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Sample records for human fetal fibroblasts

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

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    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.

  2. Ontogeny of expression of basic fibroblast growth factor and its receptors in human fetal skin

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    CHEN Wei; FU Xiao-bing; GE Shi-li; SUN Tong-zhu; SHENG Zhi-yong

    2005-01-01

    Objective : To investigate the expression characteristics of basic fibroblast growth factor (bFGF)and its receptors, flg ( FGFR1 ) and bek ( FGFR2), in fetal skin at different gestational ages underlying the relevance of these 3 proteins to skin development and the mechanisms underlying the phenotypic transition from scarless to scarforming healing.Methods: Eighteen specimens of fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages of 13-32 weeks. Gene expression of bFGF, bek and flg was examined with reverse transcription-polymerase chain reaction (RT-PCR). The dynamic expression and distribution of these 3 proteins were detected with streptavidin peroxidase ( SP )immunohistochemical staining method.Results: In the early gestational fetal skin, genes of bFGF and flg were strongly expressed and more protein contents of these 2 proteins were found as compared with the genes at late gestation fetal skin (2.446 ± 0.116 and 2.066 ± 0. 152 versus 2.157 ± 0. 101 and 1.818 ± 0.086,respectively, P < 0.05). On the contrary, the levels of gene expression and protein content of bek were not differently expressed in the early gestational fetal skin versus the late ones. Protein particles of bFGF were mainly distributed in the epidermal cells and some fibroblasts. Bek was mainly located in the cell membrane and cytoplasm of epidermal cells while flg protein was principally located in the epidermal cells, endothelial cells and some fibroblasts.Conclusions: The endogenous bFGF and their receptors might be involved in the cutaneous development at fetal stage. The differently expressing levels of bFGF and flg during gestation may be related to scarless or scarforming repair during gestation.

  3. LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

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    Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

    2015-02-15

    Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

  4. Effects of extremely low frequency electromagnetic fields on human fetal scleral fibroblasts.

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    Zhu, Huang; Wang, Jie; Cui, Jiefeng; Fan, Xianqun

    2016-06-01

    This study investigated the effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human fetal scleral fibroblasts (HFSFs). HFSFs were subjected to 50 Hz artificial ELF-EMFs generated by Helmholtz coils with 0.1, 0.2, 0.5, and 1.0 mT field intensities for 6 to 48 h. The viability and factors involved in scleral structuring of HFSFs were determined. The growth rate of HFSFs significantly decreased after only 24 h of exposure to ELF-EMFs (0.2 mT). The messenger RNA (mRNA) expression of collagen type I (COL1A1) decreased and expression of matrix metalloproteinase-2 (MMP-2) increased significantly. There was a decrease in tissue inhibitor of MMP-2 mRNA levels between treated and control cells only at the 1.0 mT intensity level. Transforming growth factor beta-2 mRNA increased in exposed cells, and, simultaneously, fibroblast growth factor-2 mRNA levels decreased. The protein expressions of COL1A1 and MMP-2 were also significantly altered subsequent to exposure (p effects on HFSFs and could cause abnormality in scleral collagen.

  5. Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts

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    Amir Amiri Yekta

    2013-01-01

    Full Text Available Objective: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.Materials and Methods: In this experimental study, a linearized recombinant vector (pBC1 entailing human coagulation factor IX (5.7 kb cDNA was introduced into goat fetal fibroblast cells (three sub passages via electroporation in four separate experiments (while other variables were kept constant, beginning with 10 μg DNA per pulse in the first experiment and increments of 10 μg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.Results: The results showed no significant difference among three first concentrations except for group 1 (10 μg/pulse and group 3 (30 μg/pulse, but there was a significant difference between these groups and the fourth group (p<0.05, as this group (40 μg/pulse statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH results indicated the transgene was integrated in a single position in PCR positive cells.Conclusion: Increasing amount of using the vector 40μg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role

  6. The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts.

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    Schultz, Francisca; Hasan, Alveera; Alvarez-Laviada, Anita; Miragoli, Michele; Bhogal, Navneet; Wells, Sarah; Poulet, Claire; Chambers, Jenny; Williamson, Catherine; Gorelik, Julia

    2016-01-01

    Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts. Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential. We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells. UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts

  7. 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.

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

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    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.

  9. Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

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    ZHANG Yu-ling; LIU Feng-jun; ZHANG Yong

    2009-01-01

    [Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%; MGE: 33.33% vs. 6.67%). Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%), confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.

  10. Mitochondrial dysfunction and transactivation of p53-dependent apoptotic genes in BaP-treated human fetal lung fibroblasts.

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    Yang, Guangtao; Jiang, Ying; Rao, Kaimin; Chen, Xi; Wang, Qian; Liu, Ailin; Xiong, Wei; Yuan, Jing

    2011-12-01

    Benzo(a)pyrene (BaP) has been shown to be an inducer of apoptosis. However, mechanisms involved in BaP-induced mitochondrial dysfunction are not well-known. In this study, human fetal lung fibroblasts cells were treated with BaP (8, 16, 32, 64 and 128 μM) for 4 and 12 h. Cell viability, intracellular level of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), mitochondrial membrane potential (ΔΨ(m)) and cytochrome c release were determined. Changes in transcriptional levels of p53-dependent apoptotic genes (p53, APAF1, CASPASE3, CASPASE9, NOXA and PUMA) were measured. At time point of 4 h, BaP induced the intracellular ROS generation in 64 (p BaP groups (p BaP groups (p BaP groups (p BaP group (p BaP groups (p BaP group a relatively little expression of p53 mRNA was observed (p BaP promoted the generation of excessive ROS and subsequently the mitochondrial depolarization, whereas transactivations of the p53-dependent apoptotic genes were significantly induced at the later period.

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

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    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.

  12. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

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    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  13. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

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    Jianhua Gong

    Full Text Available Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency, hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  14. Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.

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    Han, X J; Liang, H; Yun, T; Zhao, Y H; Zhang, M L; Zhao, L H; Li, R F; Li, X L

    2015-09-28

    The neomycin-resistance (neo(r)) gene is widely used as a selectable marker in eukaryotic expression vectors; however, its expression often affects that of target genes. Cre recombinase recognizes LoxP sites, leading to site-specific recombination and deletion of DNA and RNA between two LoxP sites. In the present study, a humanized Fat-1 gene (hFat-1) was generated by DNA Works and used to construct a pC-PGK-neo(r)-hfat-1 expression vector, in which PGK-neo(r) was flanked by two LoxP sites. The pC-PGK-neo(r)-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 μg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neo(r), which was integrated into the genome. hFat-1-neo(r) negative cells were obtained following puromycin selection. Real-time quantitative polymerase chain reaction data indicated that neomycin-resistant cells had higher hFat-1 expression than neomycin-sensitive cells. High performance gas chromatography data suggested that the n-6/n-3 ratio was significantly lower in transfected cells than in wild-type cells. The n-6/n-3 ratio in Cre-treated hFat-1-transfected cells was higher than that in untreated cells, suggesting that deletion of PGK-neo(r) decreased hFat-1 expression.

  15. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

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    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  16. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

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    Li, Xiaoou [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Liu, Lian [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Wen, Fuqiang, E-mail: wenfuqiang.scu@gmail.com [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China)

    2014-11-28

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.

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

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

    2016-03-01

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

  18. Fetal intestinal fibroblasts respond to insulin-like growth factor (IGF-II better than adult intestinal fibroblasts

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    Fillenwarth Michael J

    2006-01-01

    Full Text Available Abstract Background We compared IGF responses of fetal and adult intestinal fibroblasts to identify a developmental difference in the IGF-axis. Intestinal fibroblasts were isolated from maternal and fetal jejunum. Media was conditioned at confluence and one week afterwards. The proliferative response at confluence to 5 nM IGF-I or -II was compared. Results There were no significant differences in IGFBP expression at confluence. Post-confluence, fetal fibroblasts had no significant changes in IGFBP-2 and IGFBP-3 expression. Post-confluent maternal fibroblasts had increased IGFBP-3 levels that were significant compared to the fetal fibroblasts. IGF-I increased in post-confluent fetal fibroblasts, while in maternal fibroblasts it decreased (p Conclusion Fetal intestinal fibroblasts respond to IGF-II with greater proliferation and do not have the increased IGFBPs seen post-confluence in adult intestinal fibroblasts.

  19. Deletion of the homeobox gene PRX-2 affects fetal but not adult fibroblast wound healing responses.

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    White, Philip; Thomas, David W; Fong, Steven; Stelnicki, Eric; Meijlink, Fritz; Largman, Corey; Stephens, Phil

    2003-01-01

    The phenotype of fibroblasts repopulating experimental wounds in vivo has been shown to influence both wound healing responses and clinical outcome. Recent studies have demonstrated that the human homeobox gene PRX-2 is strongly upregulated in fibroblasts within fetal, but not adult, mesenchymal tissues during healing. Differential homeobox gene expression by fibroblasts may therefore be important in mediating the scarless healing exhibited in early fetal wounds. RNase protection analysis demonstrated that murine Prx-2 expression was involved in fetal but not adult wound healing responses in vitro. Using fibroblasts established from homozygous mutant (Prx-2-/-) and wild-type (Prx-2+/+) murine skin tissues it was demonstrated that Prx-2 affected a number of fetal fibroblastic responses believed to be important in mediating scarless healing in vivo; namely cellular proliferation, extracellular matrix reorganization, and matrix metalloproteinase 2 and hyaluronic acid production. These data demonstrate how Prx-2 may contribute to the regulation of fetal, but not adult, fibroblasts and ultimately the wound healing phenotype. This study provides further evidence for the importance of homeobox transcription factors in the regulation of scarless wound healing. A further understanding of these processes will, it is hoped, enable the targeting of specific therapies in wound healing, both to effect scarless healing and to stimulate healing in chronic, nonhealing wounds such as venous leg ulcers.

  20. Stimulation of 11β-HSD1 expression by IL-1β via a C/EBP binding site in human fetal lung fibroblasts.

    Science.gov (United States)

    Yang, Zhen; Zhu, Xiaoou; Guo, Chunming; Sun, Kang

    2009-12-01

    Proinflammatory cytokines, just like glucocorticoids (GCs), have been reported to upregulate 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression in many cell types. This concerted regulation of 11β-HSD1 by interleukin-1β (IL-1β) and GCs is in marked contrast to their antagonistic effects on inflammation. Further, the molecular mechanisms underlying the induction of 11β-HSD1 by IL-1β are not very well understood. In this study, we demonstrated that IL-1β dramatically stimulated 11β-HSD1 expression and enzyme activity as well as promoter activity including the -64 bp fragment upstream to the transcription start site in human fetal lung fibroblasts (HFL-1). Nucleotide mutations of the proximal CCAAT box within this region abolished the induction of 11β-HSD1 promoter activity by IL-1β. Western blotting analysis demonstrated that IL-1β induced the expression of C/EBPβ dramatically while C/EBPα was barely detectable in HFL-1 cells. Global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression plasmid (CMV500-A-C/EBP) significantly attenuated the induction of 11β-HSD1 by IL-1β, whereas over-expression of C/EBPβ enhanced the expression of 11β-HSD1. Chromatin immunoprecipitation assay revealed the recruitment of C/EBPβ to the promoter region containing the C/EBP binding site. In conclusion, IL-1β induces the expression of 11β-HSD1 mRNA in the fetal lung tissue through mechanisms that involve C/EBPβ binding to the promoter. This impact of IL-1β on the expression of 11β-HSD1 in human fetal lung cells may explain the alternate mechanism for the lung maturation that appears to occur when there is a risk of premature delivery of the fetus due to the presence of infection.

  1. Dogs cloned from fetal fibroblasts by nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Jang, Goo; Kim, Min Kyu; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Dae Yong; Lee, Byeong Chun

    2009-10-01

    Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9-77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2-1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.

  2. 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.

  3. Comparative analysis of the expression of surface markers on fibroblasts and fibroblast-like cells isolated from different human tissues.

    Science.gov (United States)

    Lupatov, A Yu; Vdovin, A S; Vakhrushev, I V; Poltavtseva, R A; Yarygin, K N

    2015-02-01

    Expression of 20 surface markers was analyzed in cultures of mesenchymal stromal cells of the umbilical cord, fibroblasts from adult and fetal human skin, and fibroblast-like cells of fetal liver was analyzed by fl ow cytometry. The studied cultures did not express hemopoietic cells markers, but were positive for CD73, CD90, and CD105 markers recommended by the International Society of Cell Therapy for the identification of the multipotent mesenchymal stromal cells. Fetal liver fibroblast-like cells were positive for CD54; this marker was absent in skin fibroblast cultures, but was expressed by umbilical cord mesenchymal stromal cells. Further study of these cells revealed a minor subpopulation of cells co-expressing CD24 and CD90 or CD24 and CD54. We hypothesized that these cells probably participate in epithelial mesenchymal transition.

  4. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Science.gov (United States)

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  5. Fetal Fibroblasts and Keratinocytes with Immunosuppressive Properties for Allogeneic Cell-Based Wound Therapy

    Science.gov (United States)

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors. PMID:23894651

  6. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Directory of Open Access Journals (Sweden)

    Thomas Zuliani

    Full Text Available Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  7. Human fetal mesenchymal stem cells.

    Science.gov (United States)

    O'Donoghue, Keelin; Chan, Jerry

    2006-09-01

    Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The

  8. The role of interleukin-10 and hyaluronan in murine fetal fibroblast function in vitro: implications for recapitulating fetal regenerative wound healing.

    Science.gov (United States)

    Balaji, Swathi; King, Alice; Marsh, Emily; LeSaint, Maria; Bhattacharya, Sukanta S; Han, Nathaniel; Dhamija, Yashu; Ranjan, Rajeev; Le, Louis D; Bollyky, Paul L; Crombleholme, Timothy M; Keswani, Sundeep G

    2015-01-01

    Mid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA) in the extracellular matrix (ECM) and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10) as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM). We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process. To evaluate the differences in functional profile, we compared metabolism (MTS assay), apoptosis (caspase-3 staining), migration (scratch wound assay) and invasion (transwell assay) between C57Bl/6J murine fetal (E14.5) and adult (8 weeks) fibroblasts. We found that fetal fibroblasts have lower rates of metabolism and apoptosis, and an increased ability to migrate and invade compared to adult fibroblasts, and that these effects were dependent on IL-10 and HA synthase activity. Further, addition of IL-10 to adult fibroblasts resulted in increased fibroblast migration and invasion and recapitulated the fetal phenotype in an HA-dependent manner. Our data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.

  9. The role of interleukin-10 and hyaluronan in murine fetal fibroblast function in vitro: implications for recapitulating fetal regenerative wound healing.

    Directory of Open Access Journals (Sweden)

    Swathi Balaji

    Full Text Available Mid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA in the extracellular matrix (ECM and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10 as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM. We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process.To evaluate the differences in functional profile, we compared metabolism (MTS assay, apoptosis (caspase-3 staining, migration (scratch wound assay and invasion (transwell assay between C57Bl/6J murine fetal (E14.5 and adult (8 weeks fibroblasts. We found that fetal fibroblasts have lower rates of metabolism and apoptosis, and an increased ability to migrate and invade compared to adult fibroblasts, and that these effects were dependent on IL-10 and HA synthase activity. Further, addition of IL-10 to adult fibroblasts resulted in increased fibroblast migration and invasion and recapitulated the fetal phenotype in an HA-dependent manner.Our data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.

  10. Generation of iPSC line MU011.A-hiPS from homozygous α-thalassemia fetal skin fibroblasts

    Directory of Open Access Journals (Sweden)

    Amornrat Tangprasittipap

    2015-11-01

    Full Text Available Human iPSC line MU011.A-hiPS was generated from homozygous α-thalassemia (−SEA/−SEA fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 contained in three episomal vectors were delivered using electroporation.

  11. Ex vivo culture of human fetal gonads

    DEFF Research Database (Denmark)

    Jørgensen, A; Nielsen, J.E.; Perlman, S

    2015-01-01

    STUDY QUESTION: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? SUMMARY ANSWER: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal...... phenotype in fetal testis cultures. WHAT IS KNOWN ALREADY: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA......-treatment mediates initiation of meiosis in human fetal ovary ex vivo. STUDY DESIGN, SIZE, DURATION: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated...

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

    Science.gov (United States)

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

    2016-02-03

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

  13. Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

    Directory of Open Access Journals (Sweden)

    Dawei Yu

    Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

  14. 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.

  15. 纳米金与细胞相互作用机理的蛋白质组学研究%Proteomic analysis of molecular mechanism of interactions between gold nanoparticles and human dermal fibroblasts-fetal

    Institute of Scientific and Technical Information of China (English)

    吕晓迎; 瞿颖华; 杨雅敏; 黄炎

    2011-01-01

    应用蛋白质组学结合生物信息学方法研究纳米金与人皮肤成纤维细胞(HDF-f )的作用机理.首先采用柠檬酸钠还原氛金酸法制备20nm的纳米金,然后应用MTT法和流式细胞术评价纳米金的细胞毒性及对细胞周期和细胞凋亡的影响.接着应用蛋白质组学技术和生物信息学方法筛选纳米金作用后细胞发生差异表达的蛋白质并进行基因本体论分析.MTT实验结果表明,浓度为200μmo1/L,粒径为20nm的纳米金与HDF-f作用1,4,8h后均未产生细胞毒性,流式细胞仪结果显示纳米金作用后S期细胞增加而G2/M期细胞减少.对蛋白质组学实验筛选出的29个功能蛋白质进行的基因本体论分析表明,纳米金可能会影响细胞RNA转录过程及能量代谢调节,诱导细胞产生免疫响应,进而对细胞的生理过程产生影响.%The aim of the present study is to investigate the mechanism of interactions between gold nanoparticles (GNPs) and human dermal fibroblasts-fetal (HDF-f) based on proteomics technology combining bioinformatics analysis. 20nm GNPs was prepared by sodium citrate reducing chloroauric acid at first. Then Methylthiazoltet-razolium (MTT) assay and flow cytomtery were applied to evaluate cytotoxicity and the influence on cell cycle and apoptosis by GNPs. Finally, differentially expressed proteins in HDF-f treated with GNPs were obtained u-sing proteomics technology and bioinformatics tool was employed to carry out the gene ontology (GO) analysis. MTT assay showed that 200μmol 20nm GNPs caused no cytotoxicity to cells after treated for 1, 4 and 8h. Flow cytometry experiment results suggested the effects of GNPs on cells were mainly on S and G2/M phase. 29 proteins were identified with different expression in all three-culture periods using proteomics technology. GO analysis of 29 differentially expressed proteins indicated that GNPs may have an influence on the HDF-f through impacting the transcription processes of RNA

  16. Expression and effect of matrix metalloproteinase 2 in human fetal scleral fibroblasts treated with an extremely low frequency electromagnetic field%极低频电磁辐射下HFSF中MMP-2的表达及作用

    Institute of Scientific and Technical Information of China (English)

    田甜; 朱煌

    2015-01-01

    Objective To observe the matrix metalloproteinase 2 (MMP-2) activity and protein expression in human fetal scleral fibroblasts (HFSFs) exposed to an extremely low frequency electromagnetic field (ELF-EMF) and its impact on collagen Ⅰ synthesis.Methods This was an experimental study.HFSFs were cultured and divided into a control group,radiation group and inhibitor group according to their exposure to 50 Hz,0.2 mT electromagnetic field or joint MMP-2 specific inhibitors (sc-204092).MMP-2 activity was measured by gelatin enzymography.MMP-2 and collagen Ⅰ protein expression were detected by Western Blot.A single factor analysis of variance was used to determine whether there was a difference among these groups.Results There were significant differences between the three groups (F=14.96,139.88,63.46,P<0.01).Compared to the control group,MMP-2 activity and protein expression increased in the radiation group (P<0.05) and collagen Ⅰ protein expression decreased (P<0.01).Compared to the radiation group,MMP-2 activity and protein expression decreased in the inhibitor group (P<0.01) and collagen Ⅰ protein expression increased (P<0.05).Conclusion ELF-EMF may regulate the synthesis of collagen Ⅰ by affecting the activity and protein expression of MMP-2.%目的 研究极低频电磁场(ELF-EMF)作用下人胚胎眼巩膜成纤维细胞(HFSF)中基质金属蛋白酶-2(MMP-2)活性与蛋白表达的变化以及ELF-EMF对Ⅰ型胶原(Collagen Ⅰ)合成的影响.方法 实验研究.根据是否暴露于50 Hz、0.2 mT电磁场或加入MMP-2特异性抑制剂(sc-204092),将实验细胞分为对照组、辐照组和抑制剂组.明胶酶谱法检测各组HFSF中MMP-2酶活性的变化;Western Blot法检测各组HFSF中MMP-2、Collagen Ⅰ蛋白水平的变化.采用单因素方差分析进行数据分析.结果 3组HFSF细胞中MMP-2酶活性、MMP-2、Collagen Ⅰ蛋白表达比较差异均具有统计学意义(F=14.96、139.88、63.46,P均<0.01);

  17. Wnt-4 expression is increased in fibroblasts after TGF-beta1 stimulation and during fetal and postnatal wound repair.

    Science.gov (United States)

    Colwell, Amy S; Krummel, Thomas M; Longaker, Michael T; Lorenz, H Peter

    2006-06-01

    Wnt-4 is a mitogen expressed during postnatal repair and scar formation; however, its expression profile during scarless repair is unknown. Transforming growth factor (TGF)-beta1 has high expression during healing with scar formation. Whether TGF-beta1 directly influences Wnt-4 expression in fetal or postnatal fibroblasts has not been examined. Primary fetal and postnatal mouse fibroblasts were stimulated with TGF-beta1 and Wnt-4 expression quantitated by real-time polymerase chain reaction. Fetal E17 and postnatal mouse excisional wounds were also analyzed for Wnt-4 expression by real-time polymerase chain reaction. In E17 fibroblasts after TGF-beta1 stimulation, Wnt-4 expression increased 4-fold at 1 hour (p stimulation, but peak expression was larger and relatively delayed, with a 17-fold increase at 12 hours (p fetal skin, Wnt-4 expression was 3.5-fold greater compared with 3-week-old mice (p fetal scarless and postnatal scarring mouse wound repair. The authors' data suggest that TGF-beta directly increases Wnt-4 expression in fetal and postnatal fibroblasts and that Wnt-4 is increased in both fetal and postnatal repair.

  18. Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Youichi Higuchi

    Full Text Available Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body.Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs and the subperitoneal layer (subperitoneal fibroblasts: SPFs. Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup.In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling.GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract.

  19. Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Science.gov (United States)

    Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

    2014-01-01

    Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

  20. Altered chromosome 6 in immortal human fibroblasts.

    Science.gov (United States)

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  1. Altered chromosome 6 in immortal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hubbard-Smith, K.; Pardinas, J.R.; Jha, K.K.; Ozer, H.L. (New Jersey Medical School, Newark, NJ (United States)); Patsalis, P.; Henderson, A.S. (City Univ. of New York, NY (United States))

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. 66 refs., 6 figs., 2 tabs.

  2. 人Sox2和Nanog基因的克隆及其对胎肺成纤维细胞分化的影响%Cloning of human Sox2 and Nanog gene and transforming fetal pneumal fibroblasts into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    孙中帅; 雷建园; 广茜茜; 畅继武

    2011-01-01

    To clone the complete coding sequences (CDS) of Homo sapiens Sox2 gene and Nanog gene in vitro, construct their recombinant eukaryotic expressing plasmids (pSG5-Sox2, pSG5-Nanog), and then transfect the plasmids into the fetal pneumal fibroblast, to observe the expression of Sox2 and Nanog and morphological change in fibroblast Methods The CDS of Sox2 gene was cloned from the brain tissue of aborted fetus aged 4 - 6w by RT-PCR. The CDS of Nanog gene was cloned from bladder carcinoma cells. The obtained sequences were then ligated with pSG5. The recombinant plasmids pSG5-Sox2 and pSG5-Nanog were then transfected into fetal pneumal fibroblast. Immunocytochemical method was used to detect the expression of Sox2 gene and Nanog gene. Results The complete CDS of Homo sapiens Sox2 gene and Nanog gene were 957bp and 934bp respectively. Immunocytochemical method revealed that rerecombinant plasmids pSG5-Sox2 and pSG5-Nanog were expressed positively in the fetal pneumal fibroblast. After transfection of these plasmids, the fetal pneumal fibroblasts were transformed into stem cell like cells,and then they were differentiated into epithelial-like cells with positive, CK (pan) staining, and finally showed a differentiated tendency towards neuron-like cell, with positive Nestin, GFAP and NSE staining. Conclusions In summary, Homo sapiens Sox2 gene and Nanog gene have been cloned, and the eukaryotic expressing plasmids have also been obtained in present study, which may lay a foundation for deriving human iPS cells. Meanwhile, without feeder layer, the fetal pneumal fibroblast can differentiate into epithelium-like and neuron-like cells after transfection in vitro.%目的 克隆人Sox2、Nanog基因编码序列,构建pSG5-Sox2及pSG5-Nanog真核表达重组质粒,转染胎肺间质成纤维细胞,观察Sox2、Nanog基因表达并鉴定其功能.方法 应用RT-PCR方法分别从4~6周龄流产胎儿脑组织和膀胱癌细胞中扩增出Sox2及Nanog基因编码

  3. Effects of sphingosine 1-phosphate and lysophospholipid acid on the apoptosis of human fetal lung fibroblasts in three dimensional collagen gels%1-磷酸鞘氨醇和溶血磷脂酸对三维胶原中肺成纤维细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    毛丽君; 陈明; 丁丽华; 李树强; 方秋红

    2013-01-01

    Objective To observe the effects of sphingosine 1-phosphate (SIP) and lysophospholipid acid (LPA) on the apoptosis of human fetal lung fibroblasts (HFL-1) in three dimensional collagen gels. Methods HFL-ls were cast into three dimensional culture system made with type I rat tail collagen, then different concentrations of S1P (10-6-10-8 mol/L) and LPA (10-5-10-7 mol/L) were added into the culture fluid. After contraction for 5 days, fibroblasts were harvested from the collagen gels by heating; then the apoptosis was assessed on cytospin preparations by TUNEL. Results Both S1P and LPA could stimulate the contraction of floating collagen gels, and could also inhibit the apoptosis of fibroblasts in 3D collagen gels in a concentration-dependent manner. Conclusions S1P and LPA may play a role in lung tissue remodeling and fibrosis formation by preventing fibroblasts apoptosis and the associated tissue contraction.%目的:观察l-磷酸鞘氨醇(S1P)和溶血磷脂酸(LPA)对三维培养肺纤维细胞HFL-1凋亡的影响.方法:利用Ⅰ型鼠尾胶原制作含有HFL-1细胞的三维培养系统,分别把不同浓度的S1P (10-6 ~ 10-8 mol/L)和LPA(10-5 ~ 10-7 mol/L)加入培养液中,待胶原收缩5d后,测量胶的面积并加热使胶原溶解,采用TUNEL染色细胞核计算肺纤维细胞凋亡的百分数.结果:S1P和LPA剂量依赖性地引起含有肺纤维细胞的胶原显著收缩,并抑制细胞凋亡.结论:SIP和LPA有引起组织收缩和抑制其内的纤维细胞凋亡的作用,提示S1P和LPA可能参与肺组织重构,与肺纤维化形成有关.

  4. Beryllium induces premature senescence in human fibroblasts.

    Science.gov (United States)

    Coates, Shannon S A; Lehnert, Bruce E; Sharma, Sunil; Kindell, Susan M; Gary, Ronald K

    2007-07-01

    After cells have completed a sufficient number of cell divisions, they exit the cell cycle and enter replicative senescence. Here, we report that beryllium causes proliferation arrest with premature expression of the principal markers of senescence. After young presenescent human fibroblasts were treated with 3 microM BeSO(4) for 24 h, p21 cyclin-dependent kinase inhibitor mRNA increased by >200%. Longer periods of exposure caused mRNA and protein levels to increase for both p21 and p16(Ink4a), a senescence regulator that prevents pRb-mediated cell cycle progression. BeSO(4) also caused dose-dependent induction of senescence-associated beta-galactosidase activity (SA-beta-gal). Untreated cells had 48 relative fluorescence units (RFU)/microg/h of SA-beta-gal, whereas 3 microM BeSO(4) caused activity to increase to 84 RFU/microg/h. In chromatin immunoprecipitation experiments, BeSO(4) caused p53 protein to associate with its DNA binding site in the promoter region of the p21 gene, indicating that p53 transcriptional activity is responsible for the large increase in p21 mRNA elicited by beryllium. Forced expression of human telomerase reverse transcriptase (hTERT) rendered HFL-1 cells incapable of normal replicative senescence. However, there was no difference in the responsiveness of normal HFL-1 fibroblasts (IC(50) = 1.9 microM) and hTERT-immortalized cells (IC(50) = 1.7 microM) to BeSO(4) in a 9-day proliferation assay. The effects of beryllium resemble those of histone deacetylase-inhibiting drugs, which also cause large increases in p21. However, beryllium produced no changes in histone acetylation, suggesting that Be(2+) acts as a novel and potent pharmacological inducer of premature senescence.

  5. 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)

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

    Science.gov (United States)

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

    1998-09-01

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

  7. Prostaglandin E₂ inhibits human lung fibroblast chemotaxis through disparate actions on different E-prostanoid receptors.

    Science.gov (United States)

    Li, Ying-Ji; Wang, Xing-Qi; Sato, Tadashi; Kanaji, Nobuhiro; Nakanishi, Masanori; Kim, Miok; Michalski, Joel; Nelson, Amy J; Sun, Jian-Hong; Farid, Maha; Basma, Hesham; Patil, Amol; Toews, Myron L; Liu, Xiangde; Rennard, Stephen I

    2011-01-01

    The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

  8. 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

  9. Distribution of melatonin receptor in human fetal brain

    Institute of Scientific and Technical Information of China (English)

    WANG Guo-quan; SHAO Fu-yuan; ZHAO Ying; LIU Zhi-min

    2001-01-01

    Objective: To study the distribution of 2 kinds of melatonin receptor subtypes (mtl and MT2) in human fetal brain. Methods: The fetal brain tissues were sliced and the distribution ofmelatonin receptors in human fetal brain were detected using immunohistochemistry and in situ hybridization. Results: Melatonin receptor mtl existed in the cerebellun and hypothalamus, melatonin receptor MT2 exists in hypothalamus, occipital and medulla. Conclusion: Two kinds of melatonin receptors, mtl and MT2 exist in the membrane and cytosol of brain cells, indicating that human fetal brain is a target organ of melatonin.

  10. Super-telomeres in transformed human fibroblasts.

    Science.gov (United States)

    Chiodi, Ilaria; Belgiovine, Cristina; Zongaro, Samantha; Ricotti, Roberta; Horard, Beatrice; Lossani, Andrea; Focher, Federico; Gilson, Eric; Giulotto, Elena; Mondello, Chiara

    2013-08-01

    Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.

  11. Hyaluronic acid production by irradiated human synovial fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yaron, M.; Yaron, I.; Levita, M.; Herzberg, M.

    1977-03-01

    Radioactive particles as well as x irradiation from an external source has been used in the treatment of rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In order to clarify effects of ionizing irradiation on synovial cells, radioactive gold (/sup 198/Au) and yttrium (/sup 90/Y) were added to fibroblast cultures derived from human synovial membranes. Other cultures were irradiated by a Picker x-ray machine. Fibroblast growth and hyaluronic acid production were measured. Radioactive gold and yttrium particles induced a significant increase of hyaluronic acid synthesis rate (pg/cell/day) and inhibited fibroblast growth. Fibroblasts continued to overproduce hyaluronic acid and to show growth inhibition 3 weeks after irradiation with radioactive gold. Hydrocortisone inhibited hyaluronic acid overproduction induced by radioactive gold. Overproduction of hyaluronic acid induced by the x-ray machine was inhibited by hydrocortisone, actinomycin-D, and cycloheximide. Fibroblasts derived from normal and rheumatoid patients responded similarly to ionizing irradiation.

  12. [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

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

    Science.gov (United States)

    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.

  14. Fetal magnetic resonance imaging and human genetics

    Energy Technology Data Exchange (ETDEWEB)

    Hengstschlaeger, Markus [Medical Genetics, Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna (Austria)]. E-mail: markus.hengstschlaeger@meduniwien.ac.at

    2006-02-15

    The use of fetal magnetic resonance imaging (MRI), in addition to prenatal genetic testing and sonography, has the potential to improve prenatal diagnosis of genetic disorders. MRI plays an important role in the evaluation of fetal abnormalities and malformations. Fetal MRI often enables a differential diagnosis, a determination of the extent of the disorder, the prognosis, and an improvement in therapeutic management. For counseling of parents, as well as to basically understand how genetic aberrations affect fetal development, it is of great importance to correlate different genotypes with fetal MRI data.

  15. Fetal Growth and Timing of Parturition in Humans

    Science.gov (United States)

    Sundaram, Rajeshwari; Sun, Wenyu; Troendle, James

    2008-01-01

    Animal studies indicate that either the fetus or the intrauterine environment, both of which set the pattern for fetal growth, may affect the timing of parturition. The authors examined the association between fetal growth and timing of spontaneous onset of labor in humans among low-risk white US women with singleton pregnancies (1987–1991). They restricted the data to pregnancies which had a reliable date of the last menstrual period, normal fetal growth in the first half of pregnancy, and no history of or current pregnancy complications that might have impaired fetal growth (n = 3,360). Subjects received ultrasound examinations at 15–22 and 31–35 weeks’ gestation. Fetal growth was adjusted for parity, fetal sex, and maternal prepregnancy weight and height. Results showed that slower or faster fetal growth in the second half of pregnancy resulted in substantially lower or higher birth weight, respectively. However, fetal growth in the second half of pregnancy, even at extremes (2 standard deviations below or above the mean), did not have a meaningful impact on the timing of parturition; neither did fetal growth acceleration or deceleration in late pregnancy. Thus, in low-risk pregnancies where fetal growth is normal in early gestation, fetal growth in the second half of pregnancy does not affect the timing of normal parturition. PMID:18775925

  16. Fetal growth and timing of parturition in humans.

    Science.gov (United States)

    Zhang, Jun; Sundaram, Rajeshwari; Sun, Wenyu; Troendle, James

    2008-10-15

    Animal studies indicate that either the fetus or the intrauterine environment, both of which set the pattern for fetal growth, may affect the timing of parturition. The authors examined the association between fetal growth and timing of spontaneous onset of labor in humans among low-risk white US women with singleton pregnancies (1987-1991). They restricted the data to pregnancies which had a reliable date of the last menstrual period, normal fetal growth in the first half of pregnancy, and no history of or current pregnancy complications that might have impaired fetal growth (n = 3,360). Subjects received ultrasound examinations at 15-22 and 31-35 weeks' gestation. Fetal growth was adjusted for parity, fetal sex, and maternal prepregnancy weight and height. Results showed that slower or faster fetal growth in the second half of pregnancy resulted in substantially lower or higher birth weight, respectively. However, fetal growth in the second half of pregnancy, even at extremes (2 standard deviations below or above the mean), did not have a meaningful impact on the timing of parturition; neither did fetal growth acceleration or deceleration in late pregnancy. Thus, in low-risk pregnancies where fetal growth is normal in early gestation, fetal growth in the second half of pregnancy does not affect the timing of normal parturition.

  17. SIRT3 mediates decrease of oxidative damage and prevention of ageing in porcine fetal fibroblasts.

    Science.gov (United States)

    Xie, Xiaoxian; Wang, Liangliang; Zhao, Binggong; Chen, Yangyang; Li, Jiaqi

    2017-05-15

    Sirtuin 3 (SIRT3) is a mitochondria-specific protein required for the deacetylation of metabolic enzymes and the action of oxidative phosphorylation by acting as a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase. SIRT3 increases oxidative stress resistance and prevents mitochondrial decay associated with ageing in response to caloric restriction. However, the effects of SIRT3 on oxidative damage and ageing are not well understood. We investigated the physiological functions of porcine SIRT3 on the damage and ageing in porcine fetal fibroblasts (PFFs). Overexpression and knockdown of SIRT3 were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. All cells were treated with three different stress reagents 12-o-tetradecanoylphorbol-13-acetate (TPA), methanesulfonic acid methylester (MMS), and tert-butylhydroperoxide (t-BHP), respectively, and then examined by flow cytometry following JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazol-carbocyanine iodide) staining. SIRT3 overexpression enhanced the ability of superoxide dismutase 2 (SOD2) to reduce cellular reactive oxygen species (ROS), which further decreased the damage to the membranes and the organelles of the cells, especially to mitochondria. It inhibited the initial decrease of mitochondrial membrane potential, and prevented the decrease of adenosine triphosphate (ATP) production and activity of Nampt. In contrast, SIRT3 knockdown reduced the ability of SOD2 to increase cellular ROS which was directly correlated with stress-induced oxidative damage and ageing in PFFs. Our findings identify one function of SIRT3 in PFFs was to dampen cytotoxicity, and, therefore, to decrease oxidative damage and attenuate ageing possibly by enhancing the activity of SOD2. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Non-saponifiable fraction of cocoa shell butter: effect on rat and human skin fibroblasts.

    Science.gov (United States)

    Warocquier-Clerout, R; Sigot, M; Ouraghi, M; Chaveron, H

    1992-02-01

    Synopsis Non-saponifiable lipid fraction (ICSB) extracted from cocoa shell butter was solubilized in dimethylformamide (DMF) and analysed for its biological activity on growth of rat and human fibroblasts. Non-saponifiables (10 mug ml(-1)) partially protected cells from toxicity of DMF (1%) and allowed the growth of fibroblasts cultivated in optimal conditions (10% fetal calf serum-FCS, 37 degrees C) or improved the survival of cells maintained in altered conditions (2.5% FCS, 35 degrees C). At higher concentration (ICSB 50 mug ml(-1), DMF 1%), the protective effect was suppressed. ICSB was fractionated by chromatography into four compounds: sterols, terpenic alcohols, tocopherols and hydrocarbons +/- carotenoids. We found that biological activity of ICSB was mostly due to the major fraction containing sterols.

  19. Culture and purification of human fetal olfactory bulb ensheathing cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To obtain high purity of human fetal olfactory bulb ensheathing cells (OB-hOECs) in vitro and to develop a simple and effective method for primary culture of OB-hOECs. Methods: OB-hOECs were cultured based on the differential rates of attachment of the various harvested cell types. Then the method was combined with arabinoside cytosine (Ara-C)inhibition, serum-free starvation or intermittent neurotrophin 3 (NT3) nutrition method to observe cell states in different cultural environments. The purity of OB-hOECs was assessed with immunocytochemical analysis. Results: OB-hOECs appeared bipolar and tripolar shape, with slender processes forming network. The purity of OECs reached 88% with the selective attachment method on day 6, and then fibroblast proliferated quickly and reduced the purity. When combined with the starvation method, the purity of OECs was 91% on day 6 and 86% on day 9, however, OECs were in a poor state. While combined with the NT3 method, the purity reached 95% on day 9 and 83% on day 12, respectively. The cells still remained in a good state. Conclusion: A combination of selective attachment and intermittent NT3 nutrition is an effective method to obtain OECs with higher purity and quality.

  20. [Biomechanical characteristics of human fetal membranes. Preterm fetal membranes are stronger than term fetal membranes].

    Science.gov (United States)

    Rangaswamy, N; Abdelrahim, A; Moore, R M; Uyen, L; Mercer, B M; Mansour, J M; Kumar, D; Sawady, J; Moore, J J

    2011-06-01

    The purpose of this study was to determine the biomechanical characteristics of human fetal membranes (FM) throughout gestation. Biomechanical properties were determined for 115 FM of 23-41 weeks gestation using our previously described methodology. The areas of membrane immediately adjacent to the strongest and weakest tested spots were sampled for histomorphometric analysis. Clinical data on the patients whose FM were examined were also collected. FM less than 28 weeks gestation were associated with higher incidence of abruption and chorioamnionitis. Topographically FM at all gestations had heterogeneous biomechanical characteristics over their surfaces with distinct weak areas. The most premature membranes were the strongest. FM strength represented by rupture force and work to rupture decreased with increasing gestation in both weak and strong regions of FM. This decrease in FM strength was most dramatic at more than 38 weeks gestation. The FM component amnion-chorion sublayers were thinner in the weak areas compared to strong areas. Compared to term FM, preterm FM are stronger but have similar heterogeneous weak and strong areas. Following a gradual increase in FM weakness with increasing gestation, there is a major drop-off at term 38 weeks gestation. The FM weak areas are thinner than the stronger areas. Whether the difference in thickness is enough to account for the strength differences is unknown.

  1. Blood pressure estimation in the human fetal descending aorta.

    NARCIS (Netherlands)

    Struijk, P.C.; Mathews, V.J.; Loupas, T.; Stewart, P.A.; Clark, E.B.; Steegers, E.A.P.; Wladimiroff, J.W.

    2008-01-01

    OBJECTIVES: The objectives of this study were to estimate fetal blood pressure non-invasively from two-dimensional color Doppler-derived aortic blood flow and diameter waveforms, and to compare the results with invasively derived human fetal blood pressures available from the literature. METHODS:

  2. Blood pressure estimation in the human fetal descending aorta

    NARCIS (Netherlands)

    P.C. Struijk (Pieter); V.J. Mathews; T. Loupas; P.A. Stewart (Patricia); E.B. Clark; R.P.M. Steegers-Theunissen (Régine); J.W. Wladimiroff (Juriy)

    2008-01-01

    textabstractObjectives: The objectives of this study were to estimate fetal blood pressure non-invasively from two-dimensional color Doppler-derived aortic blood flow and diameter waveforms, and to compare the results with invasively derived human fetal blood pressures available from the literature.

  3. Differentiation and functional regulation of human fetal NK cells.

    Science.gov (United States)

    Ivarsson, Martin A; Loh, Liyen; Marquardt, Nicole; Kekäläinen, Eliisa; Berglin, Lena; Björkström, Niklas K; Westgren, Magnus; Nixon, Douglas F; Michaëlsson, Jakob

    2013-09-01

    The human fetal immune system is naturally exposed to maternal allogeneic cells, maternal antibodies, and pathogens. As such, it is faced with a considerable challenge with respect to the balance between immune reactivity and tolerance. Here, we show that fetal natural killer (NK) cells differentiate early in utero and are highly responsive to cytokines and antibody-mediated stimulation but respond poorly to HLA class I-negative target cells. Strikingly, expression of killer-cell immunoglobulin-like receptors (KIRs) did not educate fetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. In addition, fetal NK cells were highly susceptible to TGF-β-mediated suppression, and blocking of TGF-β signaling enhanced fetal NK cell responses to target cells. Our data demonstrate that KIR-mediated hyporesponsiveness and TGF-β-mediated suppression are major factors determining human fetal NK cell hyporesponsiveness to HLA class I-negative target cells and provide a potential mechanism for fetal-maternal tolerance in utero. Finally, our results provide a basis for understanding the role of fetal NK cells in pregnancy complications in which NK cells could be involved, for example, during in utero infections and anti-RhD-induced fetal anemia.

  4. 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.

  5. 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.

  6. Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas.

    Science.gov (United States)

    Chen, Xiaochuan; Rozance, Paul J; Hay, William W; Limesand, Sean W

    2012-05-01

    Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Keiko Miyoshi

    2015-01-01

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

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

    Science.gov (United States)

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

    2012-09-01

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

  10. SV40-mediated immortalization of human fibroblasts.

    Science.gov (United States)

    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  11. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    Science.gov (United States)

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

    2015-05-01

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

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

    Science.gov (United States)

    Carpenter, G; Cohen, S

    1976-06-01

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

  13. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

    Science.gov (United States)

    Bishop, J E; Mitchell, J J; Absher, P M; Baldor, L; Geller, H A; Woodcock-Mitchell, J; Hamblin, M J; Vacek, P; Low, R B

    1993-08-01

    Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

  14. Effects of trichostatin A on human Tenon capsule fibroblast

    Directory of Open Access Journals (Sweden)

    Xiao-Yan Li

    2014-11-01

    Full Text Available AIM:To investigate the effect of trichostatin A(TSAon cell proliferation and the expressions of histone deacetylase 1(HDAC1and HDAC2 in cultured human Tenon capsule fibroblast(HTF.METHODS: Human Tenon capsule fibroblasts were cultured in vitro after glaucoma filtration surgery. The third to sixth passage of cell were treated by 600nmol/L TSA or none. Cell viability measured by MTT assay after 1, 2 and 3d respectively. The expressions of HDAC1 and HDAC2 were analyzed by Western blot 2d after TSA treatment.RESULTS: Compared to the control, cell viability decreased significantly after treatment with TSA at 1d(PCONCLUSION: TSA inhibits the proliferation of Tenon capsule fibroblast by inhibiting the expression of HDAC1 and HDAC2, and reduces subconjunctival scar formation.

  15. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  16. Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain

    Directory of Open Access Journals (Sweden)

    Chongyuan Luo

    2016-12-01

    Full Text Available Organoids derived from human pluripotent stem cells recapitulate the early three-dimensional organization of the human brain, but whether they establish the epigenomic and transcriptional programs essential for brain development is unknown. We compared epigenomic and regulatory features in cerebral organoids and human fetal brain, using genome-wide, base resolution DNA methylome and transcriptome sequencing. Transcriptomic dynamics in organoids faithfully modeled gene expression trajectories in early-to-mid human fetal brains. We found that early non-CG methylation accumulation at super-enhancers in both fetal brain and organoids marks forthcoming transcriptional repression in the fully developed brain. Demethylated regions (74% of 35,627 identified during organoid differentiation overlapped with fetal brain regulatory elements. Interestingly, pericentromeric repeats showed widespread demethylation in multiple types of in vitro human neural differentiation models but not in fetal brain. Our study reveals that organoids recapitulate many epigenomic features of mid-fetal human brain and also identified novel non-CG methylation signatures of brain development.

  17. Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

    Directory of Open Access Journals (Sweden)

    Zigman Warren B

    2006-03-01

    Full Text Available Abstract Background Down syndrome (DS is caused by trisomy 21 (+21, but the aberrations in gene expression resulting from this chromosomal aneuploidy are not yet completely understood. Methods We used oligonucleotide microarrays to survey mRNA expression in early- and late-passage control and +21 fibroblasts and mid-gestation fetal hearts. We supplemented this analysis with northern blotting, western blotting, real-time RT-PCR, and immunohistochemistry. Results We found chromosome 21 genes consistently over-represented among the genes over-expressed in the +21 samples. However, these sets of over-expressed genes differed across the three cell/tissue types. The chromosome 21 gene MX1 was strongly over-expressed (mean 16-fold in senescent +21 fibroblasts, a result verified by northern and western blotting. MX1 is an interferon target gene, and its mRNA was induced by interferons present in +21 fibroblast conditioned medium, suggesting an autocrine loop for its over-expression. By immunohistochemistry the p78MX1 protein was induced in lesional tissue of alopecia areata, an autoimmune disorder associated with DS. We found strong over-expression of the purine biosynthesis gene GART (mean 3-fold in fetal hearts with +21 and verified this result by northern blotting and real-time RT-PCR. Conclusion Different subsets of chromosome 21 genes are over-expressed in different cell types with +21, and for some genes this over-expression is non-linear (>1.5X. Hyperactive interferon signaling is a candidate pathway for cell senescence and autoimmune disorders in DS, and abnormal purine metabolism should be investigated for a potential role in cardiac defects.

  18. Identification of human fibroblast cell lines as a feeder layer for human corneal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Rong Lu

    Full Text Available There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4 in a 35-mm dish (9.6 cm(2 grew to confluence (about 1.87-2.41 × 10(6 cells in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.

  19. DETACHMENT OF HUMAN FIBROBLASTS FROM FEP-TEFLON SURFACES

    NARCIS (Netherlands)

    VANKOOTEN, TG; SCHAKENRAAD, JM; VANDERMEI, HC; BUSSCHER, HJ

    1991-01-01

    In this study a comparison is made between the detachment behavior of human fibroblasts adhered to hydrophobic FEP-Teflon (water contact angle 109 degrees) and to hydrophilic glass (water contact angle smaller than 15 degrees) during exposure to a laminar, incrementally loaded flow. Detachment from

  20. Aminoguanidine delays the replicative senescence of human diploid fibroblasts

    Institute of Scientific and Technical Information of China (English)

    WANG Pei-chang; ZHANG Jian; ZHANG Zong-yu; TONG Tan-jun

    2007-01-01

    advanced glycation end products; comet assayBackground The accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro.Methods The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS).Results Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17-21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 μmol/L H2O2 for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 μmol/L H2O2 had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11kb longer than that of the control cells.Conclusion Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation

  1. Basic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor-1in Human Meningiomas

    Institute of Scientific and Technical Information of China (English)

    YI Wei; CHEN Jian; Filimon H. Golwa; XUE Delin

    2005-01-01

    The expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in human meningiomas and the relationships between their expression and the tumors' histological features and angiogenesis were investigated by means of immunohistochemical technique. The expression of bFGF and FGFR-1 was detected by antibody of bFGF or FGFR-1.The tumors' angiogenesis was evaluated by microvascular density (MVD) and, which was observed by use of CD34-antibody immunohistochemically. The results showed that there were varied degrees of the expression of bFGF and FGFR-1 proteins in meningiomas. The expression was correlated with the tumors' histological characters and angiogenesis. It was concluded that bFGF and FGFR-1 might play important roles in meningiomas' angiogenesis and proliferation. The expression positive rate of bFGF and FGFR-1 may provide an indication of evaluating the histological and malignant degree of the tumor.

  2. Single Colony Preparation and Amplification Culture of Fetal Fibroblast and Mammary Gland Epithelial Cell of Human Lactoferrin Transgene from Goat%山羊转人乳铁蛋白基因成纤维细胞和乳腺上皮细胞单克隆的制备及扩大培养

    Institute of Scientific and Technical Information of China (English)

    张玉玲; 刘凤军; 张涌

    2009-01-01

    [ Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C, which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [ Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells. Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture ( FF: 53.33% vs. 10.00% ;MGE; 33. 33% vs. 6. 67% ) , confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [ Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.%[目的]为探索单个转基因阳性细胞快速扩大培养成为细胞克隆的技术体系.[方法]对转染人乳铁蛋白(Human lactoferrin,hLF)基因乳腺特异性表达载体pBLM-C1的单个山羊胎儿成纤维细胞(Fetal Fibroblasts,FF)和乳腺上皮细胞(Mammary Gland Epithelial,MGE)细胞进行克隆.在96孔板中,首先用3种浓度(V/V: 0 、50%和100%)的适应性条件培养基对单个转染细胞进行细胞单克隆的制备,进而把转染细胞单克隆与非转

  3. 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.

  4. Generation of human induced pluripotent stem cells from dermal fibroblasts

    OpenAIRE

    2008-01-01

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ...

  5. Effect of placental factors on growth and function of the human fetal adrenal in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Riopel, L.; Branchaud, C.L.; Goodyer, C.G.; Zweig, M.; Lipowski, L.; Adkar, V.; Lefebvre, Y. (McGill Univ.-Montreal Children' s Hospital Research Institute, Quebec (Canada))

    1989-11-01

    Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: (1) maximal response to PM was 2-5 times greater; (2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; (3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.

  6. Human embryonic and fetal mesenchymal stem cells differentiate toward three different cardiac lineages in contrast to their adult counterparts.

    Science.gov (United States)

    Ramkisoensing, Arti A; Pijnappels, Daniël A; Askar, Saïd F A; Passier, Robert; Swildens, Jim; Goumans, Marie José; Schutte, Cindy I; de Vries, Antoine A F; Scherjon, Sicco; Mummery, Christine L; Schalij, Martin J; Atsma, Douwe E

    2011-01-01

    Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

  7. Distinct functional programming of human fetal and adult monocytes.

    Science.gov (United States)

    Krow-Lucal, Elisabeth R; Kim, Charles C; Burt, Trevor D; McCune, Joseph M

    2014-03-20

    Preterm birth affects 1 out of 9 infants in the United States and is the leading cause of long-term neurologic handicap and infant mortality, accounting for 35% of all infant deaths in 2008. Although cytokines including interferon-γ (IFN-γ), interleukin-10 (IL-10), IL-6, and IL-1 are produced in response to in utero infection and are strongly associated with preterm labor, little is known about how human fetal immune cells respond to these cytokines. We demonstrate that fetal and adult CD14(+)CD16(-) classical monocytes are distinct in terms of basal transcriptional profiles and in phosphorylation of signal transducers and activators of transcription (STATs) in response to cytokines. Fetal monocytes phosphorylate canonical and noncanonical STATs and respond more strongly to IFN-γ, IL-6, and IL-4 than adult monocytes. We demonstrate a higher ratio of SOCS3 to IL-6 receptor in adult monocytes than in fetal monocytes, potentially explaining differences in STAT phosphorylation. Additionally, IFN-γ signaling results in upregulation of antigen presentation and costimulatory machinery in adult, but not fetal, monocytes. These findings represent the first evidence that primary human fetal and adult monocytes are functionally distinct, potentially explaining how these cells respond differentially to cytokines implicated in development, in utero infections, and the pathogenesis of preterm labor.

  8. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

    2011-01-01

    We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  9. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chan-Jung Chang

    Full Text Available We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  10. Involvement of the mitochondrial compartment in human NCL fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Pezzini, Francesco; Gismondi, Floriana [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Tessa, Alessandra [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Tonin, Paola [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Carrozzo, Rosalba [IRCCS Bambino Gesu Hospital-Molecular Medicine Unit, Roma (Italy); Mole, Sara E. [MRC Laboratory for Molecular Cell Biology, Molecular Medicines Unit, UCL Institute of Child Health and Department of Genetics, Evolution and Environment, University College London (United Kingdom); Santorelli, Filippo M. [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Simonati, Alessandro, E-mail: alessandro.simonati@univr.it [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy)

    2011-12-09

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

  11. Human fetal bone cells in delivery systems for bone engineering.

    Science.gov (United States)

    Tenorio, Diene M H; Scaletta, Corinne; Jaccoud, Sandra; Hirt-Burri, Nathalie; Pioletti, Dominique P; Jaques, Bertrand; Applegate, Lee Ann

    2011-11-01

    The aim of this study was to culture human fetal bone cells (dedicated cell banks of fetal bone derived from 14 week gestation femurs) within both hyaluronic acid gel and collagen foam, to compare the biocompatibility of both matrices as potential delivery systems for bone engineering and particularly for oral application. Fetal bone cell banks were prepared from one organ donation and cells were cultured for up to 4 weeks within hyaluronic acid (Mesolis®) and collagen foams (TissueFleece®). Cell survival and differentiation were assessed by cell proliferation assays and histology of frozen sections stained with Giemsa, von Kossa and ALP at 1, 2 and 4 weeks of culture. Within both materials, fetal bone cells could proliferate in three-dimensional structure at ∼70% capacity compared to monolayer culture. In addition, these cells were positive for ALP and von Kossa staining, indicating cellular differentiation and matrix production. Collagen foam provides a better structure for fetal bone cell delivery if cavity filling is necessary and hydrogels would permit an injectable technique for difficult to treat areas. In all, there was high biocompatibility, cellular differentiation and matrix deposition seen in both matrices by fetal bone cells, allowing for easy cell delivery for bone stimulation in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

  12. Cytosolic malate dehydrogenase regulates senescence in human fibroblasts.

    Science.gov (United States)

    Lee, Seung-Min; Dho, So Hee; Ju, Sung-Kyu; Maeng, Jin-Soo; Kim, Jeong-Yoon; Kwon, Ki-Sun

    2012-10-01

    Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated β-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.

  13. Effect of periodontal dressings on human gingiva fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E. (Ohio State Univ. College of Dentistry, Columbus (USA))

    1989-08-01

    In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings.

  14. Materno-Fetal Transmission of Human Immune Deficiency Virus

    Directory of Open Access Journals (Sweden)

    Axel Schäfer

    1997-01-01

    Full Text Available Mother-to-child transmission of human immune deficiency virus (HIV is a multifactorial event highly associated with advanced maternal HIV disease and obstetric incidents taking place during parturition. Thus, various approaches to prevention may be beneficial. Although the time and the route of materno-fetal HIV transmission are still not sufficiently clear, much speaks in favor of a late HIV transmission, most probably taking place during parturition or the phase before the delivery. The fetus is remarkably protected by the placenta and the intact fetal membranes against many viral infections during gestation. These conditions change at parturition and the chance for a transition of HIV-infected carrier cells or virus into the fetal compartment increases. Proinflammatory cytokines secreted at the materno-fetal interface accumulate in amniotic fluid and may chemoattract and stimulate potentially HIV-infected immunocytes. After rupture of membranes, maternal cells of the decidua are directly exposed to the amniotic fluid. Aside from the contamination of the fetal skin at vaginal delivery as a debatable route of infection, blood-to-blood contacts and the fetal swallowing of contaminated amniotic fluid may be the major path of fetal HIV infection. For the fetal prophylaxis of an intrauterine infection, the application of zidovudine is recommended. However, cesarian section before the onset of labor leads also to a diminution of the transmission rate. As the transmission seems to have both systemic and local causes, it makes sense to combine different intervention strategies. Whether a combination of zidovudine and elective cesarean section can lower the transmission risk further has to be evaluated.

  15. Biocompatibility of Mineral Trioxide Aggregate with TiO2 Nanoparticles on Human Gingival Fibroblasts

    Science.gov (United States)

    Samiei, Mohammad; Aghazadeh, Marzieh; Divband, Baharak; Akbarzadeh, Farzaneh

    2017-01-01

    Background The New compositions of white mineral trioxide aggregate (WMTA) or use of various additives like nanoparticles might affect MTA’s ideal characteristics This study was performed to evaluate the cytotoxicity of WMTA and WMTA with Titanium dioxide (TiO2) nanoparticles (1% weight ratio) at different storage times after mixing on human gingival fibroblasts (HGFs). Material and Methods HGFs were obtained from the attached gingiva of human premolars. HGFs were cultured in Dulbecco’s Modified Eagle medium, supplemented with 10% fetal calf serum, penicillin and streptomycin. The cells were exposed to WMTA (groups 1 and 2) and WMTA+TiO2 (groups 3 and 4). The fifth and sixth groups served as controls. Each group contained 15 wells. After 24h (groups 1, 3 and 5) and 48 h (groups 2, 4 and 6) of exposure, HGF viability was determined by Mosmann’s tetrazolium toxicity (MTT) assay. Statistical analysis of the data was performed by using one-way analysis of variance and Tukey post hoc test, with significance of p 0.05). Conclusions Under the limitations of the present study, incorporation of TiO2 nanoparticles into MTA at 1 wt% had no negative effect on its biocompatibility. Key words:Cytotoxicity, fibroblast, MTA, MTT assay, nanoparticle, TiO2. PMID:28210432

  16. Inhibition of normal human lung fibroblast growth by beryllium.

    Science.gov (United States)

    Lehnert, N M; Gary, R K; Marrone, B L; Lehnert, B E

    2001-03-07

    Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset ( approximately 1-6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO(4) (0.1-100 microM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be(2+)-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G(0)-G(1)/pre-S phase arrest. Western blot analyses indicated that the Be-induced G(0)-G(1)/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21(Waf-1,Cip1)). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

  17. 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.

  18. Evidence that neomycin inhibits human cytomegalovirus infection of fibroblasts.

    Science.gov (United States)

    Lobert, P E; Hober, D; Delannoy, A S; Wattré, P

    1996-01-01

    The effect of phosphoinositide-binding aminoglycosides, such as neomycin, gentamicin and streptomycin, on human cytomegalovirus (HCMV) infection of human fibroblasts MRC-5 was studied. The inhibition of HCMV infection was obtained with all of these molecules but neomycin was more effective than the others. We showed that the inoculation of the cells with cell-free viral suspension in presence of neomycin concentrations above 5 mM at 37 degrees C, inhibited more than 98% the HCMV infection. However, the preincubation of the fibroblasts with neomycin at 4 degrees C, before the removal of the drug and the inoculation of the cells, induced only a 30% decrease in the number of infected cells. Addition of neomycin after the HCMV-binding at 4 degrees C or the infection of the cells was less efficient to inhibit HCMV infection than the standard incubation of neomycin during inoculation of the fibroblasts. Indeed, 1 hour after the inoculation of the cells at 37 degrees C, neomycin still inhibited HCMV infection, but 4 hours after the inoculation, this drug had no effect on HCMV infection. Our findings demonstrated that neomycin must be present at the time of infection in order to exert a full inhibiting effect. The effect of neomycin on the HCMV infection was almost immediate upon the addition of the drug (binding and/or internalization) and after the virus internalization (inhibition of immediate-early events). We suggest that neomycin and other aminoglycoside antibiotics may interact with HCMV glycoproteins for binding to similar structural features of cell surface heparan sulfate proteoglycans and may inhibit HCMV infection in fibroblasts by disrupting phosphoinositide-mediated events in the cells.

  19. The ontogeny of scarless healing II: EGF and PDGF-B gene expression in fetal rat skin and fibroblasts as a function of gestational age.

    Science.gov (United States)

    Peled, Z M; Rhee, S J; Hsu, M; Chang, J; Krummel, T M; Longaker, M T

    2001-10-01

    Twenty years ago, surgeons noted the ability of early-gestation fetal skin to heal in a scarless manner. Since that time, numerous investigators have attempted to elucidate the mechanisms behind this phenomenon. As a result of this effort, it is now well established that many animals undergo a transition late in development from scarless cutaneous healing to a scar-forming, adultlike phenotype. The authors have been interested in the role played by cytokines known to be involved in the adult wound-healing process and how they relate to scarless repair. They therefore asked the following question: Are genes for epidermal growth factor (EGF) and platelet-derived growth factor-B (PDGF-B) expressed differentially as a function of gestational age in fetal rat skin and dermal fibroblasts? To answer this question, skin from fetal Sprague-Dawley rats (N = 56) at time points that represented both the scarless and scar-forming periods of rat gestation was harvested. In addition, fibroblasts derived from fetal rat skin were cultured in vitro at similar times. These cells were expanded in culture and, when confluent, total ribonucleic acid from both fibroblasts and whole skin was extracted and subjected to Northern blot analysis with probes for EGF and PDGF-B. Results demonstrated that neither EGF nor PDGF-B gene expression changed markedly as a function of gestational age in fetal fibroblasts alone. In whole skin, however, both EGF and PDGF-B demonstrated a marked decrease in gene expression with increasing gestational age. Furthermore, the most striking decrease in gene expression for both cytokines came between 16 and 18 days of gestation-the transition point between scarless and scar-forming repair in the fetal rat. These data suggest that EGF and PDGF may play a role in the mechanism of scarless cutaneous repair. Moreover, it appears that fetal fibroblasts are not the cell type responsible for this differential gene expression. These results raise questions about the

  20. Differentiated human stem cells resemble fetal, not adult, β cells.

    Science.gov (United States)

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  1. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    Science.gov (United States)

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

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

    Science.gov (United States)

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

    2011-08-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  4. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  5. Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

    Science.gov (United States)

    Lopez, Ana D.; Kayali, Ayse G.; Hayek, Alberto; King, Charles C.

    2014-01-01

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells. PMID:24895054

  6. Isolation, culture, and imaging of human fetal pancreatic cell clusters.

    Science.gov (United States)

    Lopez, Ana D; Kayali, Ayse G; Hayek, Alberto; King, Charles C

    2014-05-18

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

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

    Science.gov (United States)

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

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

  8. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

    DEFF Research Database (Denmark)

    Lepekhin, Eugene; Grøn, Birgitte; Berezin, Vladimir

    2002-01-01

    at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer...... displacement of periodontal and skin fibroblasts. The decreased cellular displacement of the buccal fibroblasts was found to be due to both lower cellular speed and less persistence in direction. The buccal fibroblasts also displayed smaller areas and longer processes. The differences in cellular morphology...

  9. Enabling research with human embryonic and fetal tissue resources

    Science.gov (United States)

    Gerrelli, Dianne; Lisgo, Steven; Copp, Andrew J.; Lindsay, Susan

    2015-01-01

    Summary Congenital anomalies are a significant burden on human health. Understanding the developmental origins of such anomalies is key to developing potential therapies. The Human Developmental Biology Resource (HDBR), based in London and Newcastle UK, was established to provide embryonic and fetal material for a variety of human studies ranging from single gene expression analysis to large scale genomic/transcriptomic studies. Increasingly HDBR material is enabling the derivation of stem cell lines and contributing towards developments in tissue engineering. Use of the HDBR and other fetal tissue resources discussed here will contribute to the long term aims of understanding the causation and pathogenesis of congenital anomalies, and developing new methods for their treatment and prevention. PMID:26395135

  10. Evaluation of functions and tissue compatibility of poly (D,L-lactic-co-glycolic acid) seeded with human dermal fibroblasts.

    Science.gov (United States)

    Yang, Won-Sun; Roh, Hye-Won; Lee, Won Kyu; Ryu, Gyu Ha

    2006-01-01

    In tissue engineering and wound-healing applications, dermal substitutes are used to provide fibroblasts with the mechanical support for their growth and then to facilitate the skin formation. In this study, three-dimensional porous poly(lactic-co-glycolic acid) (PLGA) 65/35 scaffolds were prepared and then the composites of the scaffolds and human fetal dermal fibroblasts were fabricated as a tissue-engineered dermal substitute. The function and tissue compatibility of the artificial dermal substitute were evaluated at the levels of gene expression (by RT-PCR) and protein expression (total collagen quantities), as well as by histological and immunohistochemical analysis. The PCR products indicated that the mRNA of type-I collagen, mainly secreted by the fibroblasts onto the PLGA scaffolds, was clearly expressed after 4 weeks. The amount of total collagen synthesized from the cells was shown to increase gradually during the initial culture period and slightly decreased afterwards. After 8 weeks of culture, the fibroblasts were well attached and migrated entirely throughout the pores of the PLGA scaffold with normal function. Furthermore, the positively stained type-I collagen was intensively detected throughout the pores. These results suggest that the function and tissue compatibility may be important criteria in evaluating an artificial tissue-engineered skin.

  11. Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

    Science.gov (United States)

    Keller, Amy C; Badani, Hussain; McClatchey, P Mason; Baird, Nicholas L; Bowlin, Jacqueline L; Bouchard, Ron; Perng, Guey-Chuen; Reusch, Jane E B; Kaufer, Benedikt B; Gilden, Don; Shahzad, Aamir; Kennedy, Peter G E; Cohrs, Randall J

    2016-10-01

    Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting. IE63 interacted with cytochrome c oxidase in bacterial 2-hybrid analyses. Confocal microscopy of VZV-infected HFL cells at multiple times after infection revealed the presence of IE63 in the nucleus, mitochondria, and cytoplasm. Our data provide the first evidence that VZV infection induces alterations in mitochondrial morphology, including fragmentation, which may be involved in cellular damage and/or death during virus infection.

  12. Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

    Science.gov (United States)

    Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

    2015-01-01

    Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals.

  13. Oxidized DNA induces an adaptive response in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kostyuk, Svetlana V., E-mail: svet.kostyuk@gmail.com [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Tabakov, Viacheslav J.; Chestkov, Valerij V.; Konkova, Marina S.; Glebova, Kristina V.; Baydakova, Galina V.; Ershova, Elizaveta S.; Izhevskaya, Vera L. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha, E-mail: abaranov@gmu.edu [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Center for the Study of Chronic Metabolic Diseases, School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2013-07-15

    Highlights: • We describe the effects of gDNAOX on human fibroblasts cultivated in serum withdrawal conditions. • gDNAOX evokes an adaptive response in human fibroblasts. • gDNAOX increases the survival rates in serum starving cell populations. • gDNAOX enhances the survival rates in cell populations irradiated at 1.2 Gy dose. • gDNAOX up-regulates NRF2 and inhibits NF-kappaB-signaling. - Abstract: Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA{sup OX}. The levels of cfDNA{sup OX} are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by H{sub 2}O{sub 2}in vitro (gDNA{sup OX}) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA{sup OX} on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA{sup OX} evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2 Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA{sup OX} leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of PSNA, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA{sup OX} and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA{sup OX} inhibits NF-κB signaling. gDNA{sup OX} is a model for oxidized cfDNA{sup OX} that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA

  14. Perivascular mesenchymal progenitors in human fetal and adult liver.

    Science.gov (United States)

    Gerlach, Jörg C; Over, Patrick; Turner, Morris E; Thompson, Robert L; Foka, Hubert G; Chen, William C W; Péault, Bruno; Gridelli, Bruno; Schmelzer, Eva

    2012-12-10

    The presence of mesenchymal stem cells (MSCs) has been described in various organs. Pericytes possess a multilineage differentiation potential and have been suggested to be one of the developmental sources for MSCs. In human liver, pericytes have not been defined. Here, we describe the identification, purification, and characterization of pericytes in human adult and fetal liver. Flow cytometry sorting revealed that human adult and fetal liver contains 0.56%±0.81% and 0.45%±0.39% of CD146(+)CD45(-)CD56(-)CD34(-) pericytes, respectively. Of these, 41% (adult) and 30% (fetal) were alkaline phosphatase-positive (ALP(+)). In situ, pericytes were localized around periportal blood vessels and were positive for NG2 and vimentin. Purified pericytes could be cultured extensively and had low population doubling times. Immunofluorescence of cultures demonstrated that cells were positive for pericyte and mesenchymal cell markers CD146, NG2, CD90, CD140b, and vimentin, and negative for endothelial, hematopoietic, stellate, muscle, or liver epithelial cell markers von Willebrand factor, CD31, CD34, CD45, CD144, CD326, CK19, albumin, α-fetoprotein, CYP3A7, glial fibrillary acid protein, MYF5, and Pax7 by gene expression; myogenin and alpha-smooth muscle actin expression were variable. Fluorescence-activated cell sorting analysis of cultures confirmed surface expression of CD146, CD73, CD90, CD10, CD13, CD44, CD105, and ALP and absence of human leukocyte antigen-DR. In vitro differentiation assays demonstrated that cells possessed robust osteogenic and myogenic, but low adipogenic and low chondrogenic differentiation potentials. In functional in vitro assays, cells had typical mesenchymal strong migratory and invasive activity. In conclusion, human adult and fetal livers harbor pericytes that are similar to those found in other organs and are distinct from hepatic stellate cells.

  15. Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhenwei Qin

    2016-01-01

    Full Text Available Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs. Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs; ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R antagonist (Diphenhydramine Hydrochloride and histamine receptor-2 (H2R antagonist (Nizatidine were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L on HPFs was lower than on HCFs (100 μmol/L, and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.

  16. Cytotoxic effects of nickel nanowires in human fibroblasts

    KAUST Repository

    Felix Servin, Laura P.

    2016-03-09

    The increasing interest in the use of magnetic nanostructures for biomedical applications necessitates rigorous studies to be carried out in order to determine their potential toxicity. This work attempts to elucidate the cytotoxic effects of nickel nanowires (NWs) in human fibroblasts WI-38 by a colorimetric assay (MTT) under two different parameters: NW concentration and exposure time. This was complemented with TEM and confocal images to assess the NWs internalization and to identify any changes in the cell morphology. Ni NWs were fabricated by electrodeposition using porous alumina templates. Energy dispersive X-Ray analysis, scanning electron microscopy and transmission electron microscopy imaging were used for NW characterization. The results showed decreased cell metabolic activity for incubation times longer than 24 hours and no negative effects for exposure times shorter than that. The cytotoxicity effects for human fibroblasts were then compared with those reported for HCT 116 cells, and the findings point out that it is relevant to consider the cellular size. In addition, the present study compares the toxic effects of equivalent amounts of nickel in the form of its salt to those of NWs and shows that the NWs are more toxic than the salts. Internalized NWs were found in vesicles inside of the cells where their presence induced inflammation of the endoplasmic reticulum.

  17. Perinatal Oxidative Stress May Affect Fetal Ghrelin Levels in Humans

    OpenAIRE

    Zhong-Cheng Luo; Jean-François Bilodeau; Anne Monique Nuyt; Fraser, William D; Pierre Julien; Francois Audibert; Lin Xiao; Carole Garofalo; Emile Levy

    2015-01-01

    In vitro cell model studies have shown that oxidative stress may affect beta-cell function. It is unknown whether oxidative stress may affect metabolic health in human fetuses/newborns. In a singleton pregnancy cohort (n = 248), we studied maternal (24–28 weeks gestation) and cord plasma biomarkers of oxidative stress [malondialdehyde (MDA), F2-isoprostanes] in relation to fetal metabolic health biomarkers including cord plasma glucose-to-insulin ratio (an indicator of insulin sensitivity), p...

  18. Schwann cell cultures from human fetal dorsal root ganglia

    Institute of Scientific and Technical Information of China (English)

    Yaping Feng; Hui Zhu; Jiang Hao; Xinmin Wang; Shengping Wu; Li Bai; Xiangming Li; Yun Zha

    2009-01-01

    BACKGROUND:Previous studies have used many methods for in vitro Schwann cells (SCs) cul-tures and purification,such as single cell suspension and cytosine arabinoside.However,it has been difficult to obtain sufficient cellular density,and the procedures have been quite tedious.OBJECTIVE:To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants.DESIGN,TIME AND SETTING:Cell culture and immunohistochemistry were performed at the Cen-tral Laboratory of Kunming General Hospital of Chinese PLA between March 2001 and October 2008.MATERIALS:Culture media containing 10% fetal bovine serum,as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco,USA;mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Bi-ological Products,China.METHODS:Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fe-tuses at 4-6 months pregnancy.Following removal of the dorsal root ganglion perineurium,the gan-glia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1),then explanted and cultured.SC purification was performed with 5 mL 10% fetal bovine serum added to the culture media,followed by differential adhesion.MAIN OUTCOME MEASURES:SCs morphology was observed under inverted phase contrast light microscopy.SC purity was evaluated according to percentage of S-100 immunostained cells.RESULTS:SCs were primarily cultured for 5-6 days and then subcultured for 4-5 passages.The highly enriched SC population reached > 95% purity and presented with normal morphology.CONCLUSION:A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.

  19. Changes of multiple biotransformation phase Ⅰ and phase Ⅱ enzyme activities in human fetal adrenals during fetal development

    Institute of Scientific and Technical Information of China (English)

    Hui WANG; Jie PING; Ren-xiu PENG; Jiang YUE; Xue-yan XIA; Qi-xiong LI; Rui KONG; Jun-yan HONG

    2008-01-01

    Aim: Fetal adrenal, which synthesizes steroid hormones, is critical to fetal growth and development. Our recent research showed that some xenobiotics could inter-fere with steroidogenesis and induce intrauterine growth retardation in rats. The study on the characteristics of biotransformation enzymes in fetal adrenals still seems to be important with respect to possible significance in xenobiotic-induced fetal development toxicity. In this study, the activities of several important xenobiotic-related phase Ⅰ and phase Ⅱ enzymes in human fetal adrenals were examined and compared with those in fetal livers. Methods: The activity and mRNA expression were determined by enzymatic analysis and RT-PCR. Results: The levels of cytochrome (CYP)2A6, CYP2E1, and CYP3A7 isozymes in fetal adrenals were 82%, 92%, and 33% of those in fetal livers, respectively. There was a good positive correlation between adrenal CYP2A6 activity and gestational time. The values of α glutathione S-transferase (GST), πGST, and μGST in adrenals were 0.5, 4.4, and 8.3-fold of those in the livers, respectively, and the activity of adrenal πGST was negatively correlated with gestational time. The uridine diphosphoglucuronyl transferase activities, which were measured using p-hydroxy-biphenyl and 7-hydroxy-4-methylcoumarin as substrates, were 9% and 3%, respectively, of those in the fetal livers. Conclusion: Our investiga-tion suggested that adrenal could be an important xenobiotic-metabolizing or-gan in fetal development and may play a potential role in xenobiotic-induced fetal development toxicity.

  20. Metallic nanoparticles reduce the migration of human fibroblasts in vitro

    Science.gov (United States)

    Vieira, Larissa Fernanda de Araújo; Lins, Marvin Paulo; Viana, Iana Mayane Mendes Nicácio; dos Santos, Jeniffer Estevão; Smaniotto, Salete; Reis, Maria Danielma dos Santos

    2017-03-01

    Nanoparticles have extremely wide applications in the medical and biological fields. They are being used in biosensors, local drug delivery, diagnostics, and medical therapy. However, the potential effects of nanoparticles on target cell and tissue function, apart from cytotoxicity, are not completely understood. Thus, the aim of this study was to investigate the in vitro effects of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) on human fibroblasts with respect to their interaction with the extracellular matrix and in cell migration. Immunofluorescence analysis revealed that treatment with AgNPs or AuNPs decreased collagen and laminin production at all the concentrations tested (0.1, 1, and 10 μg/mL). Furthermore, cytofluorometric analysis showed that treatment with AgNPs reduced the percentage of cells expressing the collagen receptor very late antigen 2, α2β1 integrin (VLA-2) and the laminin receptor very late antigen 6, α6β1 integrin (VLA-6). In contrast, AuNP treatment increased and decreased the percentages of VLA-2-positive and VLA-6-positive cells, respectively, as compared to the findings for the controls. Analysis of cytoskeletal reorganization showed that treatment with both types of nanoparticles increased the formation of stress fibres and number of cell protrusions and impaired cell polarity. Fibroblasts exposed to different concentrations of AuNPs and AgNPs showed reduced migration through transwell chambers in the functional chemotaxis assay. These results demonstrated that metal nanoparticles may influence fibroblast function by negatively modulating the deposition of extracellular matrix molecules (ECM) and altering the expression of ECM receptors, cytoskeletal reorganization, and cell migration.

  1. Ibuprofen results in alterations of human fetal testis development

    Science.gov (United States)

    Ben Maamar, Millissia; Lesné, Laurianne; Hennig, Kristin; Desdoits-Lethimonier, Christèle; Kilcoyne, Karen R.; Coiffec, Isabelle; Rolland, Antoine D.; Chevrier, Cécile; Kristensen, David M.; Lavoué, Vincent; Antignac, Jean-Philippe; Le Bizec, Bruno; Dejucq-Rainsford, Nathalie; Mitchell, Rod T.; Mazaud-Guittot, Séverine; Jégou, Bernard

    2017-01-01

    Among pregnant women ibuprofen is one of the most frequently used pharmaceutical compounds with up to 28% reporting use. Regardless of this, it remains unknown whether ibuprofen could act as an endocrine disruptor as reported for fellow analgesics paracetamol and aspirin. To investigate this, we exposed human fetal testes (7–17 gestational weeks (GW)) to ibuprofen using ex vivo culture and xenograft systems. Ibuprofen suppressed testosterone and Leydig cell hormone INSL3 during culture of 8–9 GW fetal testes with concomitant reduction in expression of the steroidogenic enzymes CYP11A1, CYP17A1 and HSD17B3, and of INSL3. Testosterone was not suppressed in testes from fetuses younger than 8 GW, older than 10–12 GW, or in second trimester xenografted testes (14–17 GW). Ex vivo, ibuprofen also affected Sertoli cell by suppressing AMH production and mRNA expression of AMH, SOX9, DHH, and COL2A1. While PGE2 production was suppressed by ibuprofen, PGD2 production was not. Germ cell transcripts POU5F1, TFAP2C, LIN28A, ALPP and KIT were also reduced by ibuprofen. We conclude that, at concentrations relevant to human exposure and within a particular narrow ‘early window’ of sensitivity within first trimester, ibuprofen causes direct endocrine disturbances in the human fetal testis and alteration of the germ cell biology. PMID:28281692

  2. DNA Methylation Landscapes of Human Fetal Development

    NARCIS (Netherlands)

    Slieker, Roderick C.; Roost, Matthias S.; van Iperen, Liesbeth; Suchiman, H. Eka D; Tobi, Elmar W.; Carlotti, Françoise; de Koning, Eelco J P; Slagboom, P. Eline; Heijmans, Bastiaan T.; Chuva de Sousa Lopes, Susana M.

    2015-01-01

    Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA

  3. RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Blichfeld, Kristian Aabo

    2017-01-01

    fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies...

  4. Prolactin receptor and osteogenic induction of prolactin in human periodontal ligament fibroblasts.

    Science.gov (United States)

    Surarit, Rudee; Krishnamra, Nateetip; Seriwatanachai, Dutmanee

    2016-04-01

    Prolactin is an important hormone involved in the interaction between maternal, extraembryonic, and fetal tissues that remains in high levels during the entire duration of pregnancy. Although many systemic alterations occur during pregnancy, such as hormonal changes, that are known to be associated with periodontitis and tooth loss, PRL function in human periodontal ligament fibroblasts (HPDLF) had never been studied. Herein, we investigated the role of PRL in the regulation of HPDLF proliferation and differentiation. HPDLF were cultured in differentiating medium with various concentrations of PRL. The present study demonstrated that HPDLF and primary human PDL cells that were extracted for orthodontic purpose expressed both short and long isoforms of PRLR mRNA and its proteins. An incubation with of high concentration of PRL (600 and 1,000 ng/mL) modestly decreased the HPDLF number. In contrast, PRL at a non-reproductive level (10 ng/mL) and pregnant level (100 ng/mL) significantly upregulated the markers of osteogenesis, such as RUNX2, BMP2, and POSTN, but not SOX9. Mineral nodule formation was induced, whereas proteoglycan accumulation was reduced by PRL suggesting that HPDLF were undergoing differentiation into preosteoblastic cells. In conclusion, the presence of hPRLR in human PDL together with PRL-induced upregulation of osteogenic markers strongly suggested a direct regulatory role of PRL in PDL and periodontal tissue development.

  5. 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.

  6. Generation of human induced pluripotent stem cells from dermal fibroblasts.

    Science.gov (United States)

    Lowry, W E; Richter, L; Yachechko, R; Pyle, A D; Tchieu, J; Sridharan, R; Clark, A T; Plath, K

    2008-02-26

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.

  7. Organization of human hypothalamus in fetal development.

    Science.gov (United States)

    Koutcherov, Yuri; Mai, Jürgen K; Ashwell, Ken W S; Paxinos, George

    2002-05-13

    The organization of the human hypothalamus was studied in 33 brains aged from 9 weeks of gestation (w.g.) to newborn, using immunohistochemistry for parvalbumin, calbindin, calretinin, neuropeptide Y, neurophysin, growth-associated protein (GAP)-43, synaptophysin, and the glycoconjugate 3-fucosyl- N-acetyl-lactosamine. Developmental stages are described in relation to obstetric trimesters. The first trimester (morphogenetic periods 9-10 w.g. and 11-14 w.g.) is characterized by differentiating structures of the lateral hypothalamic zone, which give rise to the lateral hypothalamus (LH) and posterior hypothalamus. The PeF differentiates at 18 w.g. from LH neurons, which remain anchored in the perifornical position, whereas most of the LH cells are displaced laterally. A transient supramamillary nucleus was apparent at 14 w.g. but not after 16 w.g. As the ventromedial nucleus differentiated at 13-16 w.g., three principal parts, the ventrolateral part, the dorsomedial part, and the shell, were revealed by distribution of calbindin, calretinin, and GAP43 immunoreactivity. The second trimester (morphogenetic periods 15-17 w.g., 18-23 w.g., and 24-33 w.g.) is characterized by differentiation of the hypothalamic core, in which calbindin- positive neurons revealed the medial preoptic nucleus at 16 w.g. abutted laterally by the intermediate nucleus. The dorsomedial nucleus was clearly defined at 10 w.g. and consisted of compact and diffuse parts, an organization that was lost after 15 w.g. Differentiation of the medial mamillary body into lateral and medial was seen at 13-16 w.g. Late second trimester was marked by differentiation of periventricular zone structures, including suprachiasmatic, arcuate, and paraventricular nuclei. The subnuclear differentiation of these nuclei extends into the third trimester. The use of chemoarchitecture in the human fetus permitted the identification of interspecies nuclei homologies, which otherwise remain concealed in the cytoarchitecture.

  8. 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

  9. Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Anne Grünewald

    Full Text Available BACKGROUND: Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD. The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7, as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and

  10. Molecular pathological changes of human fetal scleral fibroblasts following exposure to extremely low frequency electromagnetic field%极低频电磁场作用下人胚胎眼巩膜成纤维细胞的分子病理改变

    Institute of Scientific and Technical Information of China (English)

    王洁; 崔杰锋; 朱煌; 赵立全; 冯婕妤; 叶胜龙

    2012-01-01

    changes of scleral tissue after exposure to ELF-EMFs as well as the relationship between these changes and myopia are still poorly understood. Objective The present study was to investigate the molecular pathological changes of human fetal scleral fibroblasts (HFSFs) after exposure to ELF-EMFs in vitro and to explore the possible mechanism in the occurrence and development of myopia.Methods HFSFs were cultured and passaged and then exposed to 50 Hz electromagnetic fields,and HFSFs that did not receive the irradiation of ELF-EMFs were used as the control group.The expression of collagen type Ⅰ (COL1A1 ) mRNA and matrix metalloproteinase-2 (MMP-2) mRNA in cultured HFSFs were detected by real-time qualitative polymerase chain reaction (real-time PCR) under different magnetic field intensites (0,0.1,0.2,0.5,1.0 mT) and different exposure time (0,6,12,24,36,48 hours).Cell proliferation assay of HFSFs was detected by the cell counting kit 8 ( CCK8 ) assay.The expression levels of COL1 A1 and MMP-2 proteins in HFSFs were further confirmed by immunofluorescence staining. Results The expression of COL1A1 mRNA was significantly down-regulated under the exposure of 0.2 mT ELF-EMFs for 6 hours,in comparison with the control group;moreover,it decreased in parallel with the increased of flux density (0.099±0.008 vs.0.050±0.004) (P =0.009 ).The expression of MMP-2mRNA was up-regulated conspicuously after exposure to 0.1 mT ELF-EMFs for 24 hours,and it increased with exposure time in comparison with the control group ( 0.009 ±0.001 vs.0.018±0.003 ) ( P =0.038 ).Proliferation of HFSFs (A450) was inhibited following the exposure to 0.2 mT ELF-EMFs for 24 hours in comparison with the control group (P =0.009 ).The expression of COL1 A1 in the experimental group was decreased,compared with the control group,but the expression of MMP-2 was increased. Conclusions ELF-EMFs inhibit the proliferation of HFSFs and expression of COL1 A1 in HFSFs,which might be one of the reasons for the

  11. Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells

    DEFF Research Database (Denmark)

    Vandekerckhove, J; Bauw, G; Vancompernolle, K

    1990-01-01

    A systematic comparison of the protein synthesis patterns of cultured normal and transformed human fibroblasts and epithelial cells, using two-dimensional gel protein analysis combined with computerized imaging and data acquisition, identified a 90-kD protein (SSP 5714) as one of the most striking...... downregulated markers typical of the transformed state. Using the information stored in the comprehensive human cellular protein database, we found this protein strongly expressed in several fetal tissues and one of them, epidermis, served as a source for preparative two-dimensional gel electrophoresis. Partial...... and by coelectrophoresis with purified human gelsolin. These results suggest that an important regulatory protein of the microfilament system may play a role in defining the phenotype of transformed human fibroblast and epithelial cells in culture. Udgivelsesdato: 1990-Jul...

  12. Cytokeratin expression in human fetal tongue and buccal mucosa

    Indian Academy of Sciences (India)

    M M Vaidya; Sharda S Sawant; Anita M Borges; N K Naresh; Manda C Purandare; A N Bhisey

    2000-09-01

    Expression of cytokeratins (CK), a subset of intermediate filament (IF) proteins in epithelia, is developmentally regulated. CK expression may also change after malignant transformation. Our earlier studies on CK expression in human oral tumours and pre-cancerous lesions have shown specific changes in CK expression. We analysed CK expression in human tongue and buccal mucosa (BM) in fetuses in the embryonic age group of 16 to 27 weeks using biochemical and immunohistochemical techniques to find out whether there is any similarity in CK expression in human oral squamous cell carcinomas (SCC) and fetal oral tissues. CK 1, 8 and 18 were detected in a majority of samples using both techniques. Our earlier studies had shown aberrant expression of CK 1 and 18 in many of the oral SCC and leukoplakias. Studies by immunohistochemistry showed that these different CK antigens were expressed in different cell layers. CK 1(2) were present in the stratified epithelial layers whereas CK 8 and 18 were restricted to glandular epithelium. Till 27 weeks of gestation, both tongue and BM expressed CK 1, 8 and 18 along with CK 6 and 16. Thus, fetal tissues showed some similarities in CK pattern with their respective SCC.

  13. Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

    Science.gov (United States)

    Yam, Gary Hin-Fai; Williams, Geraint P.; Setiawan, Melina; Yusoff, Nur Zahirah Binte M.; Lee, Xiao-wen; Htoon, Hla Myint; Zhou, Lei; Fuest, Matthias; Mehta, Jodhbir S.

    2017-01-01

    Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density. PMID:28349952

  14. Cytotoxic Effects of Nickel Nanowires in Human Fibroblasts

    KAUST Repository

    Felix Servin, Laura P.

    2014-04-01

    There is an increasing interest for the use of nanostructures as potential tools in areas that include biology and medicine, for applications spanning from cell separation to treatments of diseases. Magnetic nanoparticles (MNPs) have been the most widely studied and utilized nanostructures in biomedical applications. Despite their popularity, the regular shape of MNPs limits their potential for certain applications. Studies have shown that magnetic nanowires (MNWs), due to their high-­‐aspect ratio and specific magnetic properties, might provide improved performance for some biomedical applications. As a consequence, MNWs have received increasing attention from researchers in the last years. However, as with any other nanostructure intended for biomedical applications, rigorous studies must be carried out to determine their potential toxicity and adverse effects before they can be successfully incorporated in clinical applications. This work attempts to elucidate the cytotoxic effects of nickel NWs (Ni NWs) in human fibroblasts by measuring cell viability under different parameters. Ni NWs of three different lengths (0.86 ± 0.02 μm, 1.1 ± 0.1 μm and 6.1 ± 0.6 μm) were fabricated by electrodeposition using porous aluminum oxide (PAO) membranes as templates. Energy dispersive X-­‐Ray analysis (EDAX) and X-­‐Ray diffraction (XRD) were used for the chemical characterization of the Ni NWs. Their physical characterization was done using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging. MTT assays were performed to assess cell viability of human fibroblasts in the presence of Ni NWs. NW length, NW/cell ratio and exposure time were changed throughout the experiments to elucidate their effects on cell viability. The results showed that NWs length has a strong effect on internalization and cytotoxicity. Smaller NWs showed higher toxicity levels at earlier times while longer NWs had stronger effects on cell viability at

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

    Directory of Open Access Journals (Sweden)

    Ji-Yong Jung

    2015-08-01

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

  16. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Lei [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Xiao, Yongsheng [Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States); Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States)

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  17. Effects of Calendula officinalis on human gingival fibroblasts.

    Science.gov (United States)

    Saini, Pragtipal; Al-Shibani, Nouf; Sun, Jun; Zhang, Weiping; Song, Fengyu; Gregson, Karen S; Windsor, L Jack

    2012-04-01

    Calendula officinalis is commonly called the marigold. It is a staple topical remedy in homeopathic medicine. It is rich in quercetin, carotenoids, lutein, lycopene, rutin, ubiquinone, xanthophylls, and other anti-oxidants. It has anti-inflammatory properties. Quercetin, one of the active components in Calendula, has been shown to inhibit recombinant human matrix metalloproteinase (MMP) activity and decrease the expression of tumor necrosis factor-α, interleukin-1β (IL), IL-6 and IL-8 in phorbol 12-myristate 13-acetate and calcium ionophore-stimulated human mast cells. To examine the effects of Calendula on human gingival fibroblast (HGF) mediated collagen degradation and MMP activity. Lactate dehydrogenate assays were performed to determine the non-toxic concentrations of Calendula, doxycycline and quercetin. Cell-mediated collagen degradation assays were performed to examine the inhibitory effect on cell-mediated collagen degradation. Gelatin zymography was performed to examine their effects on MMP-2 activity. The experiments were repeated three times and ANOVA used for statistical analyses. Calendula at 2-3% completely inhibited the MMP-2 activity in the zymograms. Doxycycline inhibited HGF-mediated collagen degradation at 0.005, 0.01, 0.02 and 0.05%, and MMP-2 activity completely at 0.05%. Quercetin inhibited HGF-mediated collagen degradation at 0.005, 0.01 and 0.02%, and MMP-2 activity in a dose-dependent manner. Calendula inhibited HGF-mediated collagen degradation and MMP-2 activity more than the same correlated concentration of pure quercetin. Calendula inhibits HGF-mediated collagen degradation and MMP-2 activity more than the corresponding concentration of quercetin. This may be attributed to additional components in Calendula other than quercetin. Published by Elsevier Ltd.

  18. Transcriptional Analysis of Normal Human Fibroblast Responses to Microgravity Stress

    Institute of Scientific and Technical Information of China (English)

    Yongqing Liu; Eugenia Wang

    2008-01-01

    To understand the molecular mechanism (s) of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the space flown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization (SSH) to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several G1-phase cell cycle traverse genes. Other genes showing up regulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the "hub" for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

  19. Regulation of bradykinin receptor gene expression in human lung fibroblasts.

    Science.gov (United States)

    Phagoo, S B; Yaqoob, M; Herrera-Martinez, E; McIntyre, P; Jones, C; Burgess, G M

    2000-06-01

    In WI-38 human fibroblasts, interleukin-1 beta and tumour necrosis factor-alpha (TNF-alpha) increased bradykinin B(1) receptor mRNA, which peaked between 2 and 4 h, remaining elevated for 20 h. Binding of the bradykinin B(1) receptor selective ligand [3H]des-Arg(10)-kallidin, also increased, peaking at 4 h and remaining elevated for 20 h. The B(max) value for [3H]des-Arg(10)-kallidin rose from 280+/-102 fmol/mg (n=3) to 701+/-147 fmol/mg (n=3), but the K(D) value remained unaltered (control, 1.04+/-0.33 nM (n=3); interleukin-1 beta, 0.88+/-0.41 nM (n=3)). The interleukin-1 beta-induced [3H]des-Arg(10)-kallidin binding sites were functional receptors, as bradykinin B(1) receptor agonist-induced responses increased in treated cells. Bradykinin B(2) receptor mRNA and [3H]bradykinin binding were upregulated by interleukin-1 beta, but not TNF-alpha. The effect of interleukin-1 beta on bradykinin B(2) receptors was smaller than for bradykinin B(1) receptors. Cycloheximide prevented interleukin-1 beta-mediated increases in B(1) and B(2) binding, but not mRNA suggesting that de novo synthesis of a transcriptional activator was unnecessary.

  20. ULTRASTRUCTURAL STUDY OF THE HEMOPOIETIC MICROENVIRONMENT IN HUMAN FETAL SPLEEN

    Institute of Scientific and Technical Information of China (English)

    刘爱莲; 朱萍; 李学均; 张丽; 迟凤琴; 吴振铎; 宋雁南; 张亚坤

    1994-01-01

    Reciprocal interations between hemopoietic stromal cells and immature hemopoietic cells in human spleens obtained from 20 fetuses of 10-28 weeks gestation were observed by transmission electron microscopy and scanning electron microscopy.The close association of stromal cells with immature hemopoietic cells was confirmed under the electron microscope and a preasumptive HIM(Hemopoietic inductive mocroenvironment) was visualized.In regions of immature hemopoietice cell-reticular cell,endothelial cell,macrophage and interdigitating cell contact,some communicating structures were found between the plasma membranes of adjacent cells.moreovcer,the cytoplasm of these four stromal cells were full of various kinds of organelles.These results suggest that reticular cells,endothelial cells,macrophages and interdigitating cells are component parts of the HIM of human fetal spleen and that these cells have a nurturing function in relation to hemopoietic cells.

  1. 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.

  2. 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.

  3. 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

  4. Human embryonic growth trajectories and associations with fetal growth and birthweight

    NARCIS (Netherlands)

    van Uitert, Evelyne M.; Exalto, Niek; Burton, Graham J.; Willemsen, Sten P.; Koning, Anton H. J.; Eilers, Paul H. C.; Laven, Joop S. E.; Steegers, Eric A. P.; Steegers-Theunissen, Regine P. M.

    2013-01-01

    How do human embryonic growth trajectories evolve in the first trimester, and is first-trimester embryonic growth associated with fetal growth and birthweight (BW)? Human embryonic growth rates increase between 9 and 10 weeks of gestation and are associated with mid-pregnancy fetal growth and BW. Fe

  5. Embryologic and Fetal Development of the Human Eyelid

    Science.gov (United States)

    Abdulhafez, Mohamed H.; Fouad, Yousef A.; Dutton, Jonathan J.

    2016-01-01

    Purpose: To review the recent data about eyelid morphogenesis, and outline a timeline for eyelid development from the very early stages during embryonic life till final maturation of the eyelid late in fetal life. Methods: The authors extensively review major studies detailing human embryologic and fetal eyelid morphogenesis. These studies span almost a century and include some more recent cadaver studies. Numerous studies in the murine model have helped to better understand the molecular signals that govern eyelid embryogenesis. The authors summarize the current findings in molecular biology, and highlight the most significant studies in mice regarding the multiple and interacting signaling pathways involved in regulating normal eyelid morphogenesis. Results: Eyelid morphogenesis involves a succession of subtle yet strictly regulated morphogenetic episodes of tissue folding, proliferation, contraction, and even migration, which may occur simultaneously or in succession. Conclusions: Understanding the extraordinary process of building eyelid tissue in embryonic life, and deciphering its underlying signaling machinery has far reaching clinical implications beyond understanding the developmental abnormalities involving the eyelids, and may pave the way for achieving scar-reducing therapies in adult mammalian wounds, or control the spread of malignancies. PMID:27124372

  6. Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields.

    Science.gov (United States)

    Golberg, Alexander; Bei, Marianna; Sheridan, Robert L; Yarmush, Martin L

    2013-06-01

    Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical-free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5-100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10(5) -1.4 ± 0.2 × 10(5)  cell/mL range. Our results suggest that IDPEFs may prove useful as a non-chemical method for fibroblast density control in human wound healing.

  7. Interleukin-19 Downregulates Interleukin-4-Induced Eotaxin Production in Human Nasal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Masaaki Higashino

    2011-01-01

    Conclusions: These results suggest that IL-19 down-regulates IL-4-induced eotaxin production via SOCS-1 in human nasal fibroblasts. In non-hematopoietic cells in AR, IL-19 might be an immunosuppressive factor.

  8. The Preliminary Study of Interferon-γGene Transfection to Human Tenon's Capsule Fibroblasts in Vitro#

    Institute of Scientific and Technical Information of China (English)

    Yuqing Lan; Jian Ge; Mingkai Lin; Jianliang Zheng; Huiyi Chen; Haiquan Liu; Jing Wei; Yanyan Li

    2000-01-01

    Purpose: To investigate the results of the interferon-gamma(IFN-y) gene transfer and transient expression in human Tenon's capsule fibroblast in vitro in order to find a way to gene therapy in vivo. Method: Using LipofectAMINE, IFN-γ gene was transferred in human Tenon's capsule fibroblasts with plasmid pcDNA3 IFN-y. Its mRNA transcription and protein expression were determined by RT-PCR and flow cytometry assay respectively.Result: The human Tenon's capsule fibroblasts transferred the IFN-γgene can express the IFN-γin transcription and protein level transiently.Conclusion: IFN-γ gene can be transferred successfully and expressed efficiently in human tenon's capsule fibroblast in vitro.

  9. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    OpenAIRE

    2011-01-01

    International audience; This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarifi...

  10. Arterial flow regulator enables transplantation and growth of human fetal kidneys in rats.

    Science.gov (United States)

    Chang, N K; Gu, J; Gu, S; Osorio, R W; Concepcion, W; Gu, E

    2015-06-01

    Here we introduce a novel method of transplanting human fetal kidneys into adult rats. To overcome the technical challenges of fetal-to-adult organ transplantation, we devised an arterial flow regulator (AFR), consisting of a volume adjustable saline-filled cuff, which enables low-pressure human fetal kidneys to be transplanted into high-pressure adult rat hosts. By incrementally withdrawing saline from the AFR over time, blood flow entering the human fetal kidney was gradually increased until full blood flow was restored 30 days after transplantation. Human fetal kidneys were shown to dramatically increase in size and function. Moreover, rats which had all native renal mass removed 30 days after successful transplantation of the human fetal kidney were shown to have a mean survival time of 122 days compared to 3 days for control rats that underwent bilateral nephrectomy without a prior human fetal kidney transplant. These in vivo human fetal kidney models may serve as powerful platforms for drug testing and discovery.

  11. Binding, uptake, and release of nicotine by human gingival fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hanes, P.J.; Schuster, G.S.; Lubas, S. (Medical College of Georgia, Augusta (USA))

    1991-02-01

    Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4{degree}C using a mixture of {sup 3}H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between {sup 3}H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37{degree}C after treating cells with {sup 3}H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours.

  12. 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...

  13. Fetal developmental programing: insights from human studies and experimental models.

    Science.gov (United States)

    Lopes, Gisele Aparecida Dionísio; Ribeiro, Vinícius Luís Bertotti; Barbisan, Luís Fernando; Marchesan Rodrigues, Maria Aparecida

    2017-03-01

    Environmental factors, particularly nutrition during pregnancy and early life can influence the risk of chronic diseases in later life. The underlying mechanism, termed "programing", postulates that an environmental stimulus during a critical window of time, early in life, has a permanent effect on subsequent structure and function of the organism. In this study we review the concept of fetal programing on chronic diseases and the proposed hypotheses for the association between early development and later disease, including epigenetic variation. We concentrate on specific aspects of maternal nutrition, particularly under-nutrition and over-nutrition, in humans and animal models. An adequate maternal nutrition during pregnancy is crucial for the health outcome of the offspring at adulthood.

  14. Trisomy 21 enhances human fetal erythro-megakaryocytic development

    Science.gov (United States)

    Chou, Stella T.; Opalinska, Joanna B.; Yao, Yu; Fernandes, Myriam A.; Kalota, Anna; Brooks, John S. J.; Choi, John K.; Gewirtz, Alan M.; Danet-Desnoyers, Gwenn-ael; Nemiroff, Richard L.

    2008-01-01

    Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes. PMID:18812473

  15. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Rong-hui, E-mail: fan_ronghuixa@163.com [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Zhu, Xiu-mei; Sun, Yao-wen [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Peng, Hui-zi [Department of Cosmetology Plastic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Wu, Hang-li; Gao, Wen-jie [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China)

    2016-07-08

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  16. 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.

  17. 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

  18. Normal human fibroblasts produce membrane-bound and soluble isoforms of FGFR-1.

    Science.gov (United States)

    Root, L L; Shipley, G D

    2000-02-01

    Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which alpha FGF and beta FGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.

  19. Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhang Dongwei

    2011-11-01

    Full Text Available Abstract Background Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, a polyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferation and reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However, there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin on the expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases. Methods We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice and human fetal lung fibroblasts (HFL-1 on the expression of cathepsins K and L which have been implicated in matrix degradation, TGF-β1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K and L, collagen, and TGF-β1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition on cell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-β1. Results Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompanied by increased expression levels of cathepsins L (25%-39% and K (41%-76% and a 30% decrease in TGF-β1 expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumin treatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here, cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and the expression of TGF-β1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation and migration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects were partly reversed by a potent cathepsin inhibitor. Conclusion This study demonstrates that

  20. Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could

  1. Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

    Institute of Scientific and Technical Information of China (English)

    WANG Hai; AO Hong; PAN QiuZhen; LI RongQi; ZHAO MengBin; LIAN ZhengXing; LI Ning; WU ChangXin

    2007-01-01

    To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation,colchicine treatment and gene transfection. Results are as follows: ( Ⅰ ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3%vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25 μm) as donor nuclei was higher than that from large cells (25-33 μm) and small cells (8-15 μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS( 11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05 μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05 μmol/L colchicine

  2. 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 Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

  3. 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...

  4. Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes

    NARCIS (Netherlands)

    El Ghalbzouri, A; Jonkman, MF; Dijkman, R; Ponec, M

    2005-01-01

    This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence

  5. Retinoic Acid signalling and the control of meiotic entry in the human fetal gonad.

    Directory of Open Access Journals (Sweden)

    Andrew J Childs

    Full Text Available The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA. Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8-9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may

  6. Regulation of NAMPT in Human Gingival Fibroblasts and Biopsies

    Directory of Open Access Journals (Sweden)

    Anna Damanaki

    2014-01-01

    Full Text Available Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT, are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.

  7. ABSORPTION-SPECTRA OF HUMAN FETAL AND ADULT OXYHEMOGLOBIN, DE-OXYHEMOGLOBIN, CARBOXYHEMOGLOBIN, AND METHEMOGLOBIN

    NARCIS (Netherlands)

    ZIJLSTRA, WG; MEEUWSENVANDERROEST, WP

    1991-01-01

    We determined the millimolar absorptivities of the four clinically relevant derivatives of fetal and adult human hemoglobin in the visible and near-infrared spectral range (450-1000 nm). As expected, spectral absorption curves of similar shape were found, but the small differences between fetal and

  8. Activation of the innate immune response against DENV in normal non-transformed human fibroblasts.

    Directory of Open Access Journals (Sweden)

    José Bustos-Arriaga

    2011-12-01

    Full Text Available BACKGROUND: When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times ("probing" before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. METHODOLOGY/PRINCIPAL FINDINGS: Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5 and β defensin 2 (HβD2. In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN regulatory factor 3 (IRF3, but not interferon regulatory factor 7 (IRF7, when compared with mock-infected fibroblasts. CONCLUSIONS/SIGNIFICANCE: In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and

  9. Preferential gene expression in quiescent human lung fibroblasts.

    Science.gov (United States)

    Coppock, D L; Kopman, C; Scandalis, S; Gilleran, S

    1993-06-01

    The exit from the proliferative cell cycle into a reversible quiescence (G0) is an active process that is not yet well understood at the molecular level. Investigation of G0-specific gene expression is an important step in studying the mechanism regulating the entrance to quiescence. Using the human embryo lung fibroblast (WI38) as a model system, we have isolated complementary DNA clones that are expressed at a higher level in quiescent cells than in logarithmically growing cells. We have identified complementary DNAs from eight genes including collagen alpha 1(VI), collagen alpha 1(III), decorin, complement C1r, collagen alpha 1(I), collagen alpha 2(I), and two novel genes, Q6 and Q10. We have named this class of quiescence-inducible genes quiescins. Expression of these genes was induced just as proliferation slowed, as indicated by the level of histone H2B mRNA, [3H]-thymidine incorporation, and cell number. The level of expression of the novel genes, Q6 and Q10, increased at the same time as the other genes. Q6 has two mRNAs of 3 and 4 kb, whereas Q10 mRNA is about 1.0 kb. The expression of the quiescins was not induced by blocking the cell cycle in S phase with aphidicolin or in G1 with lovastatin. However, the genes were highly induced by trypsinization or scraping of the cells during logarithmic growth. This induction was not blocked by inhibitors of RNA synthesis. The expression of decorin and Q6 was very low in SV40-transformed cells (VA13) either in logarithmic growth or at high density, whereas the gene Q10 was expressed more highly in VA13 than in WI38 cells. The finding that expression of some components of the extracellular matrix is induced as cells enter G0 suggests that they may have a role in both the induction and the maintenance of the quiescent state. The quiescins will serve as molecular markers for the investigation of mechanisms that regulate the onset of quiescence.

  10. Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

    Directory of Open Access Journals (Sweden)

    Dongyuan Lü

    Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

  11. Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

    Institute of Scientific and Technical Information of China (English)

    Ma; Shu-hua; Wang; Dun-cheng; 等

    2003-01-01

    79 ESTs fragments with represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fetal kidney cDNA library. Microarray was prepared by using these novel EST fragments by automatic spotting. Expression patters of 79 ESTs of novel genes from human fetal kidney were analyzed in fetal brain and fetal heart tissues of 20-week-and 26-week-age fetus by performing of cDNA chip hybridization. This provides clues for studying exact functions of the novel genes. 8 genes were obtained which were expressed differentially in the fetal brain and heart of 20-week-and 26-week-age respectively. Then differentially expressed genes were identified by Northern analysis. The more exact function of the novel genes is under study.

  12. Prolonged exposure to acetaminophen reduces testosterone production by the human fetal testis in a xenograft model

    DEFF Research Database (Denmark)

    van den Driesche, Sander; Macdonald, Joni; Anderson, Richard A

    2015-01-01

    Most common male reproductive disorders are linked to lower testosterone exposure in fetal life, although the factors responsible for suppressing fetal testosterone remain largely unknown. Protracted use of acetaminophen during pregnancy is associated with increased risk of cryptorchidism in sons......, but effects on fetal testosterone production have not been demonstrated. We used a validated xenograft model to expose human fetal testes to clinically relevant doses and regimens of acetaminophen. Exposure to a therapeutic dose of acetaminophen for 7 days significantly reduced plasma testosterone (45......% reduction; P = 0.025) and seminal vesicle weight (a biomarker of androgen exposure; 18% reduction; P = 0.005) in castrate host mice bearing human fetal testis xenografts, whereas acetaminophen exposure for just 1 day did not alter either parameter. Plasma acetaminophen concentrations (at 1 hour after...

  13. Myocardial bridges of the coronary arteries in the human fetal heart.

    Science.gov (United States)

    Cakmak, Yusuf Ozgür; Cavdar, Safiye; Yalin, Aymelek; Yener, Nuran; Ozdogmus, Omer

    2010-09-01

    During the last century, many investigators reported on myocardial bridges in the adult human heart. In the present study, 39 human fetal hearts (the mean gestastional age was 30 weeks) were studied for myocardial bridging, and the results were correlated with adult data. Among the 39 (27 male and 12 female) fetal hearts studied, 26 bridges were observed on 18 fetal hearts (46.2%). Ten of the bridges had one myocardial bridge, whereas double myocardial bridges were observed in eight fetal hearts. The most frequent myocardial bridges were observed on the left anterior descending artery (LAD), which had 13 bridges (50%). Eight (30.7%) myocardial bridges were on the diagonal artery, and on the posterior descending artery there were five (19.3%). Myocardial bridges were not observed on the circumflex artery. The data presented in this study may provide potentially useful information for the preoperative evaluation of the newborn and may have a clinical implication for sudden fetal death.

  14. 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.

  15. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Mona Alibolandi

    2011-01-01

    Full Text Available This work describes the integration of expanded bed adsorption (EBA and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

  16. Centrifugal force induces human ligamentum flavum fibroblasts inflammation through activation of JNK and p38 pathways.

    Science.gov (United States)

    Chao, Yuan-Hung; Tsuang, Yang-Hwei; Sun, Jui-Sheng; Sun, Man-Ger; Chen, Ming-Hong

    2012-01-01

    Inflammation has been proposed to be an important causative factor in ligamentum flavum hypertrophy. However, the mechanisms of mechanical load on inflammation of ligamentum flavum remain unclear. In this study, we used an in vitro model of human ligamentum flavum fibroblasts subjected to centrifugal force to elucidate the effects of mechanical load on cultured human ligamentum flavum fibroblasts; we further studied its molecular and biochemical mechanisms. Human ligamentum flavum fibroblasts were obtained from six patients undergoing lumbar spine surgery. Monolayer cultures of human ligamentum flavum fibroblasts were subjected to different magnitudes of centrifugal forces. Cell viability, cell death, biochemical response, and molecular response to centrifugal forces were analyzed. It was found that centrifugal stress significantly suppressed cell viability without inducing cell death. Centrifugal force at 67.1 g/cm(2) for 60 min significantly increases the production of prostaglandin E2 and nitric oxide as well as gene expression of proinflammatory cytokines, including interleukin (IL)-1α, IL-1β and IL-6, showed that centrifugal force-dependent induction of cyclooxygense-2 and inducible NO synthase required JNK and p38 mitogen-activated protein kinase, but not ERK 1/2 activities. This study suggested that centrifugal force does induce inflammatory responses in human ligamentum flavum fibroblasts. The activation of both JNK and p38 mitogen-activated protein kinase mechanotransduction cascades is a crucial intracellular mechanism that mediates cyclooxygense-2/prostaglandin E2 and inducible NO synthase/nitric oxide production.

  17. The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Lucia Pagani

    Full Text Available BACKGROUND: Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype ("larks" and "owls", clock properties measured in human fibroblasts correlated with extreme diurnal behavior. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer. CONCLUSIONS/SIGNIFICANCE: We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.

  18. Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

    Science.gov (United States)

    Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

    2011-09-15

    The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P fusion (P fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.

  19. Preferential attachment of human gingival fibroblasts to the resin ionomer Geristore.

    Science.gov (United States)

    Al-Sabek, Fuwad; Shostad, Sandra; Kirkwood, Keith L

    2005-03-01

    The resin ionomer Geristore has been used extensively for root perforation repairs. The purpose of this study was to evaluate oral in vitro biocompatibility of the resin ionomer Geristore compared to two other dental perforation repair materials, Ketac-Fil and Immediate Restorative Material (IRM). Growth and morphology of human gingival fibroblasts (HGFs) was determined using scanning electron microscopy (SEM) of HGFs cells grown on test materials as well as cytotoxicity assays using eluates from test materials. SEM analysis showed that HGFs attached and spread well over Geristore with relatively normal morphology. SEM showed that fibroblasts did not attach and spread well over Ketac-Fil or IRM as cells appeared much fewer with rounded and different morphology than fibroblasts grown on Geristore. Cytotoxicity assays indicated that HGFs proliferated in the presence of Geristore eluates and not in the presence of Ketac-Fil or IRM eluates. In vitro interpretation indicates that Geristore is less cytotoxic to gingival fibroblasts.

  20. Human Fetal Liver: An In Vitro Model of Erythropoiesis

    Directory of Open Access Journals (Sweden)

    Guillaume Pourcher

    2011-01-01

    Full Text Available We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES cells or induced pluripotent stem cell (iPS are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34+ cells. In this in vitro model, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i displayed a dramatic in vitro expansion (100-fold more when compared to CB CD34+ and (ii 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10–15% cloning efficiency for adult CD34+ cells. This work supports the idea that FL remains a model of study and is not a candidate for ex vivo RBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Maucksch C

    2012-01-01

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

  3. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

    Science.gov (United States)

    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  4. Possible identity of IL-8 converting enzyme in human fibroblasts as a cysteine protease.

    Science.gov (United States)

    Ohashi, Kensaku; Sano, Emiko; Nakaki, Toshio; Naruto, Masanobu

    2003-04-01

    A converting activity was characterized in human diploid fibroblasts, which secrete 72IL-8 and 77IL-8 in treatment with IFN-beta and poly I: poly C. 77IL-8 was significantly converted to 72IL-8 by a partially purified fraction of the culture supernatant of human diploid fibroblasts. The converting activity, which was temperature-dependent and optimal at pH 6, was completely inhibited by cysteine protease inhibitors, antipain dihydrochloride and E-64, but not by other types of protease inhibitors. These data clearly show that human diploid fibroblasts are capable of processing IL-8 to produce a mature IL-8 and that the putative converting enzyme appears to be a cysteine protease.

  5. The human protooncogene product p33pim is expressed during fetal hematopoiesis and in diverse leukemias.

    OpenAIRE

    Amson, R; Sigaux, F; Przedborski, S; Flandrin, G; Givol, D; Telerman, A

    1989-01-01

    We measured the human pim-1 protooncogene (PIM) expression during fetal development and in hematopoietic malignancies. Our data indicate that during human fetal hematopoiesis the 33-kDa pim product, p33pim, is highly expressed in the liver and spleen. In contrast, at the adult stage it is only slightly expressed in circulating granulocytes. Out of 70 hematopoietic malignancies analyzed, 51 patients and 19 cell lines, p33pim was overexpressed in approximately 30% of the samples, particularly i...

  6. Blood flow in the human fetal descending aorta : a pulsed Doppler study

    OpenAIRE

    Pijpers, Leendert

    1985-01-01

    textabstractIn 1628 William Harvey introduced his concept ofthe human circulation. Although a lot of studies concerning the fetal circulation were done before it was not until the 1930s that Barcroft (1934, 1939) and associates performed radiograpbic studies on the feta! goal and lamb to establish the feta! circulation. Later in 1964 Lind, Stern and Wegelius used cine-angiographic studies to describe the human fetal circulation. Volume flow measurements were already carried out in 1884 by Coh...

  7. Human fetal cardiac progenitors: The role of stem cells and progenitors in the fetal and adult heart.

    Science.gov (United States)

    Bulatovic, Ivana; Månsson-Broberg, Agneta; Sylvén, Christer; Grinnemo, Karl-Henrik

    2016-02-01

    The human fetal heart is formed early during embryogenesis as a result of cell migrations, differentiation, and formative blood flow. It begins to beat around gestation day 22. Progenitor cells are derived from mesoderm (endocardium and myocardium), proepicardium (epicardium and coronary vessels), and neural crest (heart valves, outflow tract septation, and parasympathetic innervation). A variety of molecular disturbances in the factors regulating the specification and differentiation of these cells can cause congenital heart disease. This review explores the contribution of different cardiac progenitors to the embryonic heart development; the pathways and transcription factors guiding their expansion, migration, and functional differentiation; and the endogenous regenerative capacity of the adult heart including the plasticity of cardiomyocytes. Unfolding these mechanisms will become the basis for understanding the dynamics of specific congenital heart disease as well as a means to develop therapy for fetal as well as postnatal cardiac defects and heart failure.

  8. Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide

    Institute of Scientific and Technical Information of China (English)

    Bing FENG; Lai-ji MA; Jin-jing YAO; Yun FANG; Yan-ai MEI; Shao-min WEI

    2013-01-01

    Oat contains different components that possess antioxidant properties;no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level.Therefore,the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide(H2O2).Kjeldahl determination,phenol-sulfuric acid method,and high-performance liquid chromatography(HPLC)analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%,of which 97.43% are peptides with a molecular range from 438.56 to 1301.01 Da.Assays for 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling(TUNEL)assay for apoptosis showed that administration of H2O2 in human dermal fibroblasts caused cell damage and apoptosis.Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H2O2,but application oat peptides with H2O2 at same time did not.Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H2O2-induced decrease of superoxide dismutase(SOD)and the inhibition of malondialdehyde(MDA).The results demonstrate that oat peptides possess antioxidant activity and are effective against H2O2-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level.Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury.

  9. Secretome Analysis of Human Primary Fibroblasts Undergoing Senescence

    DEFF Research Database (Denmark)

    Rogowska-Wrzesinska, Adelina; Micutkova, Lucia; Diener, Thomas

    complementary approaches: 1)  Two-dimensional gel electro-phoresis (2DGE) was used to separate proteins and quantitate their expression; selected proteins were identified using mass spectrometry; 2)  Proteins were digested into peptides and analysed using high performance liquid chromatography (HPLC) and mass...... forms of the same protein. Using the gel free approach we were able to identify over 400 proteins and 29 of them showed significant differences in secretion between young and old fibroblasts. Among them MMP-1 was found up regulated and several ECM proteins were found down regulated: lamin A, collagen...

  10. Secretome Analysis of Human Primary Fibroblasts Undergoing Senescence

    DEFF Research Database (Denmark)

    Rogowska-Wrzesinska, Adelina; Micutkova, Lucia; Diener, Thomas

    metalloproteinases (MMP1, 2 and 3), secreted protein acidic and rich in cysteine (SPARC) and two isoforms of metalloproteinase inhibitors (TIMP1 and 2). Major proteins decreased in senescent secretome were follistatin-like 1 and nucleobindin-1. Over 800 spots were separated by 2DGE of which many represent modified...... alpha-1(XII) chain and fibulin 3. Results obtained until now are in agreement with the suggested shift from matix - synthesizing to matrix - degrading phenotype in senescent fibroblasts except the increased secretion of metalloproteinase inhibitors. Work is going on to identify the remaining...

  11. GLI3 Links Environmental Arsenic Exposure and Human Fetal Growth

    Directory of Open Access Journals (Sweden)

    Emily F. Winterbottom

    2015-06-01

    Full Text Available Although considerable evidence suggests that in utero arsenic exposure affects children's health, these data are mainly from areas of the world where groundwater arsenic levels far exceed the World Health Organization limit of 10 μg/L. We, and others, have found that more common levels of in utero arsenic exposure may also impact children's health. However, the underlying molecular mechanisms are poorly understood. To address this issue, we analyzed the expression of key developmental genes in fetal placenta in a birth cohort of women using unregulated water supplies in a US region with elevated groundwater arsenic. We identified several genes whose expression associated with maternal arsenic exposure in a fetal sex-specific manner. In particular, expression of the HEDGEHOG pathway component, GLI3, in female placentae was both negatively associated with arsenic exposure and positively associated with infant birth weight. This suggests that modulation of GLI3 in the fetal placenta, and perhaps in other fetal tissues, contributes to arsenic's detrimental effects on fetal growth. We showed previously that arsenic-exposed NIH3T3 cells have reduced GLI3 repressor protein. Together, these studies identify GLI3 as a key signaling node that is affected by arsenic, mediating a subset of its effects on developmental signaling and fetal health.

  12. GLI3 Links Environmental Arsenic Exposure and Human Fetal Growth.

    Science.gov (United States)

    Winterbottom, Emily F; Fei, Dennis L; Koestler, Devin C; Giambelli, Camilla; Wika, Eric; Capobianco, Anthony J; Lee, Ethan; Marsit, Carmen J; Karagas, Margaret R; Robbins, David J

    2015-06-01

    Although considerable evidence suggests that in utero arsenic exposure affects children's health, these data are mainly from areas of the world where groundwater arsenic levels far exceed the World Health Organization limit of 10 μg/L. We, and others, have found that more common levels of in utero arsenic exposure may also impact children's health. However, the underlying molecular mechanisms are poorly understood. To address this issue, we analyzed the expression of key developmental genes in fetal placenta in a birth cohort of women using unregulated water supplies in a US region with elevated groundwater arsenic. We identified several genes whose expression associated with maternal arsenic exposure in a fetal sex-specific manner. In particular, expression of the HEDGEHOG pathway component, GLI3, in female placentae was both negatively associated with arsenic exposure and positively associated with infant birth weight. This suggests that modulation of GLI3 in the fetal placenta, and perhaps in other fetal tissues, contributes to arsenic's detrimental effects on fetal growth. We showed previously that arsenic-exposed NIH3T3 cells have reduced GLI3 repressor protein. Together, these studies identify GLI3 as a key signaling node that is affected by arsenic, mediating a subset of its effects on developmental signaling and fetal health.

  13. LR8 Expression in fibroblasts of healthy and fibrotic human tissues.

    Science.gov (United States)

    Etikala, Anusha; Bruce, Greg; Hudkins, Kelly; Narayanan, A S

    2017-07-01

    LR8 gene was first reported in a subpopulation of cultured human lung fibroblasts expressing the receptor for C1q-globular domain, and it was not detectable in cultured endothelial cells and smooth muscle cells. LR8 mRNA levels were higher in fibrotic lungs. In this study we assessed LR8 production in human tissues and determined if the distribution of fibroblasts producing LR8 is affected in fibrosis. Normal and fibrotic tissue sections from human liver, lung and kidneys were immunostained with antibodies to LR8 and examined for the presence of fibroblasts staining positively and negatively. The cells were also examined for co-expression of α-smooth muscle actin (SMA), a marker for myofibroblasts. The results showed that LR8 was expressed by fibroblasts, smooth muscle cells, endothelial cells, bile duct cells, pulmonary alveolar cells and distal and proximal kidney tubule cells. Connective tissues of normal and fibrotic tissues contained fibroblasts staining positively and negatively with anti- LR8 antibody. The number of LR8-positive cells was higher in fibrotic tissues, but differences were not statistically significant. Fibroblasts producing both LR8 and SMA were present in higher numbers in fibrotic tissues as compared to normal tissues and the differences were statistically significant (phuman tissues, and that in fibrotic tissues cells co-expressing LR8 and SMA are present. Our results indicate that LR8 expressing cells may participate in the early stages of fibrotic diseases and that fibroblasts expressing LR8, not LR8 negative cells, have potential to become myofibroblasts in fibrotic tissues.

  14. Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds.

    Directory of Open Access Journals (Sweden)

    Howard Y Chang

    2004-02-01

    Full Text Available Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

  15. Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency.

    Science.gov (United States)

    Karring, Henrik; Thøgersen, Ida B; Klintworth, Gordon K; Enghild, Jan J; Møller-Pedersen, Torben

    2004-07-01

    The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.

  16. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

    Science.gov (United States)

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

  17. Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

    Science.gov (United States)

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

    2015-01-01

    Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

  18. 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.

  19. 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.

  20. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  1. Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices

    Directory of Open Access Journals (Sweden)

    Ruiz Pedro A

    2012-02-01

    Full Text Available Abstract Background The two discoidin domain receptors (DDRs, DDR1 and DDR2 are receptor tyrosine kinases (RTKs with the unique ability among RTKs to respond to collagen. We have previously shown that collagen I induces DDR1 and matrix metalloproteinase (MMP-10 expression through DDR2 activation and a Janus kinase (JAK2 and extracellular signal-regulated kinase (ERK1/2-mediated mechanism in primary human lung fibroblasts suggesting that these signaling pathways play a role in fibroblast function. Fibroblasts can traverse basement membrane barriers during development, wound healing and pathological conditions such as cancer and fibrosis by activating tissue-invasive programs, the identity of which remain largely undefined. In the present work, we investigated the role of DDRs and DDR-associated signal transduction in these processes. Results Transwell migration experiments showed that normal human lung fibroblast (NHLF transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1. Additionally, experiments with specific small interfering (siRNAs revealed that collagen I-induced expression of MMP-10 and MMP-2 is DDR2 but not DDR1 dependent in NHLFs. Our data showed that collagen I increases NHLF migration through collagen IV, the main component of basement membranes. Furthermore, basal and collagen I-induced NHLF migration through collagen IV-coated inserts was both DDR2 and DDR1 dependent. Finally, DDR2, but not DDR1 was shown to be involved in fibroblast proliferation. Conclusions Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression. This work provides new insights into the role of DDRs in fibroblast function.

  2. MORPHOLOGICAL FEATURES AND MORPHOMETRIC PARAMETERS OF HUMAN FETAL THYMUS GLANDS

    Directory of Open Access Journals (Sweden)

    S. Havila Hasini

    2014-03-01

    Full Text Available Introduction: Thymus is one of the central lymphoid organs. It plays an important role in the differentiation, selection and maturation of T-lymphocytes. In the recent years morphology and morphometry of the thymus gland in the newborn is gaining significance as it demonstrates great variability between individual infants and in the same infant at different times. Materials and methods: In the present study 45 thymus specimens from aborted human fetuses of 16 to 40 weeks gestational age and both sexes were studied by autopsy for morphological and morphometric features. The morphometric parameters were measured using pachymeter. Results: The thymus gland was located in the superior mediastinum. 60% (27/45 specimens showed cervical extensions. Brachiocephalic vein anterior to thymus was observed in 3 cases which is an important anomaly to be observed in thymectomy procedure. Thymuses were greyish pink to greyish brown in colour. Variations were also observed in the number of lobes of glands in which one is single lobed, most of the glands are bilobed and few are trilobed. There is progressive increase in all morphometric dimensions of the thymus in relation to gestational age. Most of the specimens were less than 4cm in length. Half of the specimens were below 2cm in width and other half were 2.0 to 5.0 cm in width. For 90% of the specimens thickness of the organ was less than 0.5cm. The thymus gland was 0.2% of fetal body weight. Conclusion: The morphological observations of thymus gland shows great variations which has to be considered in thymectomy. In addition to anthropometric parameters of fetus, morphometric parameters of thymus glands present significant relation to the gestational age of fetuses. It is possible to determine the thymic morphometric parameters in relation to gestational age.

  3. Effect of Supracervical Apposition and Spontaneous Labour on Apoptosis and Matrix Metalloproteinases in Human Fetal Membranes

    Directory of Open Access Journals (Sweden)

    Mahalia Chai

    2013-01-01

    Full Text Available Background. Apoptosis and matrix metalloproteinase (MMP-9 are capable of hydrolysing components of the extracellular matrix and weakening the fetal membranes which leads to eventual rupture, a key process of human parturition. The aim of this study was to determine the effect of supracervical apposition and spontaneous labour on apoptosis and MMP-9 in human fetal membranes at term. Methods. Fetal membranes were obtained from term non-labouring supracervical site (SCS and compared to (i a paired distal site (DS or (ii site of rupture (SOR after spontaneous labour onset. Results. The expression of the proapoptotic markers Bax, Smac, Fas, FasL, caspase-3, and PARP, was significantly higher in the non-labouring SCS chorion compared to paired DS. Bax, Smac, FasL, caspase-3, and PARP staining was higher in the non-labouring SCS fetal membranes than that in the post-labour SOR. MMP-9 expression and activity were higher in the post-labour SOR fetal membranes compared to non-labouring SCS fetal membranes. Conclusion. Components of the apoptotic signalling pathways and MMP-9 may play a role in rupture and labour. Non-labouring SCS fetal membranes display altered morphology and altered apoptotic biochemical characteristics in preparation for labour, while the laboured SOR displays unique MMP characteristics.

  4. Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sascha Schäuble

    Full Text Available Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD, fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P via reversibly cell cycle arrested (C to irreversibly arrested senescent cells (S. In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

  5. 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.

  6. 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.

  7. Assessment of human gingival fibroblast interaction with dental implant abutment materials.

    Science.gov (United States)

    Rutkunas, Vygandas; Bukelskiene, Virginija; Sabaliauskas, Vaidotas; Balciunas, Evaldas; Malinauskas, Mangirdas; Baltriukiene, Daiva

    2015-04-01

    The biocompatibility of dental implant abutment materials depends on numerous factors including the nature of the material, its chemical composition, roughness, texture, hydrophilicity and surface charge. The aim of the present study was to compare the viability and adhesion strength of human gingival fibroblasts (HGFs) grown on several dental materials used in implant prosthodontics. Surfaces of the tested materials were assessed using an optical imaging profiler. For material toxicity and cellular adhesion evaluation, primary human gingival fibroblast cells were used. To evaluate the strength of cellular adhesion, gingival fibroblasts were cultured on the tested materials and subjected to lateral shear forces by applying 300 and 500 rpm shaking intensities. Focal adhesion kinase (FAK) expression and phosphorylation in cells grown on the specimens were registered by cell-based ELISA. There was a tendency of fibroblast adhesion strength to decrease in the following order: sandblasted titanium, polished titanium, sandblasted zirconium oxide, polished zirconium oxide, gold-alloy, chrome-cobalt alloy. Higher levels of total as well as phospho-FAK protein were registered in HGFs grown on roughened titanium. Material type and surface processing technique have an impact on gingival fibroblast interaction with dental implant abutment materials.

  8. Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Wei ZHAO; Zhong Xiang LIN; Zhi Qian ZHANG

    2004-01-01

    To examine the role of gap junctions in cell senescence,the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore,cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis. p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin (10 mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis,elevation of p53 expression,loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.

  9. Development of human skin equivalents mimicking skin aging : contrast between papillary and reticular fibroblasts as a lead

    NARCIS (Netherlands)

    Janson, D.

    2017-01-01

    This thesis describes the development of human skin equivalents that show characteristics of skin aging. The type of skin equivalent used was a fibroblast derived matrix equivalent, in which the dermal compartment is generated by fibroblasts and thus is fully of human origin. Two strategies are

  10. Identification of CYP3A7 for Glyburide Metabolism in Human Fetal Livers

    Science.gov (United States)

    Shuster, Diana L.; Risler, Linda J.; Prasad, Bhagwat; Calamia, Justina C.; Voellinger, Jenna L.; Kelly, Edward J.; Unadkat, Jashvant D.; Hebert, Mary F.; Shen, Danny D.; Thummel, Kenneth E.; Mao, Qingcheng

    2014-01-01

    Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1-M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers. PMID:25450675

  11. Malignant transformation of diploid human fibroblasts by transfection of oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, J.J.

    1992-01-01

    This document consist of brief reports prepared by postdoctoral students supported by the project, each describing his accomplishments under the grant. Topics include (1) Malignant Transformation of MSU-1. 1 Cells by Gamma Radiation, (2) Correlation between Levels of ras Expression and Presence of Transformed Phenotypes Including Tumorigenicity, Using a Modulatable Promoter, (3) Relation between Specific rad Oncogene Expression, (4) Correlation of Genetic Changes in Fibroblastic Tumors with Malignancies, (5)Transformation of MSU-1.1 Cells by sis Oncogene, (6) Malignant Transformation of MSU-1.0 Cells, (7) Correlation of Urokinase Plasminogen Activation (mu-PA) with Malignant Phenotype, (8)Two Dimensional Gel Electrophoresis Studies of the Proteins of the Major Cell Strains of the MSU-1 Family of Cells, and (9) Correlation between Proteinase Activity Levels and Malignancy.

  12. Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

    OpenAIRE

    1986-01-01

    Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting th...

  13. Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

    2011-01-01

    the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...

  14. Expression of stem cell markers in the human fetal kidney.

    Directory of Open Access Journals (Sweden)

    Sally Metsuyanim

    Full Text Available In the human fetal kidney (HFK self-renewing stem cells residing in the metanephric mesenchyme (MM/blastema are induced to form all cell types of the nephron till 34(th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2 are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24 in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (50% of HFK cells and predominantly co-express EpCAM(bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM(+EpCAM(- and to a lesser extent in NCAM(+EpCAM(+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM(+EpCAM(+FZD7(+, MM stem cells (NCAM(+EpCAM(-FZD7(+ or both (NCAM(+FZD7(+. These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.

  15. Salidroside protects human fibroblast cells from premature senescence induced by H(2)O(2) partly through modulating oxidative status.

    Science.gov (United States)

    Mao, Gen-xiang; Wang, Yan; Qiu, Qiang; Deng, Hong-bin; Yuan, Long-guo; Li, Rui-guo; Song, Dan-qing; Li, Yi-yang Yvonne; Li, Dian-dong; Wang, Zhen

    2010-01-01

    Although salidroside and salidroside-like compounds are considered as most critical constitutes needed and responsible for multiple therapeutic benefits of Rhodiola rosea L., including anti-aging, direct demonstration regarding the role of salidroside in anti-aging process is still deficient. In this study, we selected the H(2)O(2)-induced premature senescence model in human fetal lung diploid fibroblasts to investigate the protection of salidroside against aging in vitro and associated molecular mechanisms. We found that salidroside considerably reversed senescence-like phenotypes in the oxidant challenged model, including alterations of morphology, cell cycle, SA-β-gal staining, DNA damage, as well as related molecules expression such as p53, p21 and p16. The protection occurred in a dose-dependent manner, with 5μM offering best efficacy. The proposed antioxidant property of the compound was confirmed in this cellular system, and thus at least partially accounted for the protection of the compound against premature senescence. Similar protection of salidroside against replicative senescence was observed as well. Interestingly, the regulation of senescence-related molecules by salidroside involved ROS-irrelevant mechanisms in both models. This finding presents salidroside as an attractive agent with potential to retard aging and attenuate age-related diseases in humans. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  16. Maturation of the human fetal startle response: evidence for sex-specific maturation of the human fetus.

    Science.gov (United States)

    Buss, Claudia; Davis, Elysia Poggi; Class, Quetzal A; Gierczak, Matt; Pattillo, Carol; Glynn, Laura M; Sandman, Curt A

    2009-10-01

    Despite the evidence for early fetal experience exerting programming influences on later neurological development and health risk, very few prospective studies of human fetal behavior have been reported. In a prospective longitudinal study, fetal nervous system maturation was serially assessed by monitoring fetal heart rate (FHR) responses to vibroacoustic stimulation (VAS) in 191 maternal/fetal dyads. Responses were not detected at 26 weeks gestational age (GA). Sex-specific, age-characteristic changes in the FHR response to VAS were observed by 31 weeks' GA. Males showed larger responses and continued to exhibit maturational changes until 37 weeks' GA, females however, presented with a mature FHR startle response by 31 weeks' GA. The results indicate that there are different rates of maturation in the male and female fetuses that may have implications for sex-specific programming influences.

  17. Maturation of the human fetal startle response: Evidence for sex-specific maturation of the human fetus1

    Science.gov (United States)

    Buss, Claudia; Davis, Elysia Poggi; Class, Quetzal A.; Gierczak, Matt; Pattillo, Carol; Glynn, Laura M.; Sandman, Curt A.

    2009-01-01

    Despite the evidence for early fetal experience exerting programming influences on later neurological development and health risk, very few prospective studies of human fetal behavior have been reported. In a prospective longitudinal study, fetal nervous system maturation was serially assessed by monitoring fetal heart rate (FHR) responses to vibroacoustic stimulation (VAS) in 191 maternal/fetal dyads. Responses were not detected at 26 weeks gestational age (GA). Sex-specific, age-characteristic changes in the FHR response to VAS were observed by 31 weeks’ GA. Males showed larger responses and continued to exhibit maturational changes until 37 weeks’ GA, females however, presented with a mature FHR startle response by 31 weeks’ GA. The results indicate that there are different rates of maturation in the male and female fetus that may have implications for sex-specific programming influences. PMID:19726143

  18. ADHESION AND SPREADING OF HUMAN FIBROBLASTS ON SUPERHYDROPHOBIC FEP-TEFLON

    NARCIS (Netherlands)

    BUSSCHER, HJ; STOKROOS, [No Value; GOLVERDINGEN, JG; SCHAKENRAAD, JM

    1991-01-01

    Adhesion and spreading of human fibroblasts was studied on hydrophobized and hydrophilized FEP-Teflon, and compared with adhesion and spreading on untreated FEP-Teflon and Tissue culