermal fibroblasts. Human dermal fibroblasts were seeded on polyglycolic acid (PGA) fibers pre-fixed on a U-shape as a mechanical loading group, or simply cultured in a dish as a tension-free group. In addition, human tenocytes were also seeded on PGA fibers with tension as a comparison to human dermal fibroblasts. The results showed that human neo-tendon tissue could be generated using dermal fibroblasts during in vitro culture under static strain and the tissue structure became more mature with the increase of culture time. Longitudinally aligned collagen fibers and spindle shape cells were observed histologically and collagen fibril diameter and tensile strength increased with time and reached a peak at 14 weeks. In contrast, the dermal fibroblast-PGA constructs failed to form neo-tendon, but formed disorganized fibrous tissue in tension-free condition with significantly weaker strength and poor collagen fiber formation. Interestingly, neo-tendon tissues generated with human dermal fibroblasts were indistinguishable from the counterpart engineered with human tenocytes, which supports the viewpoint that human dermal fibroblasts is likely to replace tenocytes for future tendon graft development in vitro with dynamic mechanical loading in a bioreactor system.

  20. Prostaglandin E2 (PGE2) effect upon ICP-23 human pulmonary fibroblasts growth in culture and on measles virus multiplication in this cell type.

    PubMed

    Babeş, L; Orăşanu, M; Petraşincu, D; Teletin, N

    1991-01-01

    The influence of PGE2 in different concentrations (10(-4)M, 10(-6)M, 10(-8)M) and of 1 mM AMPc upon ICP-23 human pulmonary fibroblasts and also the influence of PGE2 upon measles virus multiplication in the same cell type were studied. PGE2 inhibited fibroblasts growth in all administered concentrations, depending on them. AMPc adding to human fibroblasts in culture progressively stimulated cells growth in the first 24 hours, produced a steady growth during 24-48-hour interval and slightly inhibited cellular divisions between 48 and 72 hours. PGE2, added in the same concentrations to measles virus--infected ICP-23 cells, concomitantly with virus administration and after 1 hour of viral adsorption influenced viral multiplication, depending on substance concentration and culturing period. Obtained data suggested that PGE2 in physiological concentrations (10(-6)M, 10(-8)M) initially has a weak inhibitory effect (titration after 6 days), but then stimulates viral production (9 days). The initial inhibition is more marked when the substance is added concomitantly with virus administration.

  1. Cell culture condition-dependent impact of AGE-rich food extracts on kinase activation and cell survival on human fibroblasts.

    PubMed

    Nass, Norbert; Weissenberg, Kristian; Somoza, Veronika; Ruhs, Stefanie; Silber, Rolf-Edgar; Simm, Andreas

    2014-03-01

    Advanced glycation end products (AGEs) are stable end products of the Maillard reaction. Effects of food extracts are often initially analysed in cellular test systems and it is not clear how different cell culture conditions might influence the results. Therefore, we compared the effects of two models for AGE-rich food, bread crust and coffee extract (CE) on WI-38 human lung fibroblasts under different cell culture conditions (sub-confluent versus confluent cells, with and without serum). WI-38 cells responded to coffee and bread crust extract (BCE) with a rapid phosphorylation of PKB (AKT), p42/44 MAPK (ERK 1/2) and p38 MAPK, strongly depending on culture conditions. BCE resulted in increased cell numbers, whereas CE appeared to be cytotoxic. When cell numbers under all culture conditions and treatments were correlated with kinase phosphorylation, the relation between phospho-p38 MAPK and phospho-AKT represented a good, cell culture condition-independent predictor of cell survival. PMID:24111510

  2. Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKT.

    PubMed

    Ranzato, Elia; Mazzucco, Laura; Patrone, Mauro; Burlando, Bruno

    2009-08-01

    There is a growing interest for the clinical use of platelet derivates in wound dressing. Platelet beneficial effect is attributed to the release of growth factors and other bioactive substances, though mechanisms are mostly unknown. We studied wound-healing processes of human primary fibroblasts, by exposing cells to a platelet lysate (PL) obtained from blood samples. Crystal violet and tetrazolium salt (MTS) assays showed dose-response increase of cell proliferation and metabolism. In scratch wound and transwell assays, a dose of 20% PL induced a significant increase of wound closure rate at 6 and 24 hrs, and had a strong chemotactic effect. BAPTA-AM, SB203580 and PD98059 caused 100% inhibition of PL effects, whereas wortmannin reduced to about one third the effect of PL on wound healing and abolished the chemotactic response. Confocal imaging showed the induction by PL of serial Ca2(+) oscillations in fibroblasts. Data indicate that cell Ca2(+) plays a fundamental role in wound healing even without PL, p38 and ERK1/2 are essential for PL effects but are also activated by wounding per se, PI3K is essential for PL effects and its downstream effector Akt is activated only in the presence of PL. In conclusion, PL stimulates fibroblast wound healing through the activation of cell proliferation and motility with different patterns of involvement of different signalling pathways.

  3. Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells.

    PubMed

    Yan, Bo; Raben, Nina; Plotz, Paul H

    2002-03-01

    Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix-loop-helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid alpha-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

  4. Antioxidant and potential anti-inflammatory activity of extracts and formulations of white tea, rose, and witch hazel on primary human dermal fibroblast cells

    PubMed Central

    2011-01-01

    Background Numerous reports have identified therapeutic roles for plants and their extracts and constituents. The aim of this study was to assess the efficacies of three plant extracts for their potential antioxidant and anti-inflammatory activity in primary human skin fibroblasts. Methods Aqueous extracts and formulations of white tea, witch hazel and rose were subjected to assays to measure anti-collagenase, anti-elastase, trolox equivalent and catalase activities. Skin fibroblast cells were employed to determine the effect of each extract/formulation on IL-8 release induced by the addition of hydrogen peroxide. Microscopic examination along with Neutral Red viability testing was employed to ascertain the effects of hydrogen peroxide directly on cell viability. Results Considerable anti-collagenase, anti-elastase, and antioxidant activities were measured for all extracts apart from the witch hazel distillate which showed no activity in the collagenase assay or in the trolox equivalence assay. All of the extracts and products tested elicited a significant decrease in the amount of IL-8 produced by fibroblast cells compared to the control (p < 0.05). None of the test samples exhibited catalase activity or had a significant effect on the spontaneous secretion of IL-8 in the control cells which was further corroborated with the microscopy results and the Neutral Red viability test. Conclusions These data show that the extracts and products tested have a protective effect on fibroblast cells against hydrogen peroxide induced damage. This approach provides a potential method to evaluate the claims made for plant extracts and the products in which these extracts are found. PMID:21995704

  5. Individual Variation of the Genetic Response to Bisphenol A in Human Foreskin Fibroblast Cells Derived from Cryptorchidism and Hypospadias Patients

    PubMed Central

    Qin, Xian-Yang; Sone, Hideko; Kojima, Yoshiyuki; Mizuno, Kentaro; Ueoka, Katsuhiko; Muroya, Koji; Miyado, Mami; Hisada, Aya; Zaha, Hiroko; Fukuda, Tomokazu; Yoshinaga, Jun; Yonemoto, Junzo; Kohri, Kenjiro; Hayashi, Yutaro; Fukami, Maki; Ogata, Tsutomu

    2012-01-01

    Background/Purpose We hypothesized that polymorphic differences among individuals might cause variations in the effect that environmental endocrine disruptors (EEDs) have on male genital malformations (MGMs). In this study, individual variation in the genetic response to low-dose bisphenol A (BPA) was investigated in human foreskin fibroblast cells (hFFCs) derived from child cryptorchidism (CO) and hypospadias (HS) patients. Methodology/Principal Findings hFFCs were collected from control children without MGMs (n = 5) and child CO and HS patients (n = 8 and 21, respectively). BPA exposure (10 nM) was found to inhibit matrix metalloproteinase-11 (MMP11) expression in the HS group (0.74-fold, P = 0.0034) but not in the control group (0.93-fold, P = 0.84) and CO group (0.94-fold, P = 0.70). Significantly lower levels of MMP11 expression were observed in the HS group compared with the control group (0.80-fold, P = 0.0088) and CO group (0.79-fold, P = 0.039) in response to 10 nM BPA. The effect of single-nucleotide polymorphism rs5000770 (G>A), located within the aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) locus, on individual sensitivity to low-dose BPA was investigated in the HS group. A significant difference in neurotensin receptor 1 (NTSR1) expression in response to 10 nM BPA was observed between AA and AG/GG groups (n = 6 and 15, respectively. P = 0.031). However, no significant difference in ARNT2 expression was observed (P = 0.18). Conclusions/Significance This study advances our understanding of the specificity of low-dose BPA effects on human reproductive health. Our results suggest that genetic variability among individuals affects susceptibility to the effects of EEDs exposure as a potential cause of HS. PMID:23285176

  6. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts.

    PubMed

    Hu, Li; Wang, Juan; Zhou, Xin; Xiong, Zehuan; Zhao, Jiajia; Yu, Ran; Huang, Fang; Zhang, Handong; Chen, Lili

    2016-09-12

    Prolonged healing and scar formation are two major challenges in the treatment of soft tissue trauma. Adipose mesenchymal stem cells (ASCs) play an important role in tissue regeneration, and recent studies have suggested that exosomes secreted by stem cells may contribute to paracrine signaling. In this study, we investigated the roles of ASCs-derived exosomes (ASCs-Exos) in cutaneous wound healing. We found that ASCs-Exos could be taken up and internalized by fibroblasts to stimulate cell migration, proliferation and collagen synthesis in a dose-dependent manner, with increased genes expression of N-cadherin, cyclin-1, PCNA and collagen I, III. In vivo tracing experiments demonstrated that ASCs-Exos can be recruited to soft tissue wound area in a mouse skin incision model and significantly accelerated cutaneous wound healing. Histological analysis showed increased collagen I and III production by systemic administration of exosomes in the early stage of wound healing, while in the late stage, exosomes might inhibit collagen expression to reduce scar formation. Collectively, our findings indicate that ASCs-Exos can facilitate cutaneous wound healing via optimizing the characteristics of fibroblasts. Our results provide a new perspective and therapeutic strategy for the use of ASCs-Exos in soft tissue repair.

  7. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts.

    PubMed

    Hu, Li; Wang, Juan; Zhou, Xin; Xiong, Zehuan; Zhao, Jiajia; Yu, Ran; Huang, Fang; Zhang, Handong; Chen, Lili

    2016-01-01

    Prolonged healing and scar formation are two major challenges in the treatment of soft tissue trauma. Adipose mesenchymal stem cells (ASCs) play an important role in tissue regeneration, and recent studies have suggested that exosomes secreted by stem cells may contribute to paracrine signaling. In this study, we investigated the roles of ASCs-derived exosomes (ASCs-Exos) in cutaneous wound healing. We found that ASCs-Exos could be taken up and internalized by fibroblasts to stimulate cell migration, proliferation and collagen synthesis in a dose-dependent manner, with increased genes expression of N-cadherin, cyclin-1, PCNA and collagen I, III. In vivo tracing experiments demonstrated that ASCs-Exos can be recruited to soft tissue wound area in a mouse skin incision model and significantly accelerated cutaneous wound healing. Histological analysis showed increased collagen I and III production by systemic administration of exosomes in the early stage of wound healing, while in the late stage, exosomes might inhibit collagen expression to reduce scar formation. Collectively, our findings indicate that ASCs-Exos can facilitate cutaneous wound healing via optimizing the characteristics of fibroblasts. Our results provide a new perspective and therapeutic strategy for the use of ASCs-Exos in soft tissue repair. PMID:27615560

  8. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts

    PubMed Central

    Hu, Li; Wang, Juan; Zhou, Xin; Xiong, Zehuan; Zhao, Jiajia; Yu, Ran; Huang, Fang; Zhang, Handong; Chen, Lili

    2016-01-01

    Prolonged healing and scar formation are two major challenges in the treatment of soft tissue trauma. Adipose mesenchymal stem cells (ASCs) play an important role in tissue regeneration, and recent studies have suggested that exosomes secreted by stem cells may contribute to paracrine signaling. In this study, we investigated the roles of ASCs-derived exosomes (ASCs-Exos) in cutaneous wound healing. We found that ASCs-Exos could be taken up and internalized by fibroblasts to stimulate cell migration, proliferation and collagen synthesis in a dose-dependent manner, with increased genes expression of N-cadherin, cyclin-1, PCNA and collagen I, III. In vivo tracing experiments demonstrated that ASCs-Exos can be recruited to soft tissue wound area in a mouse skin incision model and significantly accelerated cutaneous wound healing. Histological analysis showed increased collagen I and III production by systemic administration of exosomes in the early stage of wound healing, while in the late stage, exosomes might inhibit collagen expression to reduce scar formation. Collectively, our findings indicate that ASCs-Exos can facilitate cutaneous wound healing via optimizing the characteristics of fibroblasts. Our results provide a new perspective and therapeutic strategy for the use of ASCs-Exos in soft tissue repair. PMID:27615560

  9. Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

    PubMed Central

    Kawakami, M.; Suzuki, K.; Matsuki, Y.; Ishizuka, T.; Hidaka, T.; Konishi, T.; Matsumoto, M.; Kataharada, K.; Nakamura, H.

    1998-01-01

    OBJECTIVES—To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.
METHODS—Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.
RESULTS—HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1.
CONCLUSION—IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

 Keywords: synovial lining cells; hyaluronan, interleukin 1β; transforming growth factor β1 PMID:9893571

  10. CD44 and hyaluronan expression in human cutaneous scar fibroblasts.

    PubMed Central

    Messadi, D. V.; Bertolami, C. N.

    1993-01-01

    Fibrotic disorders of skin and other organs are typically associated with an abnormal accumulation of extracellular matrix. This study focuses on a matrix constituent, hyaluronan-which is known to be altered in fibrotic disorders of skin- and on CD44, a cell adhesion molecule and putative receptor for hyaluronan. Tissue samples were obtained from biopsies of human normal skin, normal cutaneous scar; and hypertrophic cutaneous scar. After culturing, cells were studied by single- and double-labeling immunohistochemistry using the two anti-CD44 monoclonal antibodies, BU-52 and J173, and a biotinylated hyaluronan binding complex probe, b-HABR. Certain cultures were pretreated with Streptomyces hyaluronidase to assess the dependency of CD44 expression on the presence of endogenous hyaluronan. CD44 expression, both in the presence and the absence of exogenous hyaluronan, was quantitated by radioimmunobinding assay. Overall glycosaminoglycan synthesis and identification of hyaluronan were accomplished by precursor incorporation assays and by quantitative cellulose acetate electrophoresis. CD44 was found to be a normal human adult fibroblastic antigen whose expression is markedly increased for hypertrophic scar fibroblasts compared with normal skin fibroblasts. Although hyaluronan was found to be the predominant glycosaminoglycan constituent of the pericellular matrix for these fibroblasts, CD44 attachment to the cell surface is neither mediated by hyaluronan nor is the presence of hyaluronan a prerequisite for CD44 expression. Exogenous hyaluronan induced a decline in measurable CD44 expression for normal skin fibroblasts but not for hypertrophic scar fibroblasts. These observations are compatible with current understanding of the way cells manage the hyaluronan economy of the extracellular matrix and emphasize phenotypic heterogeneities between fibroblasts derived from normal versus scar tissues. Images Figure 1 Figure 4 PMID:8475990

  11. 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.

  12. Differential Response and Priming Dose Effect on the Proteome of Human Fibroblast and Stem Cells Induced by Exposure to Low Doses of Ionizing Radiation.

    PubMed

    Hauptmann, Monika; Haghdoost, Siamak; Gomolka, Maria; Sarioglu, Hakan; Ueffing, Marius; Dietz, Anne; Kulka, Ulrike; Unger, Kristian; Babini, Gabriele; Harms-Ringdahl, Mats; Ottolenghi, Andrea; Hornhardt, Sabine

    2016-03-01

    It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation. PMID:26934482

  13. Scaffold-Free Coculture Spheroids of Human Colonic Adenocarcinoma Cells and Normal Colonic Fibroblasts Promote Tumorigenicity in Nude Mice123

    PubMed Central

    Park, Jong-il; Lee, Jisu; Kwon, Ju-Lee; Park, Hong-Bum; Lee, Su-Yel; Kim, Ji-Yeon; Sung, Jaekye; Kim, Jin Man; Song, Kyu Sang; Kim, Kyung-Hee

    2016-01-01

    The aim of this study was to form a scaffold-free coculture spheroid model of colonic adenocarcinoma cells (CACs) and normal colonic fibroblasts (NCFs) and to use the spheroids to investigate the role of NCFs in the tumorigenicity of CACs in nude mice. We analysed three-dimensional (3D) scaffold-free coculture spheroids of CACs and NCFs. CAC Matrigel invasion assays and tumorigenicity assays in nude mice were performed to examine the effect of NCFs on CAC invasive behaviour and tumorigenicity in 3D spheroids. We investigated the expression pattern of fibroblast activation protein-α (FAP-α) by immunohistochemical staining. CAC monocultures did not form densely-packed 3D spheroids, whereas cocultured CACs and NCFs formed 3D spheroids. The 3D coculture spheroids seeded on a Matrigel extracellular matrix showed higher CAC invasiveness compared to CACs alone or CACs and NCFs in suspension. 3D spheroids injected into nude mice generated more and faster-growing tumors compared to CACs alone or mixed suspensions consisting of CACs and NCFs. FAP-α was expressed in NCFs-CACs cocultures and xenograft tumors, whereas monocultures of NCFs or CACs were negative for FAP-α expression. Our findings provide evidence that the interaction between CACs and NCFs is essential for the tumorigenicity of cancer cells as well as for tumor propagation. PMID:26947885

  14. 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.

  15. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.

    PubMed

    Ostenfeld, Thor; Svendsen, Clive N

    2004-01-01

    Epidermal growth factor (EGF)- and fibroblast growth factor-2 (FGF-2)-responsive human neural stem cells may provide insight into mechanisms of neural development and have applications in cell-based therapeutics for neurological disease. However, their biology after expansion in vitro is currently poorly understood. Cells grown in either EGF or FGF-2 or a combination of both mitogens displayed characteristically similar levels of transcriptional activation and comparable proliferative profiles with linear cell-cycle kinetics and possessed similar neuronal differentiation capabilities. These data support the view that human neurospheres at later stages of expansion (>10 weeks) are comprised overwhelmingly of a single type of stem cell responsive to both EGF and FGF-2. After mitogen withdrawal and neurosphere plating, bromodeoxyuridine pulse-chase experiments revealed that the stem cells did not undergo differentiation directly into neurons. Instead, most immature neurons arose via the division of emerging progenitor cells in the absence of exogenous EGF or FGF-2. Neurogenesis was abolished by application of high concentrations of either EGF/FGF-2 or the mitotic inhibitor cytosine-b-arabinofuranoside, suggesting that there is an obligatory requirement for at least one round of cell division in the absence of mitogens as a prelude to terminal neuronal differentiation. The differentiation of human neurospheres provides a useful model of human neurogenesis, and the data presented indicate that it proceeds through the division of committed neuronal progenitor cells rather than directly from the neural stem cell. PMID:15342944

  16. Human epidermal growth factor and the proliferation of human fibroblasts.

    PubMed

    Carpenter, G; Cohen, S

    1976-06-01

    The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of 3H-thymidine incorporation after 20-24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.

  17. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    SciTech Connect

    Story, M.T. )

    1989-05-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue.

  18. Human dermal stem/progenitor cell-derived conditioned medium ameliorates ultraviolet a-induced damage of normal human dermal fibroblasts.

    PubMed

    Shim, Joong Hyun; Park, Ju-Yearl; Lee, Mi-Gi; Kang, Hak Hee; Lee, Tae Ryong; Shin, Dong Wook

    2013-01-01

    Adult skin stem cells are considered an attractive cell resource for therapeutic potential in aged skin. We previously reported that multipotent human dermal stem/progenitor cells (hDSPCs) can be enriched from (normal human dermal fibroblasts (NHDFs) using collagen type IV. However, the beneficial effects of hDSPCs on aged skin remain to be elucidated. In the present study, we analyzed the growth factors secreted from hDSPCs in conditioned medium (CM) derived from hDSPCs (hDSPC-CM) and found that hDSPCs secreted higher levels of bFGF, IGFBP-1, IGFBP-2, HGF, VEGF and IGF-1 compared with non-hDSPCs. We then investigated whether hDSPC-CM has an effect on ultraviolet A (UVA)-irradiated NHDFs. Real-time RT-PCR analysis revealed that the treatment of UVA-irradiated NHDFs with hDSPC-CM significantly antagonized the UVA-induced up-regulation of the MMP1 and the UVA-induced down-regulation of the collagen types I, IV and V and TIMP1 mRNA expressions. Furthermore, a scratch wound healing assay showed that hDSPC-CM enhanced the migratory properties of UVA-irradiated NHDFs. hDSPC-CM also significantly reduced the number of the early and late apoptotic cell population in UVA-irradiated NHDFs. Taken together, these data suggest that hDSPC-CM can exert some beneficial effects on aged skin and may be used as a therapeutic agent to improve skin regeneration and wound healing.

