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Sample records for fibroblasts culture differential

  1. Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells.

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    Ishii, Kenichiro; Mizokami, Atsushi; Tsunoda, Toshiyuki; Iguchi, Kazuhiro; Kato, Manabu; Hori, Yasuhide; Arima, Kiminobu; Namiki, Mikio; Sugimura, Yoshiki

    2011-12-01

    In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.

  2. Ras Activated ERK and PI3K Pathways Differentially Affect Directional Movement of Cultured Fibroblasts

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    Leandra Sepe

    2013-01-01

    Full Text Available Background: Cell migration is essential in physiological and pathological processes, such as wound healing and metastasis formation. Ras involvement in these processes has been extensively demonstrated. This work attempts to characterize Ras regulation of the phenomena determining directional cell migration by separately analyzing the role of its principal effector pathways, MAPK and PI3K. Methods: NIH3T3 and NIHRasV12 fibroblasts were followed in wound healing assays to study, in time and under a directional stimulus, cell migration both under standard conditions and in presence of MAPK and PI3K inhibitors. Several parameters, descriptive of specific aspects of cell motion, were evaluated by coupling dynamic microscopy with quantitative and statistical methods. Quantitative Western Blots coupled with immunofluorescence stainings, were used to evaluate ERK activation. Results: Constitutive RasV12 activation confers to NIH3T3 the ability to close the wound faster. Neither increased cell proliferation nor higher speed explains the accelerated healing, but the increased directional migration drives the wound closure. Inhibition of ERK activation, which occurs immediately after wound, greatly blocks the directional migration, while inhibition of PI3K pathway reduces cell speed but does not prevent wound closure. Conclusion: Ras is greatly involved in determining and regulating directionality, ERK is its key effector for starting, driving and regulating directional movement.

  3. The differential proliferative response of fetal and adult human skin fibroblasts to TGF-β is retained when cultured in the presence of fibronectin or collagen.

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    Armatas, Andreas A; Pratsinis, Harris; Mavrogonatou, Eleni; Angelopoulou, Maria T; Kouroumalis, Anastasios; Karamanos, Nikos K; Kletsas, Dimitris

    2014-08-01

    Transforming growth factor-β is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-β inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-β on fetal and adult cells. Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-β was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. Fetal skin fibroblast-proliferation was inhibited by TGF-β, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-β on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK-ERK pathway. Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-β when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. The interplay between TGF-β and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Physiological ER Stress Mediates the Differentiation of Fibroblasts.

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    Shinsuke Matsuzaki

    Full Text Available Recently, accumulating reports have suggested the importance of endoplasmic reticulum (ER stress signaling in the differentiation of several tissues and cells, including myoblasts and osteoblasts. Secretory cells are easily subjected to ER stress during maturation of their secreted proteins. Skin fibroblasts produce and release several proteins, such as collagens, matrix metalloproteinases (MMPs, the tissue inhibitors of metalloproteinases (TIMPs and glycosaminoglycans (GAGs, and the production of these proteins is increased at wound sites. Differentiation of fibroblasts into myofibroblasts is one of the key factors for wound healing and that TGF-β can induce fibroblast differentiation into myofibroblasts, which express α-smooth muscle actin. Well-differentiated myofibroblasts show increased production of collagen and TGF-β, and bring about wound healing. In this study, we examined the effects of ER stress signaling on the differentiation of fibroblasts, which is required for wound healing, using constitutively ER stress-activated primary cultured fibroblasts. The cells expressed positive α-smooth muscle actin signals without TGF-β stimulation compared with control fibroblasts. Gel-contraction assays suggested that ER stress-treated primary fibroblasts caused stronger shrinkage of collagen gels than control cells. These results suggest that ER stress signaling could accelerate the differentiation of fibroblasts to myofibroblasts at injured sites. The present findings may provide important insights for developing therapies to improve wound healing.

  5. Alkaline phosphatase expression/activity and multilineage differentiation potential are the differences between fibroblasts and orbital fat-derived stem cells--a study in animal serum-free culture conditions.

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    Martins, Thaís Maria da Mata; de Paula, Ana Cláudia Chagas; Gomes, Dawidson Assis; Goes, Alfredo Miranda

    2014-10-01

    Human orbital fat tissues are a potential source to isolate stem cells for the development of regenerative medicine therapies. For future safe clinical application of these cells, it is critical to establish animal component-free culture conditions as well as to clearly define the stem cell population characteristics differentiating them from other cell types, such as fibroblasts. Therefore, the present study aimed to compare phenotypic and functional characteristics of orbital fat-derived stem cells (OFSCs) and fibroblasts resident in the eyelid skin in donor-matched samples grown in culture medium supplemented with pooled allogeneic human serum (HS) replacing fetal bovine serum (FBS). We first investigated the proliferative effects of OFSCs on HS, and then we compared the alkaline phosphatase (AP) expression and activity, immunophenotypic profile, and in vitro multilineage differentiation potential of OFSCs side-by-side with fibroblasts. The results showed that HS enhanced OFSCs proliferation without compromising their immunophenotype, AP activity, and osteogenic, adipogenic, and chondrogenic differentiation capacities. In contrast to OFSCs, the fibroblasts did not exhibit AP expression and activity and did not have multilineage differentiation potential. The results enabled us to successfully distinguish OFSCs from fibroblasts populations, suggesting that AP expression/activity and multilineage differentiation assays can be used reliably to discriminate mesenchymal stem cells from fibroblasts. Our findings also support the feasibility of pooled allogeneic HS as a safer and more effective alternative to FBS for clinical applications.

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

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    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.

  7. Establishing Primary Adult Fibroblast Cultures From Rodents

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    Seluanov, Andrei; Vaidya, Amita; Gorbunova, Vera

    2010-01-01

    The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel. PMID:20972406

  8. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...... from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three...... immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells....

  9. Multiple Directional Differentiation Difference of Neonatal Rat Fibroblasts from Six Organs

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    Yuqiao Chang

    2016-06-01

    Full Text Available Background/Aims: Fibroblasts are abundantly distributed throughout connective tissues in the body and are very important in maintaining the structural and functional integrity. Recent reports have proved that fibroblasts and mesenchymal stem cells share much more in common than previously recognized. The aim of this study was to investigate comparative studies in fibroblasts on the differences in the expression of molecular markers and differentiation capacity from different organs. Methods: Combined trypsin/collagenase enzymes digestion method was used to isolate and culture the fibroblasts derived from heart, liver, spleen, lung, kidney and skin. Cell activity was determined by methyl thiazolyl tetrazolium (MTT assay. Common molecular markers for fibroblasts such as vimentin, DDR2 and FSP1, stem cell markers nanog, c-kit and sca-1 were detected by RT-PCR, immunofluorescence and western blotting. The osteogenic, adipogenic and cardiogenic differentiations of fibroblasts were performed by inductive culture in special mediums, and analyzed by Alizarin red, Oil red O and immunofluorescence staining of cTnT respectively. Results: The proliferation rate of fibroblasts in lung was faster than in other five organs. Common molecular markers for fibroblasts were expressed differently in different organs. DDR2 was strongly expressed in fibroblasts in the heart, partly expressed in the heart, skin, liver and spleen. Interestingly, no expression of DDR2 was detected in liver and kidney. However, vimentin and FSP1 were consistently expressed in fibroblasts from skin, liver, kidney, spleen and lung. nanog expression in fibroblasts from lung was less than that from heart, skin, liver and spleen (P . c-kit expression in fibroblasts from heart, skin and kidney was higher than that from spleen (P , while the c-kit positive fibroblasts from liver was obviously higher than that from spleen (P . But sca-1 expression in fibroblasts from lung was the lowest among six

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

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    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. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

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    Emond Mary

    2007-09-01

    Full Text Available Abstract Background Marfan syndrome (MFS is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value -6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status. An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.

  12. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

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    Yao, Zizhen; Jaeger, Jochen C; Ruzzo, Walter L; Morale, Cecile Z; Emond, Mary; Francke, Uta; Milewicz, Dianna M; Schwartz, Stephen M; Mulvihill, Eileen R

    2007-01-01

    Background Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 × 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater. PMID:17850668

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

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    Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

    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

  14. Biologic characteristics of fibroblast cells cultured from the knee ligaments

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    陈鸿辉; 唐毅; 李斯明; 沈雁; 刘向荣; 钟灿灿

    2002-01-01

    Objective: To culture fibroblast cells from the kneeligaments and to study the biological characteristics of thesecells.Methods: Cells of the anterior cruciate ligament(ACL) and the medial collateral ligament (MCL) fromNew Zealand white rabbit were cultured in vitro. Cellulargrowth and expression of the collagen were analyzed.Moreover, an in vitro wound closure model was establishedand the healing of the ACL and the MCL cells wascompared.Results: Maximal growth for all these cells wereobtained with Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, but RPMI 1640and Ham's F12 media were not suitable to maintain thesecells. Morphology of both ACL and MCL cells from NewZealand white rabbit was alike in vitro, but the MCL cellsgrew faster than the ACL cells. Both cell types producedsimilar amount of collagen in culture, but the ratio ofcollage type I to type III produced by ACL cells was higherthan that produced by MCL cells. Wound closure assayshowed that at 36 hours after injury, cell-free zones createdin the ACL cultures were occupied partially by the ACLcells; in contrast, the wounded zone in the MCL cultureswas almost completely covered by the cells.Conclusions: Although the ACL cells and the MCLcells from New Zealand white rabbit show similarappearance in morphology in culture, the cellular growthand the biochemical synthesis of collagen as well as thehealing in vitro were significantly different. Thesedifferences in intrinsic properties of the two types of cells invitro might contribute to the differential healing potentialsof these ligaments in vivo.

  15. Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

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    Jon M Carthy

    Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

  16. Inductive role of fibroblastic cell lines in development of the mouse thymus anlage in organ culture.

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    Itoi, M; Amagai, T

    1998-01-10

    Previously, we have shown that embryonic day 12 thymus anlage cultured alone cannot develop into the mature organ but degenerates. In the present study, we investigated the cause of this insufficient organogenesis of embryonic day 12 thymus anlage in organ culture. We cocultured embryonic day 12 thymus anlages with various cell lines as pellets formed by centrifugation. In coculture with fibroblastic cell lines, but not with thymic epithelial cell lines, embryonic day 12 thymus anlages developed to support full T cell differentiation, and expressed mature stromal cell markers, Ia and Kb. By pellet culture of thymus anlages and fibroblastic cell lines transfected with a beta-galactosidase expression vector, we analyzed the distribution of added fibroblastic cells in pellets. The added fibroblastic cells constituted neither thymic capsule nor septa but disappeared after about 2 weeks in culture. Moreover, immunohistochemical studies indicated that added fibroblastic cells were adjacent to mesenchymal cells of thymus anlage. Our results strongly suggest that added fibroblastic cells support the development of the thymus anlage through interaction with its mesenchymal cells.

  17. Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model.

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    Majety, Meher; Pradel, Leon P; Gies, Manuela; Ries, Carola H

    2015-01-01

    In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME.

  18. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

    2002-01-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...

  19. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

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    Ikari, Jun; Smith, Lynette M; Nelson, Amy J; Iwasawa, Shunichiro; Gunji, Yoko; Farid, Maha; Wang, Xingqi; Basma, Hesham; Feghali-Bostwick, Carol; Liu, Xiangde; DeMeo, Dawn L; Rennard, Stephen I

    2015-04-01

    In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

  20. Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Jiang Lu; Dongsheng Li; Kehuan Lu

    2012-01-01

    The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor,fibroblast growth factor receptor-3,in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro.Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo.Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence.Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro.Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain,namely the substantia nigra,compact part,dorsal tier,substantia nigra and reticular part,but was not detected in the forebrain comprising the caudate putamen and striatum.Unusual results were obtained in retrosplenial locations of adult rat brain.We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses.We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8,a secretory factor.Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells.In contrast,addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons.Our study may help delineate the important roles of fibroblast growth factor-8 in brain

  1. Clinical Experience with Chitosan Matrix and Cultured Fibroblasts for Burns

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    Gaziza Danlybayeva

    2014-12-01

    Full Text Available Introduction. Burns are an important public health challenge due to the frequency of getting burns in day-to-day life, occupational hazards, and catastrophes. Treatment of burns is complex and is associated with high morbidity and mortality. Duration and complexity of burn treatment require finding new ways of curing and rehabilitating burns. The result of burn treatment plays a significant role in post-traumatic status of a patient and his or her consequent adaptation in society. Chitosan is a natural safe non-toxic product compatible with human tissues, characterized by hydrosorbid, anticoagulant, antibacterial, and wound healing features. The study aims to  show a clinical application of chitosan-pectin scaffold with cultured human skin fibroblasts in the treatment of deep burns.Methods. The substrate was prepared by dissolving 3% chitosan in 0.5N acetic acid, which was then mixed with 3% solution of pectin dissolved in distillated water. Chitosan film was formed in a Petri dish for 20-24 hours at 20-25 °C. After drying the film, cultured allogeneic fibroblasts (patent number RK-25091 were seeded on its surface.Results. The results from an in vitro culture study showed that human allogeneic fibroblasts could adhere well and grow on the selected scaffold with a typical morphology. During autodermoplasty surgery, cultured allogeneic fibroblasts were applied on granulating wounds of 9 patients with IIIA to IVB degree burns and limited donor resources. Wounds treated with the fibroblast-seeded scaffold among all patients provided the highest level of re-epithelialization (day 5, in comparison to cell-free scaffold (day 7 and untreated surface of wounds (day 10.Conclusion. Our results indicate the potential use of chitosan for wound healing due to its allogenic fibroblast adherence to scaffolding as well as high epithelization. This warrants further studies on chitosan for use in wounds resulting from third and fourth degree burns.

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

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    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.

  3. Cellular interactions regulate stem cell differentiation in tri-culture.

    Science.gov (United States)

    Wang, I-Ning E; Bogdanowicz, Danielle R; Mitroo, Siddarth; Shan, Jing; Kala, Sonam; Lu, Helen H

    2016-11-01

    Currently, the mechanism governing the regeneration of the soft tissue-to-bone interface, such as the transition between the anterior cruciate ligament (ACL) and bone, is not known. Focusing on the ACL-to-bone insertion, this study tests the novel hypothesis that interactions between cells from the ligament (fibroblasts) and bone (osteoblasts) initiate interface regeneration. Specifically, these heterotypic cell interactions direct the fibrochondrogenic differentiation of interface-relevant cell populations, defined here as ligament fibroblasts and bone marrow stromal cells (BMSC). The objective of this study is to examine the effects of heterotypic cellular interactions on BMSC or fibroblast growth and biosynthesis, as well as expression of fibrocartilage-relevant markers in tri-culture. The effects of cell-cell physical contact and paracrine interactions between fibroblasts and osteoblasts were also determined. It was found that, in tri-culture with fibroblasts and osteoblasts, BMSC exhibited greater fibrochondrogenic potential than ligament fibroblasts. The growth of BMSC decreased while proteoglycan production and TGF-β3 expression increased. Moreover, tri-culture regulated BMSC response via paracrine factors, and interestingly, fibroblast-osteoblast contact further promoted proteoglycan and TGF-β1 synthesis as well as induced SOX9 expression in BMSC. Collectively, the findings of this study suggest that fibroblast-osteoblast interactions play an important role in regulating the stem cell niche for fibrocartilage regeneration, and the mechanisms of these interactions are directed by paracrine factors and augmented with direct cell-cell contact.

  4. Radiation-Induced Differentiation in Human Lung Fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sa-Rah; Ahn, Ji-Yeon; Han, Young-Soo; Shim, Jie-Young; Yun, Yeon-Sook; Song, Jie-Young [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-10-15

    One of the most common tumors in many countries is lung cancer and patients with lung cancer may take radiotherapy. Although radiotherapy may have its own advantages, it can also induce serious problems such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of {alpha}-SMA and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-{beta}), tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are related to fibrosis. Among them TGF-{beta} with Smad signaling is known to be the main stream and other signaling molecules such as MAPK, ERK and JNK (3) also participates in the process. In addition to those above factors, it is thought that more diverse and complicate mechanisms may involve in the radiationinduced fibrosis. Therefore, to investigate the underlying mechanisms in radiation induced fibrosis, first of all, we confirmed whether radiation induces trans differentiation in human normal lung fibroblasts. Here, we suggest that not only TGF-{beta} but also radiation can induce trans differentiation in human lung fibroblast WI-38 and IMR-90.

  5. Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis.

    Science.gov (United States)

    Pilling, Darrell; Zheng, Zhichao; Vakil, Varsha; Gomer, Richard H

    2014-12-23

    Monocytes leave the blood and enter tissues. In healing wounds and fibrotic lesions, some of the monocytes differentiate into fibroblast-like cells called fibrocytes. In healthy tissues, even though monocytes enter the tissue, for unknown reasons, very few monocytes differentiate into fibrocytes. In this report, we show that fibroblasts from healthy human tissues secrete the neuronal guidance protein Slit2 and that Slit2 inhibits human fibrocyte differentiation. In mice, injections of Slit2 inhibit bleomycin-induced lung fibrosis. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, Slit2 has a widespread distribution whereas, in patients with advanced disease, there is less Slit2 in the fibrotic lesions. These data may explain why fibrocytes are rarely observed in healthy tissues, may suggest that the relative levels of Slit2 present in healthy tissue and at sites of fibrosis may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and may indicate that modulating Slit2 signaling may be useful as a therapeutic for fibrosis.

  6. Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

    Directory of Open Access Journals (Sweden)

    Liu Wei

    2010-06-01

    Full Text Available Abstract Background We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. Results Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. Conclusions This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

  7. Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

    Science.gov (United States)

    Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser

    2016-01-01

    Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

  8. Histamine inhibits differentiation of skin fibroblasts into myofibroblasts.

    Science.gov (United States)

    Lin, Lin; Yamagata, Kaoru; Nakayamada, Shingo; Sawamukai, Norifumi; Yamaoka, Kunihiro; Sakata, Kei; Nakano, Kazuhisa; Tanaka, Yoshiya

    2015-07-31

    Histamine and TGF-β, major mediators secreted by mast cells, are involved in skin inflammation and play critical roles in the pathogenesis of systemic sclerosis. However, the roles of signaling mechanisms in the development of skin fibrosis remain largely unclear. Here we show that histamine suppressed the expression of α smooth muscle actin (αSMA), a marker of myofibroblasts, induced by TGF-β1 in skin fibroblasts. Histamine H1-receptor (H1R), but not H2-receptor (H2R) or H4-receptor (H4R), was expressed on skin fibroblasts at both mRNA and protein levels. Interestingly, an H1R antagonist, but not H2R or H4R antagonists, antagonized the histamine-mediated suppression of αSMA expression by TGF-β1. Correspondingly, phosphorylated Smad2 was detected after treatment with TGF-β1, whereas the addition of histamine inhibited this phosphorylation. Taken together, histamine-H1R decreased TGF-β1-mediated Smad2 phosphorylation and inhibited differentiation of skin fibroblasts into myofibroblasts.

  9. Sp3 controls fibroblast growth factor receptor 4 gene activity during myogenic differentiation.

    Science.gov (United States)

    Cavanaugh, Eric; DiMario, Joseph X

    2017-03-27

    Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is a critical component in the regulation of myoblast proliferation and differentiation. The transient FGFR4 gene expression during the transition from proliferating myoblasts to differentiated myotubes indicates that FGFR4 regulates this critical phase of myogenesis. The Specificity Protein (SP) family of transcription factors controls FGFR family member gene activity. We sought to determine if members of the Sp family regulate mouse FGFR4 gene activity during myogenic differentiation. RT-PCR and western blot analysis of FGFR4 mRNA and protein revealed transient expression over 72h, with peak expression between 24 and 36h after addition of differentiation medium to C2C12 myogenic cultures. Sp3 also displayed a transient expression pattern with peak expression occurring after 6h of differentiation. We cloned a 1527bp fragment of the mouse FGFR4 promoter into a luciferase reporter. This FGFR4 promoter contains eight putative Sp binding sites and directed luciferase gene activity comparable to native FGFR4 expression. Overexpression of Sp1 and Sp3 showed that Sp1 repressed FGFR4 gene activity, and Sp3 activated FGFR4 gene activity during myogenic differentiation. Mutational analyses of multiple Sp binding sites within the FGFR4 promoter revealed that three of these sites were transcriptionally active. Electromobility shift assays and chromatin immunoprecipitation of the area containing the activator sites showed that Sp3 bound to this promoter location.

  10. Metformin Inhibits Angiotensin II-Induced Differentiation of Cardiac Fibroblasts into Myofibroblasts

    OpenAIRE

    Jian Bai; Na Zhang; Ying Hua; Bingjian Wang; Lin Ling; Albert Ferro; Biao Xu

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibr...

  11. Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens

    Energy Technology Data Exchange (ETDEWEB)

    Montani, Claudia [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Steimberg, Nathalie; Boniotti, Jennifer [Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia (Italy); Biasiotto, Giorgio; Zanella, Isabella [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Department of Molecular and Translational Medicine, University of Brescia, Brescia (Italy); Diafera, Giuseppe [Integrated Systems Engineering (ISE), Milan (Italy); Biunno, Ida [IRGB-CNR, Milan (Italy); IRCCS-Multimedica, Milan (Italy); Caimi, Luigi [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Department of Molecular and Translational Medicine, University of Brescia, Brescia (Italy); Mazzoleni, Giovanna [Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia (Italy); Di Lorenzo, Diego, E-mail: diego.dilorenzo@yahoo.it [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy)

    2014-11-01

    Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential. - Highlights: • We here characterized the first cell line derived from an estrogen reporter mouse. • In the RCCS cells express an immortalized behavior but not a transformed phenotype. • The RCCS provides a system for

  12. Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age.

    Science.gov (United States)

    Ivanov, Nikolay A; Tao, Ran; Chenoweth, Joshua G; Brandtjen, Anna; Mighdoll, Michelle I; Genova, John D; McKay, Ronald D; Jia, Yankai; Weinberger, Daniel R; Kleinman, Joel E; Hyde, Thomas M; Jaffe, Andrew E

    2016-02-01

    Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), "block" (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts-83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation--while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p = 0

  13. Differential Gene Expression of Fibroblasts: Keloid versus Normal

    Directory of Open Access Journals (Sweden)

    Michael F. Angel

    2002-11-01

    Full Text Available Abstract: This study investigated gene regulation and unique gene products in both keloid (KDF and normal (NDF dermal fibroblasts in established cell lines. For gene regulation, NDF versus KDF were compared using Clontech's Atlas™ Human cDNA Expression Array while unique gene products were studied using RNA Fingerprinting Kit. RNA from each sample was converted to cDNA using oligo-dT primers. Down-regulated genes using Atlas Array in KDF were 1 60 S ribosomal protein, 2 Thioredoxin dependent peroxidase, 3 Nuclease sensitive element DNA binding protein, 4 c-myc purine-binding transcription factor, 5 c-AMP dependent protein kinase, and, 6 Heat Shock Protein 90 kDa. Genes that are up regulated in KDF were 1 Tubulin and 2 Heat Shock Protein 27 kDa. With the differential display, we found 17 bands unique to both KDF and NDF. The specific gene and the manner in which they were differentially regulated have direct implications to understanding keloid fibroblast proliferation.

  14. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  15. Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sylvie Séguier

    Full Text Available 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.

  16. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures

    Science.gov (United States)

    Parameswaran, Sreejit; Sharma, Rajendra K.

    2016-01-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  17. Cultural Differentiation of Negotiating Agents

    NARCIS (Netherlands)

    Hofstede, G.J.; Jonker, C.M.; Verwaart, D.

    2012-01-01

    Negotiations proceed differently across cultures. For realistic modeling of agents in multicultural negotiations, the agents must display culturally differentiated behavior. This paper presents an agent-based simulation model that tackles these challenges, based on Hofstede’s model of national cultu

  18. Cultural differentiation of negotiating agents

    NARCIS (Netherlands)

    Hofstede, G.J.; Jonker, C.M.; Verwaart, T.

    2010-01-01

    Negotiations proceed differently across cultures. For realistic modeling of agents in multicultural negotiations, the agents must display culturally differentiated behavior. This paper presents an agent-based simulation model that tackles these challenges, based on Hofstede’s model of national cultu

  19. Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium.

    Science.gov (United States)

    Hur, Woojune; Lee, Hoon Young; Min, Hye Sook; Wufuer, Maierdanjiang; Lee, Chang-Won; Hur, Ji An; Kim, Sang Hyon; Kim, Byeung Kyu; Choi, Tae Hyun

    2017-04-20

    Fibroblasts are ubiquitous cells in the human body and are absolutely necessary for wound healing such as for injured skin. This role of fibroblasts was the reason why we aimed to differentiate human adipose-derived stem cells (hADSCs) into fibroblasts and to test their wound healing potency. Recent reports on hADSC-derived conditioned medium have indicated stimulation of collagen synthesis as well as migration of dermal fibroblasts in wound sites with these cells. Similarly, human fibroblast-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important for growth of skin. However, it remains unknown whether and how F-CM can stimulate hADSCs to secrete type I collagen. In this study, we obtained F-CM from the culture of human skin fibroblast HS27 cells in DMEM media. For an in-vivo wound healing assay using cell transplantation, balb/c nude mice with full-thickness skin wound were used. Our data showed that levels of type I pro-collagen secreted by hADSCs cultured in F-CM increased significantly compared with hADSCs kept in normal medium for 72 h. In addition, from a Sircol collagen assay, the amount of collagen in F-CM-treated hADSC conditioned media (72 h) was markedly higher than both the normal medium-treated hADSC conditioned media (72 h) and the F-CM (24 h). We aimed to confirm that hADSCs in F-CM would differentiate into fibroblast cells in order to stimulate wound healing in a skin defect model. To investigate whether F-CM induced hADSCs into fibroblast-like cells, we performed FACS analysis and verified that both F-CM-treated hADSCs and HS27 cells contained similar expression patterns for CD13, CD54, and CD105, whereas normal medium-treated hADSCs were significantly different. mRNA level  analysis for Nanog, Oct4A, and Sox2 as undifferentiation markers and vimentin, HSP47, and desmin as matured fibroblast markers supported the characterization that hADSCs in F-CM were highly differentiated into fibroblast

  20. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malpass, Gloria E., E-mail: gloria.malpass@gmail.com [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Prasad, G.L., E-mail: prasadg@rjrt.com [R and D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102 (United States); Howlett, Allyn C., E-mail: ahowlett@wakehealth.edu [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States)

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  1. Substrate stiffness influences TGF-β1-induced differentiation of bronchial fibroblasts into myofibroblasts in airway remodeling.

    Science.gov (United States)

    Shi, Yanling; Dong, Yuhui; Duan, Yiyuan; Jiang, Xuemei; Chen, Cheng; Deng, Linhong

    2013-02-01

    Chronic inflammation and remodeling of the bronchial wall are basic hallmarks of asthma. During the process of bronchial wall remodeling, inflammatory factors, such as transforming growth factor-β1 (TGF-β1), are known to induce the differentiation of fibroblasts into myofibroblasts, which leads to excessive synthesis and secretion of extracellular matrix (ECM) proteins, thus thickening and stiffening the basement membrane. However, it has not been thoroughly studied whether or not substrate stiffening affects the TGF-β1‑induced myofibroblast differentiation. In the present study, the influence of substrate stiffness on the process of bronchial fibroblast differentiation into myofibroblasts in the presence of TGF-β1 was investigated. To address this question, we synthesized polydimethylsiloxane (PDMS) substrates with varying degrees of stiffness (Young's modulus of 1, 10 and 50 kPa, respectively). We cultured bronchial fibroblasts on the substrates of varying stiffness in media containing TGF-β1 (10 ng/ml) to stimulate the differentiation of fibroblasts into myofibroblasts. Myofibroblast differentiation was examined using semi-quantitative RT-PCR for the expression of α-smooth muscle actin (α-SMA) mRNA and collagen I mRNA, the enzyme-linked immunosorbent assay method was used to assess the expression of collagen I protein and western blotting to assess the expression of α-SMA protein. The optical magnetic twisting cytometry (OMTC) method was used for the changing of cell mechanical properties. Our findings suggest that when fibroblasts were incubated with TGF-β1 (10 ng/ml) on substrate of varying stiffness, the differentiation of fibroblasts into myofibroblasts was enhanced by increasing substrate stiffness. Compared with those cultured on substrate with Young's modulus of 1 kPa, the mRNA and protein expression of collagen I and α-SMA of fibroblasts cultured on substrates with Young's modulus of 10 and 50 kPa were increased. Furthermore, with the

  2. Hepatocyte differentiation of human fibroblasts from cirrhotic liver in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Yu-Ling Sun; Sheng-Yong Yin; Lin Zhou; Hai-Yang Xie; Feng Zhang; Li-Ming Wu; Shu-Sen Zheng

    2011-01-01

    BACKGROUND: Mesenchymal stem cells (MSCs) and fibro-blasts have intimate relationships, and the phenotypic homology between fibroblasts and MSCs has been recently described. The aim of this study was to investigate the hepatic differentiating potentialofhumanfibroblastsincirrhoticliver. METHODS: The phenotypes of fibroblasts in cirrhotic liver were labeled by biological methods. After that, the differentiation potential of these fibroblasts in vitro was characterized in terms of liver-specific gene and protein expression. Finally, an animal model of hepatocyte regeneration in severe combined immunodeficient (SCID) mice was created by retrorsine injection and partial hepatectomy, and the expression of human hepatocyte proteins in SCID mouse livers was checked by immunohistochemicalanalysisafterfibroblastadministration. RESULTS: Surface immunophenotyping revealed that a minority of fibroblasts expressed markers of MSCs and hepatic epithelial cytokeratins as well as alpha-smooth muscle actin, but homogeneously expressed vimentin, desmin, prolyl 4-hydroxylase and fibronectin. These fibroblasts presented the characteristics of hepatocytes in vitro and differentiated directly into functional hepatocytes in the liver of hepatecto-mized SCID mice. CONCLUSIONS: This study demonstrated that fibroblasts in cirrhotic liver have the potential to differentiate into hepatocyte-like cells in vitro and in vivo. Our findings infer that hepatic differentiation of fibroblasts may serve as a new target for reversion of liver fibrosis and a cell source for tissue engineering.

  3. Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

    Science.gov (United States)

    Fleischmajer, R; MacDonald, E D; Contard, P; Perlish, J S

    1993-09-01

    Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

  4. Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis

    Science.gov (United States)

    Thirdborough, Steve; Mellows, Toby; Garcia, Edwin; Woo, Jeongmin; Tod, Joanne; Frampton, Steve; Jenei, Veronika; Moutasim, Karwan A.; Kabir, Tasnuva D.; Brennan, Peter A; Venturi, Giulia; Ford, Kirsty; Herranz, Nicolas; Lim, Kue Peng; Clarke, James; Lambert, Daniel W.; Prime, Stephen S.; Underwood, Timothy J.; Vijayanand, Pandurangan; Eliceiri, Kevin W.; Woelk, Christopher; King, Emma V.; Gil, Jesus; Ottensmeier, Christian H.; Thomas, Gareth J.

    2017-01-01

    Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo, showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro, we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-β signaling. Similar to TGF-β1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes. PMID:27992856

  5. Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

    Science.gov (United States)

    Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2011-01-01

    Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

  6. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2015-12-01

    Full Text Available BACKGROUND Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. METHODS In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. RESULTS Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1- fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity CONCLUSIONS Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  7. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2014-08-01

    Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  8. Transforming growth factor-β1 involved in urotensin Ⅱ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yong-gang; HU Yan-chao; MAO Yan-yan; WEI Rui-hong; BAO Shi-lin; WU Li-biao; KUANG Ze-jian

    2010-01-01

    Background Urotensin Ⅱ (UⅡ) is a new vasoconstrictive peptide that may activate the adventitial fibroblasts.Transforming growth factor-β1 (TGF-β1) is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UⅡ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.Methods Adventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of α-smooth nuscle actin (α-SM-actin), the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR (real-time RT-PCR) and Western blotting, respectively.Results UⅡ stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% (P <0.01), than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10-8 mol/L of UⅡ (P <0.01), which was increased by 59.9%,compared with in the control group (P <0.01). The secretion of TGF-β1 induced by UⅡ was significantly blocked by SB-710411 (10-7 mol/L), a specific antagonist of UⅡ receptor. In addition, both UⅡ (10-8 mol/L) and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UⅡ was inhibited by the specific neutralizing antibody (20 μg/ml) of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 (20 ng/ml)was inhibited by SB-710411 (10-7 mol/L), the UⅡ receptor antagonist.Conclusion This study suggests that UⅡ could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UⅡ, and TGF-β1

  9. TNF-α-stimulated fibroblasts secrete lumican to promote fibrocyte differentiation.

    Science.gov (United States)

    Pilling, Darrell; Vakil, Varsha; Cox, Nehemiah; Gomer, Richard H

    2015-09-22

    In healing wounds and fibrotic lesions, fibroblasts and monocyte-derived fibroblast-like cells called fibrocytes help to form scar tissue. Although fibrocytes promote collagen production by fibroblasts, little is known about signaling from fibroblasts to fibrocytes. In this report, we show that fibroblasts stimulated with the fibrocyte-secreted inflammatory signal tumor necrosis factor-α secrete the small leucine-rich proteoglycan lumican, and that lumican, but not the related proteoglycan decorin, promotes human fibrocyte differentiation. Lumican competes with the serum fibrocyte differentiation inhibitor serum amyloid P, but dominates over the fibroblast-secreted fibrocyte inhibitor Slit2. Lumican acts directly on monocytes, and unlike other factors that affect fibrocyte differentiation, lumican has no detectable effect on macrophage differentiation or polarization. α2β1, αMβ2, and αXβ2 integrins are needed for lumican-induced fibrocyte differentiation. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, lumican is present at low levels throughout the tissue, whereas patients with advanced disease have pronounced lumican expression in the fibrotic lesions. These data may explain why fibrocytes are increased in fibrotic tissues, suggest that the levels of lumican in tissues may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and indicate that modulating lumican signaling may be useful as a therapeutic for fibrosis.

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

    Science.gov (United States)

    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.

  11. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Bai, Jian; Zhang, Na; Hua, Ying; Wang, Bingjian; Ling, Lin; Ferro, Albert; Xu, Biao

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10-200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  12. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Jian Bai

    Full Text Available Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM in the presence or absence of metformin (10-200 µM. Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  13. Beryllium toxicity testing in the suspension culture of mouse fibroblasts.

    Science.gov (United States)

    Rössner, P; Bencko, V

    1980-01-01

    Suspension culture of mouse fibroblast cell line L-A 115 was used to test beryllium toxicity in the presence of magnesium ions. Beryllium added to the MEM cultivation medium was bound in a complex with sulphosalicylic acid BeSSA complex, because the use of beryllium chloride turned out to yield ineffective beryllium phosphate that formed macroscopically detectable insoluble opacities. The BeSSA complex was used in the concentration range: 10(-3)--10(-9)M, magnesium was used in 3 concentrations: 10(-1)M, 5 x 10(-2)M and 10(-2)M. Growth curve analysis revealed pronounced beryllium toxicity at the concentration of 10(-3)M, magnesium-produced toxic changes were observed only at the concentration of 10(-1)M. No competition between the beryllium and magnesium ions was recorded. It is assumed that the possible beryllium-magnesium competition was significantly modified by the use of BeSSA complex-bound beryllium.

  14. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  15. Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells*

    Institute of Scientific and Technical Information of China (English)

    Yan Zhang; Junmei Zhou; Zhenfu Fang; Manxi Jiang; Xuejin Chen

    2013-01-01

    The difference between Noggin and basic fibroblast growth factor for the neural precursor differen-tiation from human embryonic stem cel s has not been studied. In this study, 100 µg/L Noggin or 20 µg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen-tiate human embryonic stem cel s H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro-scope. Immunofluorescence staining revealed expression levels of Nestin,β-III Tubulin and Sox-1 were higher in the induced cel s and reverse-transcription PCR showed induced cel s expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cel differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in-creases the differentiation of neural precursors.

  16. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    Science.gov (United States)

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  17. Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts.

    Directory of Open Access Journals (Sweden)

    Zhenxiang Wang

    Full Text Available Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11(th to 15(th day, keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF, IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

  18. Effects of the supernatant from ankylosing spondylitis synovial cells culture on the osteogenic differentiation of ligament fibroblasts%强直性脊柱炎滑膜细胞培养上清液对韧带成纤维细胞成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    徐卫东; 梁志民; 袁恒锋

    2012-01-01

    Objective To investigate the effects of synovial cells culture fluid on the differentiation of ligament fibroblasts into osteoblasts. Methods Specimens of ligament and synovium were obtained from the hip joints with ankylosing spondylitis respectively. The primary cell culture of ligament fibroblasts and synovial cells were processed and then the supernatant of synovial cells in the first passage was collected and used to induce ligament cells. Osteogenic phenotype and alkaline phosphatase (ALP) activity were determined at the days of 0, 5,10,15 and 20 respectively. Calcium deposit was detected by Von Kossa staining at the 25th day. Results With time extending, the expression of ALP and osteocalcin increased significantly at day 10 and reached a peak at day 15. Calcium deposit could be detected at day 25. Conclusions The supernatant of synovial cells cuture plays a certain role on the differentiation of ligament fibroblasts into osteoblasts.%目的 探讨强直性脊柱炎(AS)滑膜细胞培养上清液对韧带成纤维细胞成骨分化的影响.方法 分别进行AS患者滑膜和韧带成纤维细胞原代培养,收集第1代的滑膜细胞培养液上清液,应用此上清液对韧带成纤维细胞进行诱导,分别于0、5、10、15、20 d检测碱性磷酸酶和骨钙素表达水平,并于25 d时进行钙结节染色.结果 在应用AS滑膜细胞培养上清液对韧带成纤维细胞进行诱导的第10天,碱性磷酸酶(ALP)和骨钙素(OC)表达水平明显升高,并在第15天达到高峰.第25天时进行钙结节Von Kossa染色,发现有钙结节形成.结论 AS滑膜细胞培养上清液对韧带成纤维细胞具有成骨诱导分化的能力.

  19. In vitro effects of pirfenidone on cardiac fibroblasts: proliferation, myofibroblast differentiation, migration and cytokine secretion.

    Directory of Open Access Journals (Sweden)

    Qiang Shi

    Full Text Available Cardiac fibroblasts (CFs are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remodeling such as proliferation, myofibroblast differentiation, migration and cytokine secretion. Fibroblasts were isolated from neonatal rat hearts and bioassays were performed to determine the effects of pirfenidone on fibroblast function. We demonstrated that treatment of CFs with pirfenidone resulted in decreased proliferation, and attenuated fibroblast α-smooth muscle actin expression and collagen contractility. Boyden chamber assay illustrated that pirfenidone inhibited fibroblast migration ability, probably by decreasing the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1. Furthermore, pirfenidone attenuated the synthesis and secretion of transforming growth factor-β1 but elevated that of interleukin-10. These direct and pleiotropic effects of pirfenidone on cardiac fibroblasts point to its potential use in the treatment of adverse myocardial remodeling.

  20. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    Energy Technology Data Exchange (ETDEWEB)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M. (Medical Univ. School of Silesia, Katowice (Poland))

    1990-04-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and (14C)thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil.

  1. Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

    Science.gov (United States)

    Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

    2015-12-01

    Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with

  2. Regenerative material for aneurysm embolization A 3-dimensional culture system of fibroblasts and calcium alginate gel

    Institute of Scientific and Technical Information of China (English)

    Jingdong Zhang; Kan Xu; Jinlu Yu; Jun Wang; Qi Luo

    2011-01-01

    Calcium alginate gel (CAG) has been shown to successfully model aneurysm embolization within a short period of time. However, gradually degrading CAG potentially results in aneurysm recanalization.In the present study, a regenerative embolic material was designed by seeding rat fibroblasts in a CAG. The study investigated the feasibility of constructing a 3-dimensional culture system. The fibroblasts grew well and firmly attached to the CAG. CAG was conducive for fibroblast growth, and resulted in a 3-dimensional culture system. Results show that CAG can be used theoretically as a vascular, regenerative, embolic material.

  3. Cultured skin keratinocytes from both normal individuals and basal cell naevus syndrome patients are more resistant to. gamma. -rays and UV light compared with cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, M.; Thacker, S.; Taylor, A.M.R. (Birmingham Univ. (UK). Cancer Research Campaign Labs.)

    1989-07-01

    Measurement of colony-forming ability following exposure to {gamma}-rays was performed on cultured skin keratinocytes and skin fibroblasts from normal individuals, basal cell naevus syndrome patients (BCNS) and ataxia telangiectasia (A-T) patients. The most striking observation was the radiation resistance of 8/8 keratinocyte strains compared with fibroblasts whether from BCNS patients or normals. The single A-T keratinocyte cell strain showed the same radiosensitivity as A-T fibroblast cell strains. The differential survival of keratinocytes and fibroblasts was also observed following exposure to 254 nm UV light. Survival curves of SV40 immortalized keratinocytes and retinoblasts derived from normal individuals or BCNS patients showed large shoulder regions following exposure to {gamma}-rays or 254 nm UV light. An increased D{sub 37} rather than an increased D{sub o} was therefore the feature of such curves, contrasting with SV40 immortalized A-T keratinocytes or fibroblasts which showed a minimal shoulder effect and an increased D{sub o}. No difference in survival was observed between BCNS and normal cells following exposure to either UV or {gamma}-rays. (author).

  4. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three...... major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10...

  5. TGF-β1和bFGF-1对单层共培养BMSCs和韧带成纤维细胞韧带特异性的影响%DIFFERENTIATION OF DIRECTLY CO-CULTURED BONE MARROW MESENCHYMAL STEM CELLS AND LIGAMENT FIBROBLASTS INTO LIGAMENT CELLS AFTER INDUCED BY TRANSFORMING GROWTH FACTOR β1 AND BASIC FIBROBLAST GROWTH FACTOR 1

    Institute of Scientific and Technical Information of China (English)

    刘毅; 张承昊; 范青洪; 孙鹏鹏; 吴术红

    2014-01-01

    Objective To investigate the effect of transforming growth factor β1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities,proliferation,and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured.Methods BMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation.LFs were isolated using collagenase.The cells at passage 3 were divided into 6 groups:non-induced BMSCs group (group A),non-induced LFs group (group B),non-induced co-cultured BMSCs and LFs group (group C),induced BMSCs group (group D),induced LFs group (group E),and induced co-cultured BMSCs and LFs group (group F).The cellular activities and proliferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types Ⅰ and Ⅲ,fibronectin,tenascin C,and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR.Results A single cell layer formed in the co-cultured cells under inverted contrast microscope.Group F had fastest cell fusion (> 90%).The MTT result indicated that group F showed the highest absorbance (A) value,followed by group D,and group B showed the lowest A value at 9 days after culture,showing significant difference (P <0.05).Moreover,the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05);the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05),but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05).The ratios of collagen type Ⅰ to type Ⅲ were 1.17,1.19,1.10,1.25,1.17,and 1.18 in groups A-F; group D was higher than the other groups.The real-time fluorescent quantitative PCR results revealed that

  6. 张力刺激皮肤成纤维细胞向肌成纤维细胞分化的实验研究%Myofibroblasts Differentiation of Skin Fibroblasts Induced by Mechanical Loading Based on 3D Culture in Vitro

    Institute of Scientific and Technical Information of China (English)

    傅士博; 温从吉; 王琛; 王丹茹

    2014-01-01

    目的:探索机械张力刺激诱导基于三维培养的皮肤成纤维细胞向肌成纤维细胞分化的可行性。方法将正常人皮肤成纤维细胞三维培养于Ⅰ型鼠尾胶原支架中,通过机械张力装置进行拉伸实验(牵拉组,n=9),并设立对照(非牵拉组,n=9),绘制细胞生长曲线,免疫荧光染色和实时定量PCR检测成纤维细胞的分化程度。结果三维培养环境下细胞增殖缓慢,牵拉组有促进细胞增殖的趋势,但与非牵拉组相比,统计学差异不显著;免疫荧光染色显示,牵拉组成纤维细胞α-SMA和fibronectin染色亮度明显高于非牵拉组;实时定量PCR检测显示,牵拉组α-SMA、纤连蛋白和Ⅰ、Ⅲ型胶原mRNA表达高于非牵拉组。结论机械张力可成功诱导基于三维培养的皮肤成纤维细胞向肌成纤维细胞分化。%Objective To explore the feasibility of inducing myofibroblasts differentiation of skin fibroblasts by mechanical loading based on three dimensional culture in vitro. Methods Human dermal fibroblasts were isolated and three dimensionally cultured in type I rat tail collagen scaffold. Then mechanical loading were added by stretching the gel (stretch group, n=9), and control group was set up (non-stretch group, n=9). The situation of myofibroblasts differentiation were tested by drawing cell growth curve, immunofluorescent staining and Real-Time PCR. Results Cell proliferation was slowed down under mechanical loading environment. The cell proliferation was promoted in stretch group, but there was no significant difference comparing with non-stretch group. Dyeing brightness of α-SMA and fibronectin were significantly higher in stretch group showed by immunofluorescent staining. Higher mRNA expression of α-SMA, fibronectin, type I and III collagen genes were observed in stretch group by real time PCR. Conclusion The model of mechanical loading inducing myofibroblasts differentiation of skin

  7. MORPHOFUNCTIONAL CHARACTERISTICS OF FIBROBLASTS (MCCOY CELL LINE CULTURED WITH MAGNESIUM PREPARATIONS

    Directory of Open Access Journals (Sweden)

    L. V. Didenko

    2015-01-01

    Full Text Available Aim. To study the effect of magnesium orotate, magnesium/pyridoxine combination and magnesium sulfate on fibroblast morphofunctional characteristics in cell culture of fibroblasts (McCoy line.Material and methods. The study of fibroblasts (McCoy line with the addition of magnesium-containing preparations (magnesium orotate, magnesium/pyridoxine combination, magnesium sulphate to the culture medium was performed using scanning and transmission electron microscopy.Results. When adding into the media magnesium orotate or magnesium/pyridoxine combination, significant changes in morphofunctional state of fibroblasts were noted, which were absent when adding magnesium sulfate. At that fibroblasts significantly promoted synthetic and secretory activity. This was expressed in the formation of amorphous and fibrous components in well-formed cell layers.Conclusion. Stimulation by magnesium ions of the proliferative activity of fibroblasts is shown in the morphological analysis of the effect of magnesium orotate or magnesium in combination with pyridoxine and magnesium sulfate on morphological and functional organization of fibroblasts. Revealed the predominant influence of magnesium orotate, and a less pronounced effect of magnesium in combination with pyridoxine on biosynthetic processes in the cells, including the synthesis of amorphous and fibrous components (protocollagen and elastin of the extracellular matrix founded.

  8. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

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    Laura N Bonifacio

    Full Text Available Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

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

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    Lucie Germain

    2013-02-01

    Full Text Available 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.

  10. Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

    Directory of Open Access Journals (Sweden)

    Rui Song

    2011-05-01

    Full Text Available Basic fibroblast growth factor (bFGF regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL. The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01. The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05, while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01. These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

  11. Effect of glutathione on arecanut treated normal human buccal fibroblast culture.

    Directory of Open Access Journals (Sweden)

    Saraswathi T

    2006-01-01

    Full Text Available BACKGROUND: Experimental studies have shown arecanut to be a cytotoxic substance with mutagenic and carcinogenic potential. OBJECTIVE: The present study was undertaken to evaluate the effect of glutathione on arecanut treated human buccal fibroblast culture and its potential as a chemopreventive agent. MATERIALS AND METHODS: Fibroblast culture was done in Dulbecco′s Modified Eagle′s Medium MEM supplemented with 10% Fetal Calf Serum (FCS and antibiotic at 370C degrees in an atmosphere of 5% carbon di-oxide and 95% air. The fibroblast cells were subjected to different concentrations of aqueous extracts of raw and boiled arecanut. Fibroblasts were plated in two 24-well culture plates and in each plate, cells were dividt,ednto 2 groups; 600gg microml of reduced glutathione was added to the first group of cells; subsequently, aqueous extracts of raw and boiled arecanut at least and highest concentrations i.e., 20j. microml and 100lg microml were added to the first group of cells in the respective plates whereas the second group served as a control. The morphological alterations and cell survival were assayed at 24, 48, 72, and 96 hours. Results Morphologically, the initial (10 hours attached fibroblast cells were converted from spheroidal shape towards hexagonal and finally to a fully extended spindle shaped configuration. The three morphological types of fibroblasts at 48 hours were F-I, F-II and F-III. Aqueous extract of raw arecanut exhibited significant cytotoxicity (p < .0 001 at all time periods studied, when compared against the control values of untreated fibroblasts. Addition of reduced glutathione to cultures showed a significant (p < 0. 001 reduction in cytotoxicity, as indicated by higher optical density values and morphological reversion to the spindle-shaped configuration. CoCONCLUSION:Addition of glutathione reduced the cytotoxic and morphological alterations of the fibroblasts treated with aqueous extracts of both raw and boiled

  12. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian;

    2013-01-01

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired...

  13. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired, in...

  14. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...... collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.......Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts...... to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned...

  15. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation.

    Directory of Open Access Journals (Sweden)

    Qi-cui Du

    Full Text Available Hypertrophic scars (HTS, the excessive deposition of scar tissue by fibroblasts, is one of the most common skin disorders. Fibroblasts derived from surgical scar tissue produce high levels of α-smooth muscle actin (α-SMA and transforming growth factor-β1 (TGF-β1. However, the molecular mechanisms for this phenomenon is poorly understood. Thus, the purpose of this study was to evaluate the molecular mechanisms of HTS and their potential therapeutic implications. Fibroblasts derived from skin HTS were cultured and characterized in vitro. The fibroblasts were synchronized and randomly assigned to two groups: cyclic stretch and cyclic stretch pre-treated with SB203580 (a p38MAPK inhibitor. Cyclic stretch at 10% strain was applied at a loading frequency of 10 cycles per minute (i.e. 5 seconds of tension and 5 seconds of relaxation for 0 h, 6 h and 12 h. Cyclic stretch on HTS fibroblasts led to an increase in the expression of α-SMA and TGF-β1 mRNA and protein and the phosphorylation of p38MAPK. SB203580 reversed these effects and caused a decrease in matrix contraction. Furthermore, HTS fibroblast growth was partially blocked by p38MAPK inhibition. Therefore, the mechanism of cyclic stretch involves p38 MAPK, and its inhibition is suggested as a novel therapeutic strategy for HTS.

  16. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation.

    Science.gov (United States)

    Du, Qi-cui; Zhang, Dai-zun; Chen, Xiu-juan; Lan-Sun, Gui; Wu, Min; Xiao, Wen-lin

    2013-01-01

    Hypertrophic scars (HTS), the excessive deposition of scar tissue by fibroblasts, is one of the most common skin disorders. Fibroblasts derived from surgical scar tissue produce high levels of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1). However, the molecular mechanisms for this phenomenon is poorly understood. Thus, the purpose of this study was to evaluate the molecular mechanisms of HTS and their potential therapeutic implications. Fibroblasts derived from skin HTS were cultured and characterized in vitro. The fibroblasts were synchronized and randomly assigned to two groups: cyclic stretch and cyclic stretch pre-treated with SB203580 (a p38MAPK inhibitor). Cyclic stretch at 10% strain was applied at a loading frequency of 10 cycles per minute (i.e. 5 seconds of tension and 5 seconds of relaxation) for 0 h, 6 h and 12 h. Cyclic stretch on HTS fibroblasts led to an increase in the expression of α-SMA and TGF-β1 mRNA and protein and the phosphorylation of p38MAPK. SB203580 reversed these effects and caused a decrease in matrix contraction. Furthermore, HTS fibroblast growth was partially blocked by p38MAPK inhibition. Therefore, the mechanism of cyclic stretch involves p38 MAPK, and its inhibition is suggested as a novel therapeutic strategy for HTS.

  17. Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation

    OpenAIRE

    Zhao, Haotian; Yang, Tianyu; Madakashira, Bhavani P.; Thiels, Cornelius A.; Bechtle, Chad A.; Garcia, Claudia M.; Zhang, Huiming; Yu, Kai; Ornitz, David M.; Beebe, David C.; Robinson, Michael L.

    2008-01-01

    The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens dev...

  18. A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

    Science.gov (United States)

    Jubin, K; Martin, Y; Lawrence-Watt, D J; Sharpe, J R

    2011-12-01

    Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

  19. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Kuh, Hyo-Jeong, E-mail: hkuh@catholic.ac.kr [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of)

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  20. Characterization of storage material in cultured fibroblasts by specific lectin binding in lysosomal storage diseases.

    Science.gov (United States)

    Virtanen, I; Ekblom, P; Laurila, P; Nordling, S; Raivio, K O; Aula, P

    1980-11-01

    The lysosomal storage material in cultured fibroblasts from patients with various lysosomal storage diseases was characterized by fluorescence microscopy using lectins specific for different saccharide moieties. In normal fibroblasts and cultured amniotic fluid cells lectins specific for mannosyl and glucosyl moieties, Con A and LcA gave a bright perinuclear cytoplasmic staining corresponding to the localization of endoplasmic reticulum in the cells. All other lectins stained the Golgi apparatus as a juxtanuclear reticular structure. In fucosidosis fibroblasts, only lectins specific for fucosyl groups LTA and UEA, distinctly stained the lysosomal inclusions. The lysosomes in mannosidosis fibroblasts did not react with Con A and LcA, both specific for mannosyl moieties of glycoconjugates, but were brightly labeled with WGA, a lectin specific for N-acetyl glucosaminyl moieties. In I-cell fibroblasts, the numerous perinuclear phase-dense granules, representing abnormal lysosomes, were labeled with every lectin used. In fibroblasts from patients with Salla disease, a newly discovered lysosomal storage disorder, the lysosomes were brightly stained only with LPA, indicating the presence of increased amounts of sialic acid residues in the lysosomal inclusions.

  1. In vitro effects of Choukroun's PRF (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures.

    Science.gov (United States)

    Dohan Ehrenfest, David M; Diss, Antoine; Odin, Guillaume; Doglioli, Pierre; Hippolyte, Marie-Pascale; Charrier, Jean-Baptiste

    2009-09-01

    The objective of this study was to analyze the effects of Choukroun's PRF (platelet-rich fibrin), a leucocyte and platelet concentrate clinically usable as fibrin membrane or clot, on human primary cultures of gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts. For the proliferation study, these cells were cultivated with or without a PRF membrane originating from the same donor as for the cells. For osteoblasts and fibroblasts, dose-dependent effect was assessed (using 2 membranes). Cell counts and cytotoxicity tests were performed at 3, 7, 14, and 21 days, and even 28 days for osteoblasts. More osteoblast cultures were prepared in differentiation conditions according to 3 modalities (without PRF, with PRF, with PRF the first day and differentiation medium applied only after the first week of culture). Osteoblast differentiation was analyzed using Von Kossa staining and alkaline phosphatase, DNA and total cell proteins dosage. PRF induced a significant and continuous stimulation of proliferation in all cell types. It was dose dependent during all the experiment with osteoblasts, but only on day 14 with fibroblasts. Moreover, PRF induced a strong differentiation in the osteoblasts, whatever the culture conditions. The analysis of osteoblast cultures in differentiation conditions with PRF, using light and scanning electron microscopy, revealed a starting mineralization process in the PRF membrane itself after 14 days. Moreover, PRF leucocytes seemed to proliferate and interact with osteoblasts. Cultures with PRF are always cocultures with leucocytes. These "chaperone leucocytes" could be the source of differential geographic regulation within the culture and explain the double contradictory effect proliferation/differentiation observed on osteoblasts.

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

    Directory of Open Access Journals (Sweden)

    Won-Gyu Bae

    2015-12-01

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

  3. Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells

    Science.gov (United States)

    Kim, Bona; Yoon, Byung Sun; Moon, Jai-Hee; Kim, Jonggun; Jun, Eun Kyoung; Lee, Jung Han; Kim, Jun Sung; Baik, Cheong Soon; Kim, Aeree; Whang, Kwang Youn

    2012-01-01

    Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic β-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus. PMID:22020533

  4. Evaluation for safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C.

    Science.gov (United States)

    Kim, Tae-im; Tchah, Hungwon; Cho, Eun Hee; Kook, Michael S

    2004-01-01

    To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction

  5. Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

    Directory of Open Access Journals (Sweden)

    Hidetatsu Outani

    Full Text Available The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4 and one chondrogenic factor (SOX9 can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon cells from human dermal fibroblast (HDF culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

  6. Integration, differentiation and ambiguity in safety cultures

    DEFF Research Database (Denmark)

    Richter, Anne; Koch, Christian

    2004-01-01

    This article discusses safety cultures, drawing on the differentiation, integration and ambiguity-scheme introduced by scholars of organizational culture. An ethnographic approach has been applied in the study of meaning and symbols relating to work, hazards, occupational accidents and prevention....... The application of this approach is demonstrated through a multifacetted analysis of safety cultures. Case studies in Danish manufacturing show that it usually is necessary to differentiate between several safety cultures dispersed throughout the shop floor and other parts of the manufacturing organization...

  7. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid

    Directory of Open Access Journals (Sweden)

    Fernanda Gonçalves BASSO

    Full Text Available Abstract Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

  8. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid.

    Science.gov (United States)

    Basso, Fernanda Gonçalves; Soares, Diana Gabriela; Pansani, Taisa Nogueira; Turrioni, Ana Paula Silveira; Scheffel, Débora Lopes; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-11-28

    Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

  9. Cytotoxicity evaluation of root repair materials in human-cultured periodontal ligament fibroblasts

    Directory of Open Access Journals (Sweden)

    Voruganti Samyuktha

    2014-01-01

    Full Text Available Aim: To evaluate the cytotoxicity of three root repair materials, mineral trioxide aggregate (MTA, Endosequence Root Repair Material and Biodentine in human periodontal ligament fibroblasts. Materials and Methods: Periodontal ligament fibroblasts were cultured from healthy premolar extracted for orthodontic purpose. Cells in the third passage were used in the study. The cultured fibroblast cells were placed in contact with root repair materials: (a Biodentine, (b MTA, (c Endosequence, (d control. The effects of these three materials on the viability of Periodontal ligament (PDL fibroblasts were determined by trypan blue dye assay after 24 hours and 48-hour time period. Cell viability was determined using inverted phase contrast microscope. Statistical Analysis: Cell viability was compared for all the experimental groups with Wilcoxons matched pair test. Results: At the 24-hour examination period, all the materials showed increased cell viability. At 48-hour time period, there is slight decrease in cell viability. Mineral trioxide aggregate showed statistically significant increase in the cell viability when compared to other root repair materials. Conclusion: Mineral trioxide aggregate was shown to be less toxic to periodontal ligament fibroblasts than Endosequence Root Repair Material and Biodentine.

  10. Cytotoxicity evaluation of root repair materials in human-cultured periodontal ligament fibroblasts

    Science.gov (United States)

    Samyuktha, Voruganti; Ravikumar, Pabbati; Nagesh, Bolla; Ranganathan, K.; Jayaprakash, Thumu; Sayesh, Vemuri

    2014-01-01

    Aim: To evaluate the cytotoxicity of three root repair materials, mineral trioxide aggregate (MTA), Endosequence Root Repair Material and Biodentine in human periodontal ligament fibroblasts. Materials and Methods: Periodontal ligament fibroblasts were cultured from healthy premolar extracted for orthodontic purpose. Cells in the third passage were used in the study. The cultured fibroblast cells were placed in contact with root repair materials: (a) Biodentine, (b) MTA, (c) Endosequence, (d) control. The effects of these three materials on the viability of Periodontal ligament (PDL) fibroblasts were determined by trypan blue dye assay after 24 hours and 48-hour time period. Cell viability was determined using inverted phase contrast microscope. Statistical Analysis: Cell viability was compared for all the experimental groups with Wilcoxons matched pair test. Results: At the 24-hour examination period, all the materials showed increased cell viability. At 48-hour time period, there is slight decrease in cell viability. Mineral trioxide aggregate showed statistically significant increase in the cell viability when compared to other root repair materials. Conclusion: Mineral trioxide aggregate was shown to be less toxic to periodontal ligament fibroblasts than Endosequence Root Repair Material and Biodentine. PMID:25298650

  11. Expression of molecules involved in B lymphocyte survival and differentiation by synovial fibroblasts.

    Science.gov (United States)

    Edwards, J C; Leigh, R D; Cambridge, G

    1997-06-01

    The synovitis of rheumatoid arthritis (RA) is one of few pathological lesions in which B lymphocyte accumulation progresses to the extent of germinal centre formation. The present study was designed to assess the ability of synovial fibroblasts to express molecules implicated in B lymphocyte survival and differentiation, both in vivo, and in response to cytokines in vitro. Normal and diseased synovia were examined by indirect immunofluorescence. In all tissues synovial intimal fibroblasts showed co-expression of vascular cell adhesion molecule-1 (VCAM-1) and complement decay-accelerating factor (DAF) comparable to that of follicular dendritic cells (FDC), but not complement receptor 2 (CR2). In rheumatoid synovia, subintimal cells showed variable expression of VCAM-1 and DAF, with bright co-expression of VCAM-1, DAF and CR2 in lymphoid follicle centres. B lymphocytes, some of which were proliferating cell nuclear antigen-positive, were present in contact with subintimal cells expressing VCAM-1 with or without DAF or CR2. B lymphocytes were rarely present in the intimal layer, and, where present, showed fragmentation. In vitro, synovial fibroblasts exposed to tumour necrosis factor-alpha (TNF-alpha) in combination with interferon-gamma (IFN-gamma) showed enhanced expression of VCAM-1, in comparison with fibroblasts from skin and lung and, unlike skin and lung fibroblasts, also expressed DAF and CR2. These findings support the hypothesis that synovial targeting in RA involves an enhanced ability of synovial fibroblasts to support B lymphocyte survival. This appears to be dependent, not on the constitutive expression of VCAM-1 and DAF on intimal cells, but on the increased ability of subintimal cells to respond to proinflammatory cytokines, perhaps critically in the expression of VCAM-1.

  12. Apoptotic markers in cultured fibroblasts correlate with brain metabolites and regional brain volume in antipsychotic-naive first-episode schizophrenia and healthy controls

    NARCIS (Netherlands)

    Batalla, A.; Bargallo, N.; Gasso, P.; Molina, O.; Pareto, D.; Mas, S.; Roca, J.M.; Bernardo, M.; Lafuente, A.; Parellada, E.

    2015-01-01

    Cultured fibroblasts from first-episode schizophrenia patients (FES) have shown increased susceptibility to apoptosis, which may be related to glutamate dysfunction and progressive neuroanatomical changes. Here we determine whether apoptotic markers obtained from cultured fibroblasts in FES and cont

  13. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-02-01

    The authors evaluated the interaction of (/sub 3/H)1,25(OH)/sup 2/D3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH)/sub 2/D (1,25(OH)/sub 2/D). They analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer. With normal fibroblasts the affinity and capacity of nuclear uptake of (/sub 3/H)1,25(OH)/sup 2/D3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. The following four patterns of interaction with (/sub 3/H)1,25(OH)/sup 2/D3 were observed with cells cultured from affected patients. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of (/sub 3/H)1,25(OH)/sup 2/D3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH)/sup 2/D resulted from five or six distinct genetic mutations in six kindreds.

  14. Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model

    Energy Technology Data Exchange (ETDEWEB)

    Horie, Masafumi [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Saito, Akira, E-mail: asaitou-tky@umin.ac.jp [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Mikami, Yu [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Ohshima, Mitsuhiro [Department of Biochemistry, Ohu University School of Pharmaceutical Sciences (Japan); Morishita, Yasuyuki [Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo (Japan); Nakajima, Jun [Department of Thoracic Surgery, Graduate School of Medicine, University of Tokyo (Japan); Kohyama, Tadashi; Nagase, Takahide [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We established three patient-paired sets of CAFs and NFs. Black-Right-Pointing-Pointer CAFs and NFs were analyzed using three-dimensional co-culture experiments. Black-Right-Pointing-Pointer CAFs clearly enhanced collagen gel contraction. Black-Right-Pointing-Pointer CAFs showed higher {alpha}-SMA expression than NFs. Black-Right-Pointing-Pointer CAFs were implicated in invasion and differentiation of lung cancer cells. -- Abstract: Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher {alpha}-smooth muscle actin ({alpha}-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

  15. IL-1β suppresses TGF-β-mediated myofibroblast differentiation in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Brønnum, Hasse; Eskildsen, Tilde; Andersen, Ditte Caroline;

    2013-01-01

    Cardiac fibrosis is a maladaptive response of the injured myocardium and is mediated through a complex interplay between molecular triggers and cellular responses. Interleukin (IL)-1β is a key inflammatory inducer in cardiac disease and promotes cell invasion and cardiomyocyte injury, but little...... a differentiation model of ventricular fibroblasts, we found that IL-1β instigated substantial anti-fibrogenic effects. In specific, IL-1β reduced proliferation, matrix activity, cell motility and α-smooth muscle actin expression, which are all hallmarks of myoFb differentiation. These findings suggest that IL-1β...

  16. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-02-01

    We evaluated the interaction of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH)/sub 2/D (1,25(OH)/sub 2/D). We analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer containing 300 mM KCl and 10 mM sodium molybdate. With normal fibroblasts the affinity and capacity of nuclear uptake of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH)/sub 2/D resulted from five or six distinct genetic mutations in six kindreds.

  17. Effect of three commercial mouth rinses on cultured human gingival fibroblast: An in vitro study

    Directory of Open Access Journals (Sweden)

    Flemingson

    2008-01-01

    Full Text Available Aim: To examine the effect of three commercial mouth rinses (Hexidine 0.2%, Listerine Cool Mint, Betadine 1% upon cultured human gingival fibroblast proliferation. Materials and Methods: Human gingival fibroblasts were cultured and incubated in Dulbecco′s Minimum Eagle′s Medium containing Chlorhexidine, Listerine, Povidone-Iodine at varying concentrations (1%, 2%, 5%, 10%, 20% and 100% of the given solution at 37°C for 1, 5 and 15 min. Control cells received an equal volume of Dulbecco′s Minimum Eagle′s Medium without adding mouth rinses, for similar duration of exposure at 37°C. Following incubation the media were removed, cells were washed twice with medium, supplemented with 10% Fetal Bovine Serum, and fibroblasts in the test and control group were allowed to recover in the same media for 24 h. Results: In all the three groups, the proliferation inhibition was dependent on the concentration of solublized mouth rinses in the cell culture but independent of the duration of exposure to all three mouth rinses. The results showed that all three solutions were toxic to cultured human gingival fibroblasts, Chlorhexidine being the most cytotoxic. It was seen that at dilute concentrations (1% and 2% of given solutions Listerine was more cytotoxic than Chlorhexidine and Povidone-Iodine. Conclusion: These results suggest that Chlorhexidine, Listerine and Povidone-Iodine are capable of inducing a dose-dependent reduction in cellular proliferation of fibroblasts. The results presented are interesting, but to know the clinical significance, further studies are needed.

  18. Dupuytren's disease: comparative growth dynamics and morphology between cultured myofibroblasts (nodule) and fibroblasts (cord).

    Science.gov (United States)

    Vande Berg, J S; Gelberman, R H; Rudolph, R; Johnson, D; Sicurello, P

    1984-01-01

    The excised palmar fascia of 11 patients with Dupuytren's disease was separated clinically into nodules and cords. Myofibroblasts were seen by light and electron microscopy in each of the nodules, but the cords generally lacked myofibroblasts. Only one cord specimen had microscopic features that were intermediate between nodule and cord. Electron microscopy demonstrated that in vivo differences between myofibroblasts from nodules and fibroblasts from cords and control skin samples could be preserved in vitro. Growth studies showed slower growth of cultured myofibroblasts (mean +/- SD generation time 68.7 +/- 15 h) than cord-derived fibroblasts (mean +/- SD generation time 51.5 +/- 0.9 h). These data suggest that the life cycle of the myofibroblasts from Dupuytren's disease nodules differs from that of fibroblasts found in cordlike tissues. These myofibroblasts have biological characteristics nearly identical to those of myofibroblasts found in other contracting tissues, such as granulating wounds and breast cancer.

  19. Adipose Tissue-Derived Stromal Cells Inhibit TGF-beta 1-Induced Differentiation of Human Dermal Fibroblasts and Keloid Scar-Derived Fibroblasts in a Paracrine Fashion

    NARCIS (Netherlands)

    Spiekman, Maroesjka; Przybyt, Ewa; Plantinga, Josee A.; Gibbs, Susan; van der Lei, Berend; Harmsen, Martin C.

    2014-01-01

    Background: Adipose tissue-derived stromal cells augment wound healing and skin regeneration. It is unknown whether and how they can also influence dermal scarring. The authors hypothesized that adipose tissue-derived stromal cells inhibit adverse differentiation of dermal fibroblasts induced by the

  20. Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture.

    Science.gov (United States)

    Pineda, Emma T; Nerem, Robert M; Ahsan, Tabassum

    2013-01-01

    Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine.

  1. TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration.

    Science.gov (United States)

    Negmadjanov, Ulugbek; Godic, Zarko; Rizvi, Farhan; Emelyanova, Larisa; Ross, Gracious; Richards, John; Holmuhamedov, Ekhson L; Jahangir, Arshad

    2015-01-01

    Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts. Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit. Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells). TGF-β1 induced differentiation

  2. TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration.

    Directory of Open Access Journals (Sweden)

    Ulugbek Negmadjanov

    Full Text Available Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.Treatment with TGF-β1 (5 ng/mL induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU. TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU, as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control. TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells.TGF-β1 induced

  3. Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan

    Science.gov (United States)

    Patrick, Alison; Seluanov, Michael; Hwang, Chaewon; Tam, Jonathan; Khan, Tanya; Morgenstern, Ari; Wiener, Lauren; Vazquez, Juan M.; Zafar, Hiba; Wen, Robert; Muratkalyeva, Malika; Doerig, Katherine; Zagorulya, Maria; Cole, Lauren; Catalano, Sophia; Lobo Ladd, Aliny AB; Coppi, A. Augusto; Coşkun, Yüksel; Tian, Xiao; Ablaeva, Julia; Nevo, Eviatar; Gladyshev, Vadim N.; Zhang, Zhengdong D.; Vijg, Jan; Seluanov, Andrei; Gorbunova, Vera

    2016-01-01

    Differences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species' lifespan. PMID:27163160

  4. Dexamethasone inhibition of IL-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

    Science.gov (United States)

    Lu, Ying; Fukuda, Ken; Liu, Yang; Kumagai, Naoki; Nishida, Teruo

    2004-09-01

    Corticosteroids regulate the functions of inflammatory cells. The purpose of the present study was to investigate the effect of dexamethasone on collagen degradation by corneal fibroblasts, an underlying cause of corneal ulceration. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of IL-1beta or dexamethasone. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was evaluated by immunoblot analysis, gelatin zymography, and reverse transcription and real-time polymerase chain reaction. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was assessed by immunoblot analysis. Dexamethasone inhibited IL-1beta-induced collagen degradation by corneal fibroblasts in a dose-dependent manner. Both the synthesis and activation of MMPs and the expression of TIMPs were inhibited by dexamethasone, as was the activity of plasmin in culture supernatants. Dexamethasone also inhibited the IL-1beta-induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not that of p38. Dexamethasone exerted multiple effects on the MMP-TIMP system in corneal fibroblasts and thereby inhibited IL-1beta-induced collagen degradation by these cells. Inhibition of the IL-1beta-induced activation of ERK and JNK may contribute to these effects of dexamethasone. Copyright Association for Research in Vision and Ophthalmology

  5. Postnatal visual deprivation in rats regulates several retinal genes and proteins, including differentiation-associated fibroblast growth factor-2.

    Science.gov (United States)

    Prokosch-Willing, Verena; Meyer zu Hoerste, Melissa; Mertsch, Sonja; Stupp, Tobias; Thanos, Solon

    2015-01-01

    Little is known about the retinal cellular basis of amblyopia, which is a developmental disease characterized by impaired visual acuity. This study examined the retinal transcripts associated with experimentally induced unilateral amblyopia in rats. Surgical tarsorrhaphy of the eyelids on one side was performed in pups prior to eye opening at postnatal day 14, thereby preventing any visual experience. This condition was maintained for over 2 months, after which electroretinograms (ERGs) were recorded, the retinal ganglion cell (RGC) arrangement and number were determined using neuroanatomical tracing, the retinal transcripts were studied using microarray analysis, regulated mRNAs were confirmed with quantitative reverse-transcriptase PCR, and proteins were stained using Western blotting and immunohistochemistry. An attenuated ERG was found in eyes that were deprived of visual experience. Retrograde neuroanatomical staining disclosed a larger number of RGCs within the retina on the visually deprived side compared to the non-deprived, control side, and a multilayered distribution of RGCs. At the retinomic level, several transcripts associated with retinal differentiation, such as fibroblast growth factor 2 (FGF-2), were either up- or downregulated. Most of the transcripts could be verified at the mRNA level. To unravel the role of a differentiation-associated protein, we tested FGF-2 in dissociated postnatal retinal cell cultures and found that FGF-2 is a potent factor triggering ganglion cell differentiation. The data suggest that visual experience shapes the postnatal retinal differentiation, whereas visual deprivation induces changes at the functional, cellular and molecular levels within the retina.

  6. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    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.

  7. An attempt to eliminate fibroblast-like cells from primary cultures of fetal human livers.

    Directory of Open Access Journals (Sweden)

    Tokiwa,Takayoshi

    1986-04-01

    Full Text Available The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF, which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC or putrescine (PUT. The growth of HuF cells was inhibited by HC at a concentration of 10(-2 M and by PUT at a concentration higher than 10(-3 M. Long-term treatment of HuF cells with 10(-3 M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5M to 10(-3 M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3 M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

  8. Biocompatibility of three bioabsorbable membranes assessed in FGH fibroblasts and human osteoblast like cells culture.

    Science.gov (United States)

    Soares, Michelle Pereira Costa Mundim; Soares, Paulo Vinícius; Pereira, Analice Giovani; Moura, Camilla Christian Gomes; Soares, Priscila Barbosa Ferreira; Naves, Lucas Zago; de Magalhães, Denildo

    2014-08-06

    Specific physical and chemical features of the membranes may influence the healing of periodontal tissues after guided tissue regeneration (GTR). The aim of the present investigation was to analyze the biological effects of three bioabsorbable membranes. The hypothesis is that all tested membranes present similar biological effects. Human osteoblast like-cells (SaOs-2) and gingival fibroblasts FGH (BCRJ -RJ) were cultured in DMEM medium. The viability of the cells cultured on the membranes was assesses using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Quantitative determination of activated human Transforming Growth Factor beta 1 (TGF-β1) on the supernatants of the cell culture was observed. Samples were examined using scanning electron microscope (SEM). SaOs2, in 24 hours, PLA group showed higher values when compared to other groups (P statistical significance values when compared two times. In 4 h and 24 h, for the fibroblasts group, significantly difference was found to PLA membrane, when compared with the other groups (p statistically significant difference (p analysis of culture supernatants of fibroblasts, in 24 hours, only PLA group presented significant difference (p = 0,008). The biomaterials analyzed did not show cytotoxicity, since no membrane presented lower results than the control group. PLA membrane presented the best performance due to its higher cell viability and absorbance levels of proliferation. Both collagen membranes showed similar results either when compared to each other or to the control group.

  9. Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.

    Science.gov (United States)

    Rufini, S; Cesaroni, M P; Balestro, N; Luly, P

    1996-12-01

    Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.

  10. Teratogenic and cytotoxic effects of VOsalen complex on chicken embryos, hepatic and fibroblastic- cell cultures

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    Abdolmaleki A

    2013-04-01

    Full Text Available Background: Salen metal complexes are used successfully in a wide range of asymmet-ric reactions and important in the pharmaceutical and industry. On the toxicity of salen vanadium oxide (VOsalen on embryo and cell cultures, little information is available. In the present study, the toxic and teratogenic effects of VOsalen was evaluated against chicken embryos as a animal model and liver and fibroblast cell cultures which was derived from the embryo.Methods: The VOsalen compound was synthesized. The compound solution was inject-ed in triplicate examination, in the air sac of the eggs, at third day of incubation. Treat-ed and control eggs, on day 19 of incubation opened and embryos were weighted, then mortality rate was recorded. The liver and fibroblast cell culture were treated by this and survival fraction was recorded.Results: The survived fraction of the embryos depends on the compound concentration. In concentration of 300μM/egg, 36/32% of the embryos survived and the Lethal dose 50% (LD50 was 226/37 μM/egg. Morphological study of the treated embryos showed retarded growth, and skeletal staining showed the deletion of caudal vertebrate. The compound was inhibited liver and fibroblast cells growth with IC50 1047/25 and 1036/82μM respectively. The cytoplasm of treated cells became dense and their interco-nnections were loosed.Conclusion: The VOsalen compound had low toxic effects against the embryos and the cultured cells at the concentrations. Significant cytotoxic effect was not observed in the treated cells. However the proliferative cells were affected significantly in comparison with the cells which their growth was stopped. The effect of VOsalen compound against replication of liver cells were lower than fibroblast cells.

  11. Transplantation of Unique Subpopulation of Fibroblasts, Muse Cells, Ameliorates Experimental Stroke Possibly via Robust Neuronal Differentiation.

    Science.gov (United States)

    Uchida, Hiroki; Morita, Takahiro; Niizuma, Kuniyasu; Kushida, Yoshihiro; Kuroda, Yasumasa; Wakao, Shohei; Sakata, Hiroyuki; Matsuzaka, Yoshiya; Mushiake, Hajime; Tominaga, Teiji; Borlongan, Cesario V; Dezawa, Mari

    2016-01-01

    Muse cells reside as pre-existing pluripotent-like stem cells within the fibroblasts, are nontumorigenic, exhibit differentiation capacity into triploblastic-lineage cells, and replenish lost cells when transplanted in injury models. Cell fate and function of human skin fibroblast-derived Muse cells were evaluated in a rat stroke model. Muse cells (30,000), collected by pluripotent surface marker stage-specific embryonic antigen-3, were injected stereotaxically into three deposits within the rat ischemic cortex at 2 days after transient middle cerebral artery occlusion, and the cells' biological effects were examined for more than 84 days. Muse cells spontaneously and promptly committed to neural/neuronal-lineage cells when cocultured with stroke brain slices. Muse-transplanted stroke rats exhibited significant improvements in neurological and motor functions compared to control groups at chronic days 70 and 84, without a reduction in the infarct size. Muse cells survived in the host brain for up to 84 days and differentiated into NeuN (∼ 65%), MAP-2 (∼ 32%), calbindin (∼ 28%), and GST-π (∼ 25%)-positive cells in the cortex, but glial fibrillary acidic protein-positive cells were rare. Tumor formation was not observed. Muse cells integrated into the sensory-motor cortex, extended their neurites into cervical spinal cord, and displayed normalized hind limb somatosensory evoked potentials. Muse cells are unique from other stem cells in that they differentiate with high ratio into neuronal cells after integration with host brain microenvironment, possibly reconstructing the neuronal circuit to mitigate stroke symptoms. Human fibroblast-derived Muse cells pose as a novel source of transplantable stem cells, circumventing the need for gene manipulations, especially when contemplating autologous cell therapy for stroke. © 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  12. Characterization of epicardial-derived cardiac interstitial cells: differentiation and mobilization of heart fibroblast progenitors.

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    Adrián Ruiz-Villalba

    Full Text Available The non-muscular cells that populate the space found between cardiomyocyte fibers are known as 'cardiac interstitial cells' (CICs. CICs are heterogeneous in nature and include different cardiac progenitor/stem cells, cardiac fibroblasts and other cell types. Upon heart damage CICs soon respond by initiating a reparative response that transforms with time into extensive fibrosis and heart failure. Despite the biomedical relevance of CICs, controversy remains on the ontogenetic relationship existing between the different cell kinds homing at the cardiac interstitium, as well as on the molecular signals that regulate their differentiation, maturation, mutual interaction and role in adult cardiac homeostasis and disease. Our work focuses on the analysis of epicardial-derived cells, the first cell type that colonizes the cardiac interstitium. We present here a characterization and an experimental analysis of the differentiation potential and mobilization properties of a new cell line derived from mouse embryonic epicardium (EPIC. Our results indicate that these cells express some markers associated with cardiovascular stemness and retain part of the multipotent properties of embryonic epicardial derivatives, spontaneously differentiating into smooth muscle, and fibroblast/myofibroblast-like cells. Epicardium-derived cells are also shown to initiate a characteristic response to different growth factors, to display a characteristic proteolytic expression profile and to degrade biological matrices in 3D in vitro assays. Taken together, these data indicate that EPICs are relevant to the analysis of epicardial-derived CICs, and are a god model for the research on cardiac fibroblasts and the role these cells play in ventricular remodeling in both ischemic or non/ischemic myocardial disease.

  13. [Effects of culture supernatant of human amnion mesenchymal stem cells on biological characteristics of human fibroblasts].

    Science.gov (United States)

    Wu, Qi'er; Lyu, Lu; Xin, Haiming; Luo, Liang; Tong, Yalin; Mo, Yongliang; Yue, Yigang

    2016-06-01

    To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts. (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post

  14. Experimental study on He- Ne laser irradiation to inhibit scar fibroblast growth in culture

    Institute of Scientific and Technical Information of China (English)

    舒彬; 吴宗耀; 郝林林; 曾登芬; 冯光锐; 林永辉

    2002-01-01

    To explore the inhibitory effect of He-Ne laser irradiation on fibroblast growth of hypertrophic scars in culture. Methods: He-Ne laser with wavelength of 632.8 nm,power density of 50 mW/cm2 and doses of 3 J/cm2,30 J/cm2, 90 J/cm2 and 180 J/cm2 was used to irradiate human scar fibroblasts in culture 1, 3 and 5 times respectively, and then the cell count and cell cycle analysis were done. Results: Repeated irradiation with He-Ne laser at dose of 180 J/cm2 three and five times led to an evident decrease in total cell number compared with that of the control group and there was a significant difference ( P <0.05). The cell cycle analysis showed after three and five times of irradiation with 180 J/cm2 He-Ne laser the cell number in S-phase decreased from 51% to 20% and 14% respectively, the cell number in G0/G1 phase increased from 28% to 55% and 60% respectively, and the cell percentage in Sub-G1 phase was 6.7% and 9.8% respectively. Conclusions: Repeated irradiation with 180 J/cm2 He-Ne laser can inhibit scar fibroblasts growth in culture.It may be that He-Ne laser irradiation causes cell stagnation in G0/G1 phase and apoptosis.

  15. Low-Dose UVA Radiation-Induced Adaptive Response in Cultured Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhongrong Liu

    2012-01-01

    Full Text Available Objective. To investigate the mechanism of the adaptive response induced by low-dose ultraviolet A (UVA radiation. Methods. Cultured dermal fibroblasts were irradiated by a lethal dose of UVA (86.4 J/cm2 with preirradiation of single or repetitive low dose of UVA (7.2 J/cm2. Alterations of cellular morphology were observed by light microscope and electron microscope. Cell cycle and cellular apoptosis were assayed by flow cytometer. The extent of DNA damage was determined by single-cell gel electrophoresis (SCGE. Results. The cultured dermal fibroblasts, with pretreatment of single or repetitive irradiation of 7.2 J/cm2 UVA relieved toxic reaction of cellular morphology and arrest of cell cycle, decreased apoptosis ratio, reduced DNA chain breakage, and accelerated DNA repair caused by subsequent 86.4 J/cm2 UVA irradiation. Compared with nonpretreatment groups, all those differences were significant (P<0.01 or P<0.05. Conclusions. The adaptation reaction might depend on the accumulated dose of low-dose UVA irradiation. Low-dose UVA radiation might induce adaptive response that may protect cultured dermal fibroblasts from the subsequent challenged dose of UVA damage. The duration and protective capability of the adaptive reaction might be related to the accumulated dose of low-dose UVA Irradiation.

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

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    Thon-Hon Vincent G

    2012-09-01

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

  17. In vitro clonogenic capacity of periodontal ligament fibroblasts cultured with Emdogain.

    Science.gov (United States)

    Ashkenazi, Malka; Shaked, Ilanit

    2006-02-01

    The aim of the present study was to evaluate the efficiency of Emdogain (EMD) in preserving the size of the periodontal ligament progenitor pool (clonogenic capacity) and in promoting their proliferation. Periodontal ligament fibroblasts (PDLF) were obtained from explants of young permanent healthy tooth. After initial outgrowth (10 days to 2 weeks following explantation), the culture medium of experimental flasks was replaced with medium supplemented with 100 microg ml(-1) EMD, whereas the other served as controls and were fed with regular medium. Following 5 weeks, the cells were washed (3x), harvested (trypsin + EDTA), and evaluated for their viability. Viable cells from each group were inoculated into six 96-well plates at a concentration of one viable cell per two wells and were allowed to grow for 5 weeks. The percentage of cells with clonogenic capacity was determined as the number of colonies formed/number of cells seeded x 100 in the experimental and control groups. Three degrees of dish area coverage were utilized: up to 25%, between 25% and 75% and higher than 75%. This experiment was repeated four times from four different donors. A total of 2328 cells were evaluated, half of which, were cultured with EMD. The mean percentage of cells (from all donors) who exhibited any clonogenic capacity in the presence of EMD was comparable with that of cells cultured in the absence of EMD: 26.6 +/- 14.3% when compared with 34.6 +/- 20.6% respectively (P = 0.186). Similarly, the percentage of clones that proliferated to cover up to 25% of the well area was comparable in the two groups 7.5 +/- 8.6 for EMD-treated clones and 7.1 +/- 7.8 for untreated clones (P = 0.674). The percentage of clones that proliferated to cover 25% up to 75% of the well area was greater EMD-treated clones as compared with the untreated cells: 8.1 +/- 6.7% vs 3.8 +/- 3%. However this difference was not statistically significant (P = 0.277). In contrast, the percentage of clones that covered

  18. Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture

    Directory of Open Access Journals (Sweden)

    Ertl Ronald F

    2001-09-01

    Full Text Available Abstract Background Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. Methods Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Results Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P P P 2 production was significantly increased by co-culture and its presence attenuated collagen degradation. Conclusion The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture.

  19. Differential gene expression in SV40-mediated immortalization of human fibroblasts.

    Science.gov (United States)

    Pardinas, J; Pang, Z; Houghton, J; Palejwala, V; Donnelly, R J; Hubbard, K; Small, M B; Ozer, H L

    1997-06-01

    Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of

  20. Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

    Science.gov (United States)

    Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

    2012-12-01

    The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

  1. Identification of differentially expressed genes in fibroblasts derived from patients with Dupuytren's Contracture

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    Hu Fen Z

    2008-04-01

    Full Text Available Abstract Dupuytren's contracture (DC is the most common inherited connective tissue disease of humans and is hypothesized to be associated with aberrant wound healing of the palmar fascia. Fibroblasts and myofibroblasts are believed to play an important role in the genesis of DC and the fibroproliferation and contraction that are hallmarks of this disease. This study compares the gene expression profiles of fibroblasts isolated from DC patients and controls in an attempt to identify key genes whose regulation might be significantly altered in fibroblasts found within the palmar fascia of Dupuytren's patients. Total RNA isolated from diseased palmar fascia (DC and normal palmar fascia (obtained during carpal tunnel release; 6 samples per group was subjected to quantitative analyses using two different microarray platforms (GE Code Link™ and Illumina™ to identify and validate differentially expressed genes. The data obtained was analyzed using The Significance Analysis of Microarrays (SAM software through which we identified 69 and 40 differentially regulated gene transcripts using the CodeLink™ and Illumina™ platforms, respectively. The CodeLink™ platform identified 18 upregulated and 51 downregulated genes. Using the Illumina™ platform, 40 genes were identified as downregulated, eleven of which were identified by both platforms. Quantitative RT-PCR confirmed the downregulation of three high-interest candidate genes which are all components of the extracellular matrix: proteoglycan 4 (PRG4, fibulin-1 (FBLN-1 transcript variant D, and type XV collagen alpha 1 chain. Overall, our study has identified a variety of candidate genes that may be involved in the pathophysiology of Dupuytren's contracture and may ultimately serve as attractive molecular targets for alternative therapies.

  2. Comparative Culturing of 3T3 Swiss J2 Mouse Embryo Fibroblasts on Modified Chitosan Matrices.

    Science.gov (United States)

    Alekhin, A I; Gaenko, G P

    2016-07-01

    Comparative culturing of mouse embryo fibroblasts on chitosan matrices and culture plastic was carried out. During the first 2 h of culturing (lag phase), cell adhesion to chitosan and chitosan-gelatin matrices was 20-30% higher than adhesion to culture plastic (control). During the stationary phase, 80% cells adhered to chitosan matrices (vs. 60% in the control). Cell culturing on chitosan matrices was carried out without medium replacement with fresh portions. The cells remained viable within 5 days of culturing. Cell death phase was observed on day 6 of culturing on chitosan matrices: cell adhesion dropped to 50%. Culturing on culture plastic was carried out with daily medium replacement with a fresh portion. Cell death phase (50% decrease in the number of adherent cell) under these condition was observed on day 5. It seems that the observed effect was a result of contact interactions of cell integrins and chitosan ligands, modulation of transmembrane signal, eventually modifying the expression of cell genes. This effect can be required in regenerative medicine for production of primary cell culture.

  3. Effects of Baicalin on Expression of Inducible Nitric Oxide Synthase in Cultured Fibroblasts Stimulated by Cytokines

    Institute of Scientific and Technical Information of China (English)

    毕新岭; 顾军; 聂本勇; 李泉; 刘辉; 米庆胜

    2004-01-01

    Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ), interleukin-8 (IL-S) in different groups. iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results: Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1. 0× 106 U/L TNF-α, 0.2× 106U/L IFN-γ, 0.2× 106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50 μg/ mi of baicalin. Conclusion: Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.

  4. Effects of Balcalin on Expression of Inducible Nitric Oxide Synthase In Cultured Fibroblasts Stimulated by Cytokines

    Institute of Scientific and Technical Information of China (English)

    毕新岭; 顾军; 聂本勇; 李泉; 刘辉; 米庆胜

    2004-01-01

    Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α((TNF-α), interferon-γ(IFN-γ), interleukin-8 (IL-8) in different groups, iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results. Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1.0×106 U/L TNF-α, 0.2×106U/L IFN-γ, 0.2×106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50μg/ ml of baicalin. Conclusion. Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.

  5. Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

    DEFF Research Database (Denmark)

    Hansen, Jacob B; Petersen, Rasmus K; Jørgensen, Claus;

    2002-01-01

    A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity....../Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte...... differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role...

  6. Turnover of ribosomal RNA in mouse fibroblasts (3T3) in culture

    Energy Technology Data Exchange (ETDEWEB)

    Kolodny, G.M.

    1975-01-01

    Growing and confluent cultures of mouse fibroblasts were labeled with /sup 3/H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.

  7. Comparative use of glucose and fructose in cultured fibroblasts from patients with hereditary fructose intolerance.

    Science.gov (United States)

    Lemonnier, F; Delhotal-Landes, B; Couturier, M; Decimo, D; Odiévre, M; Gautier, M; Lemonnier, A

    1987-01-01

    The utilization of fructose and glucose by fibroblast cultures obtained from patients with hereditary fructose intolerance (HFI) was studied in comparison with fibroblast controls. The cell growth, the time course of D-glucose or D-fructose uptake and the consumption of fructose were similar for both HFI and control cells. Some results showed significant differences between these two cell types: HFI cells consumed less glucose, produced less lactate and contained less glycogen than control cells. Furthermore, significantly less [U-14C]D-glucose and [U-14C]D-fructose was incorporated into lipids in HFI cells than in control cells. The mechanisms responsible for these differences observed between the two cell types are not known.

  8. Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts.

    Science.gov (United States)

    Nagel, Marie-Danielle; Verhoef, René; Schols, Henk; Morra, Marco; Knox, J Paul; Ceccone, Giacomo; Della Volpe, Claudio; Vigneron, Pascale; Bussy, Cyrill; Gallet, Marlène; Velzenberger, Elodie; Vayssade, Muriel; Cascardo, Giovanna; Cassinelli, Clara; Haeger, Ash; Gilliland, Douglas; Liakos, Ioannis; Rodriguez-Valverde, Miguel; Siboni, Stefano

    2008-01-01

    Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-alpha), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR-alpha causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR-alpha show the opposite behaviour to the one observed on MHR-B and -alpha-grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-alpha. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-alpha. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.

  9. Induction of Neuronal Differentiation of Rat Muscle-Derived Stem Cells in Vitro Using Basic Fibroblast Growth Factor and Ethosuximide

    Directory of Open Access Journals (Sweden)

    Jin Seon Kwon

    2013-03-01

    Full Text Available Several studies have demonstrated that basic fibroblast growth factor (bFGF can induce neural differentiation of mesenchymal stem cells. In this study, we investigated the neural differentiation of muscle-derived stem cells (MDSCs following treatment with bFGF and ethosuximide, a small molecule used as an anticonvulsant in humans. Stem cells isolated from rat skeletal muscle (rMDSCs were pre-induced by culturing with 25 ng/mL bFGF for 24 h and then were transferred to a medium supplemented with or without 4 mM ethosuximide. Neuronal differentiation was assessed by immunocytochemical and western blotting analyses of marker expression. Immunocytochemistry of rMDSCs treated with bFGF and ethosuximide identified abundant cells expressing neuronal markers (TuJ1, neuron-specific class III β-tubulin; NeuN, neuronal nuclear antigen; and NF-MH; neurofilament M and H. Olig2 (oligodendrocyte transcription factor 2-positive cells were also observed, indicating the presence of oligodendrocyte lineage cells. These findings were substantiated by western blotting analysis of marker proteins. In particular, the expression of NeuN and TuJ1 was significantly higher in rMDSCs treated with ethosuximide and bFGF than in cells stimulated with bFGF alone (NeuN, p < 0.05 and TuJ1, p < 0.001. Expression of the astrocyte marker GFAP (glial fibrillary acidic protein was not detected in this study. Collectively, the results showed that treatment with bFGF and ethosuximide induced effective transdifferentiation of rMDSCs into cells with a neural-like phenotype. Notably, rMDSCs treated with a combination of bFGF plus ethosuximide showed enhanced differentiation compared with cells treated with bFGF alone, implying that ethosuximide may stimulate neuronal differentiation.

  10. Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation.

    Science.gov (United States)

    Zhang, Jinlong; Lian, Min; Cao, Peipei; Bao, Guofeng; Xu, Guanhua; Sun, Yuyu; Wang, Lingling; Chen, Jiajia; Wang, Yi; Feng, Guijuan; Cui, Zhiming

    2017-04-01

    Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, βIII-tubulin and GFAP were the most highest in the DPSCs + bFGF + NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF + NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.

  11. Optimized “In Vitro” Culture Conditions for Human Rheumatoid Arthritis Synovial Fibroblasts

    Directory of Open Access Journals (Sweden)

    Claudia Casnici

    2014-01-01

    Full Text Available The composition of synovial fluid in rheumatoid arthritis (RA is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, “in vitro” studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform “in vitro” studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in “in vitro” studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

  12. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

    Science.gov (United States)

    Zhang, Xi; Shen, Man-Ru; Xu, Zhen-Dong; Hu, Zhe; Chen, Chao; Chi, Ya-Li; Kong, Zhen-Dong; Li, Zi-Fu; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Zhang, Chuan-Sen

    2014-05-01

    Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the

  13. Quiescent and proliferative fibroblasts exhibit differential p300 HAT activation through control of 5-methoxytryptophan production.

    Directory of Open Access Journals (Sweden)

    Huei-Hsuan Cheng

    Full Text Available Quiescent fibroblasts possess unique genetic program and exhibit high metabolic activity distinct from proliferative fibroblasts. In response to inflammatory stimulation, quiescent fibroblasts are more active in expressing cyclooxygenase-2 and other proinflammatory genes than proliferative fibroblasts. The underlying transcriptional mechanism is unclear. Here we show that phorbol 12-myristate 13-acetate (PMA and cytokines increased p300 histone acetyltransferase activity to a higher magnitude (> 2 fold in quiescent fibroblasts than in proliferative fibroblasts. Binding of p300 to cyclooxygenase-2 promoter was reduced in proliferative fibroblasts. By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts. Addition of 5-methoxytryptophan and its metabolic precursor, 5-hydroxytryptophan, to quiescent fibroblasts suppressed PMA-induced p300 histone acetyltransferase activity and cyclooxygenase-2 expression to the level of proliferative fibroblasts. Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan. Our findings indicate that robust inflammatory gene expression in quiescent fibroblasts vs. proliferative fibroblasts is attributed to uncontrolled p300 histone acetyltransferase activation due to deficiency of 5-methoxytryptophan production. 5-methoxytryptophan thus is a potential valuable lead compound for new anti-inflammatory drug development.

  14. Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakahara, Takehiro; Sato, Hiroko; Shimizu, Takehisa; Tanaka, Toru; Matsui, Hiroki; Kawai-Kowase, Keiko; Sato, Mahito; Iso, Tatsuya; Arai, Masashi [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kurabayashi, Masahiko, E-mail: mkuraba@med.gunma-u.ac.jp [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2010-04-02

    Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.

  15. Differential regulation of collagen secretion by kinin receptors in cardiac fibroblast and myofibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Catalán, Mabel; Smolic, Christian [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Contreras, Ariel [Instituto Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile (Chile); Ayala, Pedro; Olmedo, Ivonne; Copaja, Miguel; Boza, Pía; Vivar, Raúl; Avalos, Yennifer [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Lavandero, Sergio [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Instituto Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile (Chile); Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX (United States); Velarde, Victoria [Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago (Chile); Díaz-Araya, Guillermo, E-mail: gadiaz@ciq.uchile.cl [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile)

    2012-06-15

    Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. Methods: CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-β1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca{sup +2} levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. Results: B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca{sup 2+} levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca{sup 2+} levels in CF; however, after preincubation for 1 h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca{sup 2+} levels. Finally, DAKD increased intracellular Ca{sup 2+} levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. Conclusion: B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was

  16. STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation.

    Science.gov (United States)

    Pedroza, Mesias; Le, Thuy T; Lewis, Katherine; Karmouty-Quintana, Harry; To, Sarah; George, Anuh T; Blackburn, Michael R; Tweardy, David J; Agarwal, Sandeep K

    2016-01-01

    Lung fibrosis is the hallmark of the interstitial lung diseases. Alveolar epithelial cell (AEC) injury is a key step that contributes to a profibrotic microenvironment. Fibroblasts and myofibroblasts subsequently accumulate and deposit excessive extracellular matrix. In addition to TGF-β, the IL-6 family of cytokines, which signal through STAT-3, may also contribute to lung fibrosis. In the current manuscript, the extent to which STAT-3 inhibition decreases lung fibrosis is investigated. Phosphorylated STAT-3 was elevated in lung biopsies from patients with idiopathic pulmonary fibrosis and bleomycin (BLM)-induced fibrotic murine lungs. C-188-9, a small molecule STAT-3 inhibitor, decreased pulmonary fibrosis in the intraperitoneal BLM model as assessed by arterial oxygen saturation (control, 84.4 ± 1.3%; C-188-9, 94.4 ± 0.8%), histology (Ashcroft score: untreated, 5.4 ± 0.25; C-188-9, 3.3 ± 0.14), and attenuated fibrotic markers such as diminished α-smooth muscle actin, reduced collagen deposition. In addition, C-188-9 decreased the expression of epithelial injury markers, including hypoxia-inducible factor-1α (HIF-1α) and plasminogen activator inhibitor-1 (PAI-1). In vitro studies show that inhibition of STAT-3 decreased IL-6- and TGF-β-induced expression of multiple genes, including HIF-1α and PAI-1, in AECs. Furthermore, C-188-9 decreased fibroblast-to-myofibroblast differentiation. Finally, TGF-β stimulation of lung fibroblasts resulted in SMAD2/SMAD3-dependent phosphorylation of STAT-3. These findings demonstrate that STAT-3 contributes to the development of lung fibrosis and suggest that STAT-3 may be a therapeutic target in pulmonary fibrosis.

  17. Zinc and propolis reduces cytotoxicity and proliferation in skin fibroblast cell culture: total polyphenol content and antioxidant capacity of propolis.

    Science.gov (United States)

    Tyszka-Czochara, Małgorzata; Paśko, Paweł; Reczyński, Witold; Szlósarczyk, Marek; Bystrowska, Beata; Opoka, Włodzimierz

    2014-07-01

    It has been demonstrated that zinc exerts its beneficial influence on skin fibroblasts. Propolis, a complex mixture of plant-derived and bees' products, was reported to stimulate cicatrization processes in skin and prevent infections. The aim of this study was to find out how zinc and propolis influence human skin fibroblasts in cell culture and to compare the effect of individual compounds to the effect of a mixture of zinc and propolis. In this study, zinc, as zinc aspartate, at a concentration of 16 μM, increased human fibroblasts proliferation in cell culture, whereas propolis at a concentration of 0.01% (w/v) revealed antiproliferative and cytotoxic action followed by mild cell necrosis. In culture, zinc was effectively transported into fibroblasts, and propolis inhibited the amount of zinc incorporated into the cells. An addition of propolis to the medium caused a decrease in the Zn(II) amount incorporated into fibroblasts. The obtained results also indicate an appreciable antioxidant property of propolis and revealed its potential as a supplement when applied at doses lower than 0.01% (w/v). In conclusion, the present study showed that zinc had a protective effect on human cultured fibroblasts' viability, although propolis revealed its antiproliferative action and caused mild necrosis.

  18. Genes Differentially Expressed in Human Lung Fibroblast Cells Transformed by Glycidyl Methacrylate

    Institute of Scientific and Technical Information of China (English)

    XUE-JUN YIN; JIAN-NING XU; CHANG-QI ZOU; FENG-SHENG HE; FU-DE FANG

    2004-01-01

    To define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls. Methods The mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis. Results Eighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor ( inducible gene (Betaig-h3), (-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells. Conclusion Analysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

  19. Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells.

    Science.gov (United States)

    Ogawa, Shin-ichiro; Tokumoto, Yasuhito; Miyake, Jun; Nagamune, Teruyuki

    2011-08-01

    Induced pluripotent stem cells (iPSCs) prepared from somatic cells might become a novel therapeutic tool in regenerative medicine, especially for the central nervous system (CNS). In this study, we attempted to induce O4-positive (O4(+)) oligodendrocytes from adult human fibroblast-derived iPSCs in vitro. We used two adult human iPSC cell lines, 201B7 and 253G1. 201B7 was induced by four-gene transduction (oct4, sox2, klf4, c-myc), and 253G1 was induced by three-gene transduction (oct4, sox2, klf4). We treated these cells with two in vitro oligodendrocyte-directed differentiation protocols that were optimized for human embryonic stem cells. One protocol used platelet-derived growth factor as the major mitogen for oligodendrocyte lineage cells, and the other protocol used epidermal growth factor (EGF) as the mitogen. Although the differentiation efficiency was low (less than 0.01%), we could induce O4(+) oligodendrocytes from 253G1 cells using the EGF-dependent differentiation protocol. This is the first report of the in vitro induction of oligodendrocytes differentiation from human iPSCs.

  20. Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

    Science.gov (United States)

    Shi, Xingmin; Cai, Jingfen; Xu, Guimin; Ren, Hongbin; Chen, Sile; Chang, Zhengshi; Liu, Jinren; Huang, Chongya; Zhang, Guanjun; Wu, Xili

    2016-04-01

    An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro. Experimental results showed that, compared with the control cells, the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis, while the treatment of 25 s plasma resulted in a remarkable decrease. Exploration of related mechanisms suggested that cold plasma could up-regulate CyclinD1 gene expression and down-regulate p27 gene expression at a low dose, while it could down-regulate CyclinD1 expression and up-regulate p27 expression at a higher dose, thus altering the cell cycle progression, and then affecting cell viability and collagen synthesis of fibroblasts. supported partly by National Natural Science Foundation of China (Nos. 81372076, 51307133 and 51221005), China National Funds for Distinguished Young Scientists (No. 51125029), the Sci-Tech Project of Shaanxi Province of China (No. 2010K16-04), and the Fundamental Research Funds for the Central Universities of China (No. xkjc2013004)

  1. Amelogenin is phagocytized and induces changes in integrin configuration, gene expression and proliferation of cultured normal human dermal fibroblasts

    DEFF Research Database (Denmark)

    Almqvist, Sofia; Werthén, Maria; Johansson, Anna

    2010-01-01

    or down-regulation of genes, of which most are involved in cellular growth, migration and differentiation. The effect of amelogenin was exemplified by increased proliferation over 7 days. In conclusion, the beneficial effects of amelogenin on wound healing are possibly conducted by stimulating fibroblast......Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100...... signalling, proliferation and migration via integrin interactions. It is hypothesized that amelogenin stimulates wound healing by providing connective tissue cells with a temporary extracellular matrix....

  2. Effects of hydrogen peroxide in a keratinocyte-fibroblast co-culture model of wound healing.

    Science.gov (United States)

    Loo, Alvin Eng Kiat; Halliwell, Barry

    2012-06-29

    Recently, there has been renewed interest in the role of reactive oxygen species (ROS), especially H(2)O(2), in wound healing. We previously showed that H(2)O(2) stimulates healing in a keratinocyte scratch wound model. In this paper, we used a more complex and physiologically relevant model that involves co-culturing primary keratinocytes and fibroblasts. We found that the two main cell types within the skin have different sensitivities to H(2)O(2) and to the widely used "antioxidant"N-acetyl-l-cysteine (NAC). Keratinocytes were very resistant to the toxicity of H(2)O(2) (250 and 500 μM) or NAC (5 mM). However, the viability of fibroblasts was decreased by both compounds. Using the co-culture model, we also found that H(2)O(2) increases re-epithelialization while NAC retards it. Our data further illustrate the possible role of ROS in wound healing and the co-culture model should be useful for screening agents that may influence the wound healing process. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Engineering: Defining and differentiating its unique culture

    Science.gov (United States)

    Pilotte, Mary K.

    The world of work for engineering professionals is changing. At a rapid pace, experienced engineers are exiting the workforce due to retirement of the Baby Boomer generation, while at the same time the problems facing engineers are increasingly complex and frequently global in nature. For firms to protect their knowledge assets, they must ensure that acquired understandings are shared among their engineering work groups. Engineering teaching and learning in the workplace (i.e., knowledge sharing), is a social activity that resides in a social context governed by the professional engineering culture. This quantitative study uses Hofstede's Organizational Cultural Values Model (Hofstede, Neuijen, Ohayv, & Sanders, 1990) to examine dimensions of engineering culture in the workplace, producing a central tendency profile of engineering's cultural practices. Further, it explores through hypotheses if demographic differentiators, including birth generation, gender, race, industry sector of employment, and engineering discipline, play roles in forming engineering cultural practices. Results both corroborate aspects of Hofstede's model and assert new understandings relative to factors influencing dimensions of engineering practice. Outcomes are discussed in terms of their potential impact on industrial knowledge sharing and formation of beneficial engineering cultures.

  4. Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

    Science.gov (United States)

    Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

    1992-01-01

    The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

  5. Accumulation of phosphorus compounds in tissues and cultured skin fibroblasts in patients with hypophosphatasia

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Y.; Tada, Y.

    1988-08-15

    Patients with hypophosphatasia caused by a deficiency of alkaline phosphatase first showed marked accumulation of phosphoethanolamine and other phosphorus compounds in kidney and liver, while in placenta and intestine contents of these compounds were within a normal range. Furthermore, /sup 32/P-incorporation in cultured skin fibroblasts of patients with hypophosphatasia was increased about two to three times of control. FPLC chromatographic analysis also indicates that the accumulated phosphorus compounds in hypophosphatasia was smaller molecular phosphorus containing compounds. These data provide new pathophysiological aspect of hypophosphatasia.

  6. Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors.

    Science.gov (United States)

    Huang, H M; Gibson, G E

    1993-07-15

    An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

  7. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension.

    Science.gov (United States)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E; Qvortrup, Klaus; Baar, Keith; Svensson, René B; Magnusson, S Peter; Krogsgaard, Michael; Koch, Manuel; Kjaer, Michael

    2010-06-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon. (c) 2010 Elsevier Ltd. All rights reserved.

  8. Differential Regulation of a Fibroblast Growth Factor-Binding Protein during Skin Carcinogenesis and Wound Healing

    Directory of Open Access Journals (Sweden)

    Andreas Kurtz

    2004-09-01

    Full Text Available The initiation of premalignant lesions is associated with subtle cellular and gene expression changes. Here we describe a severe combined immunodeficient mouse xenograft model with human adult skin and compare chemical carcinogenesis and wound healing. We focus on a secreted binding protein for fibroblast growth factors (FGF-BP that enhances the activity of locally stored FGFs and is expressed at high levels in human epithelial cancers. Carcinogen treatment of murine skin induced papilloma within 6 weeks, whereas the human skin grafts displayed no obvious macroscopic alterations. Microscopic studies of the human skin, however, showed p53-positive keratinocytes in the epidermis, increased angiogenesis in the dermis of the treated skin, enhanced proliferation of keratinocytes in the basal layer, and an increase of FGF-BP protein and mRNA expression. In contrast, after surgical wounding of human skin grafts or of mouse skin, FGF-BP expression was upregulated within a few hours and returned to control levels after 2 days with wound closure. Enhanced motility of cultured keratinocytes and dermal fibroblasts by FGF-BP supports a role in wound healing. We conclude that adult human skin xenografts can be used to identify early molecular events during malignant transformation as well as transient changes during wound healing.

  9. Dickkopf-1 has an Inhibitory Effect on Mesenchymal Stem Cells to Fibroblast Differentiation

    Institute of Scientific and Technical Information of China (English)

    Yan Li; Sang-Sang Qiu; Yan Shao; Hong-Huan Song; Gu-Li Li; Wei Lu; Li-Mei Zhu

    2016-01-01

    Background:Mesenchymal stem cells (MSCs) are bone marrow stem cells which play an important role in tissue repair.The treatment with MSCs will be likely to aggravate the degree of fibrosis.The Wnt/β-catenin signaling pathway is involved in developmental and physiological processes,such as fibrosis.Dickkopfs (DKKs) are considered as an antagonist to block Wnt/β-catenin signaling pathway by binding the receptor of receptor-related protein (LRP5/6).DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/β-catenin signaling pathway.Methods:Stable MSCs were randomly divided into four groups:MSCs control,MSCs + transforming growth factor-β (TGF-β),MSCs + DKK1,and MSCs + TGF-β + DKK1.Flow cytometry was used to identify MSCs.Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test.Immunofluorescence was used to detect protein expression in the Wnt/β-catenin signaling pathways.Western blotting analysis was employed to test expression of fibroblast surface markers and,finally,real-time reverse transcription polymerase chain reaction was employed to test mRNA expression of fibroblast surface markers and Wnt/β-catenin signaling proteins.Results:Cultivated MSCs were found to conform to the characteristics of standard MSCs:expression of cluster of differentiation (CD) 73,90,and 105,not expression of 34,45,and 79.We found that DKK1 could maintain the normal cell morphology of MSCs.Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs + TGF-β.However,the expression was lower in the MSCs + TGF-β + DKK1.Immunofluorescence showed high expression of all Wnt/β-catnin molecules in the MSCs + TGF-β group but expressed in lower quantities in MSCs + TGF-β + DKK1 group.Finally,mRNA expression offibroblast markers vimentin,α-smooth muscle actin and Wnt/β-catenin signaling proteins β-catenin,T-cell factor,and glycogen synthase kinase-3β was

  10. The chalcone butein from Rhus verniciflua Stokes inhibits clonogenic growth of human breast cancer cells co-cultured with fibroblasts

    Directory of Open Access Journals (Sweden)

    Tan Jenny

    2005-03-01

    Full Text Available Abstract Background Butein (3,4,2',4'-tetrahydroxychalone, a plant polyphenol, is a major biologically active component of the stems of Rhus verniciflua Stokes. It has long been used as a food additive in Korea and as an herbal medicine throughout Asia. Recently, butein has been shown to suppress the functions of fibroblasts. Because fibroblasts are believed to play an important role in promoting the growth of breast cancer cells, we investigated the ability of butein to inhibit the clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts in vitro. Methods We first measured the clonogenic growth of small numbers of the UACC-812 human breast cancer cell line co-cultured on monolayers of serum-activated, human fibroblasts in the presence of butein (2 μg/mL or various other modulators of fibroblast function (troglitazone-1 μg/mL; GW9662-1 μM; meloxican-1 μM; and 3,4 dehydroproline-10 μg/mL. In a subsequent experiment, we measured the dose-response effect on the clonogenic growth of UACC-812 breast cancer cells by pre-incubating the fibroblasts with varying concentrations of butein (10 μg/ml-1.25 μg/mL. Finally, we measured the clonogenic growth of primary breast cancer cells obtained from 5 clinical specimens with normal fibroblasts and with fibroblasts that had been pre-treated with a fixed dose of butein (2.5 μg/mL. Results Of the five modulators of fibroblast function that we tested, butein was by far the most potent inhibitor of clonogenic growth of UACC-812 breast cancer cells co-cultured with fibroblasts. Pre-treatment of fibroblasts with concentrations of butein as low as 2.5 μg/mL nearly abolished subsequent clonogenic growth of UACC-812 breast cancer cells co-cultured with the fibroblasts. A similar dose of butein had no effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Significantly, clonogenic growth of the primary breast cancer cells was also

  11. Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Karlinsky, J.B.; Goldstein, R.H. (Boston Univ. School of Medicine, MA (USA))

    1989-08-01

    Prostaglandin E2 (PGE2) has been shown to increase the synthesis of hyaluronic acid in cultured fibroblasts by increasing the activity of hyaluronate synthetase, a group of plasma membrane-bound synthetic enzymes. We examined whether PGE2 also increased the activity of those enzyme systems involved in the synthesis of sulfated glycosaminoglycan in the human embryonic lung fibroblast. Exposure of cells to PGE2 resulted in dose-dependent increases in glucosamine incorporation into all sulfated glycosaminoglycan subtypes. PGE2 at 10(-7) mol/L increased total glycosaminoglycan per dish to 21.6 +/- 3.1 micrograms versus 12.0 +/- 2.5 micrograms in control untreated cultures. Stimulation of endogenous PGE2 production by bradykinin had a similar effect on glycosaminoglycan synthesis. To examine whether PGE2 affected sulfated glycosaminoglycan protein core production, cells were labeled with tritiated glucosamine in the presence of cycloheximide. Under these conditions, incorporation of radiolabel into all glycosaminoglycan subtypes was reduced. However, when exogenous sulfated glycosaminoglycan chain initiator (p-nitrophenyl beta-D-xyloside) was added, incorporation of tritiated glucosamine into sulfated glycosaminoglycan increased but not to levels found in control cultures. Application of PGE2 to cultures treated with cycloheximide alone, or to cultures treated with cycloheximide plus xyloside, increased tritiated glucosamine incorporation into chondroitin, dermatan sulfate, and to a lesser extent into heparan sulfate. We conclude that PGE2 stimulates synthesis of all sulfated glycosaminoglycan even in the absence of new protein core production, probably by increasing activities of sulfated glycosaminoglycan synthetase enzymes. PGE2 stimulation of heparan sulfate synthesis is partially dependent on the availability of heparan sulfate-specific protein core.

  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.

    Science.gov (United States)

    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.

  13. Gingival fibroblast responsiveness is differentially affected by Porphyromonas gingivalis: implications for the pathogenesis of periodontitis

    NARCIS (Netherlands)

    Scheres, N.; Crielaard, W.

    2013-01-01

    In periodontitis, tissue damage results mainly from aberrant host responses to oral microorganisms. Fibroblasts can play an important role in this. Gingival fibroblasts do not develop tolerance against the lipopolysaccharide of Porphyromonas gingivalis, a keystone pathogen in periodontitis, which ma

  14. Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation.

    Science.gov (United States)

    Zhao, Haotian; Yang, Tianyu; Madakashira, Bhavani P; Thiels, Cornelius A; Bechtle, Chad A; Garcia, Claudia M; Zhang, Huiming; Yu, Kai; Ornitz, David M; Beebe, David C; Robinson, Michael L

    2008-06-15

    The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27(kip1) and p57(kip2), increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and alpha-, beta- and gamma-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.

  15. Effects of mibefradil on intracellular Ca2+ release in cultured rat cardiac fibroblasts and human platelets.

    Science.gov (United States)

    Eberhard, M; Miyagawa, K; Hermsmeyer, K; Erne, P

    1995-12-01

    The Ca2+ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca2+ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K+ depolarization and Ca2+ channel block by verapamil or other Ca2+ antagonists. At concentrations above 10 microM, mibefradil elevated substantially cytosolic [Ca2+] without affecting the peak level of agonist-induced Ca2+ transients. These Ca2+-mobilizing actions of 10 or 100 microM mibefradil stand in contrast to the Ca2+ antagonism and relaxation of vascular muscle at 1 microM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca2+ was independent of extracellular Ca2+, and in order to define better the mechanism of Ca2+ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 microM activated dose-dependent Ca2+ release from intracellular Ca2+ stores. Verapamil had no effect at concentrations of up to 100 microM. Mibefradil-induced Ca2+ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca2+ - nor disulfide reagent-induced Ca2+ release were involved and that mibefradil did not release Ca2+ by inhibition of the Ca2+-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca2+ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca2+ release. Depletion of Ca2+ stores of permeabilized cells inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca2+ release, and depletion of Ca2+ stores by mibefradil prevented further Ca2+ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (> or

  16. Comparative effects of posterior eye cup tissues from myopic and hyperopic chick eyes on cultured scleral fibroblasts.

    Science.gov (United States)

    Christian, Parul G; Harkin, Damien G; Rayner, Cassie; Schmid, Katrina L

    2013-02-01

    The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post-hatch day 5-8) (n = 6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9 ± 2.6 μg and 8.4 ± 1.1 μg, p = 0.12; GAG 11.2 ± 0.6 μg and 10.1 ± 1.0 μg, p = 0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2 ± 1.7 μg versus 23.2 ± 1.5 μg, p = 0.21; GAG 28.1 ± 1.7 μg versus. 28.7 ± 1.2 μg, p = 0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA LIM 46.9 ± 6.4 versus LIH 51.5 ± 4.7 μg, p = 0.31). In contrast, scleral fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA

  17. Ciliated cultured dermal fibroblasts in a patient with hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome.

    Science.gov (United States)

    Haust, M D

    1995-10-01

    Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH)-syndrome is a rare autosomal recessive disorder of the urea cycle, probably caused by a defect in ornithine transport across the hepatic inner mitochondrial membrane. Single rudimentary cilia were present in approximately ten percent of post-divisional or dividing fibroblasts cultured from the skin of a patient with the HHH-syndrome, whereas no such organelles were observed in dermal fibroblasts cultured from normal controls. Since single rudimentary ("primary," "oligo," "solitary") cilia have been observed in a variety of cells in animals and men but the stimuli for their formation and their significance remain controversial, a brief report on their presence in the as yet unreported condition (HHH-syndrome) was considered of interest; hopefully, it might contribute to the ultimate unravelling of some of the unresolved problems. It is of note that unlike the author's previous findings of these unusual organelles in cells affected by a pathological process (atherosclerosis), the rudimentary cilia were observed in the present instance in dividing or postdivisional cells. The implications of these (and other) observations must await further work.

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

    Directory of Open Access Journals (Sweden)

    Kevin Dzobo

    2016-08-01

    Full Text Available 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.

  19. Content of Androgen Receptor in Cultured Genital Skin Fibroblast From Different Ages of Chinese Normal Men

    Institute of Scientific and Technical Information of China (English)

    卢建; 何立敏; 张金山; 杨震; 周云

    1995-01-01

    A ratpid, simple, reliable method is described for assaying androgen receptor (AR) in dispersed, whole, cultured human genital skin fibroblasts (GSF) with a synthetic androgen, 3H-methyltrienolone (3H-R1881). Receptors for androgen in GSF exhiblt high affinity (Kd=3.0±0.1 nmol/L), low binding capacity and androgen specificity. The content of AR in cultured GSF from 40 normal men varying in age from 1.5—60 years u:as also investigated by this assay. Scatchard analysis and slngle plot revealed the presence of 4.500-8500 binding sites per cell, mean number of AR in GSF of these men is 6288±1082 binding sites/cell. No significant difference was observed in the content of AR in different age groups. This result showed that the content of AR in these ceils did not change with age.

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

    Science.gov (United States)

    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.

  1. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

    OpenAIRE

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-01-01

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single c...

  2. Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

    Science.gov (United States)

    Benjamin, Aimee L; Green, Benjamin B; Crooker, Brian A; McKay, Stephanie D; Kerr, David E

    2016-03-24

    We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis. The Holstein breed displayed a more robust response to LPS than the Angus breed based on RNA-Seq analysis of cultures challenged with LPS for 0, 2, and 8 h. Several immune-associated genes were expressed at greater levels (FDR Angus fibroblasts, and two of these regions fell within the promoter region (-2500 to +500 bp of the transcription start site) of the genes NTRK2 and ADAMTS5. Fibroblasts isolated from Holstein cattle display a more robust response to LPS in comparison to cultures from Angus cattle. Different selection strategies and management practices exist between these two breeds that likely give rise to genetic and epigenetic factors contributing to the different immune response phenotypes.

  3. Isolation, culture, and purification of olfactory mucosa-derived olfactory ensheathing cells using modified differential attachment with low concentration serum

    Institute of Scientific and Technical Information of China (English)

    Huaqing Yang; Qiang Li; Kunzheng Wang; Bin Wang; Hui Qiang; Wei Wang; Jianxiang Yao

    2008-01-01

    BACKGROUND: Studies have demonstrated that olfactory mucosa can promote the regeneration and formation of axonal medullary sheath of injured neurons. To date, olfactory ensheathing cells (OECs) utilized in basic and clinical research arise primarily from the olfactory bulb mucosa. However, little is known regarding culture, purification, and biological properties of OECs.OBJECTIVE: To isolate and culture OECs utilized modified, differential attachment in combination with neurotrophic factor 3 (NT3) and low concentration serum to explore an optimal in vitro culture method for OECs.DESIGN, TIME AND SETFING: Single-sample observation was performed at the Medical Experimental Center of Stomatology College, Xi'an Jiaotong University between March 2006 and December 2007.MATERIALS: Twelve samples from aborted embryos, 4-6 months, were used to isolate OECs;rabbit-anti-human p75NIR and glial fibrillary acidic protein (GFAP) antibody were provided by Sigma, USA. METHODS: The differential time was six hours. This was repeated twice, based on Nash's differential attachment. Attached OECs were cultured in DMEM-F12 culture medium containing 10% fetal bovine serum (FBS) or 2.5% FBS and NT3.MAIN OUTCOME MEASURES: OEC morphology was observed, and p75NTR and GFAP immunocytochcmistry was used for identification and purity detection. RESULTS: Some cells attached after three days in culture. Several cells possessed short neurites with good refractivity. Some shuttle-shaped fibroblasts could be seen. On day six, more cells attached, exhibiting a three-dimensional appearance. Many cells appeared dipolar or tripolar, with slender neurites, and fibroblasts were clustered. On day nine, the number of dipolar or tripolar cell bodies with slender neurites was increased,and fibroblasts were clustered. On day 15, fibroblasts occupied the majority of the bottom of the culture bottle, with several OECs surviving at the upper layer. OECs were positive for P75NTR and GFAP expression,as identified by

  4. Fibroblasts isolated from human middle turbinate mucosa cause neural progenitor cells to differentiate into glial lineage cells.

    Directory of Open Access Journals (Sweden)

    Xingjia Wu

    Full Text Available Transplantation of olfactory ensheathing cells (OECs is a potential therapy for repair of spinal cord injury (SCI. Autologous transplantation of OECs has been reported in clinical trials. However, it is still controversial whether purified OECs or olfactory mucosa containing OECs, fibroblasts and other cells should be used for transplantation. OECs and fibroblasts were isolated from olfactory mucosa of the middle turbinate from seven patients. The percentage of OECs with p75(NTR+ and GFAP(+ ranged from 9.2% to 73.2%. Fibroblasts were purified and co-cultured with normal human neural progenitors (NHNPs. Based on immunocytochemical labeling, NHNPs were induced into glial lineage cells when they were co-cultured with the mucosal fibroblasts. These results demonstrate that OECs can be isolated from the mucosa of the middle turbinate bone as well as from the dorsal nasal septum and superior turbinates, which are the typical sites for harvesting OECs. Transplantation of olfactory mucosa containing fibroblasts into the central nervous system (CNS needs to be further investigated before translation to clinical application.

  5. Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

    Science.gov (United States)

    Singh, Mahipal; Sharma, Anil K

    2011-02-01

    Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

  6. Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species.

    Science.gov (United States)

    Mestre-Citrinovitz, Ana Cecilia; Sestelo, Adrián Jorge; Ceballos, María Belén; Barañao, José Lino; Saragüeta, Patricia

    2016-10-10

    Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult-to-find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar. The fibroblasts obtained are a part of the Genetic Bank of Buenos Aires Zoo with the 6700 samples, including tissues such as muscle, ovarian, testicular, blood, fibroblast cultures, sperm, hair, and fluids and cells from 450 individuals of 87 different species. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  7. Differential Expression of Matrix Metalloproteases in Human Fibroblasts with Different Origins

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    Diana Lindner

    2012-01-01

    Full Text Available Fibroblasts are widely distributed cells and are responsible for the deposition of extracellular matrix (ECM components but also secrete ECM-degrading matrix metalloproteases. A finely balanced equilibrium between deposition and degradation of ECM is essential for structural integrity of tissues. In the past, fibroblasts have typically been understood as a uniform cell population with comparable functions regardless of their origin. Here, we determined growth curves of fibroblasts derived from heart, skin, and lung and clearly show the lowest proliferation rate for cardiac fibroblasts. Furthermore, we examined basal expression levels of collagen and different MMPs in these three types of fibroblasts and compared these concerning their site of origin. Interestingly, we found major differences in basal mRNA expression especially for MMP1 and MMP3. Moreover, we treated fibroblasts with TNF-α and observed different alterations under these proinflammatory conditions. In conclusion, fibroblasts show different properties in proliferation and MMP expression regarding their originated tissue.

  8. Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

    Science.gov (United States)

    Wei, Qinzhi; Huang, Haiyan; Yang, Linqing; Yuan, Jianhui; Yang, Xiaohua; Liu, Yungang; Zhuang, Zhixiong

    2014-04-01

    Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type. Copyright © 2012 Wiley Periodicals, Inc.

  9. Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen.

    Science.gov (United States)

    Skibinski, G; Skibinska, A; Stewart, G D; James, K

    1998-12-01

    Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-mu stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blaststromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.

  10. Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Crimi Nunzio

    2006-09-01

    Full Text Available Abstract Rationale Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition. Methods We investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC in 5 primary lung fibroblast lines derived from normal subjects (NF and from 3 histologically proven IPF patients (FF. Results Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF. Conclusion These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

  11. In Vitro Wound Healing Improvement by Low-Level Laser Therapy Application in Cultured Gingival Fibroblasts

    Directory of Open Access Journals (Sweden)

    Fernanda G. Basso

    2012-01-01

    Full Text Available The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory effects on human gingival fibroblast culture. Cells (3×104 cells/cm2 were seeded on 24-well acrylic plates using plain DMEM supplemented with 10% fetal bovine serum. After 48-hour incubation with 5% CO2 at 37°C, cells were irradiated with a InGaAsP diode laser prototype (LASERTable; 780±3 nm; 40 mW with energy doses of 0.5, 1.5, 3, 5, and 7 J/cm2. Cells were irradiated every 24 h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT assay and the two most effective doses (0.5 and 3 J/cm2 were selected to evaluate the cell number (trypan blue assay and the cell migration capacity (wound healing assay; transwell migration assay. Data were analyzed by the Kruskal-Wallis and Mann-Whitney nonparametric tests with statistical significance of 5%. Irradiation of the fibroblasts with 0.5 and 3 J/cm2 resulted in significant increase in cell metabolism compared with the nonrradiated group (P<0.05. Both energy doses promoted significant increase in the cell number as well as in cell migration (P<0.05. These results demonstrate that, under the tested conditions, LLLT promoted biostimulation of fibroblasts in vitro.

  12. Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

    Directory of Open Access Journals (Sweden)

    Hui-Xian Wang

    2017-07-01

    Full Text Available AIM: To explore the possibility of human umbilical cord mesenchymal stem cells (hUCMSCs, human umbilical vein endothelial cells (hUVECs, human dental pulp stem cells (hDPSCs and human periodontal ligament stem cells (hPDLSCs serving as feeder cells in co-culture systems for the cultivation of limbal stem cells. METHODS: Different feeder layers were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12 and were treated with mitomycin C. Rabbits limbal stem cells (LSCs were co-cultured on hUCMSCs, hUVECs, hDPSCs, hPDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency (CFE assay and immunofluorescence (IPO13,CK3/12. RESULTS: The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But hUCMSCs, hDPSCs and hPDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to hUVECs and feeder-cell-free culture. CONCLUSION: hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.

  13. Bifunctional Effect of Human IFN-γ on Cultured Human Fibroblasts from Tenon‘s Capsule

    Institute of Scientific and Technical Information of China (English)

    YanGuo; JianGe; 等

    2002-01-01

    Purpose:To study the effect of human IFN-γ on in ivtro cultured human fibroblasts from Tenon's capsule.Materials and methods:The effect of different concentrations of human IFN-γand mitomycin-C (MMC),5-fluorouracil(5-Fu) on cultured human Tenon's capsule fibroblasts(HTCF) was measured using a MTT[3-(4,5-dimethylthiazo-2-yI)]-2,5-diphenylterazolium bromide;Thiazolyl blue) colorimetric assay.The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0 version.The difference was considered to be significant if P<0.05.Results:The effects of MMC and 5-Fu on the growth of HTCF were negative,while the effects of IFN-γon the growth of HTCF were both negative(102-104 units/ml in two experiments)and positive(106,105,10 units /ml in two experiments).The inhibition rate of MMC ranged from 5.73% to 46.9% ,which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92%(P=0.351).The inhibition rate of IFN-γ in two experiments was smaller than MMC and 5-Fu (P<0.05).Conclusion: IFN-γ has bifunctional effect (both enhancement and inhibition)on proliferation of cultured HTCF.The antiproliferative effect of IFN-γ was weaker than MMC and 5-Fu.Further study has to be carried out to document theinhibition of scar formation of filtration bleb by IFN-γ and the molecular mechanisms of its bifunctional effect on HTCF proliferation.Eye Science 2000;16:43-47.

  14. Bifunctional Effect of Human IFN-γon Cultured Human Fibroblasts from Tenon's Capsule

    Institute of Scientific and Technical Information of China (English)

    Yan Guo; Jian Ge; Haiquan Liu; Yanyan Li; Jianliang Zheng; Xiangkun Huang; Yuqing Lan

    2000-01-01

    Purpose: To study the effect of human IFN-γ on in vitro cultured human fibroblasts from Tenon's capsuleMaterials and methods: The effect of different concentrations of human IFN-γ and mitomycin-C (MMC), 5-fluorouracil (5-Fu) on cultured human Tenon's capsule fibroblasts (HTCF) was measured using a MIT [3-(4, 5-dimethylthiazo-2-yl)] -2,5-diphenyltetrazolium bromide; Thiazolyl blue) colorimetric assay. The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0version. The difference was considered to be significant if P < 0. 05.Results: The effects of MMC and 5-Fu on the growth of HTCF were negative, while the effects of IFN-γ on the growth of HTCF were both negative (102 ~ l04 units/ml in two experiments) and positive (106, 105, 10 units/ml in two experiments) . The inhibition rate of MMC ranged from 5.73% to 46. 9%, which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92% ( P= 0. 351) . The inhibition rate of IFN-γ in two experiments was smaller than MMC and 5-Fu ( P < 0.05).Conclusion: IFN-γ has bifunctional effect (both enhancement and inhibition) on proliferation of cultured HTCF. The antiproliferative effect of IFN-γ was weaker than MMC and 5-Fu. Further study has to be carried out to document the inhibition of scar formation of filtration bleb by IFN-γ and the molecular mechanisms of its bifunctional effect on HTCF proliferation. Eye Science 2000; 16: 43~ 47.

  15. Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines.

    Science.gov (United States)

    Kono, Kiyomi; Maeda, Hidefumi; Fujii, Shinsuke; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Monnouchi, Satoshi; Teramatsu, Yoko; Hamano, Sayuri; Koori, Katsuaki; Akamine, Akifumi

    2013-05-01

    Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

  16. Differential Bystander Signaling Between Radioresistant Chondrosarcoma Cells and Fibroblasts After X-Ray, Proton, Iron Ion and Carbon Ion Exposures

    Energy Technology Data Exchange (ETDEWEB)

    Wakatsuki, Masaru, E-mail: wa@mbe.nifty.com [Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts (United States); Magpayo, Nicole; Kawamura, Hidemasa; Held, Kathryn D. [Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts (United States)

    2012-09-01

    Purpose: Chondrosarcoma is well known as a radioresistant tumor, but the mechanisms underlying that resistance are still unclear. The bystander effect is well documented in the field of radiation biology. We investigated the bystander response induced by X-rays, protons, carbon ions, and iron ions in chondrosarcoma cells using a transwell insert co-culture system that precludes physical contact between targeted and bystander cells. Methods and Materials: Human chondrosarcoma cells were irradiated with 0.1-, 0.5-, 1-, and 2-Gy X-rays, protons, carbon ions or iron ions using a transwell insert co-culture system. Formation of micronuclei and p53 binding protein 1 staining in bystander and irradiated cells were analyzed and bystander signaling between mixed cultures of chondrosarcoma cells, and normal human skin fibroblasts was investigated. Results: In this study, we show that the fraction of cells with DNA damages in irradiated chondrosarcoma cells showed dose-dependent increases with all beams. However, the fraction of cells with DNA damages in all bystander chondrosarcoma cells did not show any change from the levels in control cells. In the bystander signaling between mixed cultures of chondrosarcoma cells and fibroblasts, the amount of micronucleus formation in all bystander chondrosarcoma cells co-cultured with irradiated fibroblasts were the same as the levels for control cells. However, all bystander fibroblasts co-cultured with irradiated chondrosarcoma cells showed significant increases in the fraction of micronucleated cells compared to the rate of control cells. Conclusions: We conclude that chondrosarcoma cells in the transwell insert co-culture system could release bystander stimulations but could not develop bystander responses.

  17. Metalloproteinase 11, potential marker and molecular target in advanced and castration-resistant prostate cancer. Culture study of peritumoral fibroblasts.

    Science.gov (United States)

    Fernandez-Gomez, J M; Eiro, N; García-Rodríguez, J J; Quintás-Blanco, A; Gonzalez-Ruiz de León, C; Perez de Haro, M L; Vizoso-Piñero, F

    To analyze the expression of metalloprotein 11 (MMP11) in cultured fibroblasts obtained from human prostate tumors with different clinical and pathological characteristics. For this study we analyzed samples of transrectal prostate biopsies from tumors with different characteristics, treated with or whithout androgen deprivation (AD). After optimization of the culture method, fibroblasts were isolated and cultured to perform the study (PCR) of MMP11 mRNA. Finally, 37 cases were studied: 5 samples of benign prostatic hyperplasia, 14 cases with localized neoplasms (7 high-risk according to the D'Amico classification), 5 with metastasic tumors (bone metastases), and 13 treated with AD therapy, of which 6 fulfilled the requirements to be defined as resistant to castration. In tumors without AD therapy, MMP11 expression was significantly higher (P=.001) in fibroblasts of higher grade tumors. A significant (P=.001) correlation was found between PSA and expression of MMP11 in fibroblast s and a significant increase of MMP11 expression in metastatic tumors. In tumors with AD therapy, a significantly greater expression of MMP11 was observed in resistant to castration patients than in those sensitive to castration (P=.003). In advanced prostate tumors or in stages of increased tumor aggressiveness, the production of MMP11 by fibroblasts is significantly greater than in non-metastatic tumors or in AD sensitive tumors. Copyright © 2016 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  18. 人成纤维细胞的原代培养及鉴定%Primary culture and identification of human fibroblasts

    Institute of Scientific and Technical Information of China (English)

    鲍荣辉; 刘先哲; 周军

    2011-01-01

    目的 建立不同组织来源成纤维细胞的培养方法并比较其培养差异.方法 用消化法和贴壁法分离人胚胸主动脉成纤维细胞与人胚肺成纤维细胞,利用差速培养法纯化成纤维细胞,并对所培养的细胞进行倒置显微镜观察和苏木素-伊红染色,观察成纤维细胞形态,并对培养的细胞行波形蛋白免疫染色鉴定.结果 接种24h后人胚胸主动脉成纤维细胞全部贴壁,48h后细胞体积增大并伸出伪足,形状以梭形或不规则形为主;而消化的人胚肺组织中细胞形态不一,经继续培养4~5代后,培养物完全由成纤维细胞构成.结论 用胶原酶I消化人胚胸主动脉成纤维细胞效果好,而用胰蛋白酶液消化人胚肺成纤维细胞效果较好,用差速生长纯化的成纤维细胞可用于实验研究.%Objective To set up the method of the culture of human fibroblasts from different sources and compare their discrepancy. Methods Human embryonic thoracic aortic fibroblasts and embryonic lung fibroblasts were isolated by enzyme digestion and adherence,and purified by means of differential attachment technique. The morphology and phenotype characterization of cultured fibroblasts was inspected by inverted microscope after hematoxylin-eosin staining and the cells were identified by immunocytochemical staining with vimentin. Results The human embryonic thoracic aortic fibroblasts were all adhere to the surface of petri dish after inoculating for 24 hours, volume of the cells enlarged and pseudopodia whose main shapes were clostridial form or irregular stretched out after inoculating for 48 hours. The shapes of human embryonic lung fibroblasts which were isolated by enzyme digestion were differ,and the culture was all composed of fibroblasts after subculturing for 4-5 generations. Conclusion The effect of the method which isolate human embryonic thoracic aortic fibroblasts by collagen I is good. The effect of the method which isolate embryonic

  19. Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Che, Xiajing; Wang, Qin; Xie, Yuanyuan; Xu, Weijia; Shao, Xinghua; Mou, Shan, E-mail: shan_mou@126.com; Ni, Zhaohui, E-mail: doctor_nzh@126.com

    2015-09-04

    Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production in a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.

  20. Stage-specific effects of fibroblast growth factor 2 on the differentiation of dental pulp cells.

    Science.gov (United States)

    Sagomonyants, Karen; Mina, Mina

    2014-01-01

    Dentinogenesis is a complex and multistep process, which is regulated by various growth factors, including members of the fibroblast growth factor (FGF) family. Both positive and negative effects of FGFs on dentinogenesis have been reported, but the underlying mechanisms of these conflicting results are still unclear. To gain a better insight into the role of FGF2 in dentinogenesis, we used dental pulp cells from various transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast differentiation. Our results showed that the continuous exposure of pulp cells to FGF2 inhibited mineralization and revealed both the stimulatory and inhibitory effects of FGF2 on the expression of markers of dentinogenesis and various transgenes. During the proliferation phase of in vitro growth, FGF2 increased the expression of markers of dentinogenesis and the percentages of dentin matrix protein 1/green fluorescent protein (DMP1-GFP)-positive functional odontoblasts and dentin sialophosphoprotein (DSPP)-Cerulean-positive odontoblasts. Additional exposure to FGF2 during the differentiation/mineralization phase of in vitro growth decreased the extent of mineralization and the expression of markers of dentinogenesis and of the DMP1-GFP and DSPP-Cerulean transgenes. Recovery experiments showed that the inhibitory effects of FGF2 on dentinogenesis were related to the blocking of the differentiation of cells into mature odontoblasts. These observations together showed the stage-specific effects of FGF2 on dentinogenesis by dental pulp cells, and they provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration.

  1. Culture of skin-derived precursors and their differentiation into neurons

    Institute of Scientific and Technical Information of China (English)

    杨立业; 郑佳坤; 刘相名; 惠国桢; 郭礼和

    2004-01-01

    Objective:To investigate the culture method of skin- derived precursors ( SKPs ) and to explore a new cell source for cell transplantation of central nervous system. Methods: Cells from skins of juvenile and adult mice were isolated and cultured in serum-free medium.A mechanical method was chosen to passage these cells and they were idemified by the immunocytochemistry assay. Results: SKPs could be isolated from adult and neonatal skins. They could be maintained in vitro for long periods with stable proliferation, and expanded as undifferentiated cells in culture for more than 12 passages. About 50% of SKPs expressed nestin and majority of these cells expressed fibronectin when they were plated on polyornithine and laminin coated plates. About 5% cells showed neuronal differentiation and expressed neurofilament-M (NF-M) and NSE when SKPs were plated in serun-containing medium, and these cells could also differentiate into adipocytes and fibroblast-like cells.Conclusions: The data support the hypothesis that adult skin contains stem cells capable of differentiating into neurons, adipocytes, and fibroblast-like cells. They may represent an alternative autologous stem cell source for CNS cell transplantation.

  2. A novel tumor-suppressor candidate gene-ndr2 is differentially expressed between osteoarthritis synovium cells and rheumatoid arthritis synovium fibroblasts

    Institute of Scientific and Technical Information of China (English)

    DENG Yan-chun; WANG Ji-cun; LIU Xin-ping; YAO Li-bo

    2004-01-01

    To test whether the novel tumor-suppressor candidate gene-ndr2 is also differentially expressed between osteoarthritis synovium cells (OASC) and rheumatoid arthritis synovium fibroblasts (RASF), and whether ndr2 can suppress the growth of RASF in vitro. Methods: Dot blotting, cell culture and gene transfection, cell cycle nalysis techniques were applied to investigate the effect of ndr2 on the cell phenotype and cell cycles. Results: ndr2 is expressed in OASC but absent in RASF. Transient transfection of ndr2 into RASF can suppress the growth of RASF from phenotype observation. Cell cycle analysis showed that apoptotic peaks can be detected in RASF cells transfected with ndr2 gene. Conclusion: Novel tumor suppressor candidate ndr2 is not only differentially expressed between OASC and RASF but also can induce the apoptosis of RASF in vitro.

  3. In Vitro Effects of Pirfenidone on Cardiac Fibroblasts: Proliferation, Myofibroblast Differentiation, Migration and Cytokine Secretion

    OpenAIRE

    Qiang Shi; Xiaoyan Liu; Yuanyuan Bai; Chuanjue Cui; Jun Li; Yishi Li; Shengshou Hu; Yingjie Wei

    2011-01-01

    Cardiac fibroblasts (CFs) are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remode...

  4. Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea.

    Science.gov (United States)

    Zhang, Yan Qing; Zhang, Wen Jie; Liu, Wei; Hu, Xiao Jie; Zhou, Guang Dong; Cui, Lei; Cao, Yilin

    2008-02-01

    Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.

  5. Differential expression of wound fibrotic factors between facial and trunk dermal fibroblasts.

    Science.gov (United States)

    Kurita, Masakazu; Okazaki, Mutsumi; Kaminishi-Tanikawa, Akiko; Niikura, Mamoru; Takushima, Akihiko; Harii, Kiyonori

    2012-01-01

    Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibroblasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts.

  6. Fibroblasts weaken the anti-tumor effect of gefitinib on co-cultured non-small cell lung cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Xiao; Wang Peiqin; Jiang Tao; Yu Wenchen; Shang Yan; Han Yiping; Zhang Pingping

    2014-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common lung malignancy worldwide.The metastatic potential of NSCLC cells has been shown to be associated with the tumor microenvironment,which consists of tumor cells,stroma,blood vessels,immune infiltrates and the extracellular matrix.Fibroblasts can produce numerous extraceilular matrix molecules and growth factors.Gefitinib has been evaluated as a first-line treatment in selected patients,and it has shown favorable efficacy especially in NSCLC,but it is not effective for everyone.Methods In this study,we examined the antitumor activity of gefitinib on lung fibroblasts co-cultured of lung cancer cells.A series of co-culture experiments that employed cell counting kit-8 (CCK8),transwells,real-time polymerase chain reaction (RT-PCR) and Western blotting with HFL-1 fibroblasts and A549 human lung carcinoma cells were performed to learn more about tumor cell proliferation,migration and invasion; and to determine any change of epithelial mesenchymal transition (EMT)-associated tumor markers vimentin,matrix metallopro-teinase 2 (MMP2) and chemotaxis cytokines receptor 4 (CXCR4) mRNA levels.Results A549 cell proliferation in the presence of HFL-1 cells was not significantly increased compared with A549 cells alone,but A549 cell spheroid body formation was increased after co-culture,and treatment with gefitinib increased further.Our study also revealed that fibroblasts attenuated the lung cancer cell inhibition ratio of migration and invasion after gefitinib treatment in vitro.To further study this mechanism,RT-PCR analysis showed that vimentin,MMP2 and CXCR4 mRNA levels were more highly expressed in the lung cancer cells after co-culture,but did not obviously decrease compared with the control cells following gefitinib treatment.This suggests the mechanism by which fibroblasts attenuate gefitinib-induced expression of EMT-associated tumor markers.Finally,our results demonstrated that co-culture with A549 lung

  7. Adhesion and morphology of fibroblastic cells cultured on different polymeric biomaterials.

    Science.gov (United States)

    Lombello, C B; Santos, A R; Malmonge, S M; Barbanti, S H; Wada, M L F; Duek, E A R

    2002-09-01

    Cell adhesion is influenced by the physical and chemical characteristics of the materials used as substrate for cell culturing. In this work, we evaluated the influence of the morphological and chemical characteristics of different polymeric substrates on the adhesion and morphology of fibroblastic cells. Cell growth on poly (L-lactic acid) [PLLA] membranes and poly(2-hydroxy ethyl methacrylate) [polyHEMA], poly(2-hydroxy ethyl methacrylate)-cellulose acetate [polyHEMA-CA] and poly(2-hydroxy ethyl methacrylate)-poly(methyl methacrylate-co-acrylic acid) [polyHEMA-poly(MMA-co-AA)] hydrogels of different densities and pore diameters was examined. Cells adhered preferentially to more negatively charged substrates, with polyHEMA hydrogels being more adhesive than the other substractes. The pores present in PLLA membranes did not interfere with adhesion, but the cells showed a distinctive morphology on each membrane.

  8. Adult human brain neural progenitor cells (NPCs and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

    Directory of Open Access Journals (Sweden)

    Thomas In-Hyeup Park

    Full Text Available The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia, and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs. These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting Ah

  9. Expression of the familial Mediterranean fever gene and activity of the C5a inhibitor in human primary fibroblast cultures.

    Science.gov (United States)

    Matzner, Y; Abedat, S; Shapiro, E; Eisenberg, S; Bar-Gil-Shitrit, A; Stepensky, P; Calco, S; Azar, Y; Urieli-Shoval, S

    2000-07-15

    Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)-8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression of MEFV and C5a/IL-8-inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8-inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1 beta. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1 beta. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8-inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation. (Blood. 2000;96:727-731)

  10. Poly[tri(ethylene glycol) ethyl ether methacrylate]-coated surfaces for controlled fibroblasts culturing.

    Science.gov (United States)

    Dworak, Andrzej; Utrata-Wesołek, Alicja; Szweda, Dawid; Kowalczuk, Agnieszka; Trzebicka, Barbara; Anioł, Jacek; Sieroń, Aleksander L; Klama-Baryła, Agnieszka; Kawecki, Marek

    2013-03-01

    Well-defined thermosensitive poly[tri(ethylene glycol) monoethyl ether methacrylate] (P(TEGMA-EE)) brushes were synthesized on a solid substrate by the surface-initiated atom transfer radical polymerization of TEGMA-EE. The polymerization reaction was initiated by 2-bromo-2-methylpropionate groups immobilized on the surface of the wafers. The changes in the surface composition, morphology, philicity, and thickness that occurred at each step of wafer functionalization confirmed that all surface modification procedures were successful. Both the successful modification of the surface and bonding of the P(TEGMA-EE) layer were confirmed by X-ray photoelectron spectroscopy (XPS) measurements. The thickness of the obtained P(TEGMA-EE) layers increased with increasing polymerization time. The increase of environmental temperature above the cloud point temperature of P(TEGMA-EE) caused the changes of surface philicity. A simultaneous decrease in the polymer layer thickness confirmed the thermosensitive properties of these P(TEGMA-EE) layers. The thermosensitive polymer surfaces obtained were evaluated for the growth and harvesting of human fibroblasts (basic skin cells). At 37 °C, seeded cells adhered to and spread well onto the P(TEGMA-EE)-coated surfaces. A confluent cell sheet was formed within 24 h of cell culture. Lowering the temperature to an optimal value of 17.5 °C (below the cloud point temperature of the polymer, TCP, in cell culture medium) led to the separation of the fibroblast sheet from the polymer layer. These promising results indicate that the surfaces produced may successfully be used as substrate for engineering of skin tissue, especially for delivering cell sheets in the treatment of burns and slow-healing wounds.

  11. Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

    Science.gov (United States)

    Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

    2015-07-01

    It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

  12. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Science.gov (United States)

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  13. Effect of hexavalent chromium on proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts.

    Science.gov (United States)

    Martini, Claudia N; Brandani, Javier N; Gabrielli, Matías; Vila, María del C

    2014-06-01

    Heavy metals contamination has become an important risk factor for public health and the environment. Chromium is a frequent industrial contaminant and is also used in orthopaedic joint replacements made from cobalt-chromium-alloy. Since hexavalent chromium (Cr(VI)) was reported as genotoxic and carcinogenic in different mammals, to further evaluate its cytotoxicity, we investigated the effect of this heavy metal in the proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts. These cells, after the addition of a mixture containing insulin, dexamethasone and methylisobutylxanthine, first proliferate, a process known as mitotic clonal expansion (MCE), and then differentiate to adipocytes. In this differentiation process a key transcription factor is induced: peroxisome proliferator-activated receptor gamma (PPAR gamma). We found that treatment of 3T3-L1 fibroblasts with potassium chromate inhibited proliferation in exponentially growing cells and MCE as well as differentiation. A decrease in PPAR gamma content, evaluated by western blot and immunofluorescence, was found in cells differentiated in the presence of chromium. On the other hand, after inhibition of differentiation with chromium, when the metal was removed, differentiation was recovered, which indicates that this may be a reversible effect. We also found an increase in the number of micronucleated cells after treatment with Cr(VI) which is associated with genotoxic effects. According to our results, Cr(VI) is able to inhibit proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts and to increase micronucleated cells, which are all indicative of alterations in cellular physiology and therefore, contributes to further elucidate the cytotoxic effects of this heavy metal. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. In vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and produce a fibrotic-like environment upon stimulation with TGF-β1: Is there a thin line between fetal scarless healing and fibrosis?

    Science.gov (United States)

    Walraven, M; Akershoek, J J; Beelen, R H J; Ulrich, M M W

    2017-03-01

    Transforming growth factor-β (TGF-β) is a cytokine occurring in three isoforms with an important function in development and wound healing. In wound healing, prolonged TGF-β signaling results in myofibroblast differentiation and fibrosis. In contrast, the developing second-trimester fetal skin contains high levels of all three TGF-β isoforms but still has the intrinsic capacity to heal without scarring. Insight into TGF-β signal transduction during fetal wound healing might lead to methods to control the signaling pathway during adult wound healing. In this study, we imitated wound healing in vitro by stimulating fibroblasts with TGF-β1 and examining myofibroblast differentiation. The aim was to gain insight into TGF-β signaling in human fibroblasts from fetal and adult dermis. First, TGF-β1 stimulation resulted in similar or even more severe upregulation of myofibroblast-associated genes in fetal fibroblasts compared to adult fibroblasts. Second, fetal fibroblasts also had higher protein levels of myofibroblast-marker α-smooth muscle actin (α-SMA). Third, stimulated fetal fibroblasts in collagen matrices had higher protein levels of α-SMA, produced more of the fibrotic protein fibronectin splice-variant extra domain A (FnEDA), and showed enhanced contraction. Finally, fetal fibroblasts also produced significant higher levels of TGF-β1. Altogether, these data show that in vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and do produce a fibrotic environment. As healthy fetal skin has high levels of TGF-β1, FnEDA, and collagen-III as well, these findings correlate with the in vivo situation. Therefore, our study demonstrates that there are similarities between fetal skin development and fibrosis and shows the necessity to discriminate between these processes.

  15. Electrochemical properties of 316L stainless steel with culturing L929 fibroblasts.

    Science.gov (United States)

    Hiromoto, Sachiko; Hanawa, Takao

    2006-08-22

    Potentiodynamic polarization and impedance tests were carried out on 316L stainless steel with culturing murine fibroblast L929 cells to elucidate the corrosion behaviour of 316L steel with L929 cells and to understand the electrochemical interface between 316L steel and cells, respectively. Potential step test was carried out on 316L steel with type I collagen coating and culturing L929 cells to compare the effects of collagen and L929 cells. The open-circuit potential of 316L steel slightly shifted in a negative manner and passive current density increased with cells, indicating a decrease in the protective ability of passive oxide film. The pitting potential decreased with cells, indicating a decrease in the pitting corrosion resistance. In addition, a decrease in diffusivity at the interface was indicated from the decrease in the cathodic current density and the increase in the diffusion resistance parameter in the impedance test. The anodic peak current in the potential step test decreased with cells and collagen. Consequently, the corrosion resistance of 316L steel decreases with L929 cells. In addition, collagen coating would provide an environment for anodic reaction similar to that with culturing cells.

  16. Effect of salicyclic acid on gluccorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma.

    Science.gov (United States)

    Koshihara, Y; Yamagishi, M; Murota, S I

    1976-06-23

    The binding activity of [3H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [3H]dexamethasone of 3.64 - 10(-8) M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 - 10(5). Total binding activity to [3H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid was at 37 degrees C. The maximum enhancement was seen at the concentration of 10(-3)M and in 3h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicyclic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin,also enhanced the total binding activity to [3H]dexamethasone of the cytoplasmic fraction at the concentration of 2 - 10(-5) M and 2 - 10(-7) M, respectively.

  17. Classical Galactosemia: Insight into Molecular Pathomechanisms by Differential Membrane Proteomics of Fibroblasts under Galactose Stress.

    Science.gov (United States)

    Staubach, Simon; Müller, Stefan; Pekmez, Murat; Hanisch, Franz-Georg

    2017-02-03

    Classical galactosemia, a hereditary metabolic disease caused by the deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), results in an impaired galactose metabolism and serious long-term developmental affection of the CNS and ovaries, potentially related in part to endogenous galactose-induced protein dysglycosylation. In search for galactose-induced changes in membrane raft proteomes of GALT-deficient cells, we performed differential analyses of lipid rafts from patient-derived (Q) and sex- and age-matched control fibroblasts (H) in the presence or absence of the stressor. Label-based proteomics revealed of the total 454 (female) or 678 (male) proteins a proportion of ∼12% in at least one of four relevant ratios as fold-changed. GALT(-) cell-specific effects in the absence of stressor revealed cell-model-dependent affection of biological processes related to protein targeting to the plasma membrane (female) or to cellular migration (male). However, a series of common galactose-induced effects were observed, among them the strongly increased ER-stress marker GRP78 and calreticulin involved in N-glycoprotein quality control. The membrane-anchored N-glycoprotein receptor CD109 was concertedly decreased under galactose-stress together with cadherin-13, GLIPR1, glypican-1, and semaphorin-7A. A series of proteins showed opposite fold-changes in the two cell models, whereas others fluctuated in only one of the two models.

  18. The effect of exposure duration of self etch dentin bonding on the toxicity of human gingival fibroblast of cell culture

    Directory of Open Access Journals (Sweden)

    Sri Lestari

    2008-06-01

    Full Text Available Self etch dentin bonding created to make light easily activate the application of composite resin on tooth surface. The monomer content has acid effect that could irritate tooth pulp. The purpose of this study was to evaluate the effect of light exposure duration of self etch dentin bonding on toxicity of human gingival fibroblast of cell culture by MTT assay. Self etch dentin bonding was used as on experimental unit and the sample was exposed by visible light curing in different duration: 10, 20, 30 seconds and immerged in artificial saliva in pH 7 for 24 hours. 100 µl artificial saliva was exposed to human gingival fibroblast of cell culture 20.000 cells/100 µl RPMI for 24 hours. Toxicity was evaluated by MTT assay, optical density was measured using 550 nm spectrophotometer. The data was analyzed using Kruskal Wallis in 5% degree of significance. The result showed that increasing exposure duration (10, 20, 30 seconds of self etch dentin bonding will reduce the toxicity of human gingival fibroblast of cell culture. It is concluded that 30 seconds-exposure of self etch dentin bonding will reduce the toxicity of human gingival fibroblast of cell culture.

  19. Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels.

    Science.gov (United States)

    Nagano, Takashi; Hao, Ji-Long; Nakamura, Masatsugu; Nishida, Teruo

    2002-08-01

    To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.

  20. Pengaruh Cairan Cultur Filtrate Fibroblast (CFF Terhadap Penyembuhan Luka; Penelitian eksperimental pada Rattus Norvegicus Galur Wistar

    Directory of Open Access Journals (Sweden)

    Oky Masir

    2012-11-01

    Full Text Available AbstrakLatar belakang:Metode penyembuhan luka telah mengalami perkembangan, baik berupa suatu produk atau stimulan terhadap proses biologis tubuh dalam menkompensasi luka. Fibroblas merupakan salah satu komponen penyembuhan yang berperan penting dalam proses fibroplasia. Culture Filtrate Fibroblast (CFF merupakan hasil kultur fibroblas yang akan dibuktikan efeknya terhadap proses percepatan penyembuhan luka pada penelitian ini. Metode. Penelitian ini menggunakan desain eksperimental dengan metode post test only control group design dan rancangan acak kelompok (RAK dengan menggunakan tikus putih wistar. Hewan coba dibagi menjadi 4 kelompok, yaitu 2 kelompok perlakuan yang diberikan CFF ke area eksisi luka dan kelompok kontrol yang diberikan larutan NaCl 0,9% ke area eksisi luka. Data diolah dengan SPSS 16.0. Data Kategori dianalisa dengan Chi-squared dan data numerik dengan Independent T-test. Hasil. Dari tingkat penyembuhan tidak ditemukan perbedaan pada kedua kelompok, namun perubahan restriksi jaringan lebih besar pada kelompok perlakuan. Pada skor pembentukan kolagen, derajat epitelisasi serta jumlah pembentukan pembuluh darah baru pada hari ke-3 tidak ditemukan perbedaan antara kedua kelompok. Namun pada pengamatan hari ke-7 memperlihatkan pembentukan kolagen, derajat epitelisasi serta jumlah pembentukan pembuluh darah baru lebih banyak pada kelompok perlakuan. Pada fibrosis hari ke-3 dan hari ke-7 memperlihatkan terjadinya fibrosis lebih banyak pada kelompok perlakuan dibanding kontrol. Pada pengamatan terjadinya infeksi hari ke-3 memperlihatkan infeksi lebih sedikit pada kelompok perlakuan dan terjadinya infeksi sama pada hari ke-7. Kesimpulan. CFF memberikan tingkat penyembuhan luka yang lebih baik dibanding NaCl.Kata kunci: CFF, NaCl 0,9 %, tingkat penyembuhan luka.Abstract Background: Wound healing methods have been developed, either a product or a stimulant to the body's biological processes in wound compensation. Fibroblasts is one

  1. Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts

    NARCIS (Netherlands)

    Lundvig, D.M.S.; Pennings, S.W.C.; Brouwer, K.M.; Mtaya-Mlangwa, M.; Mugonzibwa, E.A.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den; Wagener, F.A.D.T.G.

    2015-01-01

    Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-beta1 play a key role in these processes. The persistence of (myo)fibroblasts and their exces

  2. Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts

    NARCIS (Netherlands)

    Lundvig, D.M.S.; Pennings, S.W.C.; Brouwer, K.M.; Mtaya-Mlangwa, M.; Mugonzibwa, E.A.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den; Wagener, F.A.D.T.G.

    2015-01-01

    Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-beta1 play a key role in these processes. The persistence of (myo)fibroblasts and their

  3. CULTURES OF FIBROBLAST-LIKE SYNOVIAL CELLS FROM PATIENTS WITH RHEUMATOID ARTHRITIS: PROPERTIES AND OPPORTUNITIES

    Directory of Open Access Journals (Sweden)

    M. A. Schneider

    2016-01-01

    Full Text Available The present review contains data from literature concerning the in vivo structure of synovial membranes in healthy people and patients with rheumatoid arthritis (RA. The properties of in vitro cultured fibroblast-like synovial cells (FLS from RA patients are considered, including FLS morphology, phenotype and function. A standard protocol of in vitro FLS culturing is described. Notably, the FLS are characterized by autonomic functioning, ability for invasive growth/migration, e.g., into non-affected joints. These FLS properties may a reason of multiple joint involvement typical to RA. Special attention is drawn to characterization of stable phenotypic profile of FLS which results from certain epigenetic disturbances, i.e., changes of the DNA methylation, histone acetylation, and micro-RNA effects.The FLS from RA patients are characterized with stable and extensive hypomethylation of genes which occurs in vivo and persists after repeated culture passages. Some promoters of genes involved into RA pathogenesis (for example, CXCL12, IL-6 are hypomethylated. By contrary, some other gene promoters (e.g., the death receptor 3 gene are shown to be hypermethylated. An increased histone acetylation of genes encoding proinflammatory mediators (such as MMP1 may be an important mechanism of persistent inflammation in RA. Changes in histone acetylation in FLS are related to high levels of ubiquitin-like SUMO-1 protein and concurrent decrease in specific protease SENP1activity. A role of histone acetylation in RA pathogenesis is supported by efficacy of a histone deacetylase inhibitor (Trichostatin A in collagen-induced murine arthritis. Local concentrations of micro RNA-155, micro-RNA-146а, and micro-RNA-203 are permanently increased in FLS cultures, synovial tissues, and PBMC of the RA patients. Expression of micro RNA-124а is decreased in FLS from RA, as compared with OA FLS.One may conclude that the fibroblast-like synovial cells are key cellular

  4. Differential Gene Expression Profiling of Enriched Human Spermatogonia after Short- and Long-Term Culture

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    Sabine Conrad

    2014-01-01

    Full Text Available This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks and long-term culture (up to more than 14 months in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen−/laminin+ matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the “spermatogonial” gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future.

  5. Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

    Science.gov (United States)

    Conte, Enrico; Gili, Elisa; Fagone, Evelina; Fruciano, Mary; Iemmolo, Maria; Vancheri, Carlo

    2014-07-16

    Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.

  6. Programming of Intestinal Epithelial Differentiation by IL-33 Derived from Pericryptal Fibroblasts in Response to Systemic Infection

    Directory of Open Access Journals (Sweden)

    Mousumi Mahapatro

    2016-05-01

    Full Text Available The intestinal epithelium constitutes an efficient barrier against the microbial flora. Here, we demonstrate an unexpected function of IL-33 as a regulator of epithelial barrier functions. Mice lacking IL-33 showed decreased Paneth cell numbers and lethal systemic infection in response to Salmonella typhimurium. IL-33 was produced upon microbial challenge by a distinct population of pericryptal fibroblasts neighboring the intestinal stem cell niche. IL-33 programmed the differentiation of epithelial progenitors toward secretory IEC including Paneth and goblet cells. Finally, IL-33 suppressed Notch signaling in epithelial cells and induced expression of transcription factors governing differentiation into secretory IEC. In summary, we demonstrate that gut pericryptal fibroblasts release IL-33 to translate bacterial infection into an epithelial response to promote antimicrobial defense.

  7. Cardiac fibroblasts regulate sympathetic nerve sprouting and neurocardiac synapse stability.

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    Céline Mias

    Full Text Available Sympathetic nervous system (SNS plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of

  8. Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

    Science.gov (United States)

    Mias, Céline; Coatrieux, Christelle; Denis, Colette; Genet, Gaël; Seguelas, Marie-Hélène; Laplace, Nathalie; Rouzaud-Laborde, Charlotte; Calise, Denis; Parini, Angelo; Cussac, Daniel; Pathak, Atul; Sénard, Jean-Michel; Galés, Céline

    2013-01-01

    Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic

  9. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  10. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  11. Endothelin receptor antagonists: effects on extracellular matrix synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients

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    B. Villaggio

    2012-12-01

    Full Text Available Endothelin-1 (ET-1 seems to enhance the pro-fibrotic protein synthesis by skin fibroblasts and its effects are mediated by endothelin-A and B (ETA and ETB receptors. This study aimed to investigate the effects of ETA and ETB receptor antagonists (ETARA-sitaxentan and ETA/BRA-bosentan on type I collagen (COL-1, fibronectin (FN and fibrillin-1 (FBL-1 synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients. Primary cultures of fibroblasts were obtained from skin biopsies of 6 female systemic sclerosis patients and were treated with ET-1 (100 nM for 24 and 48 hrs with or without pre-treatment (1 hr with ETARA (2 μM or ETA/BRA (10 μM. Primary culture of human scleroderma skin fibroblasts not treated with ET-1 or ET receptor antagonists (ETARA and ETA/BRA were used as controls. COL-1, FN and FBL-1 synthesis was evaluated by immunocytochemistry and Western blot analysis. Immunocytochemistry and Western blot analysis showed that ET-1 significantly increased COL-1 and FN synthesis at 24 and 48 hrs and FBL-1 synthesis at 48 hrs vs untreated cells. ETARA significantly contrasted the ET-1-mediated increase in COL-1 and FN at 24 hrs as well as COL-1 and FBL-1 at 48 hrs, but not FN synthesis vs ET-1-treated fibroblasts. Conversely, ETA/BRA significantly antagonized the ET-1-mediated overproduction of COL-1 and FN both at 24 and 48 hrs and the FBL-1 synthesis at 48 hrs vs ET-1-treated cells. The single ETARA treatment seems to contrast significantly the increase in COL-1 synthesis, whereas the dual ETA/BRA treatment seems active in significantly antagonizing both COL-1 and FN overproduction induced by ET-1. In conclusion, ET-1 antagonism might have positive effects in contrasting the profibrotic activity of systemic sclerosis skin fibroblasts.

  12. Metallomics approach to changes in element concentration during differentiation from fibroblasts into adipocytes by element array analysis.

    Science.gov (United States)

    Ogra, Yasumitsu; Nagasaki, Shu; Yawata, Ayako; Anan, Yasumi; Hamada, Koichi; Mizutani, Akihiro

    2016-04-01

    We aimed to establish an element array analysis that involves the simultaneous detection of all elements in cells and the display of changes in element concentration before and after a cellular event. In this study, we demonstrated changes in element concentration during the differentiation of 3T3-L1 mouse fibroblasts into adipocytes. This metallomics approach yielded unique information of cellular response to physiological and toxicological events.

  13. MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

    Science.gov (United States)

    Wu, Honglu; Lu, Tao; Jeevarajan, John; Rohde, Larry; Zhang, Ye

    2014-01-01

    Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. 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. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

  14. Lunasin Inhibits Cell Proliferation via Apoptosis and Reduces the Production of Proinflammatory Cytokines in Cultured Rheumatoid Arthritis Synovial Fibroblasts

    Directory of Open Access Journals (Sweden)

    Shaohui Jia

    2015-01-01

    Full Text Available Lunasin, a peptide with 43 amino acid residues and initially isolated and identified in soybean cotyledon, has gained extensive attention due to its anti-inflammatory and anticancer properties. However, its treatment efficacy on rheumatoid arthritis (RA and corresponding mechanisms have not been reported. Herein, the synovial fibroblasts harvested and isolated from patients with RA were treated with lunasin at various concentrations to examine the proliferation, apoptosis status, and corresponding cell cycle of cultured RA synovial fibroblasts. Meanwhile, the underlying mechanisms of lunasin for RA treatment are explored through Western blot, real-time PCR, ELISA, and luciferase reporter assays. Lunasin significantly inhibited the proliferation and induced the apoptosis of cultured RA synovial fibroblasts. In addition, lunasin reduced the production of interleukin-6 (IL-6, IL-8, and matrix metalloproteinase-3 (MMP-3 and suppressed the activation of NF-κB in cultured RA synovial fibroblasts but did not reveal obvious modulation on the secretion and gene expression of MMP-1. Therefore, lunasin will have promising potential as a novel nutritional supplement or drug candidate for RA due to its potency of suppressing synovial cell proliferation and decreasing the production of proinflammatory cytokines and MMPs in synovial cells.

  15. Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon.

    Science.gov (United States)

    Pfefferkorn, E R; Guyre, P M

    1984-01-01

    The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma interferon at concentrations of 8 to 16 U/ml. The interferon was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma interferon for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma interferon were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma interferon had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma interferon had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% 3 days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant alpha and beta interferons had no effect on the growth of T. gondii. Images PMID:6425215

  16. Elasticity Modulation of Fibroblast-Derived Matrix for Endothelial Cell Vascular Morphogenesis and Mesenchymal Stem Cell Differentiation.

    Science.gov (United States)

    Du, Ping; Suhaeri, Muhammad; Subbiah, Ramesh; Van, Se Young; Park, Jimin; Kim, Sang Heon; Park, Kwideok; Lee, Kangwon

    2016-03-01

    Biophysical properties of the microenvironment, including matrix elasticity and topography, are known to affect various cell behaviors; however, the specific role of each factor is unclear. In this study, fibroblast-derived matrix (FDM) was used as cell culture substrate and physically modified to investigate the influence of its biophysical property changes on human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) behavior in vitro. These FDMs were physically modified by simply storing them at different temperatures: the one stored at 4°C, maintained its original properties, was considered natural FDM, whereas the ones stored at -20°C or -80°C, exhibited a distinct surface morphology, were considered physically modified FDM. Physical modification induced matrix fiber rearrangement in FDM, forming different microstructures on the surface as characterized by focused ion beam (FIB)-cryoSEM. A significant increase of matrix elasticity was found with physically modified FDMs as determined by atomic force microscopy. HUVEC and hMSC behaviors on these natural and physically modified FDMs were observed and compared with each other and with gelatin-coated coverslips. HUVECs showed a similar adhesion level on these substrates at 3 h, but exhibited different proliferation rates and morphologies at 24 h; HUVECs on natural FDM proliferated relatively slower and assembled to capillary-like structures (CLSs). It is observed that HUVECs assembled to CLSs on natural FDMs are independent on the exogenous growth factors and yet dependent on nonmuscle myosin II activity. This result indicates the important role of matrix mechanical properties in regulating HUVECs vascular morphogenesis. As for hMSCs multilineage differentiation, adipogenesis is improved on natural FDM that with lower matrix elasticity, while osteogenesis is accelerated on physically modified FDMs that with higher matrix elasticity, these results further confirm the crucial

  17. Regulation and inhibition of collagenase expression by long-wavelength ultraviolet radiation in cultured human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, Marta; Hamilton, Tiffani; Haili Li [Utah Univ., Salt Lake City, UT (United States). Dept. of Internal Medicine

    1995-09-01

    The cellular mechanisms responsible for the connective tissue changes produced by chronic exposure to UV light are poorly understood. collagenase, a metalloproteinase, initiates degradation of types I and III collagen and thus plays a key role in the remodeling of dermal collagen. Collagenase synthesis by fibroblasts and keratinocytes involves the protein kinase C (PKC) second messenger system, and corticosteroids have been shown to suppress its synthesis at the level of gene transcription. Long-wavelength UV light (UVA, 320-400 nm) stimulates the synthesis of interstitial collagenase, as well as increasing PKC activity, in human skin fibroblasts in vitro. This study explores the regulation of collagenase expression by UVA in cultured human skin fibroblasts. Specifically, the time course, the effect of actinomycin D, an inhibitor of RNA synthesis, as well as the effect of PKC inhibitors and dexamethansone on expression of collagenase following UVA irradiation were examined. (Author).

  18. AGEs and Glucose Levels Modulate Type I and III Procollagen mRNA Synthesis in Dermal Fibroblasts Cells Culture

    Directory of Open Access Journals (Sweden)

    Serban Iren Andreea

    2008-01-01

    Full Text Available In the dermis, fibroblasts play an important role in the turnover of the dermal extracellular matrix. Collagen I and III, the most important dermal proteins of the extracellular matrix, are progressively altered during ageing and diabetes. For mimicking diabetic conditions, the cultured human dermal fibroblasts were incubated with increasing amounts of AGE-modified BSA and D-glucose for 24 hours. The expression of procollagen α2(I and procollagen α1(III mRNA was analyzed by quantitative real-time PCR. Our data revealed that the treatment of fibroblasts with AGE-modified BSA upregulated the expression of procollagen α2(I and procollagen α1(III mRNA in a dose-dependent manner. High glucose levels mildly induced a profibrogenic pattern, increasing the procollagen α2(I mRNA expression whereas there was a downregulation tendency of procollagen α1(III mRNA.

  19. Bifunctional bioceramics stimulating osteogenic differentiation of a gingival fibroblast and inhibiting plaque biofilm formation.

    Science.gov (United States)

    Shen, Ya; Wang, Zhejun; Wang, Jiao; Zhou, Yinghong; Chen, Hui; Wu, Chengtie; Haapasalo, Markus

    2016-04-01

    Gingival recession is a common clinical problem that results in esthetic deficiencies and poor plaque control and predominantly occurs in aged patients. In order to restore the cervical region, ideal biomaterials should possess the ability to stimulate proliferation and osteogenesis/cementogenesis of human gingival fibroblasts (HGF) and have a strong antibiofilm effect. The aim of the present study was to investigate the interactions of HGF and oral multispecies biofilms with Ca, Mg and Si-containing bredigite (BRT, Ca7MgSi4O16) bioceramics. BRT extract induced osteogenic/cementogenic differentiation of HGF and its inhibition of plaque biofilm formation were systematically studied. BRT extract in concentrations lower than <200 mg mL(-1) presented high biocompatibility to HGF cells in 3 days. Ion extracts from BRT also stimulated a series of bone-related gene and protein expressions in HGF cells. Furthermore, BRT extract significantly inhibited oral multispecies plaque biofilm growth on its surface and contributed to over 30% bacterial cell death without additional antibacterial agents in two weeks. A planktonic killing test showed that BRT suppressed 98% plaque bacterial growth compared to blank control in 3 days. The results also revealed that BRT extract has an osteostimulation effect on HGF. The suppression effect on plaque biofilms suggested that BRT might be used as a bioactive material for cervical restoration and that the synergistic effect of bioactive ions, such as Ca, Mg and Si ions, played an important role in the design and construction of bifunctional biomaterials in combination with tissue regeneration and antibiofilm activity.

  20. Aislamiento y cultivo de fibroblastos endoneurales Endoneural fibroblasts isolation and culture

    Directory of Open Access Journals (Sweden)

    Spinel Clara

    2004-12-01

    time remodel tissues that are being repaired. Dermal fibroblasts are well studied both in vitro and in vivo, and are used to regenerate dermal EMC which in turn supports the regeneration of the epidermis. Confluence of dermal or periodontal fibroblasts takes place between 8 and 10 days of culture. In the process of regeneration of damaged peripheral nerves, Schwann's cells secrete neurotrophic and neurotropic growth factors and some of the EMC elements needed for regeneration to take place, which makes them the most studied and used cells in culture. So far, endoneural fibroblasts (EF have not been considered as important elements in nerve regeneration, mainly because they may occasionally form fibromes that hinder regeneration. But there is evidence that they may play a role in the remodeling of the EMC, through the secretion of metalloproteins that modify the pre-Nerve Growth Factor (preNGF secreted by Schwann's cells into active NGF, which promotes neurites regeneration. The aim of this study was is to isolate EF from sciatic nerves taken from mature rats, and to obtain them in purified culture. A number of methods of dissection and digestion were developed to obtain primary pure EF cultures as well as to study them in the way Schwann's cells have been studied. Selective isolation of EF was accomplished, reaching confluence between the fourth and the fifth day in monolayer primary culture. Producing a population of EF will make it possible to carry out studies in tridimensional culture and in prosthesis in order to define and develop new alternatives for the regeneration of peripheral nerves.

  1. Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

    Institute of Scientific and Technical Information of China (English)

    LiGU; Yuan-jueZHU; Zi-jianGUO; Xing-xiangXU; Wen-bingXU

    2004-01-01

    AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-β1 and the role of interferon (IFN)-γ, dexamethasone (DEX) in the fibroblast-myofibroblast differentiation. METHODS: α-Smooth muscle actin (α-SMA), Smad2/3, and Smad7 protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline, α-SMA and collagen III mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-β1, IFN-γ 200 μg/L markedly blocked TGF-β1-induced α-SMA protein expression (PO.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-β1-induced α-SMA expression (P<0.01). IFN-γ 200 μg/L did not block TGF-β1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-131 induced fibroblastmyofibroblast differentiation in a Smad proteins-dependent manner. IFN-γ could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism withTGF-β1. Differentiated myofibroblasts, however, were resistant to both IFN-γ and DEX.

  2. 21 CFR 866.2320 - Differential culture medium.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Differential culture medium. 866.2320 Section 866... medium. (a) Identification. A differential culture medium is a device that consists primarily of liquid... biochemical component(s) to the medium. Microorganisms are identified by a visible change (e.g., a color...

  3. Influence of the fatty acid composition of high-density lipoprotein phospholipids on the cholesterol efflux from cultured fibroblasts.

    Science.gov (United States)

    Esteva, O; Baudet, M F; Lasserre, M; Jacotot, B

    1986-02-12

    The purpose of this work was to determine whether the changes induced by dietary manipulations in the chemical composition of high-density lipoproteins (HDL) (particularly phospholipid fatty acid composition) modified their capacity to promote [3H]cholesterol efflux from cultured fibroblasts. Plasma HDL were obtained from subjects fed for six successive long periods on diets consisting of one predominant fat: peanut oil, corn oil, olive oil, soybean oil, low erucic acid rapeseed oil or milk fats. The [3H]cholesterol efflux from cells in the presence of plasma HDL was studied by means of normal adult human fibroblasts in culture. The [3H]cholesterol efflux from fibroblasts appeared to be independent of the overall composition of HDL and of the degree of saturation of the HDL phospholipid fatty acids, but it was correlated with the phospholipid fatty acid chain length. The [3H]cholesterol efflux from fibroblasts is highly and positively correlated with the sum of the HDL phospholipid C20, C22, C24 fatty acids, and negatively correlated with the sum of the HDL phospholipid C18 fatty acids.

  4. Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

    Science.gov (United States)

    Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

    2003-01-01

    In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

  5. Detection and identification of cancerous murine fibroblasts, transformed by murine sarcoma virus in culture, using Raman spectroscopy and advanced statistical methods.

    Science.gov (United States)

    Salman, A; Shufan, E; Zeiri, L; Huleihel, M

    2013-03-01

    Cancer is one of the leading worldwide causes of death. It may be induced by a variety of factors, including carcinogens, radiation, genetic factors, or DNA and RNA viruses. The early detection of cancer is critical for its successful therapy, which can result in complete recovery from some types of cancer. Raman spectroscopy has been widely used in medicine and biology. It is a noninvasive, nondestructive, and water-insensitive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer. In this study, Raman spectroscopy was used for the identification and characterization of murine fibroblast cell lines (NIH/3T3) and malignant fibroblast cells transformed by murine sarcoma virus (NIH-MuSV) cells. Using principal component analysis and LDA it was possible to differentiate between the NIH/3T3 and NIH-MuSV cells with an 80-85% success rate based on their Raman shift spectra. The best results for differentiation were achieved from spectra that were obtained from the rich membrane sites. Because of its homogeneity and complete control of most factors affecting its growth, cell culture is a preferred model for the detection and identification of specific biomarkers related to cancer transformation or other cellular modifications.

  6. Transport of dibasic amino acids, cystine, and tryptophan by cultured human fibroblasts: absence of a defect in cystinuria and Hartnup disease.

    Science.gov (United States)

    Groth, U; Rosenberg, L E

    1972-08-01

    Transport of lysine, arginine, cystine, and tryptophan was studied in cultured skin fibroblasts from normal controls and from patients with cystinuria and Hartnup disease. Each of these amino acids was accumulated against concentration gradients by energy-dependent, saturable mechanisms. Lysine and arginine were each transported by two distinct processes which they shared with each other and with ornithine. In contrast, cystine was taken up by a different transport system with no demonstrable affinity for the dibasic amino acids. The time course and Michaelis-Menten kinetics of lysine and cystine uptake by cells from three cystinuric patients differed in no way from those found in control cells. Similarly, the characteristics of tryptophan uptake by cells from a child with Hartnup disease were identical to those noted in control cells. These findings indicate that the specific transport defects observed in gut and kidney in cystinuria and Hartnup disease are not expressed in cultured human fibroblasts, thus providing additional evidence of the important role that cellular differentiation plays in the regulation of expression of the human genome.

  7. Effects of bosentan on collagen type I synthesis on in vitro culture of scleroderma skin fibroblasts

    Directory of Open Access Journals (Sweden)

    S. Soldano

    2011-01-01

    Full Text Available The present study evaluated the effects of a non-selective endothelin (ETA/B receptors antagonist, on collagen type I (COLI synthesis on in vitro culture of scleroderma (SSc skin fibroblasts (Fb. Fb were obtained from skin biopsies of 6 female SSc patients (mean age 64. 1±6 years, after informed consent and Ethical Committee Approval. Cells were treated with endothelin-I [ET-I, 100nM] for 24 and 48 hrs, pre-treated for I hr with ETA/B receptors antagonist [10nM] alone or followed by ET-I for 24 and 48 hrs. Untreated Fb were used as controls. Immunocytochemistry and western blot analysis were performed to evaluate COLI synthesis. ET-I increased COLI synthesis both at 24 and 48 hrs when compared to controls. ETA/B receptor antagonost blocks the increased COLI synthesis ET-I-mediated both at 24 and 48 hrs vs. ET-I. Results showed that ET-I receptors blockage by ETA/B receptors antagonist might prevent the excessive synthesis of COLI, supporting its positive action in the management of skin fibrosis.

  8. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    Science.gov (United States)

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.

  9. Tissue culture: the unrealized potential

    OpenAIRE

    Sato, Gordon

    2007-01-01

    Lack of differentiated functions of the tissue of origin in tissue culture thought to be due to dedifferentiation was shown to be due to selective overgrowth of fibroblasts. Enrichment culture techniques, (alternate animal and culture passage), designed to give the functionally differentiated cells selective advantage over the fibroblasts resulted in a large number of functionally differentiated clonal strains. Thus the dogma of dedifferentiation was destroyed. It is proposed to substitute th...

  10. Culturing aerobic and anaerobic bacteria and mammalian cells with a microfluidic differential oxygenator.

    Science.gov (United States)

    Lam, Raymond H W; Kim, Min-Cheol; Thorsen, Todd

    2009-07-15

    In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential oxygenator. A gas-filled microchannel network functioning as an oxygen-nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated).

  11. Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three-dimensional magnetic cell culture array.

    Science.gov (United States)

    Okochi, Mina; Matsumura, Taku; Yamamoto, And Shuhei; Nakayama, Eiichi; Jimbow, Kowichi; Honda, Hiroyuki

    2013-01-01

    A three-dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force-based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array-like multicellular patterns of melanoma were developed using a pin-holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel-coated surface as array-like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct-interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect-interaction model) of melanoma spheroids, and (iii) fibroblast-sheets coexisting under melanoma spheroids (fibroblast-sheet model). The fibroblast-sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast-sheet model, the expression of IL-8 and MMP-2 increased by 24-fold and 2-fold, respectively, in real time RT-PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment.

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

    Science.gov (United States)

    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. Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates

    NARCIS (Netherlands)

    Ruiz-Zapata, A.M.; Kerkhof, M.H.; Zandieh-Doulabi, B.; Brölmann, H.A.M.; Smit, T.H.; Helder, M.N.

    2013-01-01

    Introduction and hypothesis Little is known about dynamic cell-matrix interactions in the context of pathophysiology and treatments for pelvic organ prolapse (POP). This study sought to identify differences between fibroblasts from women with varying degrees of prolapse in reaction to mechanical sti

  14. Raman and infrared spectroscopy differentiate senescent from proliferating cells in a human dermal fibroblast 3D skin model.

    Science.gov (United States)

    Eberhardt, Katharina; Matthäus, Christian; Winter, Doreen; Wiegand, Cornelia; Hipler, Uta-Christina; Diekmann, Stephan; Popp, Jürgen

    2017-08-15

    Senescent cells contribute to tissue aging and dysfunction. Therefore, detecting senescent cells in skin is of interest for skin tumor diagnostics and therapy. Here, we studied the transition into senescence of human dermal fibroblasts (HDFs) in a three-dimensional (3D) human fibroblast-derived matrix (FDM). Senescent and proliferating cells were imaged by Raman spectroscopy (RS) and Fourier transform infrared (FTIR) spectroscopy. The obtained averaged spectra were analyzed using PLS-LDA. For these 3D cultured cells, RS and FTIR could clearly distinguish senescent from proliferating cells. For both techniques, we detected senescence-associated alterations in almost all cellular macromolecules. Furthermore, we identified different biochemical properties of 3D compared to two-dimensional (2D) cultured cells, indicating that cells in their natural, skin-like 3D environment act differently than in (2D) cell cultivations in vitro. Compared to 2D cultured cells, cells grown in 3D models displayed a sharper contrast between the proliferating and senescent state, also affecting the abundance of biomolecules including nucleic acids. The training accuracies of both vibrational spectroscopic techniques were >96%, demonstrating the suitability of these label-free measurements for detecting these cellular states in 3D skin models.

  15. FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

    Science.gov (United States)

    Joannes, Audrey; Brayer, Stéphanie; Besnard, Valérie; Marchal-Sommé, Joëlle; Jaillet, Madeleine; Mordant, Pierre; Mal, Hervé; Borie, Raphael; Crestani, Bruno; Mailleux, Arnaud A

    2016-04-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

  16. Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models.

    Directory of Open Access Journals (Sweden)

    Kyung Eun Sung

    Full Text Available The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com. We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

  17. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  18. INFLUENCE OF QUERCETIN AND X-RAY ON COLLAGEN SYNTHESIS OF CULTURED HUMAN KELOID-DERIVED FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    Xiao Long; Xuan Zeng; Fu-quan Zhang; Xiao-jun Wang

    2006-01-01

    Objective To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism.Methods Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen Ⅰ and Ⅲ expression. mRNA expression of collagen Ⅰ and Ⅲ, and transforming growth factor (TGF)-β1 were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR.Results Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent manner. Immunocytochemical staining indicated that collagen Ⅰ and Ⅲ were down-regulated by quercetin and X-ray ( P<0.05), particularly collagen Ⅰ (P<0.05). mRNA expression of both collagen Ⅰ and Ⅲ in quercetin groups significantly decreased compared with that in control group (P<0.05 ), especially in the group treated with both quercetin and X-ray( P<0.01). mRNA level of TGF-β1 gene was down-regulated by quercertin (P<0.05).Conclusions Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.

  19. PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.

    Science.gov (United States)

    Sriram, Sriniwas; Tran, Jennifer A; Guo, Xiaoqing; Hutcheon, Audrey E K; Lei, Hetian; Kazlauskas, Andrius; Zieske, James D

    2017-02-01

    The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing.

  20. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts.

    Science.gov (United States)

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-06-14

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.

  1. Age-dependent decline in mouse lung regeneration with loss of lung fibroblast clonogenicity and increased myofibroblastic differentiation.

    Directory of Open Access Journals (Sweden)

    Julia A Paxson

    Full Text Available While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days, and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a and more alpha smooth muscle actin (αSMA positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.

  2. The effect of activated alveolar macrophages on experimental lung emphysema development. II. The study of fibroblast and alveolar macrophage co-culture.

    Science.gov (United States)

    Sulkowska, M; Wołczyński, S; Sulkowski, S; Sobaniec-Lotowska, M; Chyczewski, L; Sulik, M; Kulikowski, M; Dziecioł, J; Berger, W

    1995-01-01

    The cell-cell interaction between fibroblasts and alveolar macrophages was examined using a co-culture system. Alveolar macrophages (AM) were harvested from the bronchoalveolar lavages (BAL) of rats with papain induced lung emphysema. The BCG-vaccine was applied as a macrophage mobilizing and activating agent. The morphological examinations carried out in scanning electron microscope (SEM) as well as the evaluation of the uptake of 3H-thymidine did not show any significant differences between respective co-cultures of fibroblasts and AM isolated both from the lungs of control and experimental animals (treated with BCG or papain, and BCG+papain). However, significant growth were noted in 3H-thymidine uptake between fibroblast cultures done with or without cells isolated from the lungs. The results obtained suggest that AM can promote fibroblast proliferation during the progression of experimental lung emphysema.

  3. Effect of IFN-γ and dexamethasone on TGF-β1-induced human fetal lung fibroblast-myofibroblast differentiation

    Institute of Scientific and Technical Information of China (English)

    Li GU; Yuan-jue ZHU; Zi-jian GUO; Xing-xiang XU; Wen-bing XU

    2004-01-01

    AIM: To study whether Smads signaling pathway was involved in human fetal lung fibroblast-myofibroblast differentiation induced by transforming growth factor (TGF)-β1 and the role of interferon (IFN)-γ, dexamethasone (DEX)in the fibroblast-myofibroblast differentiation. METHODS: α-Smooth muscle actin (α-SMA), Smad2/3, and Smad7protein were assessed by Western blot. Collagen protein was analyzed by measuring hydroxyproline. α-SMA and collagen Ⅲ mRNA were assessed by RT-PCR. Myofibroblasts morphology and Smad2/3 nuclear translocation were assessed by immunohistochemistry. The overexpression of Smad7, a negative mediator of Smads signaling pathway, was acquired by transfection of Smad7 vector. RESULTS: During fibroblast-myofibroblast differentiation induced by TGF-β1, IFN-γ 200 μg/L markedly blocked TGF-β1-induced α-SMA protein expression (P<0.01),collagen protein (P<0.01) and mRNA (P<0.05) expression, and myofibroblasts morphological transformation, but DEX 10 μmol/L augmented TGF-β1-induced α-SMA expression (P<0.01). For myofibroblasts, both IFN-γ 200 μg/L and DEX 10 μmol/L did not inhibit α-SMA expression (P>0.05) and collagen protein (P>0.05) and mRNA expression (P>0.05) and did not change myofibroblasts morphology. Transient transfection of Smad7 vector resulted in significant inhibition of TGF-β1-induced α-SMA expression (P<0.01). IFN-γ 200 μg/L did not block TGF-β1-stimulated Smad2/3 phosphorylation and their nuclear translocation. CONCLUSION: TGF-β1 induced fibroblastmyofibroblast differentiation in a Smad proteins-dependent manner. IFN-γ could block this process but it was not mediated by interrupting smad2/3 phosphorylation and their nuclear translocation and DEX played a synergism with TGF-β1. Differentiated myofibroblasts, however, were resistant to both IFN-γ and DEX.

  4. Neural cell adhesion molecule differentially interacts with isoforms of the fibroblast growth factor receptor.

    Science.gov (United States)

    Christensen, Claus; Berezin, Vladimir; Bock, Elisabeth

    2011-10-26

    The fibroblast growth factor receptor (FGFR) can be activated through direct interactions with various fibroblast growth factors or through a number of cell adhesion molecules, including the neural cell adhesion molecule (NCAM). We produced recombinant proteins comprising the ligand-binding immunoglobulin-like modules 2 and 3 of FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4, and found that all FGFR isoforms, except for FGFR4, interacted with NCAM. The binding affinity of NCAM-FGFR interactions was considerably higher for splice variant 'b' than for splice variant 'c'. We suggest that the expression pattern of various FGFR isoforms determines the cell context-specific effects of NCAM signaling through FGFR.

  5. Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.

    Science.gov (United States)

    Tansriratanawong, Kallapat; Tamaki, Yuichi; Ishikawa, Hiroshi; Sato, Soh

    2014-10-01

    In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.

  6. Shoot Differentiation in Callus Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1973-01-01

    promoted shoot differentiation. Gibberellic acid inhibited shoot formation weakly, but inhibited proper leaf blade formation. Root differentiation was rare. The callus cultures of Datura innoxia grew rapidly (100-fold in 4 weeks) on a slightly modified Murashige and Skoog medium (0.5 mg/l thiamin · HCl, p...

  7. Early cellular responses of BMSCs genetically modified with bFGF/BMP2 co-cultured with ligament fibroblasts in a three-dimensional model in vitro.

    Science.gov (United States)

    Li, Bin; Jha, Ramesh Kumar; Qi, Yong-Jian; Ni, Qu-Bo; Wang, Hui; Chen, Biao; Chen, Liao-Bin

    2016-11-01

    Currently, a number of strategies including the implantation of bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors have been developed to regenerate the tendon-to-bone interface after performing anterior cruciate ligament reconstruction. However, the mechanisms behind the interactions of the implanted BMSCs and tendon cells remain to be elucidated. The aim of this study was to evaluate the early cellular responses of BMSCs genetically modified with basic growth factor growth factor (bFGF)/bone morphogenic protein 2 (BMP2) and ligament fibroblasts in a three-dimensional co-culture model. BMSCs and ligament fibroblasts were both isolated from male Wistar rats. The BMSCs were then transfected with an adenoviral vector carrying bFGF or BMP2. The transfected BMSCs and ligament fibroblasts both encapsulated in alginate beads were co-cultured for 6 days in three-dimensional model. On days 0, 3 and 6, cell proliferation was assayed. On day 6, the expression of several tendon-bone related markers was evaluated. In the co-culture system, bFGF and BMP2 were highly expressed at the mRNA and protein level. During the process, bFGF significantly promoted cell proliferation, as well as the expression of scleraxis (SCX) and collagen (COL) type Ⅰ (COL1) in the BMSCs; however, it markedly decreased the expression of phenotype markers in the ligament fibroblasts, including COL1 and COL3. BMP2 markedly increased the expression of alkaline phosphatase and osteocalcin in the BMSCs and ligament fibroblasts, whereas it had no obvious effect on cell proliferation and collagen synthesis in the ligament fibroblasts. The combination of bFGF and BMP2 resulted in the similarly enhanced proliferation of BMSCs and ligament fibroblasts as observed with bFGF alone; however, this combination more potently promoted osteogenic differentiation than did BMP2 alone. The findings of our study demonstrate the superiority of the combined use of growth factors in inducing

  8. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    Energy Technology Data Exchange (ETDEWEB)

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

    1985-08-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. (/sup 14/C)Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of (/sup 14/C)proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

  9. Human ovarian neoplasm cell CD147 stimulates production and activation of matrix metalloproteinases in co-cultures with mouse fibroblasts

    Institute of Scientific and Technical Information of China (English)

    YANG Hong; ZOU Wei; XIN Xiao-yan

    2005-01-01

    Objective: To investigate the expression of CD147 on human ovarian neoplasm cell lines and its influence on production and activation of matrix metallproteinases(MMPs). Methods: The expression of CD147 on different human ovarian neoplasm cell lines was studied by western blotting. Co-culture was carried out to investigate the stimulative effect of the positive expression CD147 cell HO-8910 on the production of MMPs of fibroblast cell in vitro. Zymography and immune blotting were used to study the production and activity of positive MMPs, at the time, to explore the relation between CD147 and MMPs. Results: CD147 was positively presented in 2 ovarian neoplasm cell lines(HO-8910,3-AO), but in SKOV3, TC-1,NIN3T3 cell was negative. MMP-2 and MMP-9 were detected by HO-8910 cell line, mouse fibroblast cell and co-culture cells; but the expression in co-culture cell is obviously higher than individual cultures of each type alone.CD147 stimulated MMPs in dose-dependent manner. Conclusion: CD147 causes increased production and activation of MMP-2, MMP-9.CD147 is probably a indirect marker of some ovarian cancer cells with invasion and metastasis.

  10. Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

    Directory of Open Access Journals (Sweden)

    Gary S. L. Peh

    2012-01-01

    Full Text Available The culture of human corneal endothelial cells (CECs is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

  11. Modulation of fibroblast growth factor receptor expression and signalling during retinoic acid-induced differentiation of Tera-2 teratocarcinoma cells.

    Science.gov (United States)

    Pertovaara, L; Tienari, J; Vainikka, S; Partanen, J; Saksela, O; Lehtonen, E; Alitalo, K

    1993-02-26

    We have analyzed the regulation of fibroblast growth factor receptors (FGFRs) during retinoic acid (RA) induced differentiation of Tera-2 human embryonal carcinoma cells. Undifferentiated Tera-2 cells expressed mRNAs for all four known FGFRs. Their differentiation led to loss of FGFR-4 mRNA expression and mRNA levels for FGFR-2 and FGFR-3 were considerably downregulated, whereas the mRNA levels for FGFR-1 remained unaltered. A substantial decrease in binding of K-FGF was found to occur upon RA-induced differentiation of the cells. In undifferentiated Tera-2 cells FGF stimulation caused an increase of c-fos mRNA, and c-jun mRNAs, but no increase of junB mRNA, whereas in the differentiated cells, FGFs strongly stimulated the expression of all three genes. Thus differentiation of the Tera-2 cells leads to marked changes in FGFR gene expression as well as to complex alterations in their responses to exogenous FGFs.

  12. Dynamic behavior of fragile X full mutations in cultured female fetal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Yu-jie SUN; Xiao HAN

    2004-01-01

    AIM: To assess mitotic stability of the fragile X full mutations and its relationship with DNA methylation. METHODS:The length change of the expanded CGG repeats was examined and correlated it with the methylation status in the DNA samples isolated from the fibroblasts derived from a fragile X female fetus and a fragile X male adult, respectively.RESULTS: A dramatic instability of the expanded CGG repeats in the female fetal fibroblasts was observed. Southem blot analysis revealed that the 6.9-kb major expanded band detected in passage 2 was completely replaced by a 7.7-kb band after passage 30. Fibroblast clones derived from the passage 3 displayed an unstable expansion of the CGG repeat during clonal proliferation, while methylation status of the CGG repeat region was maintained. In contrast, in fragile X male fibroblasts the expanded CGG repeats were stable during clonal proliferation.CONCLUSION: The mitotic instability of expanded CGG repeat is not always restricted in early development window as proposed previously and other elements rather than DNA methylation could affect the stability of the expanded CGG repeats in fragile X female fetal fibroblast cells.

  13. Bone Morphogenetic Protein (BMP-4 and BMP-7 regulate differentially Transforming Growth Factor (TGF-β1 in normal human lung fibroblasts (NHLF

    Directory of Open Access Journals (Sweden)

    Lloyd Clare M

    2010-06-01

    Full Text Available Abstract Background Airway remodelling is thought to be under the control of a complex group of molecules belonging to the Transforming Growth Factor (TGF-superfamily. The Bone Morphogenetic Proteins (BMPs belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-β-induced profibrotic functions in lung fibroblasts. Methods Cell cultures were exposed to TGF-β1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of α-smooth muscle actin (α-SMA by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP activity was assessed by zymography. Results We have demonstrated TGF-β1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-β1-induced extracellular matrix protein production. TGF-β1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-β1-induced MMP-13 release compared to untreated and TGF-β1-treated cells. TGF-β1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4. Conclusions Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for

  14. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    Science.gov (United States)

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  15. Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice.

    Science.gov (United States)

    Watson, Walter H; Burke, Tom J; Zelko, Igor N; Torres-González, Edilson; Ritzenthaler, Jeffrey D; Roman, Jesse

    2016-01-01

    Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as EhCySS. Cultured cells condition their media to reproduce physiological EhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the media EhCySS before and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellular EhCySS than young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

  16. Culture of proliferating and differentiating fat-storing cells in 3T3-conditioned medium.

    Science.gov (United States)

    Mendoza-Figueroa, T; Argüello, C; Kuri-Harcuch, W

    1988-01-01

    There is growing evidence suggesting that hepatic fat-storing cells (FSC) or Ito cells have an important function in vitamin A storage and metabolism and in the synthesis of connective tissue components in normal liver and during fibrogenesis. The purified FSC acquire a fibroblastic morphology and their vitamin A content decreases in culture. We cultivated cells under in vitro conditions that allowed the expression of FSC morphological and functional characteristics for 3-4 weeks of primary culture. Cells were isolated from rat liver by the collagenase-perfusion method without further purification and cultured with 3T3-conditioned medium, which seemed to stimulate the selective proliferation of the FSC. After 8-10 days, round and stellate cells grew actively from a few precursor cells in the primary culture and were not subcultivated; the stellate cells had the ability to become round and vice versa and were highly motile. The cells had intracytoplasmic lipid droplets, a well developed rough endoplasmic reticulum, Golgi complex, numerous vesicles filled with electron-dense material, and extracellular matrix (ECM) components on their surface. Both stellate and round cells showed the presence of desmin by immunofluorescence and vitamin A autofluorescence, but lacked peroxidase activity. The culture conditions we describe allowed the selective proliferation of cells with morphological and functional characteristics of the FSC in the normal liver, raising the possibility of studying FSC proliferation and differentiation.

  17. Growth Differentiation Factor-9 Promotes Fibroblast Proliferation and Migration in Keloids through the Smad2/3 Pathway

    Directory of Open Access Journals (Sweden)

    Zhaohua Jiang

    2016-11-01

    Full Text Available Background: Keloids are fibroproliferative scars that develop as a result of a dysregulated wound healing process; however, the molecular mechanisms of keloid pathogenesis remain unclear. Keloids are characterized by the ability to spread beyond the original boundary of the wound, and they represent a significant clinical challenge. Previous work from our group suggested that growth differentiation factor (GDF-9 plays a role in the invasive behavior of keloids. Here, we examined the involvement of GDF-9 in keloid formation and spread and elucidated a potential underlying mechanism. Methods: The expression of GDF-9, cyclooxygenase (COX-2, vascular epidermal growth factor (VEGF-C, matrix metalloprotease (MMP-2, MMP-9, transforming growth factor (TGF-β1, and the related signaling pathway components in human keloid tissues or keloid fibroblasts (kFBs was monitored by qRT-PCR and western blot. A series of overexpression and silencing experiments in normal and keloid fibroblasts were used to modify the expression of GDF-9. The effects of GDF-9 on kFB proliferation and migration were assessed using the CCK-8, cell cycle and scratch wound healing assays. Results: GDF-9 promotes fibroblast proliferation and migration. GDF-9 silencing in kFBs decreased cell proliferation, blocked cell cycle progression, downregulated the angiogenic markers COX-2 and VEGF-C, and downregulated MMP-2 and MMP-9 expression, whereas it had no effect on the levels of TGF-β1. GDF-9 silencing significantly inhibited Smad2 and Smad3 phosphorylation in kFBs. Conclusions: GDF-9 promotes the proliferation and migration of kFBs via a mechanism involving the Smad2/3 pathway.

  18. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  19. Haemophilus ducreyi hemolysin acts as a contact cytotoxin and damages human foreskin fibroblasts in cell culture.

    OpenAIRE

    Alfa, M J; DeGagne, P; Totten, P A

    1996-01-01

    Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, produces several factors that damage human cells. We used isogenic mutants of H. ducreyi 35000 to demonstrate that the hemolytic activity and the cytotoxic effect of H. ducreyi on human foreskin fibroblasts are due to the same toxin.

  20. Longitudinal study of mammary epithelial and fibroblast co-cultures using optical coherence tomography reveals morphological hallmarks of pre-malignancy.

    Directory of Open Access Journals (Sweden)

    Raghav K Chhetri

    Full Text Available The human mammary gland is a complex and heterogeneous organ, where the interactions between mammary epithelial cells (MEC and stromal fibroblasts are known to regulate normal biology and tumorigenesis. We aimed to longitudinally evaluate morphology and size of organoids in 3D co-cultures of normal (MCF10A or pre-malignant (MCF10DCIS.com MEC and hTERT-immortalized fibroblasts from reduction mammoplasty (RMF. This co-culture model, based on an isogenic panel of cell lines, can yield insights to understand breast cancer progression. However, 3D cultures pose challenges for quantitative assessment and imaging, especially when the goal is to measure the same organoid structures over time. Using optical coherence tomography (OCT as a non-invasive method to longitudinally quantify morphological changes, we found that OCT provides excellent visualization of MEC-fibroblast co-cultures as they form ductal acini and remodel over time. Different concentrations of fibroblasts and MEC reflecting reported physiological ratios [1] were evaluated, and we found that larger, hollower, and more aspherical acini were formed only by pre-malignant MEC (MCF10DCIS.com in the presence of fibroblasts, whereas in comparable conditions, normal MEC (MCF10A acini remained smaller and less aspherical. The ratio of fibroblast to MEC was also influential in determining organoid phenotypes, with higher concentrations of fibroblasts producing more aspherical structures in MCF10DCIS.com. These findings suggest that stromal-epithelial interactions between fibroblasts and MEC can be modeled in vitro, with OCT imaging as a convenient means of assaying time dependent changes, with the potential for yielding important biological insights about the differences between benign and pre-malignant cells.

  1. The use of a biostatic fascia lata thigh allograft as a scaffold for autologous human culture of fibroblasts--An in vitro study.

    Science.gov (United States)

    Żurek, Jarek; Dominiak, Marzena; Botzenhart, Ute; Bednarz, Wojciech

    2015-05-01

    The method for covering gingival recession defects and augmenting keratinized gingiva involves the use of autogenuous connective tissue grafts obtained from palatal mucosa in combination with various techniques of flap repositioning or tunnel techniques. In the case of multiple gingival recession defects the amount of connective tissue available for grafting is insufficient. Therefore, the use of substitutes is necessary. The most widely used material in recent years has been the acellular dermal matrix allograft. The disadvantage of its application lies in the absence of cells and blood vessels, which increases incorporation time. Primary cultured human autologic fibroblasts are commonly used to optimize the healing process. The aim of this study was to examine the in vitro biocompatibility of human fascia lata allograft as a new scaffold for primary cultured human autologic fibroblasts. For that, a fibroblast culture obtained from a fragment of gingival tissue taken from the hard palate mucosa of a subject was used. After 14 days the colony cells were inoculated on a fragment of human fascia lata allograft. After a further 7 days of incubation the material was frozen, cut and prepared for histochemical examination. After two weeks of incubation, and 7 days after inoculation on a fragment of fascia lata allograft numerous accumulations of the cultured fibroblast were found that had a typical structure and produced collagen fibres. A human fascia lata allograft can be used as a scaffold for primary cultured human autologic fibroblasts. Further studies should confirm the clinical efficacy of this solution.

  2. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    DEFF Research Database (Denmark)

    Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus;

    2011-01-01

    this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs...... of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis......Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate...

  3. Differential gene expression before and after ionizing radiation of subcutaneous fibroblasts identifies breast cancer patients resistant to radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Alsner, Jan; Rødningen, Olaug K.; Overgaard, Jens

    2007-01-01

    -induced changes in gene expression in fibroblasts, whether differential expression is more pronounced when looking at the fold induction levels, taking into account the differences in background expression levels between patients, and whether there is a linear correlation between individual risk of RIF...... and changes in radiation-induced gene expression in fibroblasts. MATERIAL AND METHODS: Gene expression was analysed by quantitative real-time PCR before and after a fractionated scheme with 3x3.5Gy/3 days in fibroblasts derived from 26 patients with breast cancer treated with post-mastectomy radiotherapy....... RESULTS: Robust radiation-induced changes in gene expression were observed, with differential gene expression between low and high risk patients being most pronounced for the fold induction level ('after' value divided by 'before' value for each patient). When including patients with intermediate risk...

  4. Differential gene expression before and after ionizing radiation of subcutaneous fibroblasts identifies breast cancer patients resistant to radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Alsner, Jan; Rødningen, Olaug K.; Overgaard, Jens

    2007-01-01

    -induced changes in gene expression in fibroblasts, whether differential expression is more pronounced when looking at the fold induction levels, taking into account the differences in background expression levels between patients, and whether there is a linear correlation between individual risk of RIF...... and changes in radiation-induced gene expression in fibroblasts. MATERIAL AND METHODS: Gene expression was analysed by quantitative real-time PCR before and after a fractionated scheme with 3x3.5Gy/3 days in fibroblasts derived from 26 patients with breast cancer treated with post-mastectomy radiotherapy....... RESULTS: Robust radiation-induced changes in gene expression were observed, with differential gene expression between low and high risk patients being most pronounced for the fold induction level ('after' value divided by 'before' value for each patient). When including patients with intermediate risk...

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

    Science.gov (United States)

    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.

  6. Treatment of Hypertrophic Scar in Human with Autologous Transplantation of Cultured Keratinocytes and Fibroblasts along with Fibrin Glue

    Directory of Open Access Journals (Sweden)

    Ehsan Taghiabadi

    2015-04-01

    Full Text Available Objective: Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can’t be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue (FG was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. Materials and Methods: In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin (H&E and Masson’s Trichrome (MT staining of the biopsies after 8 weeks. Results: Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. Conclusion: These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate.

  7. Genotoxicity test of propolis extract, mineral trioksida aggregat, and calcium hydroxide on fibroblast BHK-21 cell cultures

    Directory of Open Access Journals (Sweden)

    Ceples Dian Kartika W.P

    2015-03-01

    Full Text Available Background: Health industry has always used natural products as an alternative. Propolis, a natural antibiotic, is a resinous yellow brown or dark brown substance derived from honey bees (Apis mellifera. The main chemical compounds contained in propolis are flavonoids, phenolics and other various aromatic compounds. Flavonoids are well known plant compounds that have antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory proprieties. Propolis is expected to be an alternative used for root canal treatment with lower toxicity compared to calcium hydroxide (Ca(OH2 . Over the last decade, a new material, mineral trioxide aggregate (MTA was developed, and has been used as the gold standard. All materials used in mouth should be biocompatible. The initial level of material biocompatibility evaluation involves toxicity and genotoxicity tests. Purpose: This research is aimed to conduct comparison test of genotoxicity effect of propolis extract, MTA and Ca(OH2 on fibroblast BHK-21 cell culture. Methods: This research was conducted with single-cell gel electrophoresis method. Results: The results indicate that propolis extract cannot cause DNA damage, while MTA can cause apoptosis and Ca(OH2 can cause neucrosis. Conclusion: It can be concluded that propolis extract has genotoxicity effect lower than MTA and Ca(OH2 , but MTA has lower effect on fibroblast BHK-21 cell culture.

  8. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

  9. Neuronal-like differentiation of single versus multiple treatments with human amnion-derived mesenchymal stem cells induced by basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongliang Jiao; Fangxia Guan; Xiang Hu; Jianbin Li; Hong Shan; Wei Li; Jun Li; Ying Du; Bo Yang; Yunfan Zhou

    2009-01-01

    BACKGROUND: Cultures from multiple portions of umbilical cord blood mesenchymal stem cells have been shown to undergo more rapid proliferation and attachment than single portions. OBJECTIVE: To observe growth of basic fibroblast growth factor (bFGF)-induced cultures of human amnion-derived mesenchymal stem cells (AMSCs) and differentiation into neuronal-like cells. DESIGN, TIME AND SETTING: Comparative observation. The study was performed at the Laboratory of Microbiology and Immunology, Basic Medical School of Zhengzhou University from January to May 2008.METHODS: Amnia from full-term, uterine-incision delivery were donated by 12 healthy women. AMSCs were obtained by cell separation and culture techniques, and were passaged and induced by bFGF. From the third passage, a total of 1 mL AMSCs, at a density of 1.0 ×10 4/mL, was separately harvested from six samples, which served as group A. A total of 1 mL AMSCs, at a density of 1.0×10 4 /mL, was harvested separately from the remaining six samples, which served as group B. A total of 0.5 mL from the six samples of group A and 0.5 mL from the six samples of group B were combined to form group C. MAIN OUTCOME MEASURES: Differences in cell quantity among the three groups were compared by cell quantification and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)analysis. Expression of a glial cell marker, neuron-specific enolase, and nestin was detected in the three groups by immunocytochemistry. RESULTS: Cell quantification and MTT analysis of live cells, as well as AMSC absorbance, were significantly greater in group C compared with groups A and B at 18 days of culture (P<0.05), and no significant difference was observed between groups A and B. Glial fibrillary acidic protein, neuron-specific enolase, and nestin were expressed in all groups following bFGF induction. CONCLUSION: Mixed AMSC cultures promoted proliferation, and bFGF-induced AMSCs differentiated into neuronal-like cells.

  10. Growth factors and feeder cells promote differentiation of human embryonic stem cell into dopaminergic neurons: a novel role of fibroblast growth factor-20

    Directory of Open Access Journals (Sweden)

    Ana Sofia Correia

    2008-07-01

    Full Text Available Human embryonic stem cells (hESCs are a potential source of dopaminergic neurons for treatment of Parkinson’s disease (PD. Dopaminergic neurons can be derived from hESCs and display a characteristic midbrain phenotype. Once transplanted, they can induce partial behavioral recovery in animal models of PD. The potential research field faces several challenges that need to be overcome before clinical application of hESCs in a transplantation therapy in PD can be considered. These include low survival of the hESC-derived, grafted dopaminergic neurons after transplantation; unclear functional integration of the grafted neurons in the host brain; and, the risk of teratoma/tumor formation from the transplanted cells. This review is focused on our recent efforts to improve the survival of hESC-dervied dopaminergic neurons. We have examined the effect of fibroblast growth factor (FGF-20 in the differentiation of hESCs into dopaminergic neurons. We supplemented cultures of hESCs with FGF-20 during differentiation on PA6 mouse stromal cells for three weeks. When we added FGF-20 the yield of neurons expressing tyrosine hydroxylase increased. We demonstrated that at least part of the effect is contributed by enhanced cell differentiation towards the dopaminergic phenotype as well as reduced cell death. We compare our results with those obtained in other published protocols using different sets of growth factors. Our data indicate that FGF-20 has potent effects to generate large number of dopaminergic neurons derived from hESCs, which may be useful for cell therapy in PD.

  11. Fibroblast growth factor receptor 2 (FGFR2) is required for corneal epithelial cell proliferation and differentiation during embryonic development.

    Science.gov (United States)

    Zhang, Jinglin; Upadhya, Dinesh; Lu, Lin; Reneker, Lixing W

    2015-01-01

    Fibroblast growth factors (FGFs) play important roles in many aspects of embryonic development. During eye development, the lens and corneal epithelium are derived from the same surface ectodermal tissue. FGF receptor (FGFR)-signaling is essential for lens cell differentiation and survival, but its role in corneal development has not been fully investigated. In this study, we examined the corneal defects in Fgfr2 conditional knockout mice in which Cre expression is activated at lens induction stage by Pax6 P0 promoter. The cornea in LeCre, Fgfr2(loxP/loxP) mice (referred as Fgfr2(CKO)) was analyzed to assess changes in cell proliferation, differentiation and survival. We found that Fgfr2(CKO) cornea was much thinner in epithelial and stromal layer when compared to WT cornea. At embryonic day 12.5-13.5 (E12.5-13.5) shortly after the lens vesicle detaches from the overlying surface ectoderm, cell proliferation (judged by labeling indices of Ki-67, BrdU and phospho-histone H3) was significantly reduced in corneal epithelium in Fgfr2(CKO) mice. At later stage, cell differentiation markers for corneal epithelium and underlying stromal mesenchyme, keratin-12 and keratocan respectively, were not expressed in Fgfr2(CKO) cornea. Furthermore, Pax6, a transcription factor essential for eye development, was not present in the Fgfr2(CKO) mutant corneal epithelial at E16.5 but was expressed normally at E12.5, suggesting that FGFR2-signaling is required for maintaining Pax6 expression in this tissue. Interestingly, the role of FGFR2 in corneal epithelial development is independent of ERK1/2-signaling. In contrast to the lens, FGFR2 is not required for cell survival in cornea. This study demonstrates for the first time that FGFR2 plays an essential role in controlling cell proliferation and differentiation, and maintaining Pax6 levels in corneal epithelium via ERK-independent pathways during embryonic development.

  12. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... proceeded via anthraquinone photochemistry to introduce amine functionalities at the surface followed by coupling of IL-4 through a bifunctional amine-reactive linker. X-ray photoelectron spectroscopy showed that undesirable multilayer formation of the photoactive compound could be avoided by reaction...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  13. Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

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    Usuki, S.; Lyu, S.C.; Sweeley, C.C.

    1988-05-15

    Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ((1-14C)N-acetylmannosamine) and a radioactive precursor of ceramide ((3,3-3H2)serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.

  14. Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: a comparative proteomics and microarray study.

    Science.gov (United States)

    Yang, Kyeong Eun; Kwon, Joseph; Rhim, Ji-Heon; Choi, Jong Soon; Kim, Seung Il; Lee, Seung-Hoon; Park, Junsoo; Jang, Ik-Soon

    2011-07-01

    The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.

  15. Uroporphyria development in cultured chick embryo fibroblasts long-term treated with chloramphenicol and ethidium bromide.

    Science.gov (United States)

    de Muys, J M; Morais, R

    1984-07-23

    Long-term chloramphenicol- and ethidium bromide-treated chick embryo fibroblasts synthesize large amounts of porphyrins from exogenously added delta-aminolevulinic acid. The porphyrins consist mainly of uro- and heptacarboxyporphyrins and are retained within cells. Uroporphyria development is a time-dependent process which accompanies a step-wise decrease in the capacity of the mitochondrial respiratory chain. Upon removal of chloramphenicol from the medium, the pattern of porphyrin production readily returns to normal (mainly proto- and coproporphyrins found in the medium) while ethidium bromide-treated cells remain uroporphyric. The results suggest that impairment of mitochondrial functions in chicken by xenobiotics leads to uroporphyria development.

  16. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  17. The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells.

    Science.gov (United States)

    Pulkkinen, Mari-Anne; Spencer-Dene, Bradley; Dickson, Clive; Otonkoski, Timo

    2003-02-01

    Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.

  18. Differential expression of fibroblast growth factor receptors in the developing murine choroid plexus.

    Science.gov (United States)

    Reid, Sarah; Ferretti, Patrizia

    2003-03-14

    The choroid plexuses (CPs) are specialised secretory organs situated within the ventricles of the brain involved in the production of cerebrospinal fluid (CSF) and the maintenance of the blood-CSF barrier. Abnormal function of the CPs can lead to hydrocephalus and raised intracranial pressure, pathologies frequently observed in certain craniofacial syndromes caused by single point mutations in fibroblast growth factor receptors (FGFRs). At present, relatively little is known about the embryonic CPs in terms of gene or protein expression, function as the brain develops or on the potential role of FGFRs within this context. Given the limited information available on the regulation of FGFRs during development of the CPs and periventricular tissues, we have carried out a detailed analysis of the localisation of FGFR1, 2, 3 and 4 proteins in these regions of the murine embryo from the time of formation of the CP in the third ventricle at E12.5 throughout the second half of gestation, and examined the expression of different FGFR isoforms at E12.5 by RT-PCR. We show here that FGFR1 and FGFR4 are expressed in murine CPs at E12.5 but not at E15.5 or E18.5, suggesting a role for the signaling pathways transduced by these receptors at early stages of CP development. In contrast, FGFR2 expression is maintained throughout CP development, indicating that this receptor may play a role in the function of immature and mature CP. Also FGFR3 is detected at each developmental stage studied, but surprisingly its expression appears confined to the nuclei of CP cells, suggesting that FGFR3 in the CP does not respond to extracellular FGFs but may act in intracrine fashion.

  19. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    Science.gov (United States)

    Kelly, B M; Yu, C Z; Chang, P L

    1989-02-01

    Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

  20. Cholesterol Metabolism Is Altered in Rett Syndrome: A Study on Plasma and Primary Cultured Fibroblasts Derived from Patients

    Science.gov (United States)

    Segatto, Marco; Trapani, Laura; Di Tunno, Ilenia; Sticozzi, Claudia; Valacchi, Giuseppe; Hayek, Joussef; Pallottini, Valentina

    2014-01-01

    Rett (RTT) syndrome is a severe neurological disorder that affects almost exclusively females. Several detectable mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are responsible for the onset of the disease. MeCP2 is a key transcription regulator involved in gene silencing via methylation-dependent remodeling of chromatin. Recent data highlight that lipid metabolism is perturbed in brains and livers of MECP2-null male mice. In addition, altered plasma lipid profile in RTT patients has been observed. Thus, the aim of the work is to investigate the protein network involved in cholesterol homeostasis maintenance on freshly isolated fibroblasts and plasma from both RTT and healthy donors. To this end, protein expression of 3-hydroxy-3methyl glutaryl Coenzyme A reductase (HMGR), sterol regulatory element binding proteins (SREBPs), low density lipoprotein receptor (LDLr) and scavenger receptor B-1 (SRB-1) was assessed in cultured skin fibroblasts from unaffected individuals and RTT patients. In addition, lipid profile and the abundance of proprotein convertase subtilisin/kexin type 9 (PCSK9) were analyzed on plasma samples. The obtained results demonstrate that the main proteins belonging to cholesterol regulatory network are altered in RTT female patients, providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology. PMID:25118178

  1. Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)-syndrome. Ultrastructural changes of mitochondria in cultured dermal fibroblasts of three patients.

    Science.gov (United States)

    Haust, M D; Dewar, R A; Gatfield, D P; Gordon, B A

    1996-03-01

    Mitochondria of fibroblasts cultured from the skin obtained at biopsy from three patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)-syndrome, one of the autosomal recessive, heritable urea cycle disorders, were studied with appropriate controls ultrastructurally. The patients were two severely retarded 10- and 12-year-old boys, and a 22-year-old sister of the former whose mental status was at the low normal range; she never had motor impairments or seizures. The mitochondria, similar in all three patients, were increased in number, very long, branching and/or "looping," and tortuous. "Spurs" or "buddings" extended from their lateral surfaces and the terminal segments were often bulbous. Other unusual configurations were also present. In addition, giant forms with large diameter contained innumerable closely-packed and parallel cristae which traversed the entire width of these mitochondria; at times they assumed a "whirled" pattern. The mitochondrial matrix was usually of high electron density. These changes were not a feature of fibroblastic mitochondria of controls. Several changes resembled those of hepatic mitochondria in this disorder. All features are interpreted as an attempt at expanding the mitochondrial volume (via structural substratum) to compensate for the metabolic incompetence of these organelles (a block in transmembranous transfer of ornithine from hyaloplasm into mitochondria for conversion to citrulline).

  2. Cholesterol metabolism is altered in Rett syndrome: a study on plasma and primary cultured fibroblasts derived from patients.

    Directory of Open Access Journals (Sweden)

    Marco Segatto

    Full Text Available Rett (RTT syndrome is a severe neurological disorder that affects almost exclusively females. Several detectable mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2 are responsible for the onset of the disease. MeCP2 is a key transcription regulator involved in gene silencing via methylation-dependent remodeling of chromatin. Recent data highlight that lipid metabolism is perturbed in brains and livers of MECP2-null male mice. In addition, altered plasma lipid profile in RTT patients has been observed. Thus, the aim of the work is to investigate the protein network involved in cholesterol homeostasis maintenance on freshly isolated fibroblasts and plasma from both RTT and healthy donors. To this end, protein expression of 3-hydroxy-3methyl glutaryl Coenzyme A reductase (HMGR, sterol regulatory element binding proteins (SREBPs, low density lipoprotein receptor (LDLr and scavenger receptor B-1 (SRB-1 was assessed in cultured skin fibroblasts from unaffected individuals and RTT patients. In addition, lipid profile and the abundance of proprotein convertase subtilisin/kexin type 9 (PCSK9 were analyzed on plasma samples. The obtained results demonstrate that the main proteins belonging to cholesterol regulatory network are altered in RTT female patients, providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology.

  3. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xiaohui [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Yang [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Jingwen [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Li, Peng [Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR (China); Liu, Yinan [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Wen, Jinhua, E-mail: jhwen@bjmu.edu.cn [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Luan, Qingxian, E-mail: kqluanqx@126.com [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China)

    2016-05-06

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  4. Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis.

    Science.gov (United States)

    Lin, Jinpiao; Zhou, Zhou; Huo, Rongfen; Xiao, Lianbo; Ouyang, Guilin; Wang, Li; Sun, Yue; Shen, Baihua; Li, Dangsheng; Li, Ningli

    2012-06-01

    Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.

  5. Neural Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells Treated with Sex Steroid Hormones and Basic Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    Kazem Parivar

    2015-04-01

    Full Text Available Objective: There are several factors, like environmental agents, neurotrophic factors, serotonin and some hormones such as estrogen, affecting neurogenesis and neural differentiation. Regarding to importance of proliferation and regeneration in central nervous system, and a progressive increase in neurodegenerative diseases, cell therapy is an attractive approach in neuroscience. The aim of the present study was to investigate the effects of sex steroid hormones and basic fibroblast growth factor (bFGF on neuronal differentiation of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs. Materials and Methods: This experimental study was established in Kharazmi University. BM was isolated from the bones of femur and tibia of 4-6-week old Naval Medical Research Institute (NMRI mice, and the cells were cultured. The cells were divided into following 4 groups based on the applied treatments: I. control (no treatment, II. steroid hormones (β-estradiol, progesterone and testosterone, III. bFGF and IV. combination of steroid hormones and bFGF. Immunocytochemistry and flow cytometery analyses were applied for beta III-tubulin (β-III tubulin and microtubule-associated proteins-2 (MAP-2 in 4 days of treatment for all groups. Results: The cells treated with combination of bFGF and steroid hormones represented more expressions of neural markers as compared to control and to other two groups treated with either bFGF or steroid hormones. Conclusion: This study showed that BM-MSCs can express specific neural markers after receiving bFGF pretreatment that was followed by sex steroid hormones treatment. More investigations are necessary to specify whether steroid hormones and bFGF can be considered for treatment of CNS diseases and disorders.

  6. Fibroblast Growth Factor Receptor-2 Contributes to the Basic Fibroblast Growth Factor-Induced Neuronal Differentiation in Canine Bone Marrow Stromal Cells via Phosphoinositide 3-Kinase/Akt Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Rei Nakano

    Full Text Available Bone marrow stromal cells (BMSCs are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2 and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR, phosphatidylinositol 3-kinase (PI3K and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs.

  7. Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

    Directory of Open Access Journals (Sweden)

    Toshiki Kanazawa

    Full Text Available Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs, CD44, hyaluronan synthase 2 (HAS2, and cyclooxygenase 2 (COX2 along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA, and prostaglandin E2 (PGE2 were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

  8. Transcriptional regulation of proteoglycans and glycosaminoglycan chain-synthesizing glycosyltransferases by UV irradiation in cultured human dermal fibroblasts.

    Science.gov (United States)

    Shin, Jeong-Eun; Oh, Jang-Hee; Kim, Yeon Kyung; Jung, Ji-Yong; Chung, Jin Ho

    2011-03-01

    Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm(2) of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, β1,3-glucuronyltransferase-1, β1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of β1,3-galactosyltransferase-6, β1,4-galactosyltransferase-3, -7, β-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.

  9. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Xin-Qin Kang; Wei-Jin Zang; Li-Jun Bao; Dong-Ling Li; Tu-Sheng Song; Xiao-Li Xu; Xiao-Jiang Yu

    2005-01-01

    AIM: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases.METHODS: vSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20±1.16 μg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28±3.11 μg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 (t = 11.748, P<0.01). Urea(4.72±1.03 μmol/L) was detected on d 20 (t = 4.272,P<0.01), and continued to increase to 10.28±1.06 μmol/L on d 28 (t = 9.276, P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCBderived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

  10. Apoptotic and necrotic influence of dental resin polymerization initiators in human gingival fibroblast cultures.

    Science.gov (United States)

    Masuki, Kouhei; Nomura, Yuji; Bhawal, Ujjal Kumar; Sawajiri, Masahiko; Hirata, Isao; Nahara, Yukinori; Okazaki, Masayuki

    2007-11-01

    The aim of this study was to examine the apoptotic and necrotic influence of four dental resin polymerization initiators--namely benzoyl peroxide (BPO), camphorquinone (CQ), dimethylaminoethyl methacrylate (DMAEMA), and dimethyl-para-toluidine (DMPT)--on human gingival fibroblast (HGF) cells. To this end, the growth inhibition of HGF cells with 1 mM BPO, CQ, and DMAEMA, and 500 microM DMPT was evaluated using Cell Counting Kit-8. Then, cell cycle analysis by flow cytometry was used to assess propidium iodide-stained cells (distribution of cells in G0/G1, S, G2/M phases). All four dental resin polymerization initiators induced G0/G1 cell cycle arrest. As for the patterns of cell death (necrosis and/or apoptosis), they were analyzed using Annexin V-FITC/PI staining with flow cytometry. All four dental resin polymerization initiators most likely induced necrosis.

  11. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Hansen Jacob B

    2011-05-01

    Full Text Available Abstract Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs, and retinoblastoma gene-deficient MEFs (Rb-/- MEFs. Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte

  12. Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms

    OpenAIRE

    1996-01-01

    Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in ...

  13. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

  14. Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode

    Science.gov (United States)

    Volpato, Luiz Evaristo Ricci; de Oliveira, Rodrigo Cardoso; Espinosa, Mariano Martinez; Bagnato, Vanderley Salvador; Machado, Maria A. A. M.

    2011-07-01

    Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm2), infrared laser (780 nm, 40 mW, 1 W/cm2), and red LED (637 +/- 15 nm, 40 mW, 1 W/cm2). All applications were punctual and performed with a spot with 0.4 mm2 of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced.

  15. The role of basic fibroblast growth factor in glioblastoma multiforme and glioblastoma stem cells and in their in vitro culture.

    Science.gov (United States)

    Haley, Elizabeth M; Kim, Yonghyun

    2014-04-28

    Glioblastoma multiforme (GBM) is the most malignant form of central nervous system tumor, and current therapies are largely ineffective at treating the cancer. Developing a more complete understanding of the mechanisms controlling the tumor is important in order to explore new possible treatment options. It is speculated that the presence of glioblastoma stem or stem-like cells (GSCs), a rare type of pluripotent cancer cell that possesses the ability to self-renew and generate tumors, could be an important factor contributing to the resistance to treatment and deadliness of the cancer. A comprehensive knowledge of the mechanisms controlling the expression and properties of GSCs is currently lacking, and one promising area for further exploration is in the influence of basic fibroblast growth factor (FGF-2) on GSCs. Recent studies reveal that FGF-2 plays a significant part in regulating GBM, and the growth factor is commonly included as a supplement in media used to culture GSCs in vitro. However, the particular role that FGF-2 plays in GSCs has not been as extensively explored. Therefore, understanding how FGF-2 is involved in GSCs and in GBMs could be an important step towards a more complete comprehension of the managing the disease. In this review, we look at the structure, signaling pathways, and specific role of FGF-2 in GBM and GSCs. In addition, we explore the use of FGF-2 in cell culture and using its synthetic analogs as a potential alternative to the growth factor in culture medium.

  16. Effects of Basic Fibroblast Growth Factor and Insulin-like Growth Factor on Cultured Cartilage Cells from Skate Raja porasa

    Institute of Scientific and Technical Information of China (English)

    樊廷俊; 晋凌云; 汪小锋

    2003-01-01

    Effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF-II) on cartilage cells from proboscis of skate, Raja porasa Günther, were investigated in this study. The cartilage cells were cultured in 20% FBS-supplemented MEM medium at 24℃. Twelve hours after culture initiation, the cartilage cells were treated with bFGF and IGF-II at different concentration combinations. It was found that 20 ng/ml of bFGF or 80 ng/ml of IGF-II was enough to have obvious stimulating effect on the growth and division of skate cartilage cells. Test of bFGF and IGF-II together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF-II together had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form a monolayer at day 7.

  17. Fibroblast growth factor-1 and-2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures

    NARCIS (Netherlands)

    Yeoh, Joyce S. G.; van Os, Ronald; Weersing, Ellen; Ausema, Albertina; Dontje, Bert; Vellenga, Edo; de Haan, Gerald

    2006-01-01

    In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 + 2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assa

  18. Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas

    Energy Technology Data Exchange (ETDEWEB)

    Yamaguchi, F.; Saya, H.; Bruner, J.M.; Morrison, R.S. (Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States))

    1994-01-18

    Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, the authors demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1[beta]) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1[alpha]) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1[alpha] to FGFR-2 and a shift in expression from FGFR-1[alpha] to FGFR-1[beta] as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.

  19. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

    2011-01-01

    at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...... by IL-4 denaturation or improper epitope presentation induced by the immobilization process, or by biological irresponsiveness of monocytes to IL-4 in immobilized formats....

  20. Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts

    Directory of Open Access Journals (Sweden)

    Olsen Charlotta J

    2010-08-01

    Full Text Available Abstract Introduction Tumour phenotype is regulated in a complex fashion as a result of interactions between malignant cells and the tumour stroma. Fibroblasts are the most abundant and perhaps most active part of the tumour stroma. A better understanding of the changes that occur in fibroblasts in response to the presence of malignant cells may lead to the development of new strategies for cancer treatment. We explored the effects of fibroblasts on the growth and invasion of mammary carcinoma tumour cells in vitro and in vivo. Methods In order to analyse secreted factors that affect invasive abilities of breast cancer cells we co-cultured human mammary fibroblasts (HMF3s and cancer cells (MCF7S1 in three-dimensional (3D growth conditions devoid of heterogeneous cell-cell contact. To study the possible influence of fibroblasts on MCF7S1 cancer cell growth in vivo we co-injected HMF3s and MCF7S1 cells in Balb/c nu/nu mice. Results In 3D co-culture both HMF3s and MCF7S1 cells demonstrated enhanced invasion into a Matrigel matrix. This was correlated with enhanced expression of the metastasis promoting S100A4 protein in fibroblasts, stimulation of the matrix metalloproteinase (MMP-2 activity, and enhanced secretion of a range of different cytokines. Orthotopic injection of oestrogen-dependent MCF7S1 cancer cells together with fibroblasts showed stimulation of tumour growth in mice without an external oestrogen supply. The resulting tumours were characterized by increased development of extracellular matrix, as well as an increase of murine S100A4 concentration and activity of MMP-2 in the tumour interstitial fluid. Conclusion Stimulation of the invasive phenotype of tumour cells in 3D co-cultures with fibroblasts could be correlated with increased production of S100A4 and MMP-2. We propose that enhanced development of mouse host-derived tumour stroma in a MCF7S1 co-injection xenograft model leads to oestrogen independency and is triggered by the

  1. Exposure of cultured fibroblasts to the peptide PR-11 for the identification of induced proteome alterations and discovery of novel potential ligands.

    Science.gov (United States)

    Breguez, Gustavo Silveira; Neves, Leandro Xavier; Silva, Karina Taciana Santos; de Freitas, Lorran Miranda Andrade; de Oliveira Faria, Gabriela; Isoldi, Mauro César; Castro-Borges, William; de Andrade, Milton Hércules Guerra

    2016-12-01

    The PR-11 peptide corresponds to the N-terminal and active region of the endogenously synthesized PR-39 molecule, of porcine origin. It is known to possess various biological effects including antimicrobial properties, angiogenic and anti-inflammatory activities. Apart from its reported activity as a proteasome inhibitor, a more comprehensive understanding of its function, at the molecular level, is still lacking. In this study, we used a label-free shotgun strategy to evaluate the proteomic alterations caused by exposure of cultured fibroblasts to the peptide PR-11. This approach revealed that more than half of the identified molecules were related to signalling, transcription and translation. Proteins directly associated to regulation of angiogenesis and interaction with the hypoxia-inducible factor 1-α (HIF-1α) were significantly altered. In addition, at least three differentially expressed molecules of the NF-κB pathway were detected, suggesting an anti-inflammatory property of PR-11. At last, we demonstrated novel potential ligands of PR-11, through its immobilization for affinity chromatography. Among the eluted molecules, gC1qR, a known complement receptor, appeared markedly enriched. This provided preliminary evidence of a PR-11 ligand possibly involved in the internalization of this peptide. Altogether, our findings contributed to a better understanding of the cellular pathways affected by PR-39 derived molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells.

    Science.gov (United States)

    Pi, Liya; Chung, Pei-Yu; Sriram, Sriniwas; Rahman, Masmudur M; Song, Wen-Yuan; Scott, Edward W; Petersen, Bryon E; Schultz, Gregory S

    2015-11-26

    fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2. CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-β. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

  3. Inhibition of aromatase activity by methyl sulfonyl PCB metabolites in primary culture of human mammary fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Berg, M. van den; Heneweer, M.; Geest, M. de; Sanderson, T. [Inst. for Risk Assessment Sciences and Utrecht Univ. (Netherlands); Jong, P. de [St. Antonius Hospital, Nieuwegein (Netherlands); Bergman, A. [Stockholm Univ., Stockholm (Sweden)

    2004-09-15

    Methyl sulfonyl PCB metabolites (MeSO2-PCBs) are persistent contaminants and are ubiquitously present in humans and the environment. Lipophilicity of MeSO2- PCB metabolites is similar to the parent compounds and they have been detected in human milk, adipose, liver and lung tissue. 4- MeSO2-PCB-149 is the most abundant PCB metabolite in human adipose tissue and milk at a level of 1.5 ng/g lipids. Human blood concentration of 4-MeSO2-PCB-149 is approximately 0.03 nM. 3- MeSO2-PCB-101 is the predominant PCB metabolite in muscle and blubber in wildlife, such as otter, mink and grey seal. In the environment, they have been linked to chronic and reproductive toxicity in exposed mink. Additionaly, some MeSO{sub 2}-PCBs have been shown to be glucocorticoid receptor (GR) antagonists. Since approximately 60% of all breast tumors are estrogen responsive, exposure to compounds that are able to alter estrogen synthesis through interference with the aromatase enzyme, can lead to changes in estrogen levels and possibly to accelerated or inhibit breast tumor growth. Therefore, it is important to identify exogenous compounds that can alter aromatase activity in addition to those compounds which have direct interaction with the estrogen receptor (ER). Aromatase (CYP19) comprises the ubiquitous flavoprotein, NADPH-cytochrome P450 reductase, and a unique cytochrome P450 that is exclusively expressed in estrogen producing cells. Previous studies have revealed that expression of the aromatase gene is regulated in a species- and tissue specific manner. In healthy breast tissue, the predominantly active aromatase promoter region I.4 is regulated by glucocorticoids and class I cytokines. Therefore, it is important to investigate possible aromatase inhibiting properties of MeSO{sub 2}-PCBs (as anti glucocorticoids?) in relevant human tissues. We used primary human mammary fibroblasts because of their role in breast cancer development. We compared the results in primary fibroblasts with

  4. In vitro culture of ear fibroblast cells from Lu-chuan pig%陆川猪耳部成纤维细胞的体外培养

    Institute of Scientific and Technical Information of China (English)

    秦小娥; 胡林林; 卢晟盛; 卢克焕

    2011-01-01

    In order to establish an isolation method and culture system for Lu-chuan pig ear fibroblast cells,Lu-chuan pig ear fibroblast cell was isolated and cultured by tissue-piece methods.Using 0.25% trypsin + 0.02% EDTA as digested medium and DMEM containing 10% FBS as culture medium for subculture,it was concluded that culture system was good enough for lu-chuan pig ear fibroblast cells.The pured fibroblast cells could be obtained after passing 3 subcultures.The cultured cells were typical fibroblast morphology,which grooved as irregular triangle or shuttle,appearing as radioactive blaze and helix in shape.The cell type was identified by fluorescent immuneocytochemistry(ICC) and the result indicated that isolated cells from Lu-chuan ear tissues were Lu-chuan ear fibroblast cells.In conclusion,Lu-chuan pig ear fibroblast cell could be isolated and cultured by tissue-piece methods.%本研究建立陆川猪耳部成纤维细胞的体外培养体系,采用组织块培养法可以获得陆川猪耳部成纤维细胞。用0.25%胰蛋白酶+0.02%EDTA消化液消化细胞、用含有10%FBS的DMEM对细胞进行培养,能很好的支持陆川猪耳部成纤维细胞的生长。传3代后,观察到培养的细胞形态逐渐均一,为典型的成纤维细胞,绝大部分呈梭形或不规则三角形。细胞生长时呈放射状、火焰状或旋涡状走势。用间接免疫荧光法对细胞类型进行鉴定。抗波形蛋白免疫荧光染色显示为阳性,抗角形蛋白免疫荧光染色为阴性。结果,分离得到的细胞为成纤维细胞。结果表明,用组

  5. Trypanosoma acomys (Wenyon, 1909): continuous culturing with a mouse fibroblast cell-line (A9).

    Science.gov (United States)

    Abdallah, M A; Abdel-Hafez, S K; al-Yaman, F M

    1990-01-01

    The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37 degrees C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 x 10(4), 1.5 x 10(5) and 7.4 x 10(5) parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 x 10(6) parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.

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

    Science.gov (United States)

    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.

  7. Hormonal regulation of dipeptidyl-aminopeptidase I activity in cultured human fibroblasts.

    Science.gov (United States)

    Davis, M H

    1987-05-01

    Human male fibroblasts, cell line GM2987, were grown in 10% Nu-Serum or fetal bovine serum. Dipeptidyl-aminopeptidase I (DAP-I) activity was higher in cells grown with Nu-Serum and cells grown in 10% fetal bovine serum purchased from Grand Island Biological Company (GIBCO) and lower in cells grown in 10% fetal bovine serum obtained from Sterile Systems, Inc. (Hyclone). The addition of 0.3 microM cortisol to all three types of sera resulted in cells that had similar levels of DAP-I activity (maximum of 800-900 nmol of beta-naphthylamine released from glycyl-L-phenylanine-beta-naphthylamine per hour per milligram of cellular protein). The addition of cortisol to Hyclone fetal bovine serum increased the DAP-I levels by up to threefold with a half-maximal response occurring at 30 nM cortisol. Triiodothyronine also could increase DAP-I levels, but only between 1.5- and 2.0-fold. Testosterone propionate increased DAP-I levels by 1.4-fold. These changes in growth media and hormones had little effect on other lysosomal enzymes or the growth characteristics of the cells.

  8. Expression of MMP-2, MT1-MMP, and TIMP-2 by cultured rabbit corneal fibroblasts under mechanical stretch.

    Science.gov (United States)

    Liu, Chengxing; Feng, Pengfei; Li, Xiaona; Song, Jie; Chen, Weiyi

    2014-08-01

    Refractive surgery not only leads to tissue injury but also evokes mechanical stress increase of the cornea. How the mechanical stress affects the corneal matrix remodeling, specifically, matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases; TIMPs) is not well understood. In this study, cultured rabbit corneal fibroblasts in vitro were subjected to regimen of 5%, 10%, or 15% equibiaxial stretch at 0.1 Hz for 3 or 24 h. MMP-2 protein level was measured by gelatin zymography and Western blotting. MMP-2, membrane type 1 MMP (MT1-MMP), and TIMP-2 mRNA levels were quantified by real-time quantitative PCR. Extracellular regulated protein kinase (ERK) phosphorylation protein levels were assessed by Western blotting. Our results showed that a 15% stretch resulted in increases in MMP-2 protein, MMP-2 mRNA, and MT1-MMP mRNA levels, but a decrease in TIMP-2 mRNA level. However, a 5% stretch caused decreases in MMP-2 protein and mRNA level, but an increase in TIMP-2 mRNA level, and no change in MT1-MMP mRNA level. A 15% stretch also caused a significant increase in ERK1/2 phosphorylation. Inhibition of the mitogenactivated protein kinase (MEK) pathway with PD98059 attenuated stretch-induced increase in MMP-2 production and ERK activity. These results suggest that small-magnitude stretching may promote corneal matrix synthetic events, whereas large-magnitude stretching promotes corneal matrix degradation by changing the balance between MMPs and TIMPs in corneal fibroblasts. Large-magnitude stretch-induced increase in pro-MMP-2 production was in an ERK-dependent manner. © 2014 by the Society for Experimental Biology and Medicine.

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

    Energy Technology Data Exchange (ETDEWEB)

    Candelario, Jose; Borrego, Stacey [Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Reddy, Sita, E-mail: sitaredd@usc.edu [Department of Biochemistry and Molecular Biology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Comai, Lucio, E-mail: comai@usc.edu [Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States)

    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

  10. A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts.

    Science.gov (United States)

    Martini, Claudia N; Gabrielli, Matías; Vila, María del C

    2012-09-01

    Glyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not only glyphosate itself but also surfactants that may also be toxic. 3T3-L1 fibroblasts are a useful tool to study adipocyte differentiation, this cell line can be induced to differentiate by addition of a differentiation mixture containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine. We used this cell line to investigate the effect of a commercial formulation of glyphosate (GF) on proliferation, survival and differentiation. It was found that treatment of exponentially growing cells with GF for 48h inhibited proliferation in a dose-dependent manner. In addition, treatment with GF dilution 1:2000 during 24 or 48h inhibited proliferation and increased cell death, as evaluated by trypan blue-exclusion, in a time-dependent manner. We showed that treatment of 3T3-L1 fibroblasts with GF increased caspase-3 like activity and annexin-V positive cells as evaluated by flow cytometric analysis, which are both indicative of induction of apoptosis. It was also found that after the removal of GF, remaining cells were able to restore proliferation. On the other hand, GF treatment severely inhibited the differentiation of 3T3-L1 fibroblasts to adipocytes. According to our results, a glyphosate-based herbicide inhibits proliferation and differentiation in this mammalian cell line and induces apoptosis suggesting GF-mediated cellular damage. Thus, GF is a potential risk factor for human health and the environment.

  11. 酸性成纤维细胞生长因子与胚胎干细胞的造血分化*★%Acidic fibroblast growth factor effect on hematopoietic differentiation of embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    许婷婷; 张海萍; 王跃嗣

    2013-01-01

      背景:有研究表明,在卵黄囊造血、胎肝造血和胚胎干细胞向造血干细胞分化过程中,酸性成纤维细胞生长因子可强烈表达。目的:探讨酸性成纤维细胞生长因子对小鼠胚胎干细胞造血分化的作用,在拟胚体培养阶段施加酸性成纤维细胞生长因子,验证酸性成纤维细胞生长因子对造血形成细胞产生的调控作用。方法:培养小鼠胚胎干细胞,将饲养层上生长状态良好的小鼠胚胎干细胞用胰酶消化成单个细胞后,利用悬滴法制备拟胚体,拟胚体继续悬浮培养,以1,2和5μg/L 酸性成纤维细胞生长因子分别培养3,5,7,9 d,通过免疫荧光法检测胚胎干细胞与拟胚体中酸性成纤维细胞生长因子的表达,流式细胞术检测Flk-1+与 CD133+阳性细胞率。结果与结论:酸性成纤维细胞生长因子在拟胚体中呈阳性表达。在5μg/L 酸性成纤维细胞生长因子作用下,Flk-1+细胞随时间增加表达增加,CD133+细胞表达模式与 Flk-1+细胞类似。在1μg/L 时,Flk-1+细胞在7 d 时表达达到高峰,随后下降。CD133+细胞表达结果类似。而在2μg/L 时,5 d 时 Flk-1+细胞表达升高,随后下降,在9 d 时表达又增高。而 CD133+细胞则总体呈现升高趋势。酸性成纤维细胞生长因子可促进 Flk-1+与 CD133+细胞的产生,证明酸性成纤维细胞生长因子能够有效促进拟胚体的扩增以及成血管血液干细胞的产生与增殖。%BACKGROUND: Studies have shown that acidic fibroblast growth factor can be strongly expressed in the yolk sac hematopoietic cel s and fetal liver hematopoietic cel s and in the differentiation process of embryonic stem cel s to hematopoietic stem cel s. OBJECTIVE: To explore the effect of acidic fibroblast growth factor on hematopoietic differentiation of mice embryonic stem cel s, to test and verify the regulation effect of acidic fibroblast growth factor in

  12. 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.

    Science.gov (United States)

    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

  13. Differences between scar and dermal cultured fibroblasts derived from a patient with recurrent abdominal incision wound herniation.

    Science.gov (United States)

    Boemi, L; Allison, G M; Graham, W P; Krummel, T M; Ehrlich, H P

    1999-10-01

    Fibroblasts were derived from dermis and scar of a 47-year-old white man with a recurrent incisional hernia as a result of fractured ribs. The scar was thin and stretched, suggesting a defect in the maturation of granulation tissue. After surgical repair, biopsy specimens of discarded scar and skin were used to generate fibroblast cell lines. Fibroblasts maintained in medium containing 10% fetal bovine serum and antibiotic were studied between their third and eighth passage. By phase contrast microscopy, no structural differences were obvious, but it was noted that to pass scar fibroblasts, a more aggressive trypsin regimen was required. Immunohistologic and Western blot analysis of patient scar fibroblasts showed (1) more a smooth muscle actin within stress fibers, (2) increased expression of the vitronectin integrin receptor alpha(v) (CD 51), and (3) reduced expression of the collagen integrin receptor alpha2 (CD 49b). The expression of vinculin from focal adhesions or a tubulin from microtubules was the same among cell lines. Contractions of scar and dermal fibroblast-populated collagen lattice were compared. At 24 hours, contractions were 69 percent with newborn fibroblasts (normal); 68 percent for patient dermal fibroblasts; and only 48 percent for patient scar fibroblasts. The retarded contraction of scar fibroblast-populated collagen lattice was significant (p > or = 0.002). Myosin ATPase activity, critical for lattice contraction, and cell migration were equivalent among all cell lines. A plausible mechanism for the retardation of scar lattice contraction is disruption of fibroblasts and collagen interactions, for which the attachment of cells to collagen is altered. It is proposed that either the decrease in the expression of collagen integrin receptor alpha2 (CD 49b), an increase in the expression of the vitronectin receptor alpha(v) (CD 51), or a combination of both is responsible for disruption of collagen fibroblast interactions.

  14. Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

    Directory of Open Access Journals (Sweden)

    Malik Kafait U

    2004-12-01

    Full Text Available Abstract Background Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α1A-adrenergic receptor (α1A-AR stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known. Results Activation of the α1A-AR in rat-1 fibroblasts by phenylephrine (PE inhibited DNA synthesis by 67% and blocked the re-entry of 81% of the cells into S phase of the cell cycle. This cell cycle blockage was associated with hypertrophy characterized by an increase in protein synthesis (64% and cell size. Elevation of cAMP levels decreased both DNA and protein synthesis. Inhibition of adenylyl cyclase or protein kinase A reversed the antiproliferative effect of cAMP analogs but not PE; the hypertrophic effect of PE was also not altered. The functional response of rat-1 cells to PE was accompanied by increased expression of cyclin-dependent kinase (Cdk inhibitors p27kip1 and p21cip1/waf1, which function as negative regulators of the cell cycle. Stimulation of α1A-AR also upregulated the cell cycle regulatory proteins pRb, cyclin D1, Cdk 2, Cdk 4, and proliferating cell nuclear antigen. The antiproliferative effect of PE was blocked by p27kip1 antisense but not sense oligonucleotide. PE also promoted expression of smooth muscle cell differentiation markers (smooth muscle alpha actin, caldesmon, and myosin heavy chain as well as the muscle development marker MyoD. Conclusions Stimulation of α1A-AR promotes cell cycle arrest, hypertrophy and differentiation of rat-1 fibroblasts into smooth muscle-like cells and expression of negative cell cycle regulators by a mechanism independent of the cAMP/PKA signaling pathway.

  15. Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture.

    Science.gov (United States)

    Gilhare, Varsha Rani; Hirpurkar, S D; Kumar, Ashish; Naik, Surendra Kumar; Sahu, Tarini

    2015-03-01

    The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE) included aggregation of cells, syncytia and plaque formation. FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level.

  16. TNFR1 and TNFR2 differentially mediate TNF-α-induced inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes.

    Science.gov (United States)

    Zhang, Hongfeng; Xiao, Weiguo

    2017-04-01

    TNF-α has long been implicated in the progression of rheumatoid arthritis (RA). However, how the receptors of TNF-α, namely TNFR1 and TNFR2, mediate TNF-α-induced inflammatory responses in fibroblast-like synoviocytes (FLS) in RA has not been elucidated. In the present study, primary FLS cells were isolated from RA patients and treated with TNF-α in vitro. The exogenous TNF-α induced the expression and release of endogenous TNF-α in FLS. In addition, TNF-α led to gradual downregulation of TNFR1 following 1 h treatment. By contrast, the expression of TNFR2 was markedly upregulated after 12 h treatment with TNF-α. Moreover, following TNF-α treatment, the expression of interleukin (IL)-2, IL-6, and IL-8 was gradually increased with time, but their mRNA levels dropped significantly at 48 h. We further investigated the differential functions of TNFR1 and TNFR2 in FLS by conducting siRNA-mediated knockdown. The TNF-α autocrine was inhibited to a greater extent in TNFR1-silenced FLS compared with TNFR2-silenced FLS. Silencing of TNFR1, not TNFR2, activated intrinsic apoptosis and inhibited TNF-α-induced cytokine production in FLS. These results suggest that TNFR1 is the major pro-inflammatory mediator of TNF-α in FLS, whereas TNFR2, which is upregulated in response to prolonged TNF-α stimulation, may act as an immunosuppressor in FLS for the prevention of overwhelming inflammatory reactions.

  17. IM-1662 Attenuates Radiation-Induced Fibroblast Differentiation through Restoration of TGF-beta type III Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sa Rah; Ahn, Ji Yeon; Kim, Mi Hyoung; Lim, Min Jin; Lee, Sae Loom; Yun, Yeon Sook; Song, Jie Young [Korea Institue of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2010-05-15

    Although pulmonary fibrosis occurs 5-20% of lung cancer patients who underwent radiotherapy, clinically standard treatment for fibrotic disease has not been developed yet. Among fibrosis mediating factors such as transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), interleukin-13 (IL-13), IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor (TNF-alpha), TGF-beta is considered as a critical mediator in normal wound healing as well as pathological fibrogenic processes. The TGF-beta transmits signals either directly or indirectly through types I, II and III (TbetaRI, II, and III) receptor complexes and activates downstream Smad signaling. The type III TGF-beta receptor (TbetaRIII or betaglycan) is a transmembrane proteoglycan without a functional kinase domain, and is regarded as a co-receptor to increase the affinity of ligand binding to TbetaRII. In addition, TbetaRIII act as a regulator in cell migration, invasion and cell growth in cancer models. However, in contrast to a great number of studies about TGF-beta ligand and TbetaRII signaling, the relationship between TGF-beta and TbetaRIII (or betaglycan) remains largely unknown. In this study, we searched for a new compound which inhibited TGF-beta responses using cell-based chemical screening and investigated the effects of the novel compound on radiation induced myofibroblast differentiation. We suggest that a novel small molecule, pyrazolopyrimidine compound IM-1662, can act as an anti-fibrotic agent through inhibiting expression of TGF-beta receptor type I and type II whereas, preserving the levels of TbetaRIII which seems to act as a negative regulator in TGF-beta signaling

  18. Expressions of Collagen I and III in Mesenchymal Stem Cells Co-cultured with Ligament Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Said; SLIMANI

    2005-01-01

    1 IntroductionThe poor or failed healing of some tendons and ligaments leads to the requirement of surgical replacement of grafts. Tissue engineering offers the appealing potential to improve the reconstruction of tissues presenting acceptable biological and mechanical properties. Mesenchymal stem cells (MSCs) have been shown to differentiate into several cells. The implantation of MSCs into damaged rabbit Achilles tendons significantly improved the structural properties of the neo-tissues, which suggests t...

  19. A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

    Energy Technology Data Exchange (ETDEWEB)

    Pan Hui; Kantharia, Sarah [Department of Biomedical Engineering, State University of New York-Stony Brook, Stony Brook, NY 11794-2580 (United States); Jiang Hongliang [Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027 (China); Chen Weiliam, E-mail: weiliam.chen@nyumc.org [Division of Wound Healing and Regenerative Medicine, Department of Surgery, New York University School of Medicine, New York, NY 10016 (United States)

    2011-12-15

    Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

  20. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    Science.gov (United States)

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  1. [Effects of Bushen Shuji Granule on IL-6 in the culture fluid sample of fibroblast cells from the synovial liquid of the ankylosing spondylitis patients].

    Science.gov (United States)

    Zhu, Jun-Ling; Yan, Xiao-Ping

    2012-02-01

    To explore the effects of Bushen Shuji Granule (BSG) on inhibiting the interleukin 6 (IL-6) level in the synovial fluid sample of fibroblast cells from the ankylosing spondylitis (AS) patients. Using serum pharmacologic method, the IL-6 level in the culture fluid sample of fibroblast cells was observed by ELISA method with different concentrations of medicated serum containing BSG. The IL-6 level at the mRNA level was detected using reverse transcriptase-polymerase chain reaction (RT-PCR). The vehicle serum and sulfasalazine (SSZ) serum were taken as controls. Results of ELISA showed the IL-6 level in the AS group was more than that in the vehicle serum group with obvious statistical difference. BSG could obviously inhibited the IL-6 level, showing statistical difference when compared with the vehicle serum group. Besides, obvious dose-dependent correlation existed between BSG and its inhibition on fibroblast proliferation. And the IL-6 level at the mRNA level in the AS group was higher than that in the vehicle serum group, showing statistical difference by semi-quantitative analysis. BSG could play its clinical role of anti-inflammation and anti-fibrosis through inhibiting the IL-6 level in the culture fluid sample of fibroblast cells.

  2. 人牙周膜成纤维细胞与牙龈成纤维细胞差异表达基因扣除文库的构建%Subtractive cDNA library construction of genes differentially xpressed in human periodontalligament fibroblast in comparison with gingival fibroblasts

    Institute of Scientific and Technical Information of China (English)

    郭希民; 吴补领; 肖明振; 蒲勤; 柴玉波; 朱峰; 赵忠良

    2000-01-01

    AIM: To construct a subtractive cDNA library of genes differentially expressed in cultured primary human PDLF in comparison with GF. METHODS: mRNAs were isolated fiom primary cultures of human PDLF and GF. Subtractive eDNA library of PDLF was constructed with a recently developed gene cloning technique that is based on PCR and subtractive hybridization. Part of randomly selected clones were identified by restriction analysis and reverse Southern dotblot. RESULTS: A subtbtetive cDNA library of PDLF was constructed with a capieity of 4×l02 clones. CONCLUSION:The gene cloning method we used in this study is both simple and effective in cloning differentially expressed gene.%目的:建立人牙周膜成纤维细胞(periodontal ligament fibroblast, PDLF) 与牙龈成纤维细胞(gin-gival fibroblast, GF)差异表达基因的扣除文库。方法:体外培养人PDLC和GF,分别提取mRNA,采用基于PCR和消减杂交的基因克隆技术构建人PDLC与GF差异表达基因的扣除文库。结果:成功构建了人PDLF与GF细胞差异表达基因的扣除文库,文库容量为4×l02。结论:基于PCR和消减杂交的基因克隆技术是构建细胞间差异表达基因扣除文库的较为简洁而有效的方法。

  3. The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs as a co-culture in vitro

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    Król Magdalena

    2012-03-01

    Full Text Available Abstract Background It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs in vitro. Results A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p Conclusion The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

  4. Human ovarian fibroblasts culture%人正常卵巢组织中成纤维细胞的培养

    Institute of Scientific and Technical Information of China (English)

    董丽华; 孙玮; 傅士龙; 韩素萍

    2011-01-01

    Objective; To obtain the purified human ovarian normal fibroblasls (NFs) preliminarily. Methods; The ovarian tissues were dissociated by 0.25% trypsin, after overgrowing on the bottom of the culture dish,the cells were adhered to the bottom of the dish repeatedly to depurate them. Then the cells were verified according to its morphology under the inverted-microscope and were further confined by a series of biological markers with immunocytochemistry. And its. Growth cure was observed. Results; The typical shape of ovarian NFs was fusiform, cords like,triangular,polyangular and irregular. Most of them contain Pseudopods-like structure. The NFs showed positive staining for vimentin.fibroblast specific protein-1, desmin and a-smooth muscle actin,negative staining for cytokeratinpan,cytokeratin7, cytokeratin 20, CD31, CD90, MMP-2, MMP-3, MMP-9, CA125, CA19-9, CEA, fibroblast activation proteina (FAP-a) and platelet-derived growth factor receptora (PDGFR-a). Conclusion; Human ovarian NFs were cultured successfully, which provides a tool for further studies on ovarian cancer and carcinoma associated fibroblasts.%目的:通过分离、纯化、培养及鉴定,获得典型人卵巢成纤维细胞.方法:采用酶消化+反复贴壁法,获得纯化成纤维细胞,通过形态学观察和免疫细胞化学染色,对所得细胞进行特征性标志及生物学特性的检测,进一步阐述细胞特征,并绘制细胞生长曲线.结果:形态学上,正常卵巢成纤维细胞形态多样,大部分为梭形、条索形,还有三角形、多角形和不规则形等,并具有伪足样外形;其生长潜伏期较长,12~14 d可传代;免疫细胞化学上,波形蛋白(vimentin)表达强阳性,结蛋白(desmin)、成纤维细胞特异性蛋白1(FSP-1)表达弱阳性;在较早期传代的细胞中,CK-7及CK-P呈少量阳性,随着细胞的纯化,其阳性表达渐行减少直至完全消失;肌动蛋白α(α-SMA)部分细胞表达阳性.而卵巢黏液性细胞标志CK-20

  5. The CLC-2 Chloride Channel Modulates ECM Synthesis, Differentiation, and Migration of Human Conjunctival Fibroblasts via the PI3K/Akt Signaling Pathway.

    Science.gov (United States)

    Sun, Lixia; Dong, Yaru; Zhao, Jing; Yin, Yuan; Zheng, Yajuan

    2016-06-09

    Recent evidence suggests that chloride channels are critical for cell proliferation, migration, and differentiation. We examined the effects of transforming growth factor (TGF)-β1 on chloride channel expression and associations with human conjunctival fibroblast (HConF) biology. To investigate the potential role of chloride channel (CLC)-2 in migration, transition to myofibroblasts and extracellular matrix (ECM) synthesis of HconF, a small interfering RNA (siRNA) approach was applied. TGF-β1-induced migration and transition of fibroblasts to myofibroblasts characterized by α-smooth muscle actin (α-SMA) expression, supported by increased endogenous expression of CLC-2 protein and mRNA transcripts. ECM (collagen I and fibronectin) synthesis in HConF was enhanced by TGF-β1. CLC-2 siRNA treatment reduced TGF-β1-induced cell migration, transition of fibroblasts to myofibroblasts, and ECM synthesis of HConF. CLC-2 siRNA treatment in the presence of TGF-β1 inhibited phosphorylation of PI3K and Akt in HConF. These findings demonstrate that CLC-2 chloride channels are important for TGF-β1-induced migration, differentiation, and ECM synthesis via PI3K/Akt signaling in HConF.

  6. Transforming growth factor type-β inhibits Mas receptor expression in fibroblasts but not in myoblasts or differentiated myotubes; Relevance to fibrosis associated to muscular dystrophies.

    Science.gov (United States)

    Cofre, Catalina; Acuña, María José; Contreras, Osvaldo; Morales, María Gabriela; Riquelme, Cecilia; Cabello-Verrugio, Claudio; Brandan, Enrique

    2015-01-01

    Duchenne muscular dystrophy is a genetic disorder characterized by myofiber degeneration, muscle weakness, and increased fibrosis. Transforming growth factor type-β (TGF-β), a central mediator of fibrosis, is upregulated in fibrotic diseases. Angiotensin-(1-7) [Ang-(1-7)] is a peptide with actions that oppose those of angiotensin-II (Ang II). Ang-(1-7) effects are mediated by the Mas receptor. Treatment with Ang-(1-7) produce positive effects in the mdx mouse, normalizing skeletal muscle architecture, decreasing local fibrosis, and fibroblasts, and improving muscle function. Mdx mice deficient for the Mas receptor showed the opposite effects. To identify the cell type(s) responsible for Mas receptor expression, and to characterize whether profibrotic effectors had any effect on its expression, we determined the effect of profibrotic agents on Mas expression. TGF-β, but not connective tissue growth factor or Ang-II, reduced the expression of Mas receptor in fibroblasts isolated from skeletal muscle cells and fibroblasts from two established cell lines. In contrast, no effects were observed in myoblasts and differentiated myotubes. This inhibition was mediated by the Smad-dependent (canonical) and the PI3K and MEK1/2 (noncanonical) TGF-β signaling pathways. When both canonical and noncanonical inhibitors of the TGF-β-dependent pathways were added together, the inhibitory effect of TGF-β on Mas expression was lost. The decrease in Mas receptor induced by TGF-β in fibroblasts reduced the Ang-(1-7) mediated stimulation of phosphorylation of AKT pathway proteins. These results suggest that reduction of Mas receptor in fibroblasts, by TGF-β, could increase the fibrotic phenotype observed in dystrophic skeletal muscle decreasing the beneficial effect of Ang-(1-7). © 2015 International Union of Biochemistry and Molecular Biology.

  7. Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture.

    Science.gov (United States)

    Mytych, Jennifer; Wnuk, Maciej; Rattan, Suresh I S

    2016-04-01

    Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 μg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells.

  8. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Yoshino A

    2016-08-01

    Full Text Available Atsushi Yoshino,1 Natalia Polouliakh,1–3 Akira Meguro,1 Masaki Takeuchi,1,4 Tatsukata Kawagoe,1 Nobuhisa Mizuki1 1Department of Ophthalmology and Visual Science, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 2Sony Computer Science Laboratories Inc., Fundamental Research Laboratories, 3Systems Biology Institute, Tokyo, Japan; 4Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Abstract: Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. Keywords: fish egg, antiaging, gene expression analysis, antioxidative gene, phylogenetic footprinting analysis

  9. Notch Signaling Mediates TNF-α-Induced IL-6 Production in Cultured Fibroblast-Like Synoviocytes from Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Zhijun Jiao

    2012-01-01

    Full Text Available It has been reported that Notch family proteins are expressed in synovium tissue and involved in the proliferation of synoviocyte from rheumatoid arthritis (RA. The aim of this paper was to investigate whether Notch signaling mediated TNF-α-induced cytokine production of cultured fibroblast-like synoviocytes (FLSs from RA. Exposure of RA FLSs to TNF-α (10 ng/ml led to increase of Hes-1, a target gene of Notch signaling, and a marked upregulation of Notch 2, Delta-like 1, and Delta-like 3 mRNA levels. Blockage of Notch signaling by a γ-secretase inhibitor (DAPT inhibited IL-6 secretion of RA FLSs in response to TNF-α while treatment with recombinant fusion protein of Notch ligand Delta-like 1 promoted such response. TNF-α stimulation also induced IL-6 secretion in OA FLSs; however, the Hes-1 level remained unaffected. Our data confirm the functional involvement of Notch pathway in the pathophysiology of RA FLSs which may provide a new target for RA therapy.

  10. Diminished internalization and action of 1,25-dihydroxyvitamin D3 in dermal fibroblasts cultured from New World primates

    Energy Technology Data Exchange (ETDEWEB)

    Adams, J.S.; Gacad, M.A.; Baker, A.J.; Kheun, G.; Rude, R.K.

    1985-06-01

    We investigated the occurrence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-resistant osteomalacia in the New World primate colony of Saguinus imperator at the Los Angeles Zoo. The mean serum concentration of 1,25-(OH)2D3 was elevated 5-fold in the New World primates compared to that in their Old World counterparts. The specific internalization of 0.6 nM (/sup 3/H)1,25-(OH)2D3 by cultured dermal fibroblasts from New World primates was reduced 75% compared to that by cells from Old World primates or man. The decrease in hormone uptake resulted from a decrease in the number of high affinity intracellular binding sites for 1,25-(OH)2D3 and apparently caused a 90-95% reduction in 1,25-(OH)2D3-induced 25-hydroxyvitamin-D3-24-hydroxylase activity. There was no alteration in the capacity or avidity of New World primate serum for 1,25-(OH)2D3 compared to that of serum from Old World primates. These data suggest that the occurrence of vitamin D-resistant osteomalacia in New World primates is the result of decreased high affinity, receptor-mediated uptake of 1,25-(OH)2D3 by the target cell.

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

    Science.gov (United States)

    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 (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

  12. Biocompatibility of potential wound management products: fungal mycelia as a source of chitin/chitosan and their effect on the proliferation of human F1000 fibroblasts in culture.

    Science.gov (United States)

    Chung, L Y; Schmidt, R J; Hamlyn, P F; Sagar, B F; Andrews, A M; Turner, T D

    1994-04-01

    Aspergillus oryzae, Mucor mucedo, and Phycomyces blakesleeanus cultures were examined as sources of chitin/chitosan. The nitrogen content of the alkali-treated mycelia/sporangiophores of A. oryzae, M. mucedo, and P. blakesleeanus was 2.52, 3.61, and 6.27% w/w, which relates to an estimated chitin content of 37, 52, and 91%, respectively. The effect of these fungal materials on the rate of proliferation of human F1000 fibroblasts in culture was examined. At 0.01% w/v, all three materials exhibited significant (P mucedo produced a significant (P mucedo, possessed cell attractant properties, again correlating with chitin content. If developed for use as wound management materials, the sporangiophores of P. blakesleeanus and the mycelium of M. mucedo could possibly promote the growth of fibroblasts and provide a matrix for their anchorage, thus contributing to the granulation phase of the healing cascade.

  13. Revelation of fibroblast protein commonalities and differences and their possible roles in wound healing and tumourigenesis using co-culture models of cells.

    Science.gov (United States)

    Jarkovska, Karla; Dvorankova, Barbora; Halada, Petr; Kodet, Ondrej; Szabo, Pavol; Gadher, Suresh Jivan; Motlik, Jan; Kovarova, Hana; Smetana, Karel

    2014-07-01

    The in vitro co-culture models of communication between normal fibroblasts and epithelial cells, such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia-induced changes in overall fibroblast proteome patterns. We were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light-chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts towards myofibroblasts or cancer-associated fibroblasts. Results showed coordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumourigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation. The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumourigenesis. In particular, epithelia-induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  14. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    Science.gov (United States)

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  15. Prostaglandin E2 Stimulates the Production of Vascular Endothelial Growth Factor through the E-Prostanoid–2 Receptor in Cultured Human Lung Fibroblasts

    Science.gov (United States)

    Nakanishi, Masanori; Sato, Tadashi; Nelson, Amy J.; Farid, Maha; Michalski, Joel; Kanaji, Nobuhiro; Wang, Xingqi; Basma, Hesham; Patil, Amol; Goraya, Jadvinder; Liu, Xiangde; Togo, Shinsaku; L. Toews, Myron; Holz, Olaf; Muller, Kai-Christian; Magnussen, Helgo

    2012-01-01

    Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E2 (PGE2) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE2 of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbecco's modified Eagle's medium, with or without PGE2, and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE2 and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE2 was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase–A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE2. The increased release of VEGF induced by PGE2 was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE2 can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall. PMID:22298530

  16. Linkages between the life-history evolution of tropical and temperate birds and the resistance of cultured skin fibroblasts to oxidative and non-oxidative chemical injury

    OpenAIRE

    Jimenez, Ana Gabriela; Harper, James M.; Simon A. Queenborough; Williams, Joseph B.

    2013-01-01

    A fundamental challenge facing physiological ecologists is to understand how variation in life history at the whole-organism level might be linked to cellular function. Thus, because tropical birds have higher annual survival and lower rates of metabolism, we hypothesized that cells from tropical species would have greater cellular resistance to chemical injury than cells from temperate species. We cultured dermal fibroblasts from 26 tropical and 26 temperate species of birds and examined cel...

  17. Inhibition by a selective IkappaB kinase-2 inhibitor of interleukin-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

    Science.gov (United States)

    Kondo, Yukiko; Fukuda, Ken; Adachi, Tadafumi; Nishida, Teruo

    2008-11-01

    Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-kappaB and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-kappaB activation is mediated by phosphorylation of the inhibitor IkappaB by IkappaB kinase (IKK)-2 and consequent IkappaB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts. Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IkappaBalpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively. IL-1beta-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration- and time-dependent manner. TPCA-1 inhibited the IL-1beta-induced expression of MMP-1, -3, and -9 in these cells at both the mRNA and protein levels and the IL-1beta-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IkappaBalpha and the nuclear translocation of NF-kappaB induced by IL-1beta. An IKK-2 inhibitor blocked IL-1beta-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-kappaB signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.

  18. Exposure to Varying Strain Magnitudes Influences the Conversion of Normal Skin Fibroblasts Into Hypertrophic Scar Cells.

    Science.gov (United States)

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Cai, Xia; Liu, Tao

    2016-04-01

    Mechanical strain is a key contributor in the pathogenesis of hypertrophic scarring, whose optimal stretch magnitudes to initiate the differentiation of normal skin fibroblasts into aberrant fibroblasts phenotype remains largely unresolved. Influence of varying cyclic strain magnitudes on cultured human normal skin fibroblasts and its transformation into hypertrophic scar fibroblast-like phenotype is investigated in this study. Cultured fibroblasts isolated from hypertrophic scar and normal skin tissue were subjected to cyclic mechanical stretching under individual 10%, 15%, and 20% strain magnitudes at a frequency of 0.1 Hz for 24 hours. Stretched normal skin fibroblasts demonstrated significantly increased rates of cell proliferation, and also apparently oriented away nearly perpendicular to the applied stretching direction. Interestingly, the applied 10% strains magnitude resulted in a markedly enhanced cell proliferative ability compared with that of 20% strain magnitude. Parameters involving the mechanotransduction signaling, such as integrin β1 and P130Cas, were significantly improved at both mRNA and protein levels in the stretched normal skin fibroblasts, which was demonstrated in a negative magnitude-dependent manner. In addition, 10% strains magnitude triggered the highest expression levels of growth factor TGF-β1 and collagen matrix in stretched normal skin fibroblasts. Collectively, these results indicate that the 10% stretching magnitude, of the 3 strain magnitudes studied, is most effective for triggering the optimal mechanotransduction effects and biological responses inside cultured skin fibroblasts. The demonstrable conversion of normal skin fibroblasts into hypertrophic scar fibroblasts was also observed when 10% stretching magnitude was applied to cultured fibroblasts in vitro.

  19. Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena A Goncharova

    Full Text Available TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1(-/- MEFs have decreased migration compared to littermate-derived Tsc1(+/+ MEFs. Migration of Tsc1(-/- MEFs with re-expressed TSC1 was comparable to Tsc1(+/+ MEF migration. In contrast, Tsc2(-/- MEFs showed an increased migration compared to Tsc2(+/+ MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1(-/- MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2(-/- MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1(-/- or Tsc2(-/- MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2(-/- MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2-null cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes

  20. Metabolites of Hypoxic Cardiomyocytes Induce the Migration of Cardiac Fibroblasts.

    Science.gov (United States)

    Shi, Huairui; Zhang, Xuehong; He, Zekun; Wu, Zhiyong; Rao, Liya; Li, Yushu

    2017-01-01

    The migration of cardiac fibroblasts to the infarct region plays a major role in the repair process after myocardial necrosis or damage. However, few studies investigated whether early hypoxia in cardiomyocytes induces the migration of cardiac fibroblasts. The purpose of this study was to assess the role of metabolites of early hypoxic cardiomyocytes in the induction of cardiac fibroblast migration. Neonatal rat heart tissue was digested with a mixture of trypsin and collagenase at an appropriate ratio. Cardiomyocytes and cardiac fibroblasts were cultured via differential adhesion. The cardiomyocyte cultures were subjected to hypoxia for 2, 4, 6, 8, 10, and 12 h. The supernatants of the cardiomyocyte cultures were collected to determine the differences in cardiac fibroblast migration induced by hypoxic cardiomyocyte metabolites at various time points using a Transwell apparatus. Meanwhile, ELISA was performed to measure TNF-α, IL-1β and TGF-β expression levels in the cardiomyocyte metabolites at various time points. The metabolites of hypoxic cardiomyocytes significantly induced the migration of cardiac fibroblasts. The induction of cardiac fibroblast migration was significantly enhanced by cardiomyocyte metabolites in comparison to the control after 2, 4, and 6 h of hypoxia, and the effect was most significant after 2 h. The expression levels of TNF-α, IL-1β, IL-6, and TGF-β were substantially increased in the metabolites of cardiomyocytes, and neutralization with anti-TNF-α and anti-IL-1β antibodies markedly reduced the induction of cardiac fibroblast migration by the metabolites of hypoxic cardiomyocytes. The metabolites of early hypoxic cardiomyocytes can induce the migration of cardiac fibroblasts, and TNF-α and IL-1β may act as the initial chemotactic inducers. © 2017 The Author(s) Published by S. Karger AG, Basel.

  1. Surface modified superparamagnetic nanoparticles: Interaction with fibroblasts in primary cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Chapa Gonzalez, Christian; Roacho Pérez, Jorge A.; Martínez Pérez, Carlos A.; Olivas Armendáriz, Imelda [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Jimenez Vega, Florinda [Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo envolvente del PRONAF y Estocolmo, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Castrejon Parga, Karen Y. [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Garcia Casillas, Perla E., E-mail: pegarcia@uacj.mx [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico)

    2014-12-05

    Highlights: • An inorganic layer before an organic material shell onto MNPs improves cell viability. • The coating type and the concentration of nanoparticles directly affect cell viability. • Modified magnetite nanoparticles with organic and inorganic materials was developed. - Abstract: The development of a variety of medical applications such as drug delivery, cell labeling, and medical imaging have been possible owing to the unique features exhibited by magnetic nanoparticles. Nanoparticle–cell interaction is related to the surface aspects of nanoparticle, which may be described based on their chemistry or inorganic/organic characteristics. The coating on particle surface reduces the inter-particle interactions and provides properties such as biocompatibility. Among the coating materials used for nanoparticles employed in biomedical applications, oleic acid is one of the most utilized due to its biocompatibility. However, a major drawback with this naturally occurring fatty acid is that it is easily oxidized by cells and this reduces their performance in biomedical applications. In order to avoid the direct contact of the cell with the magnetite particle, coating with an inorganic material prior to the oleic acid shell would be effective. This would retard the magnetite dissociation thereby improve the cell viability. Here we report our investigation on the effect of surface modified magnetite nanoparticles (MNPs) on the cell viability using primary cultures incubated with those particles. We prepared magnetite nanoparticles by chemical co-precipitation method; nanoparticle surface was first modified by silanol condensation followed by chemisorption of oleic acid. All nanostructures have a particle size less than 100 nm, depending on the material coating and superparamagnetic behavior. The saturated magnetizations (M{sub s}) of the magnetite samples coated with oleic acid (MAO; 49.15 emu/g) and double shell silica-oleic acid (MSAO; 46.16 emu/g) are

  2. Substratum topography modulates corneal fibroblast to myofibroblast transformation.

    Science.gov (United States)

    Myrna, Kathern E; Mendonsa, Rima; Russell, Paul; Pot, Simon A; Liliensiek, Sara J; Jester, James V; Nealey, Paul F; Brown, Donald; Murphy, Christopher J

    2012-02-01

    The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFβ-induced myofibroblast transformation of corneal cells. Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFβ at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed. Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.

  3. Diet, social differentiation and cultural change in Roman Britain

    DEFF Research Database (Denmark)

    Cheung, Christina; Schroeder, Hannes; Hedges, R. E. M.

    2012-01-01

    This study uses stable isotope analyses (d 13 C and d 15 N) of human bone collagen to reconstruct the diet of three Romano-British (first to early fifth century AD) populations from Gloucestershire in South West England. Gloucestershire was an important part of Roman Britain with two major admini...... sensitive, if settlement-specific, indicator of social differentiation and culture change.......This study uses stable isotope analyses (d 13 C and d 15 N) of human bone collagen to reconstruct the diet of three Romano-British (first to early fifth century AD) populations from Gloucestershire in South West England. Gloucestershire was an important part of Roman Britain with two major...

  4. Differential gene expression in human fibroblasts after alpha-particle emitter (211)At compared with (60)Co irradiation

    DEFF Research Database (Denmark)

    Danielsson, Anna; Claesson, Kristina; Parris, Toshima Z;

    2013-01-01

    trastuzumab monoclonal antibody (0.25, 0.5, and 1 Gy) and (60)Co (1, 2, and 3 Gy). Results: We report gene expression profiles that distinguish the effect different radiation qualities and absorbed doses have on cellular functions in human fibroblasts. In addition, we identified commonly expressed transcripts...

  5. Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

    Science.gov (United States)

    Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

    2016-10-01

    Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

  6. Transcriptional and Posttranscriptional Inhibition of Lysyl Oxidase Expression by Cigarette Smoke Condensate in Cultured Rat Fetal Lung Fibroblasts

    Science.gov (United States)

    Gao, Song; Chen, Keyang; Zhao, Yinzhi; Rich, Celeste B.; Chen, Lijun; Li, Sandy J.; Toselli, Paul; Stone, Phillip; Li, Wande

    2005-01-01

    Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0–120 μg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80–120 μg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 μg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 μg CSC/ml. Moreover, 80–120 μg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema. PMID:15933228

  7. Simultaneous Expression of Cancer Stem Cell-Like Properties and Cancer-Associated Fibroblast-Like Properties in a Primary Culture of Breast Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, Mami; Inoue, Takahiro; Shirai, Takuma; Takamatsu, Kazuhiko; Kunihiro, Shiori; Ishii, Hirokazu [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Nishikata, Takahito, E-mail: nisikata@konan-u.ac.jp [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe 650-0047 (Japan)

    2014-07-31

    The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.

  8. Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    Liya; Pi; Pei-Yu; Chung; Sriniwas; Sriram; Masmudur; M; Rahman; Wen-Yuan; Song; Edward; W; Scott; Bryon; E; Petersen; Gregory; S; Schultz

    2015-01-01

    .01). Cells containing the cystine knot of BMP-4 expressed the second most activity,with a 24.77 ± 0.47 fold increase(P < 0.01). Cells that contained the cystine knot of TGF-β1 had a 3.80 ± 0.66 fold increase(P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of β-galactosidase activity(P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant(KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGFBB ligand and PDGFRβ receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition,CTGF potentiated phosphorylation of PDGFRβ and AKT in rabbit corneal fibroblast cells stimulated by PDGFBB in tissue culture condition. In contrast,CTGF did not affect PDGF-B induced phosphorylation of ERK1/2.CONCLUSION:CTGF has a differential binding affinity to VEGF-A,PDGF-B,BMP-4,and TGF-β. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

  9. Highly cytocompatible and flexible three-dimensional graphene/polydimethylsiloxane composite for culture and electrochemical detection of L929 fibroblast cells.

    Science.gov (United States)

    Waiwijit, Uraiwan; Maturos, Thitima; Pakapongpan, Saithip; Phokharatkul, Ditsayut; Wisitsoraat, Anurat; Tuantranont, Adisorn

    2016-08-01

    Recently, three-dimensional graphene interconnected network has attracted great interest as a scaffold structure for tissue engineering due to its high biocompatibility, high electrical conductivity, high specific surface area and high porosity. However, free-standing three-dimensional graphene exhibits poor flexibility and stability due to ease of disintegration during processing. In this work, three-dimensional graphene is composited with polydimethylsiloxane to improve the structural flexibility and stability by a new simple two-step process comprising dip coating of polydimethylsiloxane on chemical vapor deposited graphene/Ni foam and wet etching of nickel foam. Structural characterizations confirmed an interconnected three-dimensional multi-layer graphene structure with thin polydimethylsiloxane scaffold. The composite was employed as a substrate for culture of L929 fibroblast cells and its cytocompatibility was evaluated by cell viability (Alamar blue assay), reactive oxygen species production and vinculin immunofluorescence imaging. The result revealed that cell viability on three-dimensional graphene/polydimethylsiloxane composite increased with increasing culture time and was slightly different from a polystyrene substrate (control). Moreover, cells cultured on three-dimensional graphene/polydimethylsiloxane composite generated less ROS than the control at culture times of 3-6 h. The results of immunofluorescence staining demonstrated that fibroblast cells expressed adhesion protein (vinculin) and adhered well on three-dimensional graphene/polydimethylsiloxane surface. Good cell adhesion could be attributed to suitable surface properties of three-dimensional graphene/polydimethylsiloxane with moderate contact angle and small negative zeta potential in culture solution. The results of electrochemical study by cyclic voltammetry showed that an oxidation current signal with no apparent peak was induced by fibroblast cells and the oxidation current at an

  10. Placental-derived stem cells: Culture, differentiation andchallenges

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Stem cell therapy is a promising approach to clinicalhealing in several diseases. A great variety of tissues(bone marrow, adipose tissue, and placenta) arepotentially sources of stem cells. Placenta-derivedstem cells (p-SCs) are in between embryonic andmesenchymal stem cells, sharing characteristics withboth, such as non-carcinogenic status and property todifferentiate in all embryonic germ layers. Moreover,their use is not ethically restricted as fetal membranesare considered medical waste after birth. In this context,the present review will be focused on the biologicalproperties, culture and potential cell therapy usesof placental-derived stem cells. Immunophenotypecharacterization, mainly for surface marker expression,and basic principles of p-SC isolation and culture(mechanical separation or enzymatic digestion ofthe tissues, the most used culture media, cell platingconditions) will be presented. In addition, somepreclinical studies that were performed in differentmedical areas will be cited, focusing on neurological,liver, pancreatic, heart, muscle, pulmonary, and bonediseases and also in tissue engineering field. Finally,some challenges for stem cell therapy applications willbe highlighted. The understanding of the mechanismsinvolved in the p-SCs differentiation and the achievementof pure cell populations (after differentiation) arekey points that must be clarified before bringing thepreclinical studies, performed at the bench, to themedical practice.

  11. [The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

    Science.gov (United States)

    Poleshko, A G; Lobanok, E S; Mezhevikina, L M; Fesenko, E E; Volotkovskiĭ, I D

    2014-01-01

    It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

  12. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  13. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.

    Science.gov (United States)

    Doi, Akiko; Park, In-Hyun; Wen, Bo; Murakami, Peter; Aryee, Martin J; Irizarry, Rafael; Herb, Brian; Ladd-Acosta, Christine; Rho, Junsung; Loewer, Sabine; Miller, Justine; Schlaeger, Thorsten; Daley, George Q; Feinberg, Andrew P

    2009-12-01

    Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P cancer-specific DMRs (C-DMRs; 3.6-fold, P cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.

  14. Helium-neon laser treatment transforms fibroblasts into myofibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Pourreau-Schneider, N.; Ahmed, A.; Soudry, M.; Jacquemier, J.; Kopp, F.; Franquin, J.C.; Martin, P.M. (CNRS Unite 1175, Marseille (France))

    1990-07-01

    The differentiation of myofibroblastic cells from normal human gingival fibroblasts in vitro has been established by transmission electron microscopy and quantitated by immunohistochemistry, using antigelsolin monoclonal antibodies. Untreated control cultures were compared to cultures exposed to Helium-Neon (He-Ne) laser irradiation. A direct and massive transformation of the cultured fibroblasts into myofibroblasts was observed as early as 24 hours after laser treatment, whereas control cultures were comprised of only resting fibroblasts and active fibroblasts. This in vitro induction of myofibroblasts may be analogous to that which occurs in vivo. Therefore we undertook a similar study using biopsies from gingival tissues after wisdom tooth extraction. Myofibroblasts were present in the connective tissue of laser-treated gums 48 hours after irradiation, but not in untreated contralateral control tissues. These data provide evidence that the primary biologic effect of the Helium-Neon laser on connective tissue is the rapid generation of myofibroblasts from fibroblasts. The induction of a phenotype with contractile properties may have clinical significance in the acceleration of the wound-healing process.

  15. Differential effect of culture temperature and specific growth rate on CHO cell behavior in chemostat culture.

    Science.gov (United States)

    Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodríguez-Moyá, María; Gonzalez, Ramon; Altamirano, Claudia

    2014-01-01

    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h(-1)) and low dilution rate (0.012 h(-1)) at two cultivation temperatures (37 and 33 °C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33 °C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.

  16. Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts.

    Science.gov (United States)

    Wagstaff, P; Kang, H Y; Mylott, D; Robbins, P J; White, M K

    1995-01-01

    Vertebrate cells that are transformed by oncogenes such as v-src or are stimulated by mitogens have increased rates of glucose uptake. In rodent cells, the mechanisms whereby glucose transport is up-regulated are well understood. Stimulation of glucose transport involves an elevation in mRNA encoding the GLUT1 glucose transporter that is controlled at the levels of both transcription and mRNA stability. Cloning and sequencing of chicken GLUT1 cDNA showed that it shares 95% amino acid sequence similarity to mammalian GLUT1s. Nevertheless, unlike mammalian GLUT1 mRNA, it was not induced by v-src, serum addition, or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in chicken embryo fibroblasts. Rather, the induction of glucose transport in chicken embryo fibroblasts by v-src, serum, and 12-O-tetradecanoylphorbol 13-acetate was associated with induction of GLUT3 mRNA level and GLUT3 transcription. Rat fibroblasts were also found to express both GLUT1 and GLUT3 isoforms, but v-src induced GLUT1 and not GLUT3. This suggests that animal cells require both a basal and an upregulatable glucose transporter and that these functions have been subsumed by different GLUT isoforms in avian and mammalian cells. Images PMID:8589457

  17. A simple and effective protocol for fast isolation of human Tenon's fibroblasts from a single trabeculectomy biopsy - a comparison of cell behaviour in different culture media.

    Science.gov (United States)

    Przekora, Agata; Zarnowski, Tomasz; Ginalska, Grazyna

    2017-01-01

    Human Tenon's fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco's modified Eagle's medium (DMEM). We used Eagle's minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM. Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 10(6) vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production. Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some

  18. Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring.

    Science.gov (United States)

    Crawford, Justin; Nygard, Karen; Gan, Bing Siang; O'Gorman, David Brian

    2015-02-01

    Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix-associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type-I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three-dimensional type-I collagen lattices. Inhibition of Rho-associated protein kinase activity significantly attenuated the effects of matrix-associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix-associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho-associated protein kinase-dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.

  19. Identification of lipid profile on γ-irradiated primary cultured human diploid fibroblasts by UPLC-ESI-QTOF-MS

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Kyeong; Yun, Hyun Jin; Kim, Sun Young; Kim, So Ra; Kim, Eun Ju; Kang, Chang Mo [Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Hwang, Geum Sook [Integrated Metabolomics Research Group, Korea Basic Science Institute, Sejong (Korea, Republic of)

    2013-11-15

    The study of biomarkers in normal-tissue radiobiology is of great importance to people subjected to non-destructive testing, to nuclear power industry, and to radiological terrorism. Cytogenetics has remained the 'gold standard' for the detection of chromosomal changes, but this method is cumbersome and time consuming. Therefore, it is necessary to establish a rapid, high-throughput biological monitoring protocol to determine the exposure radiation dose in order to rapidly triage mass casualties. Metabolomics studies is an effective platform for identification and quantification of the complete set of metabolites in a biological system, and it has been successfully provides important insights into physiological and disease states and facilitate in depth understanding of underlying biochemical pathways. Radiation metabolomics has been used to identify radiation effects at the metabolite levels in cells and in urine or serum collected from laboratory animals such as mouse, rat and monkey. Recently, a few radiation metabolomics studies showed that multiple metabolites related to the purine, inositol or pyrimidine metabolism, fatty acid biosynthesis and glycolysis/gluconeogenesis could be potential minimal-invasive markers for radiation exposure. However, there is little consistency in these studies due to differences in analyzed sample (cells, urine, serum), animal models (mouse, rat and monkey). Furthermore, the biological effects of ionizing radiation (IR) were differently regulated in the different absorbed doses, dose-rates and type of IR. Thus, it is necessary to explore the entire biosphere profiling of metabolic markers to make reasonable biological explanation under radiation exposure. We hypothesized that the variations in lipid expression in IR-induced cells would result in a unique lipid profile in human diploid fibroblasts (HDF), which can be detected with the targeted and quantitative metabolomics approach. Irradiated HDF cells showed the dose

  20. In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

    Science.gov (United States)

    Chen, X C; Liu, H; Li, H; Cheng, Y; Yang, L; Liu, Y F

    2016-06-27

    In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.

  1. Apoptotic markers in cultured fibroblasts correlate with brain metabolites and regional brain volume in antipsychotic-naive first-episode schizophrenia and healthy controls.

    Science.gov (United States)

    Batalla, A; Bargalló, N; Gassó, P; Molina, O; Pareto, D; Mas, S; Roca, J M; Bernardo, M; Lafuente, A; Parellada, E

    2015-08-25

    Cultured fibroblasts from first-episode schizophrenia patients (FES) have shown increased susceptibility to apoptosis, which may be related to glutamate dysfunction and progressive neuroanatomical changes. Here we determine whether apoptotic markers obtained from cultured fibroblasts in FES and controls correlate with changes in brain glutamate and N-acetylaspartate (NAA) and regional brain volumes. Eleven antipsychotic-naive FES and seven age- and gender-matched controls underwent 3-Tesla magnetic resonance imaging scanning. Glutamate plus glutamine (Glx) and NAA levels were measured in the anterior cingulate (AC) and the left thalamus (LT). Hallmarks of apoptotic susceptibility (caspase-3-baseline activity, phosphatidylserine externalization and chromatin condensation) were measured in fibroblast cultures obtained from skin biopsies after inducing apoptosis with staurosporine (STS) at doses of 0.25 and 0.5 μM. Apoptotic biomarkers were correlated to brain metabolites and regional brain volume. FES and controls showed a negative correlation in the AC between Glx levels and percentages of cells with condensed chromatin (CC) after both apoptosis inductions (STS 0.5 μM: r = -0.90; P = 0.001; STS 0.25 μM: r = -0.73; P = 0.003), and between NAA and cells with CC (STS 0.5 μM induction r = -0.76; P = 0.002; STS 0.25 μM r = -0.62; P = 0.01). In addition, we found a negative correlation between percentages of cells with CC and regional brain volume in the right supratemporal cortex and post-central region (STS 0.25 and 0.5 μM; P < 0.05 family-wise error corrected (FWEc)). We reveal for the first time that peripheral markers of apoptotic susceptibility may correlate with brain metabolites, Glx and NAA, and regional brain volume in FES and controls, which is consistent with the neuroprogressive theories around the onset of the schizophrenia illness.

  2. Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Su, Xiaohu; Ling, Yu; Liu, Chunxia; Meng, Fanhua; Cao, Junwei; Zhang, Li; Zhou, Huanmin; Liu, Zongzheng; Zhang, Yanru

    2015-08-01

    We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.

  3. Biomedical advances from tissue culture

    OpenAIRE

    Okamoto, Tetsuji; Sato, J. Denry; Barnes, David W.; Sato, Gordon H.

    2013-01-01

    The demonstration that the “dedifferentiation” of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that...

  4. Celiac anti-type 2 transglutaminase antibodies induce differential effects in fibroblasts from celiac disease patients and from healthy subjects.

    Science.gov (United States)

    Paolella, Gaetana; Lepretti, Marilena; Barone, Maria Vittoria; Nanayakkara, Merlin; Di Zenzo, Marina; Sblattero, Daniele; Auricchio, Salvatore; Esposito, Carla; Caputo, Ivana

    2017-03-01

    Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

  5. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  6. Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

    Science.gov (United States)

    Spooner, B. S.; Hardman, P.; Paulsen, A.

    1994-01-01

    Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

  7. Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors.

    Directory of Open Access Journals (Sweden)

    Verónica Ramos-Mejía

    Full Text Available Human induced pluripotent stem cells (hiPSC have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM. The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.

  8. Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

    Science.gov (United States)

    Mackey, Abigail L; Magnan, Mélanie; Chazaud, Bénédicte; Kjaer, Michael

    2017-08-01

    Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable

  9. Human 45,X Fibroblast Transcriptome Reveals Distinct Differentially Expressed Genes Including Long Noncoding RNAs Potentially Associated with the Pathophysiology of Turner Syndrome

    Science.gov (United States)

    Patowary, Ashok; Scaria, Vinod; Sivasubbu, Sridhar; Deobagkar, Deepti D.

    2014-01-01

    Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s) in the establishment of Turner syndrome phenotypes. PMID:24932682

  10. Human 45,X fibroblast transcriptome reveals distinct differentially expressed genes including long noncoding RNAs potentially associated with the pathophysiology of Turner syndrome.

    Directory of Open Access Journals (Sweden)

    Shriram N Rajpathak

    Full Text Available Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s in the establishment of Turner syndrome phenotypes.

  11. Human 45,X fibroblast transcriptome reveals distinct differentially expressed genes including long noncoding RNAs potentially associated with the pathophysiology of Turner syndrome.

    Science.gov (United States)

    Rajpathak, Shriram N; Vellarikkal, Shamsudheen Karuthedath; Patowary, Ashok; Scaria, Vinod; Sivasubbu, Sridhar; Deobagkar, Deepti D

    2014-01-01

    Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s) in the establishment of Turner syndrome phenotypes.

  12. Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide.

    Science.gov (United States)

    Balzer, Julian; Heuer, Kiara; Demir, Erhan; Hoffmanns, Martin A; Baldus, Sabrina; Fuchs, Paul C; Awakowicz, Peter; Suschek, Christoph V; Opländer, Christian

    2015-01-01

    The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal "cold" atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60-300 s; ~77-66%) or exposure to freshly DBD-treated PBS (60-300 s; ~75-55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in

  13. Non-Thermal Dielectric Barrier Discharge (DBD Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide.

    Directory of Open Access Journals (Sweden)

    Julian Balzer

    Full Text Available The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal "cold" atmospheric plasma (CAP. Dielectric barrier discharge (DBD devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2 and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS led to a treatment time-dependent acidification (pH 6.7; 300 s and coincidently accumulation of nitrite (~300 μM, nitrate (~1 mM and H2O2 (~200 μM. Fibroblast viability was reduced by single DBD treatments (60-300 s; ~77-66% or exposure to freshly DBD-treated PBS (60-300 s; ~75-55%, accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well

  14. Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide

    Science.gov (United States)

    Demir, Erhan; Hoffmanns, Martin A.; Baldus, Sabrina; Fuchs, Paul C.; Awakowicz, Peter; Suschek, Christoph V.; Opländer, Christian

    2015-01-01

    The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well

  15. Optimal primary culture conditions of human gastric mucosal fibroblasts%人胃黏膜成纤维细胞原代培养条件的优化

    Institute of Scientific and Technical Information of China (English)

    张浩; 申晓军; 唐古生; 毕建威

    2009-01-01

    目的 通过优化人胃黏膜成纤维细胞的原代培养条件,提高培养成功率,为进一步研究胃癌相关成纤维细胞奠定基础.方法 原代培养采用植块法,利用免疫组织化学、细胞形态学和电镜等方法进行细胞鉴定;分析不同培养表面、培养基和培养液pH值对原代细胞培养游出情况的影响,筛选适合的培养条件.结果 Logistic逐步回归分析提示RPMI 1640培养基的Waldχ2值=32.4533,P0.05),而且各培养条件之间无交互作用.结论 通过优化培养表面、培养基种类和pH值等培养条件,可以提高人胃黏膜成纤维细胞原代培养的成功率.%Objective To optimize primary culture conditions of human gastric mucosal fibre- blasts for further investigations of gastric cancer associated fibroblasts. Methods The fibroblasts were cul-tured primarily by explant method, and identified by immunohistochemistry, morphology and electron mi-croscopy. The effects of different culture conditions on emigration ratios of ceils were investigated to opti-mize the culture surface, medium and pH in primary culture of gastric mucosal fibroblasts. Results When the other conditions were the same,no significandy different emigration rate was found on the mouse tail-coated surface,fetal bovine serum-coated surface ,and nothing coated surface (P > 0.05 ). As to the role of different culture media including RPMI 1640 medium, DMEM and high-gluco DMEM,there was signifi-cantly higher emigration rate in the RPMI 1640 medium compared with other two media (P < 0.01, stand- ardized estimate = 0. 4688 ). Different pH values(pH = 7.0,7.4 and 7.8)of culture solution led to differ-ent emigration rate,and the best emigration rate was found in pH = 7.4 solution(P < 0.01, standardized estimate =0.5857 ). Furthermore, there was no interaetional effect on the cell emigration rate among cul- ture conditions. Conclusion The primary culture of human gastric mucosal fibroblasts can be greatly im-proved by

  16. Prenatal diagnosis of Zellweger syndrome by measurement of very long chain fatty acid (C26 : 0) β-oxidation in cultured chorionic villous fibroblasts: Implications for early diagnosis of other peroxisomal disorders

    NARCIS (Netherlands)

    Wanders, R.J.A.; Wijland, M.J.A. van; Roermund, C.W.T. van; Schutgens, R.B.H.; Bosch, H. van den; Tager, J.M.; Nijenhuis, A.; Tromp, A.

    1987-01-01

    In this paper we show that cultured chorionic villous fibroblasts efficiently catalyse the peroxisomal β-oxidation of hexacosanoic acid (cerotic acid), a saturated very long chain fatty acid containing 26 carbon atoms. Hexacosanoic β-oxidation was found to be strongly impaired in cultured chorionic

  17. Differential expression of insulin like growth factor I and other fibroblast mitogens in porcine colostrum and milk

    Energy Technology Data Exchange (ETDEWEB)

    Tan, T.J.; Simmen, R.C.M.; Simmen, F.A.

    1987-05-01

    Sow mammary secretions contain at least 3 distinct growth factor activities, distinguished by their size and relative abundance in colostrum or later milk. Gel filtration of colostrum in Sephadex G-200 columns, followed by acid-ethanol extraction and radioimmunoassay (RIA) for insulin like growth factor I (IGF-I) revealed high levels of this factor in the 150K and 50K MW regions, characteristic of IGF-I: binding protein complexes. Acid treatment of these fractions yielded free IGF-I peptide (7.5K). Parallel mitogen assays with a fibroblast cell line (AKR-2B) demonstrated a predominant peak of high MW activity (sow colostral growth factor-I, SCGF-I) eluting near the column void volume (MW > 150K). Treatment of SCGF-I with 1M acetic acid resulted in a size reduction of the mitogenic activity (MW < 10K), suggesting association of SCGF-I with a binding protein. The SCGF-I peptide was noncompetitive in IGF-I RIA, was distinct in MW from free IGF-I, and was not mitogenic for chick embryo fibroblasts. Sow milk contains less IGF-I and SCGF-I but does display a predominant peak of small MW (approx. 3K) AKR-2B activity. The changes in expression of these growth factors during lactation may reflect differing roles in lactogenesis and/or neonatal growth and development.

  18. Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts.

    Science.gov (United States)

    Ghayad, Sandra E; Rammal, Ghina; Ghamloush, Farah; Basma, Hussein; Nasr, Rihab; Diab-Assaf, Mona; Chelala, Claude; Saab, Raya

    2016-11-17

    Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes.

  19. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  20. Culturally Responsive Differentiated Instruction: Narrowing Gaps between Best Pedagogical Practices Benefiting All Learners

    Science.gov (United States)

    Santamaria, Lorri J.

    2009-01-01

    Background/Context: Because of its special education association, differentiated instruction (DI) is a topic of concern for many educators working with culturally and linguistically diverse (CLD) learners, whereby bilingual, multicultural, and culturally responsive teaching (CRT) is considered more appropriate for responding to cultural and…

  1. A Co-Culture Model of Fibroblasts and Adipose Tissue-Derived Stem Cells Reveals New Insights into Impaired Wound Healing After Radiotherapy

    Directory of Open Access Journals (Sweden)

    Frank Haubner

    2015-10-01

    Full Text Available External radiation seems to be associated with increased amounts of cytokines and other cellular modulators. Impaired microcirculation and fibrosis are examples of typical long term damage caused by radiotherapy. Adipose tissue-derived stem cells (ASC are discussed to enhance wound healing, but their role in wounds due to radiotherapy is poorly understood. Normal human fibroblasts (NHF and ASCs were co-cultured and external radiation with doses from 2–12 Gray (Gy was delivered. Cell proliferation and mRNA levels of matrix metalloproteinases (MMP1, MMP2 and MMP13 were determined 48 h after irradiation of the co-cultures by qPCR. Additionally, tissue inhibitors of matrix metalloproteinases (TIMP1, TIMP2 were determined by enzyme-linked immunosorbent assay (ELISA. There was a reduction of cell proliferation after external radiation in mono-cultures of NHFs and ASCs compared to controls without irradiation. The co-culture of ASCs and NHFs showed reduced impairment of cell proliferation after external radiation. Gene expression of MMP1 and MMP13 was reduced after external irradiation in NHF. MMP2 expression of irradiated NHFs was increased. In the co-culture setting, MMP1 and MMP2 gene expression levels were upregulated. TIMP1 and TIMP2 protein expression was increased after irradiation in NHFs and their co-cultures with ASCs. ASCs seem to stimulate cell proliferation of NHFs and modulate relevant soluble mediators as well as proteinases after external radiation.

  2. A Co-Culture Model of Fibroblasts and Adipose Tissue-Derived Stem Cells Reveals New Insights into Impaired Wound Healing After Radiotherapy.

    Science.gov (United States)

    Haubner, Frank; Muschter, Dominique; Pohl, Fabian; Schreml, Stephan; Prantl, Lukas; Gassner, Holger G

    2015-10-29

    External radiation seems to be associated with increased amounts of cytokines and other cellular modulators. Impaired microcirculation and fibrosis are examples of typical long term damage caused by radiotherapy. Adipose tissue-derived stem cells (ASC) are discussed to enhance wound healing, but their role in wounds due to radiotherapy is poorly understood. Normal human fibroblasts (NHF) and ASCs were co-cultured and external radiation with doses from 2-12 Gray (Gy) was delivered. Cell proliferation and mRNA levels of matrix metalloproteinases (MMP1, MMP2 and MMP13) were determined 48 h after irradiation of the co-cultures by qPCR. Additionally, tissue inhibitors of matrix metalloproteinases (TIMP1, TIMP2) were determined by enzyme-linked immunosorbent assay (ELISA). There was a reduction of cell proliferation after external radiation in mono-cultures of NHFs and ASCs compared to controls without irradiation. The co-culture of ASCs and NHFs showed reduced impairment of cell proliferation after external radiation. Gene expression of MMP1 and MMP13 was reduced after external irradiation in NHF. MMP2 expression of irradiated NHFs was increased. In the co-culture setting, MMP1 and MMP2 gene expression levels were upregulated. TIMP1 and TIMP2 protein expression was increased after irradiation in NHFs and their co-cultures with ASCs. ASCs seem to stimulate cell proliferation of NHFs and modulate relevant soluble mediators as well as proteinases after external radiation.

  3. Development of a chemically defined in vitro culture system to effectively stimulate the proliferation of adult human dermal fibroblasts.

    Science.gov (United States)

    Kim, Min Seong; Yun, Jung Im; Gong, Seung Pyo; Ahn, Ji Yeon; Lim, Jeong Mook; Song, Young Han; Park, Kyu Hyun; Lee, Seung Tae

    2015-07-01

    Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 μg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 μg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.

  4. A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

    Directory of Open Access Journals (Sweden)

    Kristín Rós Kjartansdóttir

    Full Text Available Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

  5. A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

    Science.gov (United States)

    Kjartansdóttir, Kristín Rós; Reda, Ahmed; Panula, Sarita; Day, Kelly; Hultenby, Kjell; Söder, Olle; Hovatta, Outi; Stukenborg, Jan-Bernd

    2015-01-01

    Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

  6. Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase+catalase and vitamins E and C.

    Science.gov (United States)

    Anane, R; Creppy, E E

    2001-09-01

    Lipid peroxidation is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity and carcinogenicity of many xenobiotics. In the present study, we investigated the possible induction of lipid peroxidation by aluminium in human foreskin fibroblast cultures by assaying the malondialdehyde (MDA) produced inside the cells. The MDA-thiobarbituric acid (TBA) adduct was assayed by HPLC using fluorometric quantification after extraction in n-butanol. Lactate dehydrogenase (LDH) release was used as a marker of aluminium toxicity. MDA production was significantly increased after 24 h incubation with aluminium and paralleled LDH release. Superoxide dismutase (SOD)+catalase and vitamins C and E added in the culture medium as oxygen radical and free radical scavengers were efficient in preventing MDA production by aluminium, indicating that oxidative processes are one of the main pathways whereby this metal induces cytotoxicity. The latter is also largely prevented, thus confirming the link between oxidative stress induced by aluminium and its cytotoxicity in human skin fibroblasts.

  7. Uptake of (/sup 3/H)vitamin D/sub 3/ from low and high density lipoproteins by cultured human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Shireman, R.B.; Williams, D.; Remsen, J.F.

    1986-03-01

    The plasma distribution and cellular uptake of (/sup 3/H)vitamin D/sub 3/ was studied in vitro using cultured human fibroblasts. Incubation of (/sup 3/H)vitamin D/sub 3/ (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from (/sup 3/H) 1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of (/sup 3/H)vitamin D/sub 3/ from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D/sub 3/ was time, concentration, and temperature dependent. At a concentration of 50 ..mu..g LDL/ml of medium, the uptake of (/sup 3/H)vitamin D/sub 3/ from LDL at 37/sup 0/C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D/sub 3/ absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver.

  8. Probing tumor-stroma interactions and response to photodynamic therapy in a 3D pancreatic cancer-fibroblast co-culture model

    Science.gov (United States)

    Glidden, Michael D.; Massodi, Iqbal; Rizvi, Imran; Celli, Jonathan P.; Hasan, Tayyaba

    2012-02-01

    Pancreatic ductal adenocarcinoma is a lethal disease that is often unresectable by the time of diagnosis and is typically non-responsive to chemo- and radiotherapy, resulting in a five year survival of only 3%. Tumors of the pancreas are characterized by a dense fibrous stroma rich in extracellular matrix proteins, which is implicated in poor therapeutic response, though its precise roles remain poorly understood. Indeed, while the use of therapeutics that target the stroma is an emerging paradigm in the clinical management of this disease, the primary focus of such efforts is to enhance drug penetration through dense fibrous stroma and it is unclear to what extent the characteristically rigid stroma of pancreatic tumors imparts drug resistance by acting as a complex signaling partner, or merely as a physical barrier for drug delivery. Here we use 3D in vitro co-cultures of pancreatic cancer cells and normal human fibroblasts as a model system to study heterotypic interactions between these populations. Leveraging this in vitro model along with image-based methods for quantification of growth and therapeutic endpoints, we characterize these co-cultures and examine the role of verteporfin-based photodynamic therapy (PDT) for targeting tumor-fibroblast interactions in pancreatic tumors.

  9. Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

    Science.gov (United States)

    Bressan, F F; Miranda, M S; Bajgelman, M C; Perecin, F; Mesquita, L G; Fantinato-Neto, P; Merighe, G F K; Strauss, B E; Meirelles, F V

    2013-04-01

    Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed

  10. Effects of lower fluence pulsed dye laser irradiation on production of collagen and the mRNA expression of collagen relative gene in cultured fibroblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    YU Hai-yan; CHEN Da-fang; WANG Qi; CHENG Hao

    2006-01-01

    Background Lower fluence of 585-nm flashlamp-pumped pulsed dye laser has been successfully used as a nonablative technique in the treatment of wrinkles. The objective of this study was to evaluate the effect of the pulsed dye laser (585 nm) on the production of collagen and the mRNA expression of collagen related gene in fibroblasts in vitro.Methods Cultured fibroblasts were treated with a 585-nm flashlamp-pumped pulsed dye laser ( fluence 3 J/cm2,4 J/cm2, spot size 7 mm, pulse duration 450 μs). The production of collagen and the mRNA expression of transforming growth factor (TGF)-β1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen α1, α2 in fibroblasts were investigated by colorimetry or real time polymerase chain reaction.Results The production of collagen was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm2 (P <0.001). The mRNA expression of TGF- β1,SMAD2, SMAD3, SMAD4, SMAD7 and procollagen I was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm2 (P <0.001). No significant difference of mRNA expression of SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen was found between controls and fibroblasts treated with pulsed dye laser with a fluence of 4 J/cm2 (P >0.05).Conclusions Lower fluence (3 J/cm2) pulsed dye laser increased the collagen production in fibroblasts by up-regulating TGF-β1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen mRNA expression. These may be the reason it can be effectively used in the treatment of wrinkles.

  11. Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA.

    OpenAIRE

    Weissmahr, R N; Schüpbach, J; Böni, J

    1997-01-01

    We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associ...

  12. Neural progenitor cell apoptosis and differentiation were affected by activated microglia in spinal cord slice culture.

    Science.gov (United States)

    Liu, Xuqing; Chu, Tak-Ho; Su, Huanxing; Guo, Anchen; Wu, Wutian

    2014-03-01

    Neural progenitor cell (NPC) transplantation offers great potential to treat spinal cord injury (SCI). NPCs may replace lost neurons or oligodendrocytes and act as a source of neurotrophic factors to support survival of remaining cells. However, their efficiency was limited by poor survival after transplantation, and they tended more to differentiate into astrocytes, but not neurons and oligodendrocytes. This study investigated whether activated microglia is a factor that contributes to this phenomenon. Organotypic spinal cord slice (SCS) culture was used to mimic the local environment after SCI, and NPCs were co-cultured with them to share the culture medium. After specific depletion of microglia in the SCSs with clodronate loaded liposome, the apoptotic rate of NPCs decreased, more NPCs differentiated into neurons, and glial differentiation was impaired. This suggested that microglia may impair NPC survival, and neuronal differentiation, but improve astrocyte differentiation. In NPC transplantation strategy for SCI, microglia would be manipulated to improve the survival and neuronal differentiation of NPCs.

  13. Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Biver, Emmanuel, E-mail: ebiver@yahoo.fr [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Soubrier, Anne-Sophie [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Thouverey, Cyril [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Cortet, Bernard [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Broux, Odile [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Caverzasio, Joseph [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Hardouin, Pierre [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France)

    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.

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

    Science.gov (United States)

    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.

  15. Induction of adipogenic differentiation in three-dimensional culture model on a novel microfabricated scaffold.

    Science.gov (United States)

    Miyagawa, Yoshitaka; Okita, Hajime; Kiyokawa, Nobutaka

    2013-01-01

    The development of a three-dimensional (3D) culture system is very important for regenerative medicine and drug discovery applications of stem cell technology because the 3D culture condition could mimic the stem cell environment in vivo and support accurate differentiation. This chapter describes a novel 3D culture technique for efficient induction of adipogenic differentiation. This 3D culture system provides an easy way to allow cells to form a 3D spheroid structure without any matrix derived from animal and chemical substances. We firstly describe the details of the 3D culture technique using human mesenchymal stem/progenitor cells (MPCs) and its optimization. Then we elaborate on the protocol of efficient induction for adipogenic differentiation and adipocyte-specific gene expression, including peroxisome proliferator-activated receptor-γ, with our culture system in human MPCs.

  16. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    Science.gov (United States)

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  17. Role of ERK1/2, Akt, and PLCy pathways in proliferation and neuronal differentiation in the adult rat spinal cord neural stem/progenitor cell culture

    Directory of Open Access Journals (Sweden)

    Wai Si eChan

    2013-08-01

    Full Text Available Proliferation of endogenous neural stem/progenitor cells (NSPCs has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2. We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2 over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2 or the phosphoinositide 3-kinase (PI3K/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase C gamma (PLCy pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCy signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.

  18. Dopaminergic neuronal differentiation from the forebrain-derived human neural stem cells induced in cultures by using a combination of BMP-7 and pramipexole with growth factors

    Directory of Open Access Journals (Sweden)

    Hongna eYang

    2016-04-01

    Full Text Available Transplantation of dopaminergic (DA neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson’s disease (PD, thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX, bone morphogenetic proteins 7 (BMP-7, and growth factors, including acidic fibroblast factor (aFGF, forskolin, and phorbol-12-myristae-13-acetate (TPA, to induce differentiation of forebrain-derived hNSCs towards DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2, and tyrosine hydroxylase (TH were significantly increased after 24h of differentiation by RT-PCR analysis (p<0.01. Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24h, about 5% of cells became VMAT2 (vascular monoamine transporter 2-positive neurons, and less than 5% of cells became DAT (dopamine transporter-positive neurons at 72h following differentiation in cultures. Importantly, these TH-, VMAT2- and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P<0.01, as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.

  19. Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Mehdi Forouzandeh-Moghadam

    2009-01-01

    Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

  20. Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Rachel C Williams

    Full Text Available Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts.We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts.We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival

  1. Culture and Differentiation of Human Orbital Preadipocytes in Thyroid-associated Ophthalmopathy

    Institute of Scientific and Technical Information of China (English)

    Lifei Wang; Zhongyao Wu; Jianhua Yan; Huasheng Yang; Zhicong Chen

    2005-01-01

    Purpose: To identify whether there are preadipocytes in the orbits of thyroid-associated ophthalmopathy (TAO) patients and whether they possess the capacity to undergo adipocytic differentiation.Methods: Orbital fat tissue from TAO patients was placed in primary culture and proliferating cells were sub-passaged. The preadiocytes were identified with immunohistochemistry. Confluent preadipocytes were subjected to a differentiation protocol. And Peroxisome proliferators-associated receptorγ (PPARγ)transcript levels were determined before and after differentiation.Result: In primary culture, cells were spindly and showed positive immunostaining for preadipocyte factor-1 (pref-1 ) protein. During the process of differentiation, orbital cells showed evidence of adipogenesis and relatively greater PPARγ gene expression with differentiation.Conclusion: There are active preadipocytes in the orbital tissue of TAO patients which might be able to undergo adipocyte differentiation. These observations suggest that the preadipocytes differentiation contributed to excess orbital adipose tissue volume in TAO.

  2. Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System.

    Science.gov (United States)

    Hu, Wentao; He, Yongpei; Xiong, Yongjie; Lu, Hong; Chen, Hong; Hou, Limin; Qiu, Zhandong; Fang, Yu; Zhang, Suming

    2016-04-01

    Induced pluripotent stem cells (iPSCs) generated from patient-derived somatic cells provides the opportunity for model development in order to study patient-specific disease states with the potential for drug discovery. However, use of lentivirus and exposure of iPSCs to animal-derived products limit their therapeutic utility and affect lineage differentiation and subsequent downstream functionality of iPSC derivatives. Within the context of this study, we describe a simple and practical protocol enabling the efficient reprogramming of terminally differentiated adult fibroblasts into integration-free human iPSCs (hiPSCs) using a combination of episomal plasmids with small molecules (SMs). Using this approach, there was a 10-fold increase in reprogramming efficiency over single plasmid vector-based methods. We obtained approximately 100 iPSCs colonies from 1 × 10(5) human adult dermal fibroblasts (HADFs) and achieved approximately 0.1% reprogramming efficiencies. Concurrently, we developed a highly conducive culture system using xeno-free media and human vitronectin. The resulting hiPSCs were free of DNA integration and had completely lost episomal vectors, maintained long-term self-renewal, featured a normal karyotype, expressed pluripotent stem cell markers, and possessed the capability of differentiating into components of all three germ layers in vivo. Finally, we demonstrate that the integration-free hiPSCs could be differentiated into motor neurons under xeno-free culture conditions. This induction method will promote the derivation of patient-specific integration-free and xeno-free iPSCs and improve the strategy for motor neuron derivation. Our approach provides a useful tool for human disease models, drug screen, and clinical applications.

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

    Directory of Open Access Journals (Sweden)

    Leslie A. Mehalick

    2015-12-01

    Full Text Available 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.

  4. Proliferative response of fibroblasts expressing internalization-deficient epidermal growth factor (EGF) receptors is altered via differential EGF depletion effect.

    Science.gov (United States)

    Reddy, C C; Wells, A; Lauffenburger, D A

    1994-01-01

    We describe experiments comparing the proliferation responses to epidermal growth factor (EGF) by NR6 fibroblasts expressing genetically engineered epidermal growth factor receptors (EGFRs). These cells present either wild-type (WT) EGFR or a cytoplasmic domain-truncated (c'973) EGFR that exhibits a decreased ligand-induced internalization rate constant. In two distinct in vitro proliferation assays, with or without medium replenishment, we measured the specific cell proliferation rate constants and EGF depletion kinetics for both WT and c'973 cells. When EGF depletion is minimized by replenishment, the EGF concentration dependencies of the two cell types are similar, whereas when EGF depletion is not prevented, maximal proliferation of WT cells requires an initial EGF concentration that is approximately 10x that required by c'973 cells. However, when EGF depletion is accounted for, the dependencies of growth rate for the two cell types on the current EGF concentration in both assays are essentially identical. Our results demonstrate that diminished depletion of EGF from the extracellular medium is a major reason for increased mitogenic sensitivity to EGF by cells possessing internalization-deficient receptors.

  5. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    Science.gov (United States)

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  6. Culture of Ear Skin Fibroblasts from the Red Fox (Vulpesvulpes) and Analysis of the Karyotype%赤狐耳成纤维细胞的体外培养及核型分析

    Institute of Scientific and Technical Information of China (English)

    鲍加荣; 王世勇; 郑军军; 杨福合

    2015-01-01

    This study is to isolate and culture skin fibroblasts from the ear of fox.It propels the research on fox somatic nuclear transfer and transgenic fox.In this study,fox ear skin fibroblasts were obtained by tissue pieced culture.The primary culture was digested by trypsin at a short time for the subculture.The fibroblasts were observed by the electron microscope,which had a good growth status.Then karyotype analysis was conduc-ted on the fibroblasts,which showed a standard karyotype of fox.The results showed that fox ear skin fibroblasts could be cultivated successfully in vitro by means of tissues culture method.The primary culture could be puri-fied and ear fibroblast cell line be formed,which can be used as donor cell for nuclear transfer and propels the research on transgenetic special animals.%建立赤狐的耳成纤维细胞的分离和培养体系,为狐的核移植,转基因狐的研究奠定基础.采用组织块培养法分离狐耳成纤维细胞的原代细胞,再利用胰酶短暂消化法进行纯化传代培养,观察成纤维细胞的形态,并进行染色体分析.结果表明,组织块培养法可获得成纤维细胞,经过纯化培养,可获得均一、稳定的狐耳成纤维细胞.可以作为核移植的供体细胞,推动特种动物的转基因研究.

  7. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or lo...

  8. Adsorption of fibronectin and vitronectin onto Primaria and tissue culture polystyrene and relationship to the mechanism of initial attachment of human vein endothelial cells and BHK-21 fibroblasts.

    Science.gov (United States)

    Steele, J G; Dalton, B A; Johnson, G; Underwood, P A

    1995-09-01

    The two cell culture substrata, tissue culture polystyrene (TCPS) and Primaria, were compared in order to determine whether a nitrogen-containing surface such as Primaria attracts cells by a different mechanism to an oxygen-containing surface (TCPS). The amounts of vitronectin (Vn) and fibronectin (Fn) which adsorb from the fetal bovine serum (FBS) component of the culture medium onto Primaria and TCPS were determined. Primaria adsorbed two- to threefold more Fn than TCPS, but adsorbed similar amounts of Vn from medium containing FBS. The Fn and Vn binding sites on Primaria were distinct, as adsorption was non-competitive between these two proteins. The amounts of Fn and Vn that adsorbed onto the two surfaces were compared to the concentration dependence of the cell attachment activities of Fn and Vn. Whereas the amounts of Fn which adsorbed onto TCPS were suboptimal for cell attachment, Primaria adsorbed an Fn surface density that was supraoptimal for attachment of human vein endothelial cells and BHK-21 fibroblasts. We conclude that Primaria differs from TCPS in that both Fn and Vn mediate initial cell attachment to Primaria when the culture medium contains FBS, whereas cell attachment to TCPS is dependent upon Vn.

  9. [Mouse teratocarcinoma: differentiation in cultures of a multipotential primitive cell line (author's transl)].

    Science.gov (United States)

    Nicolas, J F; Dubois, P; Jakob, H; Gaillard, J; Jacob, F

    1975-01-01

    A line of primitive cells of teratocarcinoma is able of differentiating during in vitro cultures. The differentiated cell types correspond to derivatives of the three germ layers. They appear according to a reproducible schedule. The differentiated cell types exhibit a normal karyotype. They have lost their tumorigenicity and have a limited life span. Agregates containing various numbers of primitive cells have been prepared and plated in culture dishes. Their study shows that the first differentiated cell types appear only when the agregates are greater than a certain critical mass. Primitive cells carry on their surface the F9 but not the H-2 antigen. During differentiation in culture, the H-2 antigen appears rapidly on certain cells. The fraction of H-2-carrying cells increases as a function of time while that of F9-carrying cells decreases.

  10. Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro.

    Science.gov (United States)

    Hu, Yang; Zhang, Yan; Tian, Kang; Xun, Chong; Wang, Shouyu; Lv, Decheng

    2016-01-01

    Recent studies regarding regenerative medicine have focused on bone marrow mesenchymal stem cells (BMSCs), which have the potential to undergo neural differentiation, and may be transfected with specific genes. BMSCs can differentiate into neuron‑like cells in certain neurotropic circumstances in vitro. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are often used to induce neural differentiation in BMSCs in vitro. However, previous studies regarding their combined actions are insufficient. The present study is the first, to the best of our knowledge, to thoroughly assess the enhancement of neural differentiation of BMSCs following transfection with bFGF and NGF. Sprague‑Dawley (SD) rat BMSCs were separated through whole bone marrow adherence, and were then passaged to the third generation. The cells were subsequently divided into five groups: The control group, which consisted of untransfected BMSCs; the plv‑blank‑transfected BMSCs group; the plv‑bFGF‑transfected BMSCs group; the plv‑NGF‑transfected BMSCs group; and the plv‑NGF‑bFGF co‑transfected BMSCs group. Cell neural differentiation was characterized in terms of stem cell molecular expression, and the neuronal morphology and expression of neural‑like molecules was detected in each of the groups. A total of 72 h post‑transfection, the expression levels of neuron‑specific enolase, glial fibrillary acidic protein, and nestin protein, were higher in the co‑transfected group, as compared with the other groups, the expression levels of β‑tubulin III were also increased in the co‑transfected cells, thus suggesting the maturation of differentiated neuron‑like cells. Furthermore, higher neuronal proliferation was observed in the co‑transfected group, as compared with the other groups at passages 2, 4, 6 and 8. Western blotting demonstrated that the transfected groups exhibited a simultaneous increase in phosphorylation of the AKT and extracellular signal

  11. An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

    Directory of Open Access Journals (Sweden)

    Kajohnkiart Janebodin

    2013-01-01

    Full Text Available Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10, decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.

  12. Focal adhesion kinase activation is required for TNF-α-induced production of matrix metalloproteinase-2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts.

    Science.gov (United States)

    Zhang, Peng; Li, Ya-jing; Guo, Liu-yun; Wang, Guo-fang; Lu, Ke; Yue, Er-li

    2015-08-01

    Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF-α) and its effects on interleukin (IL)-6 and IL-8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP-2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real-time PCR. Tumor necrosis factor alpha dose-dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF-α-induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL-6, IL-8, and MMP-2 in a dose-dependent manner. Knockdown of FAK significantly suppressed TNF-α-induced expression of IL6 and IL8 mRNA and release of IL-6 and IL-8 protein in HPDLFs. Similarly, MMP-2 down-regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF-α-induced IL-6, IL-8, and MMP-2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.

  13. [The influence of fibroblast growth factor (FGF2) on cardiomyocytes differentiation of mesenchymal stem cells of bone marrow ex vivo].

    Science.gov (United States)

    Lobanok, E S; Kvacheva, Z B; Pinchuk, S V; Volk, M V; Mezhevkina, L M; Fesenko, E E; Volotovski, I D

    2014-01-01

    The influence of FGF2 on the efficiency of cardiomyocytes differentiation of mesenchymal stem cells (MSC) of bone marrow induced by 5-azacetidine (5-aza) was studied. The effect of FGF2 developing by the 14th day after the combined action of a differentiating agent and growth factor was manifested in an increase in Mef2A, Mef2D and gene transcription and a rise of ionized Ca2+ concentration in cytoplasm keeping cell viability and proliferation activity. In the presence of FGF2 this approach provided cardiomyogenesis and the increase in the formation of early precursors of cardiomyocytes.

  14. Differential translocation of heat shock factor-1 after mild and severe stress to human skin fibroblasts undergoing aging in vitro.

    Science.gov (United States)

    Demirovic, Dino; de Toda, Irene Martinez; Nizard, Carine; Rattan, Suresh I S

    2014-12-01

    Repeated exposure to mild heat shock (HS) has been shown to induce a wide range of health promoting hormetic effects in various biological systems, including human cells undergoing aging in vitro. In order to understand how cells distinguish between mild and severe stress, we have investigated the extent of early and immediate HS response by analyzing the nuclear translocation of the transcription factor heat shock factor-1 (HSF1), in serially passaged normal adult human facial skin fibroblasts exposed to mild (41 °C) or severe (43 °C) HS. Cells respond differently when exposed to mild and severe HS at different passage levels in terms of the extent of HSF1 translocation. In early passage young cells there was a 5-fold difference between mild and severe HS in the extent of HSF1 translocation. However, in near senescent late passage cells, the difference between mild and severe stress in terms of the extent of HSF1 translocation was reduced to less than 2-fold. One of the reasons for this age-related attenuation of heat shock response is due to the fact there was a higher basal level of HSF1 in the nuclei of late passage cells, which is indicative of increased intrinsic stress during cellular aging. These observations are consistent with previously reported data that whereas repeated mild stress given at younger ages can slow down aging and increase the lifespan, the same level of stress given at older ages may not provide the same benefits. Therefore, elucidating the early and immediate steps in the induction of stress response can be useful in deciding whether a particular level of stress is potentially hormetically beneficial or not.

  15. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells.

    Science.gov (United States)

    Kuroda, Yasumasa; Wakao, Shohei; Kitada, Masaaki; Murakami, Toru; Nojima, Makoto; Dezawa, Mari

    2013-01-01

    Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4-6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

  16. Effects of connective tissue growth factor antisense oligonucleotides on the proliferation and collagen synthesis of the cultured human keloid fibroblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    刘剑毅; 李世荣; 纪淑兴

    2004-01-01

    Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P < 0.01 ). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.

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

    Science.gov (United States)

    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. PMID:23107315

  18. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-05-15

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approx. 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

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

    Science.gov (United States)

    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.

  20. Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    María J. Pérez

    2017-10-01

    Full Text Available The identification of an early biomarker to diagnose Alzheimer's disease (AD remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients.

  1. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Science.gov (United States)

    2011-01-01

    Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within

  2. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Shafa Mehdi

    2011-12-01

    Full Text Available Abstract Background Embryonic stem cells (ESCs can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs. However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC

  3. Biological responses of human gingival fibroblasts (HGFs in an innovative co-culture model with Streptococcus mitis to thermosets coated with a silver polysaccharide antimicrobial system.

    Directory of Open Access Journals (Sweden)

    Silvia Sancilio

    Full Text Available This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6 and prostaglandin E2 (PGE2 secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE2 production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE2 secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.

  4. Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

    Energy Technology Data Exchange (ETDEWEB)

    Silbert, C.K.; Humphries, D.E.; Palmer, M.E.; Silbert, J.E. (Veterans Administration Outpatient Clinic, Boston, MA (USA))

    1991-02-15

    Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with (3H)glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of (3H)chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

  5. Globalization, differentiation and drinking cultures, an anthropological perspective

    Directory of Open Access Journals (Sweden)

    Thomas Wilson

    2004-12-01

    Full Text Available L’alcool et sa consommation ne renvoient pas simplement au domaine économique. L’alcool est devenu aujourd’hui une partie intégrale des relations sociales dans différentes cultures au point où son importance globale est souvent sous-estimée par ses plus ardents critiques. En dépit de ses conséquences directes sur la santé, sa consommation a pris une certaine ampleur dans le monde industriel développé. Certainement son rôle central dans la construction des identités individuelles explique sa position clé au sein des sociétés. Que nous dit le saké à propos du Japon ou le vin de Bourgogne sur la France? Que nous dit la consommation ou l’abstinence d’alcool sur les questions d’identité individuelle, d’ethnicité, de classe et de culture? Quelle place tient l’alcool dans la définition de soi et dans la notion de résistance? Répondre à ces questions et à d’autres est le but essentiel de cet article qui examine la consommation d’alcool à travers différentes cultures et ce que boire signifie pour ceux qui choisissent de consommer ou de s’abstenir. De l’Irlande à Hong-Kong, Mexico à l’Allemagne, l’alcool occupe un certain nombre de fonctions sociales, religieuses, politiques et familiales. Les cultures du boire définissent ces consommations dans le cadre plus large des pratiques sociales et montrent comment classes sociales, ethnicité et nationalisme peuvent s’exprimer à travers cette commodité. En partant d’approches de terrain, les contributeurs analysent l’interface entre culture et pouvoir dans les bars et pubs, la signification des images publicitaires, le rôle de ces boissons dans la vie quotidienne. Le résultat est la première publication comparative sur les questions de l’impact que la consommation d’alcool a sur l’identité nationale dans le monde aujourd’hui.Alcohol is not only big business, it has become an essential part of social relations in so many cultures that

  6. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2012-01-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  7. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2013-02-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  8. Differentiation in teaching : Moving towards a cross-cultural perspective

    NARCIS (Netherlands)

    Isac, Maria; Maulana, Ridwan; Lorenz, Michelle; van de Grift, Willem

    2016-01-01

    Worldwide, educators, researchers and policy-makers struggle to understand the changes needed for responding to the increased diversity in classrooms. Teachers applying different teaching strategies that can be used to differentiate in classrooms, intend to meet the variation in students’ abilities,

  9. Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    S Montagnani

    2009-12-01

    Full Text Available