  19. Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation.

    PubMed

    de Esch, Celine E F; Ghazvini, Mehrnaz; Loos, Friedemann; Schelling-Kazaryan, Nune; Widagdo, W; Munshi, Shashini T; van der Wal, Erik; Douben, Hannie; Gunhanlar, Nilhan; Kushner, Steven A; Pijnappel, W W M Pim; de Vrij, Femke M S; Geijsen, Niels; Gribnau, Joost; Willemsen, Rob

    2014-10-14

    Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene. PMID:25358783

  20. Epigenetic Characterization of the FMR1 Promoter in Induced Pluripotent Stem Cells from Human Fibroblasts Carrying an Unmethylated Full Mutation

    PubMed Central

    de Esch, Celine E.F.; Ghazvini, Mehrnaz; Loos, Friedemann; Schelling-Kazaryan, Nune; Widagdo, W.; Munshi, Shashini T.; van der Wal, Erik; Douben, Hannie; Gunhanlar, Nilhan; Kushner, Steven A.; Pijnappel, W.W.M. Pim; de Vrij, Femke M.S.; Geijsen, Niels; Gribnau, Joost; Willemsen, Rob

    2014-01-01

    Summary Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene. PMID:25358783

  1. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

    PubMed Central

    Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

    2011-01-01

    Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

  2. Analysis of Chromosomal Aberrations after Low and High Dose Rate Gamma Irradiation in ATM or NBS Suppressed Human Fibroblast Cells

    NASA Technical Reports Server (NTRS)

    Hada, M.; Huff, J. L.; Patel, Z.; Pluth, J. M.; George, K. A.; Cucinotta, F. A.

    2009-01-01

    A detailed understanding of the biological effects of heavy nuclei is needed for space radiation protection and for cancer therapy. High-LET radiation produces more complex DNA lesions that may be non-repairable or that may require additional processing steps compared to endogenous DSBs, increasing the possibility of misrepair. Interplay between radiation sensitivity, dose, and radiation quality has not been studied extensively. Previously we studied chromosome aberrations induced by low- and high- LET radiation in several cell lines deficient in ATM (ataxia telangactasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. We found that the yields of both simple and complex chromosomal aberrations were significantly increased in the DSB repair defective cells compared to normal cells. The increased aberrations observed for the ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex aberrations, while the linear dose-response term was significantly higher in NBS cells only for simple exchanges. These results point to the importance of the functions of ATM and NBS in chromatin modifications that function to facilitate correct DSB repair and minimize aberration formation. To further understand the sensitivity differences that were observed in ATM and NBS deficient cells, in this study, chromosomal aberration analysis was performed in normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, or Mirin, an MRN complex inhibitor involved in activation of ATM. We are also testing siRNA knockdown of these proteins. Normal and ATM or NBS suppressed cells were irradiated with gamma-rays and chromosomes were collected with a premature chromosome

  3. Polysome profiling shows the identity of human adipose-derived stromal/stem cells in detail and clearly distinguishes them from dermal fibroblasts.

    PubMed

    Zych, Jaiesa; Spangenberg, Lucia; Stimamiglio, Marco A; Abud, Ana Paula R; Shigunov, Patrícia; Marchini, Fabricio K; Kuligovski, Crisciele; Cofré, Axel R; Schittini, Andressa V; Aguiar, Alessandra M; Senegaglia, Alexandra; Brofman, Paulo R S; Goldenberg, Samuel; Dallagiovanna, Bruno; Naya, Hugo; Correa, Alejandro

    2014-11-15

    Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells. Contrary to previous reports, fibroblasts were not able to differentiate into adipocytes, osteoblasts, or chondrocytes. Polysome-bound mRNA profiling revealed that ∼ 1,547 genes were differentially expressed (DE) in the two cell types; the genes were related to cell adhesion, the extracellular matrix, differentiation, and proliferation. These findings were confirmed by functional analyses showing that ADSCs had a greater adhesion capacity than fibroblasts; the proliferation rate of fibroblasts was also higher than that of ADSCs. Importantly, 185 DE genes were integral to the plasma membrane and, thus, candidate markers for ADSC isolation and manipulation. We also observed that an established marker of fibroblasts and ADSCs, CD105, was overexpressed in ADSCs at both mRNA and protein levels. CD105 expression seemed to be related to differentiation capacity, at least for adipogenesis. This study shows that ADSCs and fibroblasts are distinct cell types. These findings should be taken into account when using these two cell types in basic and therapeutic studies.

  4. Studies in porphyria. IV. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells: prenatal diagnosis of the porphyric trait

    PubMed Central

    1975-01-01

    The gene lesion of the porphyrin-heme synthetic pathway in acute intermittent porphyria (AIP) is reflected in a deficient level of activity of the cytosol enzyme uroporphyrinogen I synthetase (URO-S). A marked URO-S deficiency has been demonstrated in the liver and in circulating erythrocytes of individuals with both active and latent AIP. This enzymic abnormality accounts for the excessive production and excretion into urine of the porphyrin precursors, lamda-aminolevulinic acid (ALA) and porphobilinogen (PBG) in AIP subjects. In this study, utilizing cell culture techniques, a marked URO-S deficiency has also been demonstrated in skin fibroblasts from AIP patients and in cells derived through aminocentesis from an approximately 17-wk old fetus. The prenatal diagnosis of the AIP trait in this fetus was confirmed postnatally by the demonstration in the child of a deficient level of erythrocyte URO-S activity which was comparable to those found in her AIP mother and affected sibling and which was approximately one-half the levels characterizing her normal father and aunt and a second unaffected sibling. The identification of the URO-S deficiency in cultured human fibroblasts from AIP patients was facilitated by a newly developed, sensitive assay for the enzyme activity. In this assay, the ability of such cells to convert ALA to protoporphyrin was quantitated; in the sequence of reactions involved in this transformation, URO-S is limiting so that the gene defect of AIP could be simply and precisely determined by appropriate spectrofluorometry of cell extracts. The technique described has distinct advantages over the direct enzymatic assay for URO-S activity in cultured human skin fibroblasts and permits clear differentiation of AIP carrier from normal individuals. PMID:1165472

  5. Studies in porphyria. IV. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells: prenatal diagnosis of the porphyric trait.

    PubMed

    Sassa, S; Solish, G; Levere, R D; Kappas, A

    1975-09-01

    The gene lesion of the porphyrin-heme synthetic pathway in acute intermittent porphyria (AIP) is reflected in a deficient level of activity of the cytosol enzyme uroporphyrinogen I synthetase (URO-S). A marked URO-S deficiency has been demonstrated in the liver and in circulating erythrocytes of individuals with both active and latent AIP. This enzymic abnormality accounts for the excessive production and excretion into urine of the porphyrin precursors, lamda-aminolevulinic acid (ALA) and porphobilinogen (PBG) in AIP subjects. In this study, utilizing cell culture techniques, a marked URO-S deficiency has also been demonstrated in skin fibroblasts from AIP patients and in cells derived through aminocentesis from an approximately 17-wk old fetus. The prenatal diagnosis of the AIP trait in this fetus was confirmed postnatally by the demonstration in the child of a deficient level of erythrocyte URO-S activity which was comparable to those found in her AIP mother and affected sibling and which was approximately one-half the levels characterizing her normal father and aunt and a second unaffected sibling. The identification of the URO-S deficiency in cultured human fibroblasts from AIP patients was facilitated by a newly developed, sensitive assay for the enzyme activity. In this assay, the ability of such cells to convert ALA to protoporphyrin was quantitated; in the sequence of reactions involved in this transformation, URO-S is limiting so that the gene defect of AIP could be simply and precisely determined by appropriate spectrofluorometry of cell extracts. The technique described has distinct advantages over the direct enzymatic assay for URO-S activity in cultured human skin fibroblasts and permits clear differentiation of AIP carrier from normal individuals.

  6. Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  7. Degradation of type IV collagen by neoplastic human skin fibroblasts

    SciTech Connect

    Sheela, S.; Barrett, J.C.

    1985-02-01

    An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion.

  8. Scaffold fiber diameter regulates human tendon fibroblast growth and differentiation.

    PubMed

    Erisken, Cevat; Zhang, Xin; Moffat, Kristen L; Levine, William N; Lu, Helen H

    2013-02-01

    The diameter of collagen fibrils in connective tissues, such as tendons and ligaments is known to decrease upon injury or with age, leading to inferior biomechanical properties and poor healing capacity. This study tests the hypotheses that scaffold fiber diameter modulates the response of human tendon fibroblasts, and that diameter-dependent cell responses are analogous to those seen in healthy versus healing tissues. Particularly, the effect of the fiber diameter (320 nm, 680 nm, and 1.80 μm) on scaffold properties and the response of human tendon fibroblasts were determined over 4 weeks of culture. It was observed that scaffold mechanical properties, cell proliferation, matrix production, and differentiation were regulated by changes in the fiber diameter. More specifically, a higher cell number, total collagen, and proteoglycan production were found on the nanofiber scaffolds, while microfibers promoted the expression of phenotypic markers of tendon fibroblasts, such as collagen I, III, V, and tenomodulin. It is possible that the nanofiber scaffolds of this study resemble the matrix in a state of injury, stimulating the cells for matrix deposition as part of the repair process, while microfibers represent the healthy matrix with micron-sized collagen bundles, thereby inducing cells to maintain the fibroblastic phenotype. The results of this study demonstrate that controlling the scaffold fiber diameter is critical in the design of scaffolds for functional and guided connective tissue repair, and provide new insights into the role of matrix parameters in guiding soft tissue healing.

  9. 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

  10. Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study.

    PubMed

    Navaeipour, Farzaneh; Afsharan, Hadi; Tajalli, Habib; Mollabashi, Mahmood; Ranjbari, Farideh; Montaseri, Azadeh; Rashidi, Mohammad-Reza

    2016-08-01

    In this experimental study, cancer and normal cells behavior during an in vitro photodynamic therapy (PDT) under exposure of continuous wave (CW) and fractionated mode of laser with different irradiation power and time intervals was compared and investigated. At the first, human fibroblast cancer cell line (SW 872) and human dermal normal (HFFF2) cell line were incubated with different concentrations of zinc phthalocyanine (ZnPc), as a PDT drug. The cells, then, were irradiated with a 675nm diode laser and the cell viability was evaluated using MTT assay. Under optimized conditions, the viability of the cancer cells was eventually reduced to 3.23% and 13.17%, and that of normal cells was decreased to 20.83% and 36.23% using CW and fractionated diode lasers, respectively. In general, the ratio of ZnPc LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractionated laser. Subsequently, cancer cells in comparison with normal ones were found to be more sensitive toward the photodynamic damage induced by ZnPc. In addition, treatment with CW laser was found to be more effective against the cancer cells with a lower toxicity to the normal cells compared with the fractionated diode laser. PMID:27318602

  11. Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study.

    PubMed

    Navaeipour, Farzaneh; Afsharan, Hadi; Tajalli, Habib; Mollabashi, Mahmood; Ranjbari, Farideh; Montaseri, Azadeh; Rashidi, Mohammad-Reza

    2016-08-01

    In this experimental study, cancer and normal cells behavior during an in vitro photodynamic therapy (PDT) under exposure of continuous wave (CW) and fractionated mode of laser with different irradiation power and time intervals was compared and investigated. At the first, human fibroblast cancer cell line (SW 872) and human dermal normal (HFFF2) cell line were incubated with different concentrations of zinc phthalocyanine (ZnPc), as a PDT drug. The cells, then, were irradiated with a 675nm diode laser and the cell viability was evaluated using MTT assay. Under optimized conditions, the viability of the cancer cells was eventually reduced to 3.23% and 13.17%, and that of normal cells was decreased to 20.83% and 36.23% using CW and fractionated diode lasers, respectively. In general, the ratio of ZnPc LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractionated laser. Subsequently, cancer cells in comparison with normal ones were found to be more sensitive toward the photodynamic damage induced by ZnPc. In addition, treatment with CW laser was found to be more effective against the cancer cells with a lower toxicity to the normal cells compared with the fractionated diode laser.

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

    PubMed Central

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

    1994-01-01

    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. Images PMID:8289799

  13. 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.

  14. Recombinant growth factor mixtures induce cell cycle progression and the upregulation of type I collagen in human skin fibroblasts, resulting in the acceleration of wound healing processes.

    PubMed

    Lee, Do Hyun; Choi, Kyung-Ha; Cho, Jae-We; Kim, So Young; Kwon, Tae Rin; Choi, Sun Young; Choi, Yoo Mi; Lee, Jay; Yoon, Ho Sang; Kim, Beom Joon

    2014-05-01

    Application of growth factor mixtures has been used for wound healing and anti-wrinkles agents. The aim of this study was to evaluate the effect of recombinant growth factor mixtures (RGFM) on the expression of cell cycle regulatory proteins, type I collagen, and wound healing processes of acute animal wound models. The results showed that RGFM induced increased rates of cell proliferation and cell migration of human skin fibroblasts (HSF). In addition, expression of cyclin D1, cyclin E, cyclin-dependent kinase (Cdk)4, and Cdk2 proteins was markedly increased with a growth factor mixtures treatment in fibroblasts. Expression of type I collagen was also increased in growth factor mixtures-treated HSF. Moreover, growth factor mixtures-induced the upregulation of type I collagen was associated with the activation of Smad2/3. In the animal model, RGFM-treated mice showed accelerated wound closure, with the closure rate increasing as early as on day 7, as well as re-epithelization and reduced inflammatory cell infiltration than phosphate-buffered saline (PBS)-treated mice. In conclusion, the results indicated that RGFM has the potential to accelerate wound healing through the upregulation of type I collagen, which is partly mediated by activation of Smad2/3-dependent signaling pathway as well as cell cycle progression in HSF. The topical application of growth factor mixtures to acute and chronic skin wound may accelerate the epithelization process through these molecular mechanisms.

  15. NOVEL SPLICED VARIANTS OF IONOTROPIC GLUTAMATE RECEPTOR GLUR6 IN NORMAL HUMAN FIBROBLAST AND BRAIN CELLS ARE TRANSCRIBED BY TISSUE SPECIFIC PROMOTERS

    PubMed Central

    Zhawar, Vikramjit K.; Kaur, Gurpreet; deRiel, Jon K.; Kaur, G. Pal; Kandpal, Raj P.; Athwal, Raghbir S.

    2010-01-01

    The members of the ionotropic glutamate receptor family, namely, a-amino-3-hydroxy-S-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors, are important mediators of the rapid synaptic transmission in the central nervous system. We have investigated the splicing pattern and expression of the kainate receptor subunit GluR6 in human fibroblast cell lines and brain tissue. We demonstrate the expression of GluR6A variant specifically in brain, and four variants, namely, GluR6B, GluR6C, GluR6D and GluR6E in fibroblast cell lines. The variants GluR6D and GluR6E have not been described before, and appear to be specific for non-neuronal cells. Genomic analysis and cloning of the sequence preceding the transcribed region led to the identification of two tissue specific promoters designated as neuronal promoter PN and non-neuronal promoter PNN. We have used RNA ligase mediated RACE and in silico analyses to locate two sets of transcription start sites, and confirmed specific transcripts initiated by PN and PNN in brain cells and fibroblasts, respectively. The domain structure of variants GluR6D and GluR6E revealed the absence of three transmembrane domains. The lack of these domains suggests that the mature receptors arising from these variant subunits may not function as active channels. Based on these structural features in GluR6D and GluR6E, and the observations that GluR6B, GluR6C, GluR6D and GluR6E are exclusively expressed in non-neuronal cells, it is likely that these receptor subunits function as non-channel signaling proteins. PMID:20230879

  16. Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

    PubMed

    Esmekaya, Meric Arda; Seyhan, Nesrin; Kayhan, Handan; Tuysuz, Mehmet Zahid; Kurşun, Ayşe Canseven; Yağcı, Münci

    2013-01-01

    In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway.

  17. Involvement of the mitochondrial compartment in human NCL fibroblasts

    SciTech Connect

    Pezzini, Francesco; Gismondi, Floriana; Tessa, Alessandra; Tonin, Paola; Carrozzo, Rosalba; Mole, Sara E.; Santorelli, Filippo M.; Simonati, Alessandro

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. Black-Right-Pointing-Pointer Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. Black-Right-Pointing-Pointer Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  18. Brief Report: Inhibition of miR-145 Enhances Reprogramming of Human Dermal Fibroblasts to Induced Pluripotent Stem Cells.

    PubMed

    Barta, Tomas; Peskova, Lucie; Collin, Joseph; Montaner, David; Neganova, Irina; Armstrong, Lyle; Lako, Majlinda

    2016-01-01

    MicroRNA (miRNAs) are short noncoding RNA molecules involved in many cellular processes and shown to play a key role in somatic cell induced reprogramming. We performed an array based screening to identify candidates that are differentially expressed between dermal skin fibroblasts (DFs) and induced pluripotent stem cells (iPSCs). We focused our investigations on miR-145 and showed that this candidate is highly expressed in DFs relative to iPSCs and significantly downregulated during reprogramming process. Inhibition of miR-145 in DFs led to the induction of "cellular plasticity" demonstrated by: (a) alteration of cell morphology associated with downregulation of mesenchymal and upregulation of epithelial markers; (b) upregulation of pluripotency-associated genes including SOX2, KLF4, C-MYC; (c) downregulation of miRNA let-7b known to inhibit reprogramming; and (iv) increased efficiency of reprogramming to iPSCs in the presence of reprogramming factors. Together, our results indicate a direct functional link between miR-145 and molecular pathways underlying reprogramming of somatic cells to iPSCs.

  19. Effect of polyphenols on reactive oxygen species production and cell growth of human dermal fibroblasts after irradiation with ultraviolet-A light.

    PubMed

    Shirai, Akihiro; Onitsuka, Masayoshi; Maseda, Hideaki; Omasa, Takeshi

    2015-01-01

    Ultraviolet-A (UV-A) can damage microbes by generating reactive oxygen species (ROS), singlet oxygen, superoxides, hydrogen peroxide and hydroxyl radicals. These species readily react with lipids, proteins, DNA and other constituents of cells, leading to oxidative deterioration and the eventual death of the microbe. However, the oxidative ability of these reactive species also harms the viability of mammalian cells such as fibroblasts and keratinocytes, as they cause both acute and chronic damage, photo-aging, and photo-carcinogenesis. This study describes a UV-A treatment that does not affect the viability or growth of human neonate dermal fibroblasts, as determined by examining the post-irradiation cell density after the addition of polyphenols as antioxidants. The results demonstrate the possible wide applicability of UV-A sterilization. The potency of polyphenols for attenuating UV-A-induced ROS generation in cells was tested using (+)-catechin hydrate, (-)- epigallocatechin gallate hydrate, morin hydrate, quercetin hydrate and resveratrol. The lowest concentration of polyphenols required to reduce ROS by 50% in cells upon exposure to a dose of 15 J cm(-2) was determined and defined as its IC50. Pre-treatment with morin hydrate at its IC50 allowed cells irradiated with 5.0 J cm(-2) UV-A to recover to the level of the specific growth rate of cells incubated without UV-A irradiation. However, the growth rate of cells exposed to 15 J cm(-2) UV-A irradiation was scarcely influenced by co-incubation with morin hydrate; this dose of UV-A also suppressed cell growth completely in the absence of morin hydrate, although co-incubation resulted in no decrease in cell viability. This study demonstrates the potential of polyphenols for protecting both the viability of cells and their ability to proliferate from damage caused by UV-A-irradiation.

  20. Disruption of the p53-mediated G{sub 1}/S cell cycle checkpoint results in elevated rates of spontaneous genetic recombination in human fibroblasts

    SciTech Connect

    Strasfeld, L.; Brainerd, E.; Meyn, M.S.

    1994-09-01

    A key feature of the cancer-prone inherited disease ataxia-telangiectasia (A-T) is genetic instability. We recently demonstrated that one aspect of genetic instability in A-T is a marked elevation in the spontaneous rates of intrachromosomal mitotic recombination. We have proposed a model for A-T that attributes these high recombination rates to a lack of DNA damage-sensitive cell cycle checkpoints. One prediction of this model is that disrupting p53 function in normal cells should increase their spontaneous rates of recombination by interfering with their p53-dependent G{sub 1}/S cell cycle checkpoint. To test this prediction, we transfected control and A-T fibroblast lines that each harbor a single integrated copy of lacZ-based recombination vector (pLrec) with derivatives of a eukaryotic expression vector (pRep5) that contain either a dominant-negative p53 mutant (143{sup val{yields}ala}) or a human papilloma virus E6 gene (HPV18 E6). Expression of either of these genes results in loss of p53 function and abolition of the G{sub 1}/S cell cycle checkpoint. Four independent p53{sup 143ala} transformants of the control line showed 25-80 fold elevations in spontaneous recombination rates when compared to their parent cell line. Elevations in spontaneous recombination rates were also detected following transfection with the HPV18 E6 gene. In contrast, four independent p53{sup 143ala} transformants of the A-T cell line showed no significant changes in their already high spontaneous recombination rates. We are now extending these observations to additional normal human fibroblast lines and carrying out molecular analyses of the products of these recombinational events. Our results support our hypothesis that the lack of a p53-dependent G{sub 1}/S cell cycle checkpoint contributes to the hyperrecombination seen in A-T.

  1. M-FISH Analysis of Chromosome Aberrations in Human Fibroblast Cells After In Vitro Exposure to Low- and High-LET Radiation

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Furusawa, Yoshiya; George, Kerry; Kawata, Tetsuya; Cucinotta, Francis

    2002-01-01

    The recently commercialized multiplex fluorescence in situ hybridization (m-FISH) technique, which allows human chromosomes to be painted in 24 different colors, was used to analyze chromosome aberrations in diploid human fibroblast cells after in vitro radiation exposure. Confluent flasks of a normal primary fibroblast cell line (AG 1522) were irradiated at high dose rates with either gamma rays or 200 MeV/nucleon Fe ions (LET = 440 keV/micron), incubated at 37 C for 24 hours after exposure, and subsequently subcultured. A chemically induced premature chromosome condensation technique was used to collect chromosome samples 32 hours after subculture. Results showed that the fraction of exchanges which were identified as complex, i.e. involving misrejoining of three or more DSB, were higher in the Fe-irradiated samples compared with the gamma-irradiated samples, as has been shown previously using FISH with one or two painted chromosomes . The ratios of complex/simple type exchanges were similar for samples irradiated with 0.7 Gy and 3 Gy of Fe ions, although exchanges involving five or more breaks were found only in 3 Gy irradiated samples. The fraction of incomplete exchanges was also higher in Fe- than gamma-irradiated samples. Data on the distribution of individual chromosome involvement in interchromosomal exchanges will be presented.

  2. 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

  3. Aqueous extract of Arbutus unedo inhibits STAT1 activation in human breast cancer cell line MDA-MB-231 and human fibroblasts through SHP2 activation.

    PubMed

    Mariotto, S; Ciampa, A R; de Prati, A Carcereri; Darra, E; Vincenzi, S; Sega, M; Cavalieri, E; Shoji, K; Suzuki, H

    2008-05-01

    Arbutus unedo L. has been for a long time employed in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes. Signal transducer and activator of transcription 1 (STAT1) is a fascinating and complex protein with multiple yet contrasting transcriptional functions. Although activation of this nuclear factor is finely regulated in order to control the entire inflammatory process, its hyper-activation or time-spatially erroneous activation may lead to exacerbation of inflammation. The modulation of this nuclear factor, therefore, has recently been considered as a new strategy in the treatment of inflammatory diseases. In this study, we present data showing that the aqueous extract of Arbutus unedo's leaves exerts inhibitory action on interferon-gamma (IFN-gamma) elicited activation of STAT1, both in human breast cancer cell line MDA-MB-231 and in human fibroblasts. This down-regulation of STAT1 is shown to result from a reduced tyrosine phosphorylation of STAT1 protein. Evidence is also presented indicating that the inhibitory effect of this extract may be mediated through enhancement of tyrosine phosphorylation of SHP2 tyrosine phosphatase. The modulation of this nuclear factor turns out into the regulation of the expression of a number of genes involved in the inflammatory response such as inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1). Taken together, our results suggest that the employment of the Arbutus unedo aqueous extract is promising, at least, as an auxiliary anti-inflammatory treatment of diseases in which STAT1 plays a critical role. PMID:18473914

  4. Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

    PubMed

    Wiederholt, Tonio; Heise, Ruth; Skazik, Claudia; Marquardt, Yvonne; Joussen, Sylvia; Erdmann, Kati; Schröder, Henning; Merk, Hans F; Baron, Jens Malte

    2009-11-01

    Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts. PMID:19397697

  5. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro.

    PubMed

    Dzobo, Kevin; Turnley, Taegyn; Wishart, Andrew; Rowe, Arielle; Kallmeyer, Karlien; van Vollenstee, Fiona A; Thomford, Nicholas E; Dandara, Collet; Chopera, Denis; Pepper, Michael S; Parker, M Iqbal

    2016-01-01

    Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell-matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures. PMID:27527147

  6. Pharmacological modulation of human subconjunctival fibroblast behavior in vitro.

    PubMed

    Damji, K F; Rootman, J; Palcic, B; Thurston, G

    1990-01-01

    The response of human subconjunctival fibroblasts to a variety of pharmacological agents was evaluated utilizing a novel in vitro wound assay and a separate proliferation assay. Both colchicine and cytochalasin B dramatically arrested wound closure at concentrations greater than or equal to 0.01 micrograms/ml and 2 micrograms/ml, respectively (p less than 0.05). At lower doses these drugs altered fibroblast morphology and inhibited directed cell migration. Dexamethasone and 6-MP delayed wound closure at concentrations greater than or equal to 100 micrograms/ml and 1000 micrograms/ml, respectively (p less than 0.05). Effective antiproliferative agents, in order of decreasing potency (based on unit weight), were Cytarabine (cytosine arabinoside), doxorubicin (Adriamycin), colchicine, 5-fluorouracil, cytochalasin B, cyclosporin (Sandimmune), 6-mercaptopurine, and dexamethasone. The antiprotease agents and methotrexate were ineffective as determined by both assays. We conclude that the wound assay is well suited for rapid screening of drugs for their effect on fibroblast morphology, motility, and proliferation, and that colchicine and cytochalasin B, in doses well below those documented to produce ocular toxicity, are effective in inhibiting directed migration and proliferation of subconjunctival fibroblasts in vitro. Differences in mechanism, onset of action, therapeutic range, and cytotoxicity of drugs could be exploited in controlling ocular fibroblast behavior in vivo. PMID:2325993

  7. 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

  8. CEMP1 Induces Transformation in Human Gingival Fibroblasts

    PubMed Central

    Bermúdez, Mercedes; Imaz-Rosshandler, Ivan; Rangel-Escareño, Claudia; Zeichner-David, Margarita; Arzate, Higinio; Mercado-Celis, Gabriela E.

    2015-01-01

    Cementum Protein 1 (CEMP1) is a key regulator of cementogenesis. CEMP1 promotes cell attachment, differentiation, deposition rate, composition, and morphology of hydroxyapatite crystals formed by human cementoblastic cells. Its expression is restricted to cementoblasts and progenitor cell subpopulations present in the periodontal ligament. CEMP1 transfection into non-osteogenic cells such as adult human gingival fibroblasts results in differentiation of these cells into a “mineralizing” cell phenotype. Other studies have shown evidence that CEMP1 could have a therapeutic potential for the treatment of bone defects and regeneration of other mineralized tissues. To better understand CEMP1’s biological effects in vitro we investigated the consequences of its expression in human gingival fibroblasts (HGF) growing in non-mineralizing media by comparing gene expression profiles. We identified several mRNAs whose expression is modified by CEMP1 induction in HGF cells. Enrichment analysis showed that several of these newly expressed genes are involved in oncogenesis. Our results suggest that CEMP1 causes the transformation of HGF and NIH3T3 cells. CEMP1 is overexpressed in cancer cell lines. We also determined that the region spanning the CEMP1 locus is commonly amplified in a variety of cancers, and finally we found significant overexpression of CEMP1 in leukemia, cervix, breast, prostate and lung cancer. Our findings suggest that CEMP1 exerts modulation of a number of cellular genes, cellular development, cellular growth, cell death, and cell cycle, and molecules associated with cancer. PMID:26011628

  9. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

    PubMed Central

    Dzobo, Kevin; Turnley, Taegyn; Wishart, Andrew; Rowe, Arielle; Kallmeyer, Karlien; van Vollenstee, Fiona A.; Thomford, Nicholas E.; Dandara, Collet; Chopera, Denis; Pepper, Michael S.; Parker, M. Iqbal

    2016-01-01

    Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures. PMID:27527147

  10. Effect of basic fibroblast growth factor on pluripotent marker expression and colony forming unit capacity of stem cells isolated from human exfoliated deciduous teeth.

    PubMed

    Sukarawan, Waleerat; Nowwarote, Nunthawan; Kerdpon, Piyarat; Pavasant, Prasit; Osathanon, Thanaphum

    2014-07-01

    Human dental pulp of exfoliated deciduous teeth contains the population of cells that exhibited mesenchymal stem cell (MSC) characters. Though, a cell amplification process is indeed required to secure an adequate cell number for such a potential employment. Several publications suggested the alteration of MSCs upon in vitro culture, for example, the decrease in proliferation and the loss of stem cell characters. Here, we investigated an influence of basic fibroblast growth factor (bFGF) on stem cells isolated from human exfoliated deciduous teeth (SHEDs) with respect to cell proliferation, colony forming unit efficiency and stem cell marker expression in both short- and long-term cultures. For short-term bFGF treatment, SHEDs were treated with bFGF for 48 h. While, in long-term bFGF supplementation, SHEDs were maintained in culture and continuous passage upon confluence in medium supplemented with bFGF. Cells at passage (P) 5 and 10 were employed for characterization. Our results showed that short-term bFGF treatment enhanced OCT4, REX1, and NANOG mRNA expression as well as colony forming unit ability. The FGFR inhibitor pretreatment was able to attenuate the influence of bFGF on pluripotent stem cell marker expression, confirming bFGF function. In addition, cells cultured in high passage number had decreased in cell proliferation, colony forming unit capacity, and pluripotent stem cell maker mRNA expression. However, bFGF supplementation in culture medium enhanced both pluripotent stem cell marker expression and colony forming unit capacity in later passage, though the effect was not robust. Together, these results indicate that high passage number may attenuate pluripotent properties of SHEDs and bFGF supplementation could be the beneficial approach to maintain SHEDs' stemness properties.

  11. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    PubMed

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting.

  12. The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts.

    PubMed

    Korade, Zeljka; Kim, Hye-Young H; Tallman, Keri A; Liu, Wei; Koczok, Katalin; Balogh, Istvan; Xu, Libin; Mirnics, Karoly; Porter, Ned A

    2016-02-11

    Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway. PMID:26789657

  13. Chamber-specific differences in human cardiac fibroblast proliferation and responsiveness toward simvastatin.

    PubMed

    Rizvi, Farhan; DeFranco, Alessandra; Siddiqui, Ramail; Negmadjanov, Ulugbek; Emelyanova, Larisa; Holmuhamedov, Alisher; Ross, Gracious; Shi, Yang; Holmuhamedov, Ekhson; Kress, David; Tajik, A Jamil; Jahangir, Arshad

    2016-08-01

    Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. A chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear whether similar differences exist in human cardiac fibroblasts (HCFs) and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than threefold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with a higher sensitivity toward inhibition compared with ventricular fibroblasts. The DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 μM) that bypasses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Chamber-specific differences exist in the human heart because atrial fibroblasts have a higher proliferative capacity and are more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury.

  14. Human Cytomegalovirus Induces JC Virus DNA Replication in Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Heilbronn, Regine; Albrecht, Ingrid; Stephan, Sonja; Burkle, Alexander; Zur Hausen, Harald

    1993-12-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML.

  15. Determination of chromium in human lung fibroblast cells using a large bore-direct injection high-efficiency nebulizer with inductively coupled plasma mass spectrometry

    SciTech Connect

    McLean, John A.; Acon, Billy W.; Montaser, Akbar; Singh, Jatinder; Pritchard, Daryl E.; Patierno, Steven R.

    2000-05-01

    A novel method for the determination of chromium in suspensions of human lung fibroblast cells is described by using a large bore-direct injection high efficiency nebulizer (LB-DIHEN) with microscale flow injection analysis and inductively coupled plasma mass spectrometric detection. Chromium (VI)-treated cells were first counted and then suspended in a phosphate buffer saline solution. With the use of the method of standard additions, the relative concentration of Cr in {approx}100 HLF cells/peak was determined at m/z=50. Because the cells tend to clump and can yield inhomogeneities in the total number analyzed, Mg was used as an internal standard to compensate for the total cell mass. The level of Cr in HLF cells grown in a medium of 100 {mu}M Na{sub 2}CrO{sub 4} for two hours is on the order of 180 fg Cr/cell after correction for the number of cells in each injection. (c) 2000 Society for Applied Spectroscopy.

  16. Highly Aligned Nanofibrous Scaffold Derived from Decellularized Human Fibroblasts

    PubMed Central

    Xing, Qi; Vogt, Caleb; Leong, Kam W.; Zhao, Feng

    2014-01-01

    Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nanofibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) were used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm–thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter was generated after removing the cellular components from the detached fibroblast sheet. The elastic modulus of the scaffold was well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) showed the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold’s biocompatibility was further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induced a significantly lower immune response compared to its unaligned counterpart, as detected by the pro-inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues. PMID:25484849

  17. Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

    PubMed

    Wang, Feng; Yang, Lijun; Shi, Lin; Li, Qian; Zhang, Gengshen; Wu, Jianliang; Zheng, Jun; Jiao, Baohua

    2015-08-28

    Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments. PMID:26056081

  18. Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

    PubMed

    Wang, Feng; Yang, Lijun; Shi, Lin; Li, Qian; Zhang, Gengshen; Wu, Jianliang; Zheng, Jun; Jiao, Baohua

    2015-08-28

    Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.

  19. Apurinic/apyrimidinic endonuclease 1 induced upregulation of fibroblast growth factor 2 and its receptor 3 induces angiogenesis in human osteosarcoma cells.

    PubMed

    Ren, Tao; Qing, Yi; Dai, Nan; Li, Mengxia; Qian, Chengyuan; Yang, Yuxin; Cheng, Yi; Li, Zheng; Zhang, Shiheng; Zhong, Zhaoyang; Wang, Dong

    2014-02-01

    Tumor angiogenesis contributes to inferior prognosis in osteosarcoma. Apurinic/apyrimidinic endonuclease 1 (APE1) and fibroblast growth factor 2 (FGF2) and its receptor 3 (FGFR3) signaling pathway plays an important role in the angiogenic process. In this study we observed that high expression of APE1, FGF2 and FGFR3, and microvessel density are positively correlated with poor prognosis of osteosarcoma patients. Furthermore, the Cox model showed that the tumor size, FGF2 and its receptor 3 (FGFR3), and microvessel density were adverse prognostic factors. Based on our clinical data, and the fact that APE1 is involved in tumor angiogenesis, we hypothesize that it is very likely that APE1 may indirectly promote angiogenesis by upregulating fibroblast FGF2 and FGFR3. Our preliminary data show small interfering RNA-mediated silence of APE1 experiments, which further supports this hypothesis. APE1-small interfering RNA significantly inhibited tumor angiogenesis by downregulating in vitro expression of FGF2 and FGFR3 in human umbilical vein endothelial cells in Matrigel tube formation assay, and further inhibited tumor growth in vivo in a mouse xenograft model. Thus, the proposed APE1-FGF2 and FGFR3 pathway may provide a novel mechanism for regulation of FGF2 and FGFR3 by APE1 in tumor angiogenesis. PMID:24329908

  20. Division of mitochondria in cultured human fibroblasts.

    PubMed

    Fujioka, Hisashi; Tandler, Bernard; Consolo, Mary C; Karnik, Pratima

    2013-12-01

    Ovate mitochondria in cultured human fibroblasts divide by pinching. In the process, as observed by transmission electron microscopy, a deep incisure of the surface membranes separates the organelle into two lobes connected by a slender isthmus. A single element of smooth endoplasmic reticulum (SER) invariably accompanies each incisure, extending deep into the cleft. When the ingrowing membranes meet and fuse with the antipodal membranes, fission occurs. Elongated mitochondria that give no indication of division often are cloaked by a single, continuous cistern of SER.

  1. Evaluation of cytotoxic, genotoxic and inflammatory responses of micro- and nano-particles of granite on human lung fibroblast cell IMR-90.

    PubMed

    Ahmad, Iqbal; Khan, Mohd Imran; Patil, Govil; Chauhan, L K S

    2012-02-01

    Occupational exposure of granite workers is well known to cause lung impairment and silicosis. Toxicological profiles of different size particles of granite dust, however, are not yet understood. Present evaluation of micro- and nano-particles of granite dust as on human lung fibroblast cells IMR-90, revealed that their toxic effects were dose-dependent, and nanoparticles in general were more toxic. In this study we first demonstrated that nanoparticles caused oxidative stress, inflammatory response and genotoxicity, as seen by nearly 2 fold induction of ROS and LPO, mRNA levels of TNF-α and IL-1β, and induction in micronuclei formation. All these were significantly higher when compared with the effect of micro particles. Thus, the study suggests that separate health safety standards would be required for granite particles of different sizes.

  2. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

    PubMed Central

    Janmaat, C. J.; de Rooij, K. E; Locher, H; de Groot, S. C.; de Groot, J. C. M. J.; Frijns, J. H. M.; Huisman, M. A.

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  3. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins.

    PubMed

    Janmaat, C J; de Rooij, K E; Locher, H; de Groot, S C; de Groot, J C M J; Frijns, J H M; Huisman, M A

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  4. Chromosomal analysis in young vs. senescent human fibroblasts by FISH

    SciTech Connect

    Mukheriee, A.B.; Thomas, S.

    1994-09-01

    Almost all previous studies on chromosomal analysis related to in vitro aging of human fibroblasts were done using only metaphase chromosomes. However, this procedure might provide only partial information since the aneuploidy presumably hidden in interphase cells would remain undetected. We, therefore, have analyzed aneuploidy both at interphase and at metaphase. Female (IMR-90) and male (IMR-91) cells were grown from the lowest to the highest population doubling levels and aneuploidy analysis was done by FISH with {alpha}-satellite DNA probes of 15 autosomes and 2 sex chromosomes. Our data on total aneuploidy in young cells indicate that significantly higher proportions of cells with aneuploidy are detected at interphase as opposed to metaphase. This presumably indicates that during active division of young cells, a greater proportion of cells with aneuploidy than diploidy is selected against entry to mitosis. In contrast, both cell strains at senescence exhibit significantly lower proportions of nuclei with aneuploidy at interphase as compared to that of young cells. This probably indicates that during senescense, a greater proportion of cells with aneuploidy than diploidy is selected against prolonged survival in culture. Our study shows that cellular dynamics with respect to aneuploidy involving various chromosomes differs significantly at interphase and at mitosis during in vitro aging of human fibroblasts.

  5. Ionizing radiation-mediated premature senescence and paracrine interactions with cancer cells enhance the expression of syndecan 1 in human breast stromal fibroblasts: the role of TGF-β

    PubMed Central

    Liakou, Eleni; Mavrogonatou, Eleni; Pratsinis, Harris; Rizou, Sophia; Evangelou, Konstantinos; Panagiotou, Petros N.; Karamanos, Nikos K.; Gorgoulis, Vassilis G.; Kletsas, Dimitris

    2016-01-01

    The cell surface proteoglycan syndecan 1 (SDC1) is overexpressed in the malignant breast stromal fibroblasts, creating a favorable milieu for tumor cell growth. In the present study, we found that ionizing radiation, a well-established treatment in human breast cancer, provokes premature senescence of human breast stromal fibroblasts in vitro, as well as in the breast tissue in vivo. These senescent cells were found to overexpress SDC1 both in vitro and in vivo. By using a series of specific inhibitors and siRNA approaches, we showed that this SDC1 overexpression in senescent cells is the result of an autocrine action of Transforming Growth Factor-β (TGF-β) through the Smad pathway and the transcription factor Sp1, while the classical senescence pathways of p53 or p38 MAPK - NF-kB are not involved. In addition, the highly invasive human breast cancer cells MDA-MB-231 (in contrast to the low-invasive MCF-7) can also enhance SDC1 expression, both in early-passage and senescent fibroblasts via a paracrine action of TGF-β. The above suggest that radiation-mediated premature senescence and invasive tumor cells, alone or in combination, enhance SDC1 expression in breast stromal fibroblasts, a poor prognostic factor for cancer growth, and that TGF-β plays a crucial role in this process. PMID:27434331

  6. Ionizing radiation-mediated premature senescence and paracrine interactions with cancer cells enhance the expression of syndecan 1 in human breast stromal fibroblasts: the role of TGF-β.

    PubMed

    Liakou, Eleni; Mavrogonatou, Eleni; Pratsinis, Harris; Rizou, Sophia; Evangelou, Konstantinos; Panagiotou, Petros N; Karamanos, Nikos K; Gorgoulis, Vassilis G; Kletsas, Dimitris

    2016-08-01

    The cell surface proteoglycan syndecan 1 (SDC1) is overexpressed in the malignant breast stromal fibroblasts, creating a favorable milieu for tumor cell growth. In the present study, we found that ionizing radiation, a well-established treatment in human breast cancer, provokes premature senescence of human breast stromal fibroblasts in vitro, as well as in the breast tissue in vivo. These senescent cells were found to overexpress SDC1 both in vitro and in vivo. By using a series of specific inhibitors and siRNA approaches, we showed that this SDC1 overexpression in senescent cells is the result of an autocrine action of Transforming Growth Factor-β (TGF-β) through the Smad pathway and the transcription factor Sp1, while the classical senescence pathways of p53 or p38 MAPK - NF-kB are not involved. In addition, the highly invasive human breast cancer cells MDA-MB-231 (in contrast to the low-invasive MCF-7) can also enhance SDC1 expression, both in early-passage and senescent fibroblasts via a paracrine action of TGF-β. The above suggest that radiation-mediated premature senescence and invasive tumor cells, alone or in combination, enhance SDC1 expression in breast stromal fibroblasts, a poor prognostic factor for cancer growth, and that TGF-β plays a crucial role in this process. PMID:27434331

  7. Effect of saccharin on metabolic cooperation between human fibroblasts

    SciTech Connect

    Mosser, D.D.; Bols, N.C.

    1983-01-01

    Autoradiography was used to study the effect of saccharin on metabolic cooperation between human diploid fibroblasts. When the donors, HGPRT+ cells, and recipients, HGPRT- cells, were plated together in the presence of saccharin, all the interactions that developed in 4 and 24 h were positive for metabolic cooperation. When saccharin was added after donor cells and recipient cells had made contact, the proportion of interactions that were positive for metabolic cooperation was unchanged but the number of grains over primary recipients was reduced. However, in donor cells saccharin caused a reduction in (/sup 3/H)hypoxanthine incorporation into both acid-soluble and acid-insoluble fractions, although the relative distribution of radioactivity between these two fractions and between the phosphorylated and non-phosphorylated derivatives of (/sup 3/H)hypoxanthine was unchanged. Metabolic cooperation was studied under conditions in which the number of grains over the nuclei of both the primary recipient and the primary recipient's donor could be counted. The change in the number of grains over these two cell types in response to saccharin was compared and found to be the same. Thus in normal human fibroblasts saccharin does not appear to affect metabolic cooperation, which is a measure of cell-to-cell communication.

  8. 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

  9. Mouse liver repopulation with hepatocytes generated from human fibroblasts

    PubMed Central

    Zhu, Saiyong; Rezvani, Milad; Harbell, Jack; Mattis, Aras N.; Wolfe, Alan R.; Benet, Leslie Z.; Willenbring, Holger; Ding, Sheng

    2014-01-01

    Human induced pluripotent stem cells (iPSCs) promise to revolutionize research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modeling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1–3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. As a solution to this problem, we report generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs, but shortcut reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells (iMPC-EPCs) and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this, we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to aHeps. Unfractionated iMPC-Heps did not form tumors, most likely because they never entered a pluripotent state. To our knowledge, this is the first demonstration of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy. PMID:24572354

  10. 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.

  11. Replicative senescence: the human fibroblast comes of age.

    PubMed

    Goldstein, S

    1990-09-01

    Human diploid fibroblasts undergo replicative senescence predominantly because of arrest at the G1/S boundary of the cell cycle. Senescent arrest resembles a process of terminal differentiation that appears to involve repression of proliferation-promoting genes with reciprocal new expression of antiproliferative genes, although post-transcriptional factors may also be involved. Identification of participating genes and clarification of their mechanisms of action will help to elucidate the universal cellular decline of biological aging and an important obverse manifestation, the rare escape of cells from senescence leading to immortalization and oncogenesis.

  12. 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

  13. Quantitative Proteomic Analysis Revealed 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone-induced Up-regulation of 20S Proteasome in Cultured Human Fibroblast Cells

    PubMed Central

    Prins, John M.; Wang, Yinsheng

    2012-01-01

    The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK), is a well-known carcinogen. Although the ability of the metabolically activated form of NNK to generate DNA adducts is well established, little is known about the cellular pathways perturbed by NNK in its native state. In this study, we utilized stable isotope labeling by amino acid in cell culture (SILAC), together with mass spectrometry, to assess the perturbation of protein expression in GM00637 human skin fibroblast cells upon NNK exposure. With this approach, we were able to quantify 1412 proteins and 137 of them were with significantly altered expression following NNK exposure, including the up-regulation of all subunits of the 20S proteasome core complex. The up-regulation of the 20S core complex was also reflected by a significant increase in 20S proteasome activities in GM00637, IMR90 and MCF-7 cells upon NNK treatment. Furthermore, the β-adrenergic receptor (β-AR) antagonist propranolol could attenuate significantly the NNK-induced increase in proteasome activity in all the three cell lines, suggesting that up-regulation of the 20S proteasome may be mediated through the β-AR. Additionally, we found that NNK treatment altered the expression levels of other important proteins including mitochondrial proteins, cytoskeleton-associated proteins, and proteins involved in glycolysis and gluconeogenesis. Results from the present study provided novel insights into the cellular mechanisms targeted by NNK. PMID:22369695

  14. 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

  15. A voltage-activated proton current in human cardiac fibroblasts

    SciTech Connect

    El Chemaly, Antoun; Guinamard, Romain; Demion, Marie; Fares, Nassim; Jebara, Victor; Faivre, Jean-Francois; Bois, Patrick . E-mail: patrick.bois@univ-poitiers.fr

    2006-02-10

    A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51 mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn{sup 2+} reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.

  16. Relationship between DNA double-strand break rejoining and cell survival after exposure to ionizing radiation in human fibroblast strains with differing ATM/p53 status: Implications for evaluation of clinical radiosensitivity

    SciTech Connect

    Mirzayans, Razmik; Severin, Diane; Murray, David . E-mail: davem@cancerboard.ab.ca

    2006-12-01

    Purpose: To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. Methods and Materials: We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Results: Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early ({<=}4 h) after exposure to 8 Gy of {gamma}-radiation and also exhibited high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX ({gamma}-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Conclusion: Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and {gamma}-H2AX assays. Our data suggest that the {gamma}-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.

  17. 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.

  18. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.

    PubMed

    Williamson, Chad D; Wong, Daniel S; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M

    2015-09-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

  19. Cell attachment of human gingival fibroblasts in vitro to porous-surfaced titanium alloy discs coated with collagen and platelet-derived growth factor.

    PubMed

    Lowenberg, B F; Pilliar, R M; Aubin, J E; Sodek, J; Melcher, A H

    1988-07-01

    The influence of biological coating, with or without the incorporation of growth factor, on the migration, attachment and orientation of human gingival fibroblasts in relation to porous-surfaced titanium alloy (Ti6AI4V) discs, was measured. Comparison was made between coating the discs with collagen and with collagen incorporating platelet-derived growth factor (PDGF); controls comprised porous-surfaced discs coated with agar or collagen containing bovine serum albumin (used as a carrier for the PDGF), uncoated porous-surfaced Ti6AI4V discs (with or without additional protein additives) exhibited significantly higher attachment indices (AI) and orientation indices (OI) compared with naked control discs (p less than 0.01); OI was also significantly higher than that of surface-demineralized root slices (p less than 0.001) on days 1, 2 and 3. Addition of PDGF to the collagen resulted in a further enhancement in OI on days 1 and 2 (p less than 0.01) over that shown by discs coated with collagen incorporating the bovine serum albumin vehicle. There was no cell attachment and consequently, no cell orientation, in relation to Ti alloy discs that had been coated with agar. These data suggest that attachment and orientation of cells following migration in relation to porous-surfaced Ti6AI4V discs can be modified by the application of biological molecules to the surface of the disc. This may have a useful application in clinical implantology.

  20. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

    PubMed Central

    Williamson, Chad D.; Wong, Daniel S.; Bozidis, Petros; Zhang, Aiping; Colberg-Poley, Anamaris M.

    2015-01-01

    Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. PMID:26331984

  1. Increased cell proliferation and differential protein expression induced by low-level Er:YAG laser irradiation in human gingival fibroblasts: proteomic analysis.

    PubMed

    Ogita, Mayumi; Tsuchida, Sachio; Aoki, Akira; Satoh, Mamoru; Kado, Sayaka; Sawabe, Masanori; Nanbara, Hiromi; Kobayashi, Hiroaki; Takeuchi, Yasuo; Mizutani, Koji; Sasaki, Yoshiyuki; Nomura, Fumio; Izumi, Yuichi

    2015-09-01

    Erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment has demonstrated favorable wound healing effect after periodontal therapy. One of the reasons may be the positive biological effect of the low-level laser on the irradiated tissues, although the mechanism remains unclear. The aim of this study was to investigate the effect of low-level Er:YAG laser irradiation on cell proliferation and laser-induced differential expression of proteins in human gingival fibroblasts (HGFs) by proteomic analysis. In the first experiment, HGFs were exposed to low-level Er:YAG laser irradiation and the laser-induced cell proliferation and damage were evaluated on day 3. In the second experiment, proteomic analysis was performed on day 1 after irradiation. The peptides prepared from HGFs were analyzed by a hybrid ion trap-Fourier transform mass spectrometer, Mascot search engine, and UniProtKB database. A significant increase in cell proliferation without cell damage after irradiation was observed. Among the total identified 377 proteins, 59 proteins, including galectin-7, which was associated with the process of wound healing, were upregulated and 15 proteins were downregulated in laser-treated HGFs. In the third experiment, the increase in messenger RNA (mRNA) and protein expression of galectin-7 in the irradiated HGFs was validated by various analytical techniques. In addition, the effect of recombinant human galectin-7 on the modulation of HGFs proliferation was confirmed. The results indicate that low-level Er:YAG laser irradiation can promote HGF proliferation and induce a significant change in protein expression and the upregulation of galectin-7 expression may partly contribute to the increase in cell proliferation.

  2. Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

    SciTech Connect

    Sowa, Marianne B.; Goetz, Wilfried; Baulch, Janet E.; Pyles, Dinah N.; Dziegielewski, J.; Yovino, Susannah; Snyder, Andrew R.; de Toledo, S. M.; Azzam, Edouard I.; Morgan, William F.

    2010-02-01

    The conventional paradigm in radiation biology has been that DNA is the primary target for energy deposition following exposure to ionizing radiation. However, studies focusing on the non-target effects of radiation, i.e. effects occurring in cells not directly exposed to radiation, imply that the target of exposure is larger than what has traditionally been assumed and could have significant implications for radiation health risks. We have conducted an extensive study of the low-LET bystander effect including multiple cell lines and endpoints and various radiation sources and exposure scenarios. In no instance do we see evidence of a low-LET induced bystander effect. However, direct comparison for alpha particle exposure showed a statistically significant media transfer bystander effect for high-LET but not for low-LET radiation. From our results it is evident that there are many confounding factors mitigating bystander responses as reported in the literature and for the cell lines we studied that there is a LET dependence for the observed responses. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

  3. Transcriptome Alterations In X-Irradiated Human Gingiva Fibroblasts

    PubMed Central

    Weissmann, Robert; Kacprowski, Tim; Peper, Michel; Esche, Jennifer; Jensen, Lars R.; van Diepen, Laura; Port, Matthias; Kuss, Andreas W.; Scherthan, Harry

    2016-01-01

    Abstract Ionizing radiation is known to induce genomic lesions, such as DNA double strand breaks, whose repair can lead to mutations that can modulate cellular and organismal fate. Soon after radiation exposure, cells induce transcriptional changes and alterations of cell cycle programs to respond to the received DNA damage. Radiation-induced mutations occur through misrepair in a stochastic manner and increase the risk of developing cancers years after the incident, especially after high dose radiation exposures. Here, the authors analyzed the transcriptomic response of primary human gingival fibroblasts exposed to increasing doses of acute high dose-rate x rays. In the dataset obtained after 0.5 and 5 Gy x-ray exposures and two different repair intervals (0.5 h and 16 h), the authors discovered several radiation-induced fusion transcripts in conjunction with dose-dependent gene expression changes involving a total of 3,383 genes. Principal component analysis of repeated experiments revealed that the duration of the post-exposure repair intervals had a stronger impact than irradiation dose. Subsequent overrepresentation analyses showed a number of KEGG gene sets and WikiPathways, including pathways known to relate to radioresistance in fibroblasts (Wnt, integrin signaling). Moreover, a significant radiation-induced modulation of microRNA targets was detected. The data sets on IR-induced transcriptomic alterations in primary gingival fibroblasts will facilitate genomic comparisons in various genotoxic exposure scenarios. PMID:27356049

  4. Transcriptome Alterations In X-Irradiated Human Gingiva Fibroblasts.

    PubMed

    Weissmann, Robert; Kacprowski, Tim; Peper, Michel; Esche, Jennifer; Jensen, Lars R; van Diepen, Laura; Port, Matthias; Kuss, Andreas W; Scherthan, Harry

    2016-08-01

    Ionizing radiation is known to induce genomic lesions, such as DNA double strand breaks, whose repair can lead to mutations that can modulate cellular and organismal fate. Soon after radiation exposure, cells induce transcriptional changes and alterations of cell cycle programs to respond to the received DNA damage. Radiation-induced mutations occur through misrepair in a stochastic manner and increase the risk of developing cancers years after the incident, especially after high dose radiation exposures. Here, the authors analyzed the transcriptomic response of primary human gingival fibroblasts exposed to increasing doses of acute high dose-rate x rays. In the dataset obtained after 0.5 and 5 Gy x-ray exposures and two different repair intervals (0.5 h and 16 h), the authors discovered several radiation-induced fusion transcripts in conjunction with dose-dependent gene expression changes involving a total of 3,383 genes. Principal component analysis of repeated experiments revealed that the duration of the post-exposure repair intervals had a stronger impact than irradiation dose. Subsequent overrepresentation analyses showed a number of KEGG gene sets and WikiPathways, including pathways known to relate to radioresistance in fibroblasts (Wnt, integrin signaling). Moreover, a significant radiation-induced modulation of microRNA targets was detected. The data sets on IR-induced transcriptomic alterations in primary gingival fibroblasts will facilitate genomic comparisons in various genotoxic exposure scenarios.

  5. Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes

    PubMed Central

    Lee, Darwin V.; Li, Dongmei; Yan, Qingyun; Zhu, Yimin; Goodwin, Bryan; Calle, Roberto; Brenner, Martin B.; Talukdar, Saswata

    2014-01-01

    Fibroblast growth factor 21 (FGF21) has evolved as a major metabolic regulator, the pharmacological administration of which causes weight loss, insulin sensitivity and glucose control in rodents and humans. To understand the molecular mechanisms by which FGF21 exerts its metabolic effects, we developed a human in vitro model of adipocytes to examine crosstalk between FGF21 and insulin signaling. Human adipose stem cell-derived (hASC) adipocytes were acutely treated with FGF21 alone, insulin alone, or in combination. Insulin signaling under these conditions was assessed by measuring tyrosine phosphorylation of insulin receptor (InsR), insulin receptor substrate-1 (IRS-1), and serine 473 phosphorylation of Akt, followed by a functional assay using 14C-2-deoxyglucose [14C]-2DG to measure glucose uptake in these cells. FGF21 alone caused a modest increase of glucose uptake, but treatment with FGF21 in combination with insulin had a synergistic effect on glucose uptake in these cells. The presence of FGF21 also effectively lowered the insulin concentration required to achieve the same level of glucose uptake compared to the absence of FGF21 by 10-fold. This acute effect of FGF21 on insulin signaling was not due to IR, IGF-1R, or IRS-1 activation. Moreover, we observed a substantial increase in basal S473-Akt phosphorylation by FGF21 alone, in contrast to the minimal shift in basal glucose uptake. Taken together, our data demonstrate that acute co-treatment of hASC-adipocytes with FGF21 and insulin can result in a synergistic improvement in glucose uptake. These effects were shown to occur at or downstream of Akt, or separate from the canonical insulin signaling pathway. PMID:25365322

  6. Fibroblast Growth Factor 2 Regulates High Mobility Group A2 Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Kalomoiris, Stefanos; Cicchetto, Andrew C; Lakatos, Kinga; Nolta, Jan A; Fierro, Fernando A

    2016-09-01

    Mesenchymal stem cells (MSCs) are an excellent source for numerous cellular therapies due to their simple isolation, low immunogenicity, multipotent differentiation potential and regenerative secretion profile. However, over-expanded MSCs show decreased therapeutic efficacy. This shortcoming may be circumvented by identifying methods that promote self-renewal of MSCs in culture. HMGA2 is a DNA-binding protein that regulates self-renewal in multiple types of stem cells through chromatin remodeling, but its impact on human bone marrow-derived MSCs is not known. Using an isolation method to obtain pure MSCs within 9 days in culture, we show that expression of HMGA2 quickly decreases during early expansion of MSCs, while let-7 microRNAs (which repress HMGA2) are simultaneously increased. Remarkably, we demonstrate that FGF-2, a growth factor commonly used to promote self-renewal in MSCs, rapidly induces HMGA2 expression in a time- and concentration-dependent manner. The signaling pathway involves FGF-2 receptor 1 (FGFR1) and ERK1/2, but acts independent from let-7. By silencing HMGA2 using shRNAs, we demonstrate that HMGA2 is necessary for MSC proliferation. However, we also show that over-expression of HMGA2 does not increase cell proliferation, but rather abrogates the mitogenic effect of FGF-2, possibly through inhibition of FGFR1. In addition, using different methods to assess in vitro differentiation, we show that modulation of HMGA2 inhibits adipogenesis, but does not affect osteogenesis of MSCs. Altogether, our results show that HMGA2 expression is associated with highly proliferating MSCs, is tightly regulated by FGF-2, and is involved in both proliferation and adipogenesis of MSCs. J. Cell. Biochem. 117: 2128-2137, 2016. © 2016 Wiley Periodicals, Inc. PMID:26888666

  7. Cigarette smoke and α,β-unsaturated aldehydes elicit VEGF release through the p38 MAPK pathway in human airway smooth muscle cells and lung fibroblasts

    PubMed Central

    Volpi, Giorgia; Facchinetti, Fabrizio; Moretto, Nadia; Civelli, Maurizio; Patacchini, Riccardo

    2011-01-01

    BACKGROUND AND PURPOSE Vascular endothelial growth factor (VEGF) is an angiogenic factor known to be elevated in the sputum of asymptomatic smokers as well as smokers with bronchitis type of chronic obstructive pulmonary disease. The aim of this study was to investigate whether acute exposure to cigarette smoke extract altered VEGF production in lung parenchymal cells. EXPERIMENTAL APPROACH We exposed human airway smooth muscle cells (ASMC), normal human lung fibroblasts (NHLF) and small airways epithelial cells (SAEC) to aqueous cigarette smoke extract (CSE) in order to investigate the effect of cigarette smoke on VEGF expression and release. KEY RESULTS Vascular endothelial growth factor release was elevated by sub-toxic concentrations of CSE in both ASMC and NHLF, but not in SAEC. CSE-evoked VEGF release was mimicked by its component acrolein at concentrations (10–100 µM) found in CSE, and prevented by the antioxidant and α,β-unsaturated aldehyde scavenger, N-acetylcysteine (NAC). Both CSE and acrolein (30 µM) induced VEGF mRNA expression in ASMC cultures, suggesting an effect at transcriptional level. Crotonaldehyde and 4-hydroxy-2-nonenal, an endogenous α,β-unsaturated aldehyde, stimulated VEGF release, as did H2O2. CSE-evoked VEGF release was accompanied by rapid and lasting phosphorylation of p38 MAPK (mitogen-activated protein kinase), which was abolished by NAC and mimicked by acrolein. Both CSE- and acrolein-evoked VEGF release were blocked by selective inhibition of p38 MAPK signalling. CONCLUSIONS AND IMPLICATIONS α,β-Unsaturated aldehydes and possibly reactive oxygen species contained in cigarette smoke stimulate VEGF expression and release from pulmonary cells through p38 MAPK signalling. PMID:21306579

  8. 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

  9. 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

  10. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

    PubMed Central

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-01-01

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements. PMID:27322248

  11. Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells

    SciTech Connect

    Kobashigawa, Shinko; Suzuki, Keiji; Yamashita, Shunichi

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We report first time that ionizing radiation induces mitochondrial dynamic changes. Black-Right-Pointing-Pointer Radiation-induced mitochondrial fission was caused by Drp1 localization. Black-Right-Pointing-Pointer We found that radiation causes delayed ROS from mitochondria. Black-Right-Pointing-Pointer Down regulation of Drp1 rescued mitochondrial dysfunction after radiation exposure. -- Abstract: Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial O{sub 2}{sup {center_dot}-} production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial O{sub 2}{sup {center_dot}-}. Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.

  12. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts.

    PubMed

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-01-01

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements. PMID:27322248

  13. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts.

    PubMed

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-06-16

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

  14. Respiratory activity and growth of human skin derma fibroblasts.

    PubMed

    Papa, F; Scacco, S; Vergari, R; Bucaria, V; Dioguardi, D; Papa, S

    1998-09-01

    A study has been made on the speed of growth and respiratory activity of fibroblast cultures from control derma, cheloid (hypertrophic) scar and stabilized scar taken from human skin. The speed of growth and the efficiency of plaque formation of fibroblasts from cheloid scar were greater in comparison with those of fibroblasts from stabilized scar and were stimulated by the addition to the culture medium of the exudate from post-traumatic ulcer. Measurement of the contents of cytochromes showed a decrease in the content of cytochromes b562 and c + c1 in the fibroblast culture from both cheloid and stabilized scar as compared to the fibroblast culture from control derma. Cytochrome aa3 content did not show significant difference among the three types of fibroblast cultures. The respiratory activities supported by pyruvate plus malate, succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine did not show, however, significant difference among the three fibroblast cultures. These observations show that the speed of growth of skin fibroblasts does not depend on the overall respiratory capacity. The exudate stimulated the activity of cytochrome c oxidase in fibroblasts from control derma, and cheloid scar. This effect and the accompanying stimulation of fibroblast growth might be correlated with the balance of oxygen free radicals.

  15. Fibroblast growth factor-10 is a mitogen for urothelial cells

    SciTech Connect

    Bagai, Shelly; Rubio, Eric; Cheng, Jang-Fang; Sweet, Robert; Thomas, Regi; Fuchs, Elaine; Grady, Richard; Mitchell, Michael; Bassuk, James A.

    2002-02-01

    Fibroblast Growth Factor (FGF)-10 plays an important role in regulating growth, differentiation, and repair of the urothelium. This process occurs through a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the epithelium (urothelium). In situ hybridization analysis demonstrated that (i) fibroblasts of the human lamina propria were the cell type that synthesized FGF-10 RNA and (ii) the FGF-10 gene is located at the 5p12-p13 locus of chromosome 5. Recombinant (r) preparations of human FGF-10 were found to induce proliferation of human urothelial cells in vitro and of transitional epithelium of wild-type and FGF7-null mice in vivo. Mechanistic studies with human cells indicated two modes of FGF-10 action: (i) translocation of rFGF-10 into urothelial cell nuclei and (ii) a signaling cascade that begins with the heparin-dependent phosphorylation of tyrosine residues of surface transmembrane receptors. The normal urothelial phenotype, that of quiescence, is proposed to be typified by negligible levels of FGF-10. During proliferative phases, levels of FGF-10 rise at the urothelial cell surface and/or within urothelial cell nuclei. An understanding of how FGF-10 works in conjunction with these other processes will lead to better management of many diseases of the bladder and urinary tract.

  16. 7-Hydroxycoumarin prevents UVB-induced activation of NF-κB and subsequent overexpression of matrix metalloproteinases and inflammatory markers in human dermal fibroblast cells.

    PubMed

    Karthikeyan, Ramasamy; Kanimozhi, Govindasamy; Prasad, Nagarajan Rajendra; Agilan, Balupillai; Ganesan, Muthusamy; Mohana, Shanmugham; Srithar, Gunaseelan

    2016-08-01

    Ultraviolet B (UVB) irradiation alters multiple molecular pathways in the skin, thereby inducing skin damage. Human dermal fibroblasts (HDFa) were subjected to single UVB-irradiation (18mJ/cm(2)) resulting in reactive oxygen species (ROS) generation, oxidative DNA damage and upregulation of nuclear factor kappa B (NF-κB) expression. Further, it has been observed that there was a significant cytokine production (TNF-α and IL-6) in UVB irradiated HDFa cells. Our results show that 7-hydroxycoumarin (7-OHC) prevents UVB-induced activation of NF-κB thereby subsequently preventing the overexpression of TNF-α and IL-6 in HDFa cells. Further, 7-OHC prevents UVB-induced activation of cyclooxygenase-2 (COX-2) expression, an inflammatory mediator in skin cells. Moreover, 7-OHC inhibited mRNA expression pattern of matrix metalloproteinases (MMP-1 and MMP-9) in UVB irradiated skin cells. Furthermore, 7-OHC restored antioxidant status, thereby scavenging the excessively generated ROS; consequently preventing the oxidative DNA damage. It has also been noticed that 7-OHC prevents UVB mediated DNA damage through activation of DNA repair enzymes such as XRCC1 and HOGG1. In this study, we treated HDFa cells with 7-OHC before and after UVB irradiation and we found that pretreatment showed better results when compared to posttreatment. Further, 7-OHC showed 9.8416 sun protection factor (SPF) value and it absorbs photons in the UVB wavelength rage. Thus, it has been concluded that sunscreen property, free radical scavenging potential and prevention of NF-κB activation play a role for photoprotective property of 7-OHC. PMID:27240190

  17. Modulation of androgen receptor protein by culture conditions of human skin fibroblasts.

    PubMed

    Palma, Marcela M; Fernandez, Mireya; Vivanco, Ximena; Pino, Ana M

    2002-10-01

    Cultures of skin fibroblasts show variation of androgen binding with culture conditions; binding variations are usually avoided by using confluent cultures. In this work, we analysed the effect of cell density and mitogenic agents on the level of androgen receptor (AR) of cultured human skin fibroblasts. Results demonstrated that in cultures of human skin fibroblasts, cellular binding of dihydrotestosterone was higher in cells grown at low than at high cell density. The reduction in binding resulted from a decrease in the number of high affinity receptors and not from a change in receptor affinity. Immunocytochemistry for AR showed greater staining intensity in cells grown at low than at high cell density. Additionally, immunoblot analysis demonstrated more AR protein in low cell density cultures. On the other hand, it was observed that cells grown at low cell density showed diminished androgen binding capacity after 24 h of treatment with insulin-like growth factor (IGF-l), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or granulocyte-colony stimulating factor (G-CSF); this effect of growth factors was not observed in cells grown at high cell density. In conclusion, we found that cell density of cultures and mitogenic agents can regulate AR binding activity in human fibroblasts. While we do not yet know how changes in cell density affect the amount of AR, we conclude that the mechanism could be mediated by activation of the tyrosine kinase pathway, as the effect was reproduced by mitogens.

  18. Respiratory-deficient human fibroblasts exhibiting defective mitochondrial DNA replication.

    PubMed Central

    Bodnar, A G; Cooper, J M; Leonard, J V; Schapira, A H

    1995-01-01

    We have characterized cultured skin fibroblasts from two siblings affected with a fatal mitochondrial disease caused by a nuclear genetic defect. Mitochondrial respiratory-chain function was severely decreased in these cells. Southern-blot analysis showed that the fibroblasts had reduced levels of mitochondrial DNA (mtDNA). The mtDNA was unstable and was eliminated from the cultured cells over many generations, generating the rho0 genotype. As the mtDNA level decreased, the cells became more dependent upon pyruvate and uridine for growth. Nuclear-encoded subunits of respiratory-chain complexes were synthesized and imported into the mitochondria of the mtDNA-depleted cells, albeit at reduced levels compared with the controls. Mitochondrial protein synthesis directed by the residual mtDNA indicated that the mtDNA was expressed and that the defect specifically involves the replication or maintenance of mtDNA. This is a unique example of a respiratory-deficient human cell line exhibiting defective mtDNA replication. Images Figure 1 Figure 2 Figure 4 Figure 5 PMID:7848281

  19. Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts.

    PubMed

    Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Zhang, Junfeng; Dong, Lei; Qin, Haiyan; Hu, Qingang

    2015-08-01

    Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson's staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson's, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

  20. Optimal Viscosity and Particle Shape of Hyaluronic Acid Filler as a Scaffold for Human Fibroblasts.

    PubMed

    Kim, Deok-Yeol; Namgoong, Sik; Han, Seung-Kyu; Won, Chang-Hoon; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2015-07-01

    The authors previously reported that cultured human fibroblasts suspended in a hyaluronic acid filler can produce human dermal matrices with extended in vivo stability in animal and clinical studies. The present study was undertaken to determine the optimal viscosity and particle shape of hyaluronic acid filler as a scaffold for cultured human dermal fibroblasts to enhance the maximal viability of injected cells. The fibroblasts were suspended in either 1 of 3 hyaluronic acid viscosities at 2 different particle shapes. The viscosities used in this study were low (600,000-800,000 centipoises), moderate (2,000,000-4,000,000 centipoises), and high (8,000,000-12,000,000 centipoises). The particle shape was evaluated by testing round and irregular shapes. The fibroblast mixed bioimplants were injected into the back of individual athymic nude mice. The levels of type I collagen were measured using fluorescent-activated cell sorting (FACS) and immunohistochemical staining at 16 weeks after the injections. Results of FACS demonstrated that the mean cell ratio with human collagens in the moderate viscosity group was greater than those of control, low, and high viscosity groups. An immunohistochemical study showed similar results. The moderate viscosity group demonstrated the highest positive staining of human collagens. However, there were no significant differences between groups of irregular and round shape particles. A hyaluronic acid bioimplant with moderate viscosity is superior to that with low or high viscosity in the viability for human fibroblasts. However, the particle shape does not influence the viability of the fibroblasts.

  1. Mapping the response of human fibroblast growth factor 21 (FGF21) promoter to serum availability and lipoic acid in HepG2 hepatoma cells.

    PubMed

    Xia, Mengna; Erickson, Anjeza; Yi, Xiaohua; Moreau, Régis

    2016-03-01

    The hormone-like polypeptide, fibroblast growth factor 21 (FGF21), is a major modulator of lipid and glucose metabolism and an exploratory treatment strategy for obesity related metabolic disorders. The costs of recombinant FGF21 and mode of delivery by injection are important constraints to its wide therapeutic use. The stimulation of endogenous FGF21 production through diet is being explored as an alternative approach. To that end, we examined the mechanism(s) by which serum manipulation and lipoic acid (a dietary activator of FGF21) induce FGF21 in human hepatocellular carcinoma HepG2 cells. Serum withdrawal markedly induced FGF21 mRNA levels (88 fold) and FGF21 secreted in the media (19 fold). Lipoic acid induced FGF21 mRNA 7 fold above DMSO-treated control cells and FGF21 secretion 3 fold. These effects were several-fold greater than those of PPARα agonist, Wy14643, which failed to induce FGF21 above and beyond the induction seen with serum withdrawal. The use of transcription inhibitor, actinomycin D, revealed that de novo mRNA synthesis drives FGF21 secretion in response to serum starvation. Four previously unrecognized loci in FGF21 promoter were nucleosome depleted and enriched in acetylated histone H3 revealing their role as transcriptional enhancers and putative transcription factor binding sites. FGF21 did not accumulate to a significant degree in induced HepG2 cells, which secreted FGF21 time dependently in media. We conclude that lipoic acid cell signaling connects with the transcriptional upregulation of FGF21 and it may prove to be a safe and affordable means to stimulate FGF21 production. PMID:26691139

  2. Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) degradation and human lung fibroblast-derived hyaluronidase.

    PubMed Central

    Sampson, P M; Rochester, C L; Freundlich, B; Elias, J A

    1992-01-01

    We characterized the mechanisms by which recombinant (r) tumor necrosis factor (TNF), IFN-gamma, and IL-1, alone and in combination, regulate human lung fibroblast hyaluronic acid (HA) production. Each cytokine stimulated fibroblast HA production. The combination of rTNF and rIFN-gamma resulted in a synergistic increase in the production of high molecular weight HA. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous decrease in HA degradation. In contrast, when rTNF and rIL-1 were combined, an additive increase in low molecular weight HA was noted. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous increase in HA degradation. Human lung fibroblasts contained a hyaluronidase that, at pH 3.7, depolymerized high molecular weight HA to 10-40 kD end products of digestion. However, hyaluronidase activity did not correlate with fibroblast HA degradation. Instead, HA degradation correlated with fibroblast-HA binding, which was increased by rIL-1 plus rTNF and decreased by rIFN-gamma plus rTNF. Recombinant IL-1 and rTNF weakly stimulated and rIL-1 and rTNF in combination further augmented the levels of CD44 mRNA in lung fibroblasts. In contrast, rIFN-gamma did not significantly alter the levels of CD44 mRNA in unstimulated or rTNF stimulated cells. These studies demonstrate that rIL-1, rTNF, and rIFN-gamma have complex effects on biosynthesis and degradation which alter the quantity and molecular weight of the HA produced by lung fibroblasts. They also show that fibroblast HA degradation is mediated by a previously unrecognized lysosomal-type hyaluronidase whose function may be regulated by altering fibroblast-HA binding. Lastly, they suggest that the CD44 HA receptor may be involved in this process. Images PMID:1401082

  3. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  4. Use of diploid human fibroblasts as a model system to culture, grow, and study human cytomegalovirus infection.

    PubMed

    Fortunato, Elizabeth A

    2014-01-01

    Primary human diploid fibroblasts are used routinely to study host/pathogen interactions of human cytomegalovirus (HCMV). Fibroblasts' ease of culture and tremendous permissiveness for infection allow the study of all facets of infection, an abbreviated list of which includes ligand/receptor interactions, activation of cell signaling responses, and dysregulation of the cell cycle and DNA repair processes. Another advantage to fibroblasts' permissiveness for HCMV is the capability to grow high titer stocks of virus in them. This chapter will discuss the production of viral stocks of HCMV in primary human fibroblasts, commencing with culturing and infection of cells and continuing through harvest, titration (determining the infectious capacity of a particular virus preparation), and storage of viral stocks for use in downstream experiments.

  5. Nanoplex-Mediated Co-delivery of Fibroblast Growth Factor and Bone Morphogenetic Protein Genes Promotes Osteogenesis in Human Adipocyte-Derived Mesenchymal Stem Cells

    PubMed Central

    Atluri, Keerthi; Seabold, Denise; Hong, Liu; Elangovan, Satheesh; Salem, Aliasger K.

    2015-01-01

    This study highlights the importance of transfection mediated coordinated bone morphogenetic protein 2 (BMP-2) and fibroblast growth factor 2 (FGF-2) signaling in promoting osteogenesis. We employed plasmids independently encoding BMP-2 and FGF-2 complexed with polyethylenimine (PEI) to transfect human adipose derived mesenchymal stem cells (hADMSCs) in vitro. The nanoplexes were characterized for size, surface charge, in vitro cytotoxicity and transfection ability in hADMSCs. A significant enhancement in BMP-2 protein secretion was observed on day 7 post-transfection of hADMSCs with PEI nanoplexes loaded with both pFGF-2 and pBMP-2 (PEI/(pFGF-2 + pBMP-2)) versus transfection with PEI nanoplexes of either pFGF-2 alone or pBMP-2 alone. Osteogenic differentiation of transfected hADMSCs was determined by measuring osteocalcin and Runx-2 gene expression using real time polymerase chain reactions. A significant increase in the expression of Runx-2 and osteocalcin was observed on day 3 and day 7 post-transfection, respectively, by cells transfected with PEI/(pFGF-2 + pBMP-2) compared to cells transfected with nanoplexes containing pFGF-2 or pBMP-2 alone. Alizarin Red staining and atomic absorption spectroscopy revealed elevated levels of calcium deposition in hADMSC cultures on day 14 and day 30 post-transfection with PEI/(pFGF-2 + pBMP-2) compared to other treatments. We have shown that co-delivery of pFGF-2 and pBMP-2 results in a significant enhancement in osteogenic protein synthesis, osteogenic marker expression and subsequent mineralization. This research points to a new clinically translatable strategy for achieving efficient bone regeneration. PMID:26121311

  6. The role of fibroblast Tiam1 in tumor cell invasion and metastasis

    PubMed Central

    Xu, Kun; Rajagopal, Soumitra; Klebba, Ina; Dong, Shumin; Ji, Yuxin; Liu, Jiewei; Kuperwasser, Charlotte; Garlick, Jonathan A.; Naber, Stephen P.; Buchsbaum, Rachel J.

    2010-01-01

    The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knock-out mouse models. Conversely, tumors arising in Tiam1 knock-out mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with premalignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may play a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets. PMID:20802514

  7. Skin punch biopsy explant culture for derivation of primary human fibroblasts.

    PubMed

    Vangipuram, Malini; Ting, Dennis; Kim, Sam; Diaz, Robert; Schüle, Birgitt

    2013-01-01

    Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease(1). Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms(2) and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2

  8. The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts.

    PubMed

    Wise, John Pierce; Wise, Sandra S; Holmes, Amie L; LaCerte, Carolyne; Shaffiey, Fariba; Aboueissa, AbouEl-Makarim

    2010-06-01

    In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes. PMID:20211760

  9. The Cytotoxicity and Genotoxicity of Hexavalent Chromium in Steller Sea Lion Lung Fibroblasts Compared to Human Lung Fibroblasts

    PubMed Central

    Wise, John Pierce; Wise, Sandra S.; Holmes, Amie L.; LaCerte, Carolyne; Shaffiey, Fariba; Aboueissa, AbouEl-Makarim

    2010-01-01

    In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes. PMID:20211760

  10. Distribution of fibroblast growth factors and their roles in skin fibroblast cell migration.

    PubMed

    Song, Yong Huan; Zhu, Yu Ting; Ding, Jian; Zhou, Fei Ya; Xue, Ji Xin; Jung, Jin Hee; Li, Zhi Jie; Gao, Wei Yang

    2016-10-01

    Fibroblast growth factor (FGF)2/basic FGF is a member of the fibroblast growth factor family. Its function in skin wound healing has been well-characterized. However, the function of other FGFs in skin tissues remains to be elucidated. In the present study, FGF expression patterns in heart, liver, skin and kidney tissues were analyzed. Notably, in contrast to other tissues, only four FGFs, FGF2, 7, 10 and 21, were dominant in the skin. To examine FGF function in the wound healing process, mouse NIH3T3 fibroblast cells were treated with FGF2, FGF10 and FGF21, and cell migration was monitored. The results revealed that FGF treatment promoted cell migration, which is an important step in wound healing. In addition, FGF treatment enhanced the activity of c-Jun N-terminal kinase (JNK), a key regulator of fibroblast cell migration. To analyze its role in cell migration, FGF7 was overexpressed in fibroblast cells via a lentivirus system; however, this did not change cell migration speed. FGF2, 7, 10 and 21 were highly expressed in skin tissue, and all except FGF7 regulated fibroblast cell migration and activated JNK. The results of the present study increase our understanding of the role of FGFs in skin wound healing. PMID:27572477

  11. Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

    PubMed Central

    Bustos-Arriaga, José; García-Machorro, Jazmín; León-Juárez, Moisés; García-Cordero, Julio; Santos-Argumedo, Leopoldo; Flores-Romo, Leopoldo; Méndez-Cruz, A. René; Juárez-Delgado, Francisco J.; Cedillo-Barrón, Leticia

    2011-01-01

    Background When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times (“probing”) before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. Methodology/Principal Findings Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts. Conclusions/Significance In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral

  12. Flavonoids Derived from Abelmoschus esculentus Attenuates UV-B Induced Cell Damage in Human Dermal Fibroblasts Through Nrf2-ARE Pathway

    PubMed Central

    Patwardhan, Juilee; Bhatt, Purvi

    2016-01-01

    Background: Ultraviolet-B (UV-B) radiation is a smaller fraction of the total radiation reaching the Earth but leads to extensive damage to the deoxyribonucleic acid (DNA) and other biomolecules through formation of free radicals altering redox homeostasis of the cell. Abelmoschus esculentus (okra) has been known in Ayurveda as antidiabetic, hypolipidemic, demulscent, antispasmodic, diuretic, purgative, etc. Objective: The aim of this study is to evaluate the protective effect of flavonoids from A. esculentus against UV-B-induced cell damage in human dermal fibroblasts. Materials and Methods: UV-B protective activity of ethyl acetate (EA) fraction of okra was studied against UV-B-induced cytotoxicity, antioxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway. Results: Flavonoid-rich EA fraction depicted a significant antioxidant potential also showing presence of rutin. Pretreatment of cells with EA fraction (10–30 μg/ml) prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: Our study demonstrated for the 1st time that EA fraction of okra may reduce oxidative stress through Nrf2-ARE pathway as well as through endogenous enzymatic antioxidant system. These results suggested that flavonoids from okra may be considered as potential UV-B protective agents and may also be formulated into herbal sunscreen for topical application. SUMMARY Flavonoid-enriched ethyl acetate (EA) fraction from A. esculentus protected against ultraviolet-B (UV-B)-induced oxidative DNA damageEA fraction prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, and intracellular reactive oxygen species

  13. Human cytomegalovirus replicates in gamma-irradiated fibroblasts

    SciTech Connect

    Shanley, J.D.

    1986-12-01

    Because of the unique interdependence of human cytomegalovirus (HCMV) and the physiological state of the host cell, we evaluated the ability of human foreskin fibroblasts (HFF), exposed to gamma radiation, to support HCMV growth. Irradiation of HFF with 2,500 rADS prevented cellular proliferation and suppressed cellular DNA, but not RNA or protein synthesis. Treatment of HFF cells with 2,500 rADS 6 or 48 hours prior to infection did not alter the time course or virus yield during HCMV replication. Virus plaquing efficiency in irradiated cells was comparable to that of nonirradiated cells. As judged by thymidine incorporation and BUdR inhibition of virus replication, HCMV infection induced both thymidine kinase activity and host cell DNA synthesis in irradiated cells. In addition, virus could be recovered from HFF exposed to radiation 0-2 days after infection with HCMV. These studies indicate that the damage to cells by gamma irradiation does not alter the capacity of host cells to support HCMV replication.

  14. Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

    SciTech Connect

    Biver, Emmanuel; Soubrier, Anne-Sophie; Thouverey, Cyril; Cortet, Bernard; Broux, Odile; Caverzasio, Joseph; Hardouin, Pierre

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer FGF modulates BMPs pathway in HMSCs by down-regulating BMP/BMPR expression. Black-Right-Pointing-Pointer This effect is mediated by ERK and JNK MAPKs pathways. Black-Right-Pointing-Pointer Crosstalk between FGF and BMPs must be taken into account in skeletal bioengineering. Black-Right-Pointing-Pointer It must also be considered in the use of recombinant BMPs in orthopedic and spine surgeries. -- Abstract: Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

  15. Monoclonal antibody specific for human colon fibroblast-derived T-PA

    SciTech Connect

    Schaumann, J.P.; Olander, J.V.; Harakas, N.K.; Feder, J

    1989-05-23

    This patent describes a murine-derived hybridoma cell line capable of producing monoclonal antibody against human colon fibroblast-derived tissue plasminogen activator and the cell line selected from the group consisting of cell lines 63-4 (ATCC HB 9155), 54-2 (ATCC HB 9157) or 79-7 (ATCC HB 9156).

  16. 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.

  17. Mesenchymal stem cells induce dermal fibroblast responses to injury.

    PubMed

    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.

  18. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons.

    PubMed

    Liu, Meng-Lu; Zang, Tong; Zou, Yuhua; Chang, Joshua C; Gibson, Jay R; Huber, Kimberly M; Zhang, Chun-Li

    2013-01-01

    Cell fate can be reprogrammed by modifying intrinsic and extrinsic cues. Here we show that two small molecules (forskolin and dorsomorphin) enable the transcription factor Neurogenin 2 (NGN2) to convert human fetal lung fibroblasts into cholinergic neurons with high purity (>90%) and efficiency (up to 99% of NGN2-expressing cells). The conversion is direct without passing through a proliferative progenitor state. These human induced cholinergic neurons (hiCN) show mature electrophysiological properties and exhibit motor neuron-like features, including morphology, gene expression and the formation of functional neuromuscular junctions. Inclusion of an additional transcription factor, SOX11, also efficiently converts postnatal and adult skin fibroblasts from healthy and diseased human patients to cholinergic neurons. Taken together, this study identifies a simple and highly efficient strategy for reprogramming human fibroblasts to subtype-specific neurons. These findings offer a unique venue for investigating the molecular mechanisms underlying cellular plasticity and human neurodegenerative diseases.

  19. Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture.

    PubMed

    Yang, Jian; Shi, Guixin; Bei, Jianzhong; Wang, Shenguo; Cao, Yilin; Shang, Qingxin; Yang, Guanghui; Wang, Wenjing

    2002-12-01

    The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering. Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity. In the present study, poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated. An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed. Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds. The results show that hydrophilicity and surface energy were improved. The polar N-containing groups and positive charged groups also were incorporated into the sample surface. A low-temperature treatment was used to maintain the plasma-modified surface properties effectively. It would do help to the further application of plasma treatment technique. Cell culture results showed that pores smaller than 160 microm are suitable for human skin fibroblast cell growth. Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol. The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided. The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds. Mass transport issues also have been considered. PMID:12209930

  20. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

    PubMed

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology. PMID:27536196

  1. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

    PubMed

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

  2. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro

    PubMed Central

    Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology. PMID:27536196

  3. Centrifugal force induces human ligamentum flavum fibroblasts inflammation through activation of JNK and p38 pathways.

    PubMed

    Chao, Yuan-Hung; Tsuang, Yang-Hwei; Sun, Jui-Sheng; Sun, Man-Ger; Chen, Ming-Hong

    2012-01-01

    Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. However, the mechanisms of mechanical load on inflammation of ligamentum flavum remain unclear. In this study, we used an in vitro model of human ligamentum flavum fibroblasts subjected to centrifugal force to elucidate the effects of mechanical load on cultured human ligamentum flavum fibroblasts; we further studied its molecular and biochemical mechanisms. Human ligamentum flavum fibroblasts were obtained from six patients undergoing lumbar spine surgery. Monolayer cultures of human ligamentum flavum fibroblasts were subjected to different magnitudes of centrifugal forces. Cell viability, cell death, biochemical response, and molecular response to centrifugal forces were analyzed. It was found that centrifugal stress significantly suppressed cell viability without inducing cell death. Centrifugal force at 67.1 g/cm(2) for 60 min significantly increases the production of prostaglandin E2 and nitric oxide as well as gene expression of proinflammatory cytokines, including interleukin (IL)-1α, IL-1β and IL-6, showed that centrifugal force-dependent induction of cyclooxygense-2 and inducible NO synthase required JNK and p38 mitogen-activated protein kinase, but not ERK 1/2 activities. This study suggested that centrifugal force does induce inflammatory responses in human ligamentum flavum fibroblasts. The activation of both JNK and p38 mitogen-activated protein kinase mechanotransduction cascades is a crucial intracellular mechanism that mediates cyclooxygense-2/prostaglandin E2 and inducible NO synthase/nitric oxide production.

  4. Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.

    PubMed

    Hendudari, Farzane; Piryaei, Abbas; Hassani, Seyedeh-Nafiseh; Darbandi, Hasan; Bayat, Mohammad

    2016-05-01

    Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm(2) and energy densities of 0.5, 1, and 2 J/cm(2)). About 10% of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm(2) was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT + HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT + hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium. PMID:26984346

  5. Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.

    PubMed

    Hendudari, Farzane; Piryaei, Abbas; Hassani, Seyedeh-Nafiseh; Darbandi, Hasan; Bayat, Mohammad

    2016-05-01

    Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm(2) and energy densities of 0.5, 1, and 2 J/cm(2)). About 10% of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm(2) was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT + HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT + hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium.

  6. EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration

    PubMed Central

    Cao, Cong; Sun, Yun; Healey, Sarah; Bi, Zhigang; Hu, Gang; Wan, Shu; Kouttab, Nicola; Chu, Wenming; Wan, Yinsheng

    2006-01-01

    AQP3 (aquaporin-3), known as an integral membrane channel in epidermal keratinocytes, facilitates water and glycerol movement into and out of the skin. Here, we demonstrate that AQP3 is also expressed in cultured human skin fibroblasts, which under normal wound healing processes migrate from surrounding tissues to close the wound. EGF (epidermal growth factor), which induced fibroblast migration, also induced AQP3 expression in a time- and dose-dependent manner. CuSO4 and NiCl2, previously known as AQP3 water transport inhibitors, as well as two other bivalent heavy metals Mn2+ and Co2+, inhibited EGF-induced cell migration in human skin fibroblasts. AQP3 knockdown by small interfering RNA inhibited EGF-induced AQP3 expression and cell migration. Furthermore, an EGFR (EGF receptor) kinase inhibitor, PD153035, blocked EGF-induced AQP3 expression and cell migration. MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK inhibitor U0126 and PI3K (phosphoinositide 3-kinase) inhibitor LY294002 also inhibited EGF-induced AQP3 expression and cell migration. Collectively, our findings show for the first time that AQP3 is expressed in human skin fibroblasts and that EGF induces AQP3 expression via EGFR, PI3K and ERK signal transduction pathways. We have provided evidence for a novel role of AQP3 in human skin fibroblast cell migration, which occurs during normal wound healing. PMID:16848764

  7. Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice

    PubMed Central

    Lee, Hyunji; Hong, Youngeun; Kwon, So Hee; Park, Jongsun; Park, Jisoo

    2016-01-01

    Background Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Objective Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. Methods PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student’s unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. Results PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. Conclusion This study

  8. Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis.

    PubMed

    Ohgo, Shiro; Hasegawa, Seiji; Hasebe, Yuichi; Mizutani, Hiroshi; Nakata, Satoru; Akamatsu, Hirohiko

    2015-07-01

    During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.

  9. Influence of shieldings or antioxidants on DNA damage and early and delyed cell death induced in human fibroblasts by accelerated 595 MeV/u Fe ions

    NASA Astrophysics Data System (ADS)

    Antonelli, Francesca; Esposito, Giuseppe; Dini, Valentina; Belli, Mauro; Campa, Alessandro; Sorrentino, Eugenio; Antonella Tabocchini, Maria; Lobascio, Cesare; Berra, Bruno

    HZE particles from space radiation raise an important protection concern during long-term astronauts' travels. As high charge, high energy particles interact with a shield, both projec-tile and target fragmentation may occurs, so that the biological properties of the emerging radiation field depend on the nature and energy of the incident particles, and on the nature and thickness of the shield. We have studied the influence of PMMA and Kevlar shielding as well as the antioxidant compounds Rosmarinic acid or Resveratrol on DNA damage induction and processing (as evaluated by the g-H2AX phosphorylation assay) and on early and delayed cell death in AG01522 human fibroblasts irradiated with Fe ions of 595 MeV/u at the NASA Space Radiation Laboratory (NSRL), Brookhaven National Laboratory (BNL, Upton, USA). Insertion of PMMA or Kevlar shields (10 g/cm2 thick) gave no substantial change in the bio-logical effect per unit dose on the sample for all the end points studied. When irradiation was performed in the presence of 300 mM Rosmarinic acid or Resveratrol no difference were found for both early and delayed cell death, while a slight protective effect was observed for the initial and residual DNA damage. For both early and delayed cell death, Fe-ions are more effective than g-rays. The number of Fe-ion induced g-H2AX foci is instead lower than that induced by g-rays, due to the presence of multiple DSB within a single focus induced by Fe-ions. From a comparison of the g-H2AX data with the results on DNA fragmentation obtained with 414 MeV/u Fe ions at the Heavy Ions Medical Accelerator (HIMAC, Chiba, Japan) and with 1 GeV/u Fe ions at BNL, in the absence or in the presence of PMMA shields (Esposito et al, Advance in Space Research 2004) we speculate that the overall effect of the shield is a balance between the contributions due to the slowing down of the primary particles and that due to the nuclear fragmentation. Acknowledgment: Financial support from ASI project

  10. 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.

  11. Lack of protection of prior heat shock against UV-induced oxidative stress in human skin fibroblasts.

    PubMed

    Jones, Sandra A; McArdle, Anne; McArdle, Francis; Jack, Catherine I A; Jackson, Malcolm J

    2003-01-01

    The effect of prior hyperthermia on UV-induced oxidative stress was studied in human skin fibroblasts. UV radiation alone induced an increased release of superoxide anions and increased lipid peroxidation in skin fibroblasts accompanied by a rise in catalase and superoxide dismutase activities. Hyperthermia was found to induce a significant rise in the cell content of heat-shock proteins, HSP60 and HSP70, but this treatment prior to UV radiation did not influence any indicators of oxidative stress in the fibroblasts. In contrast, the combination of heat shock prior to UV-exposure reduced fibroblast cell viability compared with UV radiation-exposure alone.

  12. Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity

    PubMed Central

    Breslin, Loretta; Prosser, Suzanna L; Cuffe, Sandra; Morrison, Ciaran G

    2014-01-01

    Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence. PMID:25486364

  13. Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity.

    PubMed

    Breslin, Loretta; Prosser, Suzanna L; Cuffe, Sandra; Morrison, Ciaran G

    2014-01-01

    Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence. PMID:25486364

  14. Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity.

    PubMed

    Breslin, Loretta; Prosser, Suzanna L; Cuffe, Sandra; Morrison, Ciaran G

    2014-01-01

    Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence.

  15. 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

  16. Binding, uptake, and release of nicotine by human gingival fibroblasts

    SciTech Connect

    Hanes, P.J.; Schuster, G.S.; Lubas, S. )

    1991-02-01

    Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4{degree}C using a mixture of {sup 3}H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between {sup 3}H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37{degree}C after treating cells with {sup 3}H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours.

  17. Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

    PubMed

    Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Mirandola, Prisco; De Conto, Flora; Covan, Silvia; Germini, Diego; Razin, Sergey; Dettori, Giuseppe; Chezzi, Carlo

    2011-01-01

    The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65. PMID:21053310

  18. Hydrogen peroxide-induced apoptosis in human gingival fibroblasts

    PubMed Central

    Gutiérrez-Venegas, Gloria; Guadarrama-Solís, Adriana; Muñoz-Seca, Carmen; Arreguín-Cano, Juan Antonio

    2015-01-01

    In the process of bleaching vital, discolored teeth, low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2. However, interest has been expressed in the assessment of pathological effects of long-term exposure to bleaching agents such as irritation and ulceration of the gingival or other soft tissues. The aim of the present study was to determine the effect of hydrogen peroxide on apoptosis in human gingival fibroblasts (HGF). Cytochrome c, Bcl-2, Bax, Bid and caspase-3 protein expression were detected by Western blotting. HGF cell apoptosis induced by H2O2 was both dose and time dependent. The addition of H2O2 resulted in the release of cytochrome c to the cytosol, and an increase of Caspase-3 cleavage. Data suggest that oxidative stress-induced apoptosis in HGF is intrinsic pathway involved the release of apoptotic signal from mitochondria. PMID:26884825

  19. 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

  20. 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.

  1. A human cytomegalovirus gO-null mutant fails to incorporate gH/gL into the virion envelope and is unable to enter fibroblasts and epithelial and endothelial cells.

    PubMed

    Wille, Paul T; Knoche, Amber J; Nelson, Jay A; Jarvis, Michael A; Johnson, David C

    2010-03-01

    Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRDeltagO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRDeltagO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRDeltagO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRDeltagO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRDeltagO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of g

  2. Genotoxic and antiapoptotic effect of nicotine on human gingival fibroblasts.

    PubMed

    Argentin, Gabriella; Cicchetti, Rosadele

    2004-05-01

    Growing evidence suggests that nicotine, the addictive component of cigarettes, can have a direct role in tumor development by enhancing cell proliferation and impairing apoptotic process in certain types of human cancer cell lines. Since the correlation between apoptosis and DNA damage is already well documented, we investigated the response of human gingival fibroblasts (HGFs) to nicotine exposure by examining its effect on DNA damage induction and apoptotic process in parallel. To assess the genotoxicity of this drug, the cytokinesis-block micronucleus (CBMN) test was performed. Treatment of HGFs with nicotine, at a concentration of 1 microM, caused a statistically significant increase of micronucleus (MN) frequency at the tested time intervals, while no change was detected in cell growth under the same conditions. Furthermore, we found that preincubation of HGFs with 1 microM nicotine strongly attenuated staurosporine (STP)-induced apoptosis. Finally, we found that cultures exposed to nicotine showed an increase of reactive oxygen species, as determined by increased levels of 2,7-dichlorofluorescein (DCF). When cells were prelabeled with N-acetyl-cysteine (NAC), a substrate for glutathione synthesis, and catalase (CAT), the oxygen free radical scavenger, a significant reduction in cytogenetic damage was observed. Thus, for the first time, we report a concomitant genotoxic and antiapoptotic effect of nicotine in HGFs. PMID:14718647

  3. Maintenance of Multipotency in Human Dermal Fibroblasts Treated with Xenopus laevis Egg Extract Requires Exogenous Fibroblast Growth Factor-2

    PubMed Central

    Kole, Denis; Ambady, Sakthikumar; Page, Raymond L.

    2014-01-01

    Abstract Direct reprogramming of a differentiated somatic cell into a developmentally more plastic cell would offer an alternative to applications in regenerative medicine that currently depend on either embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSCs). Here we report the potential of select Xenopus laevis egg extract fractions, in combination with exogenous fibroblast growth factor-2 (FGF2), to affect life span, morphology, gene expression, protein translation, and cellular localization of OCT4 and NANOG transcription factors, and the developmental potential of human dermal fibroblasts in vitro. A gradual change in morphology is accompanied by translation of embryonic transcription factors and their nuclear localization and a life span exceeding 60 population doublings. Cells acquire the ability to follow adipogenic, neuronal, and osteogenic differentiation under appropriate induction conditions in vitro. Analysis of active extract fractions reveals that Xenopus egg protein and RNAs as well as exogenously supplemented FGF2 are required and sufficient for induction and maintenance of this phenotypic change. Factors so far identified in the active fractions include FGF2 itself, transforming growth factor-β, maskin, and nucleoplasmin. Identification of critical factors needed for reprogramming may allow for nonviral, chemically defined derivation of human-induced multipotent cells that can be maintained by exogenous FGF2. PMID:24405062

  4. Role of fibroblast growth factor receptors in astrocytic stem cells

    PubMed Central

    Galvez-Contreras, Alma Y.; Gonzalez-Castaneda, Rocio E; Luquin, Sonia; Gonzalez-Perez, Oscar

    2012-01-01

    There are two well-defined neurogenic regions in the adult brain, the subventricular zone (SVZ) lining the lateral wall of the lateral ventricles and, the subgranular zone (SGZ) in the dentate gyrus at the hippocampus. Within these neurogenic regions, there are neural stem cells with astrocytic characteristics, which actively respond to the basic fibroblast growth factor (bFGF, FGF2 or FGF-β) by increasing their proliferation, survival and differentiation, both in vivo and in vitro. FGF2 binds to fibroblast growth factor receptors 1 to 4 (FGFR1, FGFR2, FGFR3, FGFR4). Interestingly, these receptors are differentially expressed in neurogenic progenitors. During development, FGFR-1 and FGFR-2 drive oligodendrocytes and motor neuron specification. In particular, FGFR-1 determines oligodendroglial and neuronal cell fate, whereas FGFR-2 is related to oligodendrocyte specification. In the adult SVZ, FGF-2 promotes oligodendrogliogenesis and myelination. FGF-2 deficient mice show a reduction in the number of new neurons in the SGZ, which suggests that FGFR-1 is important for neuronal cell fate in the adult hippocampus. In human brain, FGF-2 appears to be an important component in the anti-depressive effect of drugs. In summary, FGF2 is an important modulator of the cell fate of neural precursor and, promotes oligodendrogenesis. In this review, we describe the expression pattern of FGFR2 and its role in neural precursors derived from the SVZ and the SGZ. PMID:22347841

  5. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    SciTech Connect

    Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S. . E-mail: hcheung@med.miami.edu

    2005-05-13

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKC{alpha}-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis.

  6. Uroporphyrin I Stimulation of Collagen Biosynthesis in Human Skin Fibroblasts

    PubMed Central

    Varigos, George; Schiltz, John R.; Bickers, David R.

    1982-01-01

    Porphyria cutanea tarda and erythropoietic porphyria are disorders of heme synthesis that originate in the liver and bone marrow, respectively. Each is characterized by increased accumulation of uroporphyrin, I, by cutaneous photosensitivity, and in some patients by indurated plaques and scarring that resemble scleroderma. These scleroderma-like lesions occur in light-exposed and light-protected body areas. In these studies we evaluated the role of uroporphyrin I and of light in evoking the scleroderma-like cutaneous changes. Normal human skin fibroblasts were exposed to uroporphyrin I and to 400 nm radiation and the effect of these agents on collagen accumulation by the cells was determined. Radioactive tracer studies showed that uroporphyrin I caused a specific increase in the accumulation of newly synthesized collagen by fibroblast monolayer cultures, as verified by [3H]hydroxyproline and collagenase digestion assays. Collagen accumulation was stimulated 1.5- to 2.7-fold by uroporphyrin I, whereas noncollagenous protein accumulation was unchanged. The increased collagen accumulation was time and uroporphyrin I-concentration-dependent, and occurred both in the presence or absence of ultraviolet light exposure. Further studies demonstrated that the increased accumulation was not the result of decreased rates of collagen degradation nor was it due to changes in cell population growth parameters (generation times and saturation densities). No changes in morphology of the treated cells occurred. These studies indicate that porphyrins possess previously undemonstrated biological effects that are independent of their photosensitizing properties. This novel dark effect of uroporphyrin I may account for the sclerodermatous lesions seen in the skin of patients with porphyria cutanea tarda and erythropoietic porphyria. PMID:7054234

  7. A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

    PubMed Central

    Ross, Jason; Busch, Julia; Mintz, Ellen; Ng, Damian; Stanley, Alexandra; Brafman, David; Sutton, V. Reid; Van den Veyver, Ignatia; Willert, Karl

    2015-01-01

    SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation. PMID:25464842

  8. Screening of medicinal and edible plants in Okinawa, Japan, for enhanced proliferative and collagen synthesis activities in NB1RGB human skin fibroblast cells.

    PubMed

    Takahashi, Makoto; Asikin, Yonathan; Takara, Kensaku; Wada, Koji

    2012-01-01

    To identify plants with bioactive potential for skin care, methanol extracts of 56 plant parts from 47 medical and edible plants cultivated in Okinawa were tested for their proliferative effects on NB1RGB skin fibroblast cells. Extracts from six plants, Bischofia javanica, Colocasia esculenta, Melaleuca alternifolia, Piper angustifolia, Jasminum sambac, and Curcuma longa, showed higher NB1RGB cell proliferation activity (>10%) than the control, at various concentrations. Among the six extracts, only the C. longa extract caused an increase in collagen synthesis in NB1RGB cells, as compared to treatment with the positive control, ascorbic acid (AsA). Expression of the collagen synthesis marker, transforming growth factor-β1, was higher after treatment with the C. longa extract than with AsA. PMID:23221723

  9. Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages.

    PubMed

    Wang, Qing-Hua; Peng, Yun; Cai, Xin-Yong; Wan, Meng; Liu, Yu; Wei, Hong

    2015-08-01

    Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

  10. Chromosome Conformation of Human Fibroblasts Grown in 3-Dimensional Spheroids

    PubMed Central

    Chen, Haiming; Comment, Nicholas; Chen, Jie; Ronquist, Scott; Hero, Alfred; Ried, Thomas; Rajapakse, Indika

    2015-01-01

    In the study of interphase chromosome organization, genome-wide chromosome conformation capture (Hi-C) maps are often generated using 2-dimensional (2D) monolayer cultures. These 2D cells have morphological deviations from cells that exist in 3-dimensional (3D) tissues in vivo, and may not maintain the same chromosome conformation. We used Hi-C maps to test the extent of differences in chromosome conformation between human fibroblasts grown in 2D cultures and those grown in 3D spheroids. Significant differences in chromosome conformation were found between 2D cells and those grown in spheroids. Intra-chromosomal interactions were generally increased in spheroid cells, with a few exceptions, while inter-chromosomal interactions were generally decreased. Overall, chromosomes located closer to the nuclear periphery had increased intra-chromosomal contacts in spheroid cells, while those located more centrally had decreased interactions. This study highlights the necessity to conduct studies on the topography of the interphase nucleus under conditions that mimic an in vivo environment. PMID:25738643

  11. Fibroblasts as Efficient Antigen-Presenting Cells in Lymphoid Organs

    NASA Astrophysics Data System (ADS)

    Kundig, Thomas M.; Bachmann, Martin F.; Dipaolo, Claudio; Simard, John J. L.; Battegay, Manuel; Lother, Heinz; Gessner, Andre; Kuhlcke, Klaus; Ohashi, Pamela S.; Hengartner, Hans; Zinkernagel, Rolf M.

    1995-06-01

    Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

  12. Flavonoids Derived from Abelmoschus esculentus Attenuates UV-B Induced Cell Damage in Human Dermal Fibroblasts Through Nrf2-ARE Pathway

    PubMed Central

    Patwardhan, Juilee; Bhatt, Purvi

    2016-01-01

    Background: Ultraviolet-B (UV-B) radiation is a smaller fraction of the total radiation reaching the Earth but leads to extensive damage to the deoxyribonucleic acid (DNA) and other biomolecules through formation of free radicals altering redox homeostasis of the cell. Abelmoschus esculentus (okra) has been known in Ayurveda as antidiabetic, hypolipidemic, demulscent, antispasmodic, diuretic, purgative, etc. Objective: The aim of this study is to evaluate the protective effect of flavonoids from A. esculentus against UV-B-induced cell damage in human dermal fibroblasts. Materials and Methods: UV-B protective activity of ethyl acetate (EA) fraction of okra was studied against UV-B-induced cytotoxicity, antioxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway. Results: Flavonoid-rich EA fraction depicted a significant antioxidant potential also showing presence of rutin. Pretreatment of cells with EA fraction (10–30 μg/ml) prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: Our study demonstrated for the 1st time that EA fraction of okra may reduce oxidative stress through Nrf2-ARE pathway as well as through endogenous enzymatic antioxidant system. These results suggested that flavonoids from okra may be considered as potential UV-B protective agents and may also be formulated into herbal sunscreen for topical application. SUMMARY Flavonoid-enriched ethyl acetate (EA) fraction from A. esculentus protected against ultraviolet-B (UV-B)-induced oxidative DNA damageEA fraction prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, and intracellular reactive oxygen species

  13. Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli.

    PubMed

    Garke, G; Deckwer, W D; Anspach, F B

    2000-01-14

    Aggregation and precipitation are major pitfalls during bioprocessing and purification of recombinant human basic fibroblast growth factor (rh-bFGF). In order to gain high yields of the soluble protein monomer with high biological activity, an efficient downstream process was developed, focussing on the combination of expanded bed adsorption (EBA) and heparin chromatography. After expression in E. coli TG1:plambdaFGFB, cells were harvested and washed; then the rh-bFGF was released via high pressure homogenization. The high viscosity of the feedstock of about 40 mPa s, showing non-newtonian behaviour, was reduced to 2 mPa s by the addition of DNase. The homogenate (5.6 l) was loaded directly on an expanded bed column (C-50) packed with the strong cation-exchanger Streamline SP. In the eluates, histone-like (HU) protein was identified as the main protein contaminant by sequence analysis. The thermodynamics and kinetics of rh-bFGF adsorption from the whole broth protein mixture were determined in view of competition and displacement effects with host-derived proteins. Optimal binding and elution conditions were developed with knowledge of the dependence of rh-bFGF adsorption isotherms on the salt concentration to allow direct application of eluates onto Heparin HyperD. This affinity support maintained selectivity and efficiency under CIP and over a wide range of flow-rates; both is advantageous for the flexibility of the purification protocol in view of a scalable process. Remaining DNA and HU protein were separated by Heparin HyperD. The endotoxin level decreased from approximately 1,000,000 EU/ml in the whole broth to 10 EU in 3 mg bFGF per ml. The final purification protocol yields >99% pure rh-bFGF as judged from SDS-PAGE and MALDI-TOF mass spectrometry with high mitogenic activity (ED50=1-1.5 ng/ml) of the lyophilized sample. In comparison to the conventional process, the overall protein recovery rose by 15% to 65% with saving time and costs. PMID:10681038

  14. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    PubMed

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  15. An evaluation of thiram toxicity on cultured human skin fibroblasts.

    PubMed

    Cereser, C; Boget, S; Parvaz, P; Revol, A

    2001-05-11

    Thiram is widely used in agriculture as a fungicide and, to a lesser extent, as a vulcanizing agent in the rubber industry. In spite of the extensive use of thiram, knowledge on its toxicity and health risk remains limited, and few investigations have been performed to assess specific damage at the cellular and subcellular level. We report here the cytotoxic effects of thiram on cultured human skin fibroblasts. Our results demonstrated that thiram exposure induced a dose- and time-dependent decrease in the viable cell recovery with 100% cell death observed with a concentration of 5.0 mg/l. As judged by morphological changes and biochemical criteria, thiram-mediated cell death was not of the apoptotic but seemed to be of the necrotic type. This cell death was not associated with a modification of gene expression of different constituents of the extracellular matrix. A late increase of lactate production was evident after thiram treatment, suggesting a mitochondrial metabolic pathway dysfunction as reported by other authors using similar compounds. However, this phenomenon appeared as a secondary response to the toxic action of thiram. The cytotoxic effect of thiram is possibly due to an oxidant effect inherent to the structure of thiram and the interaction between thiram and vital cellular molecules.

  16. 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.

  17. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  18. Characterization of membrane antigens on human cytomegalovirus-infected fibroblasts recognized by human antibodies

    SciTech Connect

    van der Voort, L.H.M.; de Leij, L.F.M.H.; The T.H.

    1989-03-01

    The antigens on the surface of human cytomegalovirus (HCMV)-infected fibroblasts which are recognized by human HCMV antibody-positive sera were characterized. Three HCMV-induced polypeptides, with apparent molecular masses of 53 to 63, 94, and 94 to 120 kilodaltons, were precipitated from /sup 125/I-surface-labeled cell extracts with different sera obtained from healthy individuals. Renal transplant recipients who were suffering from active HCMV infections recognized the same set of antigens. By the use of monoclonal antibodies, these antigens were identified as polypeptides belonging to the gcI and gcIII families of HCMV glycoproteins.

  19. Inhibition of matrix metalloproteinase-1 and type-I procollagen expression by phenolic compounds isolated from the leaves of Quercus mongolica in ultraviolet-irradiated human fibroblast cells.

    PubMed

    Kim, Han Hyuk; Kim, Dong Hee; Oh, Myeong Hwan; Park, Kwang Jun; Heo, Jun Hyeok; Lee, Min Won

    2015-01-01

    The aim of this study was to investigate the effect of Quercus mongolica (QM) which induce anti-photoaging process of skin in vitro. Bioassay-guided isolation of 80 % Me2CO extract of the leaves of QM led to the isolation and identification of six known phenolic compounds: pedunculagin (1), (-)-epigallocatechin (2), (+)-catechin (3), quercetin 3-O-(6″-O-galloyl)-β-D-glucopyranoside (4), kaempferol-3-O-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside (5) and kaempferol 3-O-(6″-galloyl)-β-D-glucopyranoside (6). The effects of compounds 1-6 on expression of matrix metalloproteinase-1 (MMP-1) and type-I procollagen were further evaluated. Among them, compound 1 showed potent inhibitory effect on MMP-1 and the increased type-I procollagen synthesis in ultraviolet B-induced human fibroblast. These results suggest that pedunculagin, an ellagitannin, is a potential candidate for the prevention and treatment of skin aging.

  20. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links.

    PubMed

    Clingen, Peter H; Arlett, Colin F; Hartley, John A; Parris, Christopher N

    2007-02-15

    Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity. PMID:17188678

  1. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

    SciTech Connect

    Clingen, Peter H. . E-mail: p.clingen@ucl.ac.uk; Arlett, Colin F.; Hartley, John A.; Parris, Christopher N.

    2007-02-15

    Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.

  2. Iron Accumulation During Cellular Senescence in Human Fibroblasts In Vitro

    PubMed Central

    KILLILEA, DAVID W.; ATAMNA, HANI; LIAO, CHARLES; AMES, BRUCE N.

    2015-01-01

    Iron accumulates as a function of age in several tissues in vivo and is associated with the pathology of numerous age-related diseases. The molecular basis of this change may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary human fibroblast cells (IMR-90) were studied in vitro as a model of cellular senescence. Total iron content increased exponentially during cellular senescence, resulting in 10-fold higher levels of iron compared with young cells. Low-dose hydrogen peroxide (H2O2) induced early senescence in IMR-90s and concomitantly accelerated iron accumulation. Furthermore, senescence-related and H2O2-stimulated iron accumulation was attenuated by N-tert-butylhydroxylamine (NtBHA), a mitochondrial antioxidant that delays senescence in vitro. However, SV40-transformed, immortalized IMR-90s showed no time-dependent changes in metal content in culture or when treated with H2O2 and/or NtBHA. These data indicate that iron accumulation occurs during normal cellular senescence in vitro. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells. PMID:14580305

  3. Transcriptional analysis of normal human fibroblast responses to microgravity stress.

    PubMed

    Liu, Yongqing; Wang, Eugenia

    2008-03-01

    To understand the molecular mechanism(s) of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the space-flown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization (SSH) to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several G1-phase cell cycle traverse genes. Other genes showing up-regulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the "hub" for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

  4. Cytotoxicity of orthodontic bonding adhesive resins on human oral fibroblasts.

    PubMed

    Ahrari, Farzaneh; Tavakkol Afshari, Jalil; Poosti, Maryam; Brook, Azam

    2010-12-01

    There is little information concerning the cytotoxic effects of no-mix and flowable adhesives used in orthodontics. The aim of the present study was to evaluate the cytotoxic effects of a no-mix (Unite), a light-cured (Tranbond XT), and a flowable (Denfil Flow) adhesives on human oral fibroblasts. Twelve discs of each adhesive were prepared and aged for 1, 3, 5, and 7 days in Dulbecco's Modified Eagle's Medium (DMEM). Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the difference between the groups was tested by analysis of variance and Tukey tests (α = 0.05). After 1 day of storage, the no-mix adhesive showed moderate cytotoxic effects (P < 0.05), while the light-cured and flowable adhesives were essentially non-cytotoxic. Ageing considerably reduced the cytotoxicity of the no-mix adhesive. On days 5 and 7 of the experiment, the cell viability of three adhesives did not differ significantly (P > 0.05), but cell viability was slightly reduced on day 7. Moderate cytotoxic effects of no-mix adhesive on the first day of the experiment suggest that care should be taken to protect dentists and patients when these adhesives are being handled. Despite higher resin components, the flowable adhesive showed excellent biocompatibility.

  5. Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation

    SciTech Connect

    Antoine, M.; Wirz, W.; Tag, C.G.; Gressner, A.M.; Wycislo, M.; Mueller, R.; Kiefer, P. . E-mail: pkiefer@ukaachen.de

    2006-07-21

    Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.

  6. 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

  7. The influence of elastin-like recombinant polymer on the self-renewing potential of a 3D tissue equivalent derived from human lamina propria fibroblasts and oral epithelial cells.

    PubMed

    Kinikoglu, Beste; Rodríguez-Cabello, José Carlos; Damour, Odile; Hasirci, Vasif

    2011-09-01

    Three-dimensional epithelial tissue equivalents tend to lose their self-renewing potential progressively during culture as their epithelial cells lose their proliferative capacity with time. Even though the tissue engineered construct can mimic the native tissue well, it rapidly degrades after implantation due to the insufficient number of proliferating cells in the equivalent. In the present study we demonstrate for the first time that the use of an elastin-like recombinant polymer (ELR) engineered to contain the cell adhesion peptide RGD can result in a 3D tissue equivalent with high self-renewing potential, containing as many proliferative cells as the native tissue itself. The 3D tissue equivalent was reconstructed by the coculture of human lamina propria fibroblasts and oral epithelial cells in the nanofibrous ELR-collagen scaffold. Histological, immunohistological and transmission electron microscopic analyses of this oral mucosa equivalent demonstrated the expression of markers characteristic of epithelial proliferation (Ki67) and differentiation (keratin 13), and also the presence of a pluristratified epithelium and an ultrastructurally well-organized basement membrane expressing laminin 332. The synthesis of new extracellular matrix by the fibroblasts was also demonstrated. The scaffold proposed here presents great potential for tissue engineering applications, and also for studies of epithelial proliferation, and epithelial disorders including carcinogenesis. PMID:21592566

  8. Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture.

    PubMed Central

    Sugiyama, A; Arakaki, R; Ohnishi, T; Arakaki, N; Daikuhara, Y; Takada, H

    1996-01-01

    Lipoteichoic acids (LTA) from various gram-positive bacteria, including oral streptococci such as Streptococcus sanguis, enhanced the production of hepatocyte growth factor (HGF) (scatter factor) by human gingival fibroblasts in culture, whereas lipopolysaccharides (LPS) from various gram-negative bacteria did not. In contrast, LPS induced interleukin 1 activity in human gingival epithelial cells in culture, while LTA had little effect. LTA and recombinant human interleukin 1 alpha enhanced synergistically the production of HGF/SF in human gingival fibroblast cultures. Recombinant human HGF, in turn, enhanced the proliferation of human gingival epithelial cells in culture. PMID:8606111

  9. In vitro activation of human fibroblasts by retrieved titanium alloy wear debris.

    PubMed

    Manlapaz, M; Maloney, W J; Smith, R L

    1996-05-01

    Titanium-aluminum-vanadium wear particles isolated from the soft-issue membrane of a failed total hip arthroplasty were added to human fibroblasts in cell culture. The cellular response to particle challenge was determined by assaying for levels of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, prostaglandin E2, basic fibroblast growth factor, platelet-derived growth factor-AB, and transforming growth factor-beta. Collagenase and gelatinase activities were analyzed by zymography and [3H]collagen degradation. Cell viability was assessed by measuring the uptake of [3H]thymidine. Over the range of particle concentrations tested, cell viability, as demonstrated by [3H]thymidine uptake, remained unaffected. Fibroblasts exhibited a dose-dependent release of interleukin-6 in response to exposure to titanium-aluminum-vanadium particles. At 6 and 48 hours, the highest concentration of titanium alloy particles (0.189% [vol/vol]) resulted in 7-fold and 16-fold increases in interleukin-6 release, respectively, when compared with negative controls. Neither interleukin-1 beta nor tumor necrosis factor-alpha was detected in the culture medium at any particle concentration tested for both dermal and foreskin fibroblasts. The pattern of prostaglandin E2 release by fibroblasts mirrored the pattern of interleukin-6 release. Fibroblasts exposed to the highest concentration of titanium alloy particles showed an increase in collagenase activity, starting at 12 hours. When medium samples were treated with amino phenylmercuric acetate to activate latent enzymes, a statistically significant increase in collagenase activity was observed as early as 6 hours (p < 0.001). Substrate gel analysis of medium from fibroblasts stimulated by high particle concentrations also showed an increase in gelatinolytic activity when compared with unstimulated controls. Analysis of medium samples for growth factors showed an increase in basic fibroblast growth factor at low particle

  10. The polypeptide in Chlamys farreri can protect human dermal fibroblasts from ultraviolet B damage

    NASA Astrophysics Data System (ADS)

    Zhang, Yujiang; Zhan, Songmei; Cao, Pengli; Liu, Ning; Chen, Xuehong; Wang, Yuejun; Wang, Chunbo

    2005-09-01

    To investigate the effect of polypeptide from Chlamys farreri (PCF) on NHDF in vitro, we modeled oxidative damage on normal human dermal fibroblasts (NHDF) exposed to ultraviolet B (UVB). In this study, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were tested to measure cell viability. Enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and xanthine oxidase (XOD) were determined biochemically. Total antioxidative capacity (T-AOC) and anti-superoxide anion capacity (A-SAC) were also determined. Ultrastructure of fibroblasts was observed under transmission electron microscope. The results showed that: UVB (1.176×10-4 J/cm2) suppressed the growth of fibroblasts and the introduction of PCF (0.25% 1%) before UVB reduced the suppression in a concentration-dependent manner. PCF could enhance the activities of SOD, GSH-PX and T-AOC as well as A-SAC. Also PCF could inhibit XOD activity, while it did not affect CAT activity. Ultrastructure of fibroblasts were damaged after UVB irradiation, concentration-dependent PCF reduced the destructive effect of UVB on cells. These results indicated that PCF can protect human dermal fibroblasts from being harmed by UVB irradiation via its antioxidant proerty.

  11. 5Z-7-Oxozeanol Inhibits the Effects of TGFβ1 on Human Gingival Fibroblasts.

    PubMed

    Kuk, Hanna; Hutchenreuther, James; Murphy-Marshman, Hannah; Carter, David; Leask, Andrew

    2015-01-01

    Transforming growth factor (TGF)β acts on fibroblasts to promote the production and remodeling of extracellular matrix (ECM). In adult humans, excessive action of TGFβ is associated with fibrotic disease and fibroproliferative conditions, including gingival hyperplasia. Understanding how the TGFβ1 signals in fibroblasts is therefore likely to result in valuable insights into the fundamental mechanisms underlying fibroproliferative disorders. Previously, we used the TAK1 inhibitor (5Z)-7-Oxozeaenol to show that, in dermal fibroblasts, the non-canonical TAK1 pathway mediates the ability of TGFβ1 to induce genes promoting tissue remodeling and repair. However, the extent to which TAK1 mediates fibroproliferative responses in fibroblasts in response to TGFβ1 remains unclear. Herein, we show that, in gingival fibroblasts, (5Z)-7-Oxozeaenol blocks the ability of TGFβ1 to induce expression of the pro-fibrotic mediator CCN2 (connective tissue growth factor, CTGF) and type I collagen protein. Moreover, genome-wide expression profiling revealed that, in gingival fibroblasts, (5Z)-7-Oxozeaenol reduces the ability of TGFβ1 to induce mRNA expression of essentially all TGFβ1-responsive genes (139/147), including those involved with a hyperproliferative response. Results from microarray analysis were confirmed using real time polymerase chain reaction analysis and a functional cell proliferation assay. Our results are consistent with the hypothesis that TAK1 inhibitors might be useful in treating fibroproliferative disorders, including that in the oral cavity.

  12. An in vitro evaluation of the cytotoxicity of various endodontic irrigants on human gingival fibroblasts.

    PubMed

    Barnhart, Brian D; Chuang, Augustine; Lucca, Jurandir J Dalle; Roberts, Steven; Liewehr, Frederick; Joyce, Anthony P

    2005-08-01

    The purpose of this study was to measure the cytotoxicity of six endodontic irrigants on cultured gingival fibroblasts using the CyQuant assay. Human gingival fibroblasts were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum at 37 degrees C and 5% CO(2). At confluence, cells were split, plated in 96-well plates and incubated for 24-h to allow attachment. The following irrigants were tested at various concentrations: Sodium hypochlorite (NaOCl); iodine potassium-iodide (IKI); Betadine scrub (BS); calcium hydroxide [Ca(OH)2]; chlorine dioxide (SCD) and DMEM (positive control). Experimental groups were compared by the logarithmic difference between the clinical and LD50 concentrations of a particular irrigant. The results showed that IKI and Ca(OH)2 were significantly less cytotoxic than SCD, NaOCl, and BS. In conclusion, IKI and Ca(OH)2 are well tolerated by human gingival fibroblasts.

  13. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

  14. Mitochondrial trifunctional protein deficiency in human cultured fibroblasts: effects of bezafibrate.

    PubMed

    Djouadi, Fatima; Habarou, Florence; Le Bachelier, Carole; Ferdinandusse, Sacha; Schlemmer, Dimitri; Benoist, Jean François; Boutron, Audrey; Andresen, Brage S; Visser, Gepke; de Lonlay, Pascale; Olpin, Simon; Fukao, Toshiyuki; Yamaguchi, Seiji; Strauss, Arnold W; Wanders, Ronald J A; Bastin, Jean

    2016-01-01

    Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 μM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes. PMID:26109258

  15. The Physiological Period Length of the Human Circadian Clock In Vivo Is Directly Proportional to Period in Human Fibroblasts

    PubMed Central

    Moriggi, Ermanno; Revell, Victoria L.; Hack, Lisa M.; Lockley, Steven W.; Arendt, Josephine; Skene, Debra J.; Meier, Fides; Izakovic, Jan; Wirz-Justice, Anna; Cajochen, Christian; Sergeeva, Oksana J.; Cheresiz, Sergei V.; Danilenko, Konstantin V.; Eckert, Anne; Brown, Steven A.

    2010-01-01

    Background Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior. Methodology/Principal Findings In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer. Conclusions/Significance We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness. PMID:21042402

  16. Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

    SciTech Connect

    Espinoza, B.; Wharton, W.

    1986-08-01

    Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition in the growth of sparse human fibrobasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3,5-cyclic monophosphate concentrations.

  17. Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis.

    PubMed Central

    Wilhelm, S M; Eisen, A Z; Teter, M; Clark, S D; Kronberger, A; Goldberg, G

    1986-01-01

    Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion. Images PMID:3012533

  18. Improved targeting and enhanced retention of the human, autologous, fibroblast-derived, induced, pluripotent stem cells to the sarcomeres of the infarcted myocardium with the aid of the bioengineered, heterospecific, tetravalent antibodies**

    PubMed Central

    Malecki, Marek

    2013-01-01

    Clinical trials, to regenerate the human heart injured by myocardial infarction, involve the delivery of stem cells to the site of the injury. However, only a small fraction of the introduced stem cells are detected at the site of the injury, merely two weeks after this therapeutic intervention. This significantly hampers the effectiveness of the stem cell therapy. To resolve the aforementioned problem, we genetically and molecularly bioengineered heterospecific, tetravalent antibodies (htAbs), which have both exquisite specificity and high affinity towards human, pluripotent, stem cells through the htAbs’ domains binding SSEA-4, SSEA-3, TRA-1-60, and TRA-1-81, as well as towards the injured cardiac muscle through the htAbs’ domains binding human cardiac myosin, α-actinin, actin, and titin. The cardiac tissue was acquired from the patients, who were receiving heart transplants. The autologous, human, induced, pluripotent stem cells (hiPSCs) were generated from the patients’ fibroblasts by non-viral delivery and transient expression of the DNA constructs for: Oct4, Nanog, Sox2, Lin28, Klf4, c-Myc. In the trials involving the htAbs, the human, induced, pluripotent stem cells anchored to the myocardial sarcomeres with the efficiency, statistically, significantly higher, than in the trials with non-specific or without antibodies (p < 0.0003). Moreover, application of the htAbs resulted in cross-linking of the sarcomeric proteins to create the stable scaffolds for anchoring of the stem cells. Thereafter, these human, induced pluripotent stem cells differentiated into cardiomyocytes at their anchorage sites. By bioengineering of these novel heterospecific, tetravalent antibodies and using them to guide and to anchor the stem cells specifically to the stabilized sarcomeric scaffolds, we demonstrated the proof of concept in vitro for improving effectiveness of regenerative therapy of myocardial infarction and created the foundations for the trials in vivo. PMID

  19. 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.

  20. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  1. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

    PubMed

    Varma, Sandeep R; Sivaprakasam, Thiyagarajan O; Mishra, Abheepsa; Kumar, L M Sharath; Prakash, N S; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  2. The roles of tumor- and metastasis-promoting carcinoma-associated fibroblasts in human carcinomas.

    PubMed

    Mezawa, Yoshihiro; Orimo, Akira

    2016-09-01

    Carcinoma-associated fibroblasts (CAFs) constitute a substantial proportion of the non-neoplastic mesenchymal cell compartment in various human tumors. These fibroblasts are phenotypically converted from their progenitors via interactions with nearby cancer cells during the course of tumor progression. The resulting CAFs, in turn, support the growth and progression of carcinoma cells. These fibroblasts have a major influence on the hallmarks of carcinoma and promote tumor malignancy through the secretion of tumor-promoting growth factors, cytokines and exosomes, as well as through the remodeling of the extracellular matrix. Coevolution of CAFs and carcinoma cells during tumorigenesis is therefore essential for progression into fully malignant tumors. Recent studies have revealed the molecular mechanisms underlying CAF functions, especially in tumor invasion, metastasis and drug resistance and have highlighted the significant heterogeneity among these cells. In this review, we summarize the impacts of recently identified roles of tumor-promoting CAFs and discuss the therapeutic implications of targeting the heterotypic interactions of these fibroblasts with carcinoma cells. Graphical Abstract ᅟ. PMID:27506216

  3. The human cytomegalovirus UL133-138 gene locus attenuates the lytic viral cycle in fibroblasts.

    PubMed

    Dutta, Nirmal; Lashmit, Philip; Yuan, Jinxiang; Meier, Jeffery; Stinski, Mark F

    2015-01-01

    The genomes of HCMV clinical strains (e.g. FIX, TR, PH, etc) contain a 15 kb region that encodes 20 putative ORFs. The region, termed ULb', is lost after serial passage of virus in human foreskin fibroblast (HFF) cell culture. Compared to clinical strains, laboratory strains replicate faster and to higher titers of infectious virus. We made recombinant viruses with 22, 14, or 7 ORFs deleted from the ULb' region using FIX and TR as model clinical strains. We also introduced a stop codon into single ORFs between UL133 and UL138 to prevent protein expression. All deletions within ULb' and all stop codon mutants within the UL133 to UL138 region increased to varying degrees, viral major immediate early RNA and protein, DNA, and cell-free infectious virus compared to the wild type viruses. The wild type viral proteins slowed down the viral replication process along with cell-free infectious virus release from human fibroblast cells.

  4. The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein.

    PubMed Central

    Keay, S; Baldwin, B

    1992-01-01

    A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor. Images PMID:1321272

  5. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  6. Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

    PubMed Central

    Gupta, S L; Carlin, J M; Pyati, P; Dai, W; Pfefferkorn, E R; Murphy, M J

    1994-01-01

    Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts. Images PMID:8188349

  7. Osteoclast-independent bone resorption by fibroblast-like cells

    PubMed Central

    Pap, Thomas; Claus, Anja; Ohtsu, Susumu; Hummel, Klaus M; Schwartz, Peter; Drynda, Susanne; Pap, Géza; Machner, Andreas; Stein, Bernhard; George, Michael; Gay, Renate E; Neumann, Wolfram; Gay, Steffen; Aicher, Wilhelm K

    2003-01-01

    To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders. PMID:12723988

  8. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

    PubMed

    Bishop, J E; Mitchell, J J; Absher, P M; Baldor, L; Geller, H A; Woodcock-Mitchell, J; Hamblin, M J; Vacek, P; Low, R B

    1993-08-01

    Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

  9. Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis.

    PubMed

    Trivedy, C; Meghji, S; Warnakulasuriya, K A; Johnson, N W; Harris, M

    2001-09-01

    Copper is implicated in the pathogenesis of several fibrotic disorders. Areca nut has been shown to have a high copper content and areca chewing is associated with oral submucous fibrosis (OSF). The effects of copper on human oral fibroblasts were investigated in vitro. Human oral fibroblasts were incubated with copper chloride (CuCl2) at concentrations ranging from 0.01 microM to 500 microM for 24 h, and in vitro cell proliferation was assayed by incorporation of tritiated-thymidine; soluble and non-soluble collagen synthesis was assayed using tritiated-proline. Addition of copper chloride at concentrations ranging from 0.1 microM to 50 microM increased the collagen synthesis by the oral fibroblasts compared with growth without copper (P<0.05). The addition of copper chloride neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. We conclude that copper upregulates collagen production in oral fibroblasts. This appears to be concentration dependent, with peak collagen synthesis at 50 microM CuCl2. These in vitro results taken together with the recent findings of copper in oral biopsies from OSF subjects support the hypothesis that copper in areca nut acts as a mediator of OSF.

  10. Imatinib mesylate inhibits proliferation and exerts an antifibrotic effect in human breast stroma fibroblasts.

    PubMed

    Gioni, Vassiliki; Karampinas, Theodoros; Voutsinas, Gerassimos; Roussidis, Andreas E; Papadopoulos, Savvas; Karamanos, Nikos K; Kletsas, Dimitris

    2008-05-01

    Tumor stroma plays an important role in cancer development. In a variety of tumors, such as breast carcinomas, a desmoplastic response, characterized by stromal fibroblast and collagen accumulation, is observed having synergistic effects on tumor progression. However, the effect of known anticancer drugs on stromal cells has not been thoroughly investigated. Imatinib mesylate is a selective inhibitor of several protein tyrosine kinases, including the receptor of platelet-derived growth factor, an important mediator of desmoplasia. Recently, we have shown that imatinib inhibits the growth and invasiveness of human epithelial breast cancer cells. Here, we studied the effect of imatinib on the proliferation and collagen accumulation in breast stromal fibroblasts. We have shown that it blocks the activation of the extracellular signal-regulated kinase and Akt signaling pathways and up-regulates cyclin-dependent kinase inhibitor p21(WAF1), leading to the inhibition of fibroblast proliferation, by arresting them at the G(0)/G(1) phase of the cell cycle. Imatinib inhibits more potently the platelet-derived growth factor-mediated stimulation of breast fibroblast proliferation. By using specific inhibitors, we have found that this is due to the inhibition of the Akt pathway. In addition, imatinib inhibits fibroblast-mediated collagen accumulation. Conventional and quantitative PCR analysis, as well as gelatin zymography, indicates that this is due to the down-regulation of mRNA synthesis of collagen I and collagen III-the main collagen types in breast stroma-and not to the up-regulation or activation of collagenases matrix metalloproteinase 2 and matrix metalloproteinase 9. These data indicate that imatinib has an antifibrotic effect on human breast stromal fibroblasts that may inhibit desmoplastic reaction and thus tumor progression.

  11. Regulation of bradykinin receptor gene expression in human lung fibroblasts.

    PubMed

    Phagoo, S B; Yaqoob, M; Herrera-Martinez, E; McIntyre, P; Jones, C; Burgess, G M

    2000-06-01

    In WI-38 human fibroblasts, interleukin-1 beta and tumour necrosis factor-alpha (TNF-alpha) increased bradykinin B(1) receptor mRNA, which peaked between 2 and 4 h, remaining elevated for 20 h. Binding of the bradykinin B(1) receptor selective ligand [3H]des-Arg(10)-kallidin, also increased, peaking at 4 h and remaining elevated for 20 h. The B(max) value for [3H]des-Arg(10)-kallidin rose from 280+/-102 fmol/mg (n=3) to 701+/-147 fmol/mg (n=3), but the K(D) value remained unaltered (control, 1.04+/-0.33 nM (n=3); interleukin-1 beta, 0.88+/-0.41 nM (n=3)). The interleukin-1 beta-induced [3H]des-Arg(10)-kallidin binding sites were functional receptors, as bradykinin B(1) receptor agonist-induced responses increased in treated cells. Bradykinin B(2) receptor mRNA and [3H]bradykinin binding were upregulated by interleukin-1 beta, but not TNF-alpha. The effect of interleukin-1 beta on bradykinin B(2) receptors was smaller than for bradykinin B(1) receptors. Cycloheximide prevented interleukin-1 beta-mediated increases in B(1) and B(2) binding, but not mRNA suggesting that de novo synthesis of a transcriptional activator was unnecessary.

  12. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

    PubMed

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G

    2012-11-23

    The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  13. Forced expression of chimeric human fibroblast tropomyosin mutants affects cytokinesis

    PubMed Central

    1995-01-01

    Human fibroblasts generate at least eight tropomyosin (TM) isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) from four distinct genes, and we have previously demonstrated that bacterially produced chimera hTM5/3 exhibits an unusually high affinity for actin filaments and a loss of the salt dependence typical for TM-actin binding (Novy, R.E., J. R. Sellers, L.-F. Liu, and J.J.-C. Lin, 1993. Cell Motil. & Cytoskeleton. 26: 248-261). To examine the functional consequences of expressing this mutant TM isoform in vivo, we have transfected CHO cells with the full-length cDNA for hTM5/3 and compared them to cells transfected with hTM3 and hTM5. Immunofluorescence microscopy reveals that stably transfected CHO cells incorporate force- expressed hTM3 and hTM5 into stress fibers with no significant effect on general cell morphology, microfilament organization or cytokinesis. In stable lines expressing hTM5/3, however, cell division is slow and sometimes incomplete. The doubling time and the incidence of multinucleate cells in the stable hTM5/3 lines roughly parallel expression levels. A closely related chimeric isoform hTM5/2, which differs only in the internal, alternatively spliced exon also produces defects in cytokinesis, suggesting that normal TM function may involve coordination between the amino and carboxy terminal regions. This coordination may be prevented in the chimeric mutants. As bacterially produced hTM5/3 and hTM5/2 can displace hTM3 and hTM5 from actin filaments in vitro, it is likely that CHO-expressed hTM5/3 and hTM5/2 can displace endogenous TMs to act dominantly in vivo. These results support a role for nonmuscle TM isoforms in the fine tuning of microfilament organization during cytokinesis. Additionally, we find that overexpression of TM does not stabilize endogenous microfilaments, rather, the hTM-expressing cells are actually more sensitive to cytochalasin B. This suggests that regulation of microfilament integrity in vivo

  14. Expression of recombinant platelet-derived growth factor A- and B-chain homodimers in rat-1 cells and human fibroblasts reveals differences in protein processing and autocrine effects.

    PubMed Central

    Bywater, M; Rorsman, F; Bongcam-Rudloff, E; Mark, G; Hammacher, A; Heldin, C H; Westermark, B; Betsholtz, C

    1988-01-01

    The autocrine effects of platelet-derived growth factor (PDGF) A- and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A- and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A- and B-chain transfectants released PDGF receptor-competing activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms. Images PMID:3405217

  15. 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

  16. Characterization of chitosan films and effects on fibroblast cell attachment and proliferation.

    PubMed

    Hamilton, V; Yuan, Y; Rigney, D A; Puckett, A D; Ong, J L; Yang, Y; Elder, S H; Bumgardner, J D

    2006-12-01

    Chitosan has been researched for implant and wound healing applications. However, there are inconsistencies in reports on the tissue and fibroblast responses to chitosan materials. These inconsistencies may be due to variations in chitosan material characteristics. The aim of this study was to correlate fibroblast responses with known chitosan material characteristics. To achieve this aim, chitosan was characterized for degree of deacetylation (DDA), molecular weight (MW), residual protein and ash contents, and then solution cast into films and characterized for hydrophilicity by water contact angle. The films were seeded with normal human dermal fibroblasts and the number of attached cells was evaluated for after 30 min. Cell proliferation was evaluated over 5 days. This study found no relationship between DDA, contact angle, cell attachment, and or proliferation. General trends were observed for increasing proliferation with increasing residual ash content and decreasing residual protein. These data indicate that chitosan characteristics other than DDA may be important to their biological performance.

  17. A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

    PubMed

    Song, Tuzz-Ying; Yeh, Shu-Lan; Hu, Miao-Lin; Chen, Mei-Yau; Yang, Nae-Cherng

    2015-12-01

    Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation.

  18. Influence of E-smoking liquids on human periodontal ligament fibroblasts

    PubMed Central

    2014-01-01

    Introduction Over the last years, electronic cigarettes (ECs) have become more popular, particularly in individuals who want to give up smoking tobacco. The aim of the present study was to assess the influence of the different e-smoking liquids on the viability and proliferation of human periodontal ligament fibroblasts. Method and materials For this study six test solutions with components from ECs were selected: lime-, hazelnut- and menthol-flavored liquids, nicotine, propylene glycol, and PBS as control group. The fibroblasts were incubated up to 96 h with the different liquids, and cell viability was measured by using the PrestoBlue® reagent, the ATP detection and the migration assay. Fluorescence staining was carried out to visualize cell growth and morphology. Data were statistically analyzed by two-tailed one-way ANOVA. Results The cell viability assay showed that the proliferation rates of the cells incubated with nicotine or the various flavored liquids of the e-cigarettes were reduced in comparison to the controls, though not all reductions were statistically significant. After an incubation of 96 h with the menthol-flavored liquid the fibroblasts were statistically significant reduced (p < 0.001). Similar results were found for the detection of ATP in fibroblasts; the incubation with menthol-flavored liquids (p < 0.001) led to a statistically significant reduction. The cell visualization tests confirmed these findings. Conclusion Within its limits, the present in vitro study demonstrated that menthol additives of e-smoking have a harmful effect on human periodontal ligament fibroblasts. This might indicate that menthol additives should be avoided for e-cigarettes. PMID:25224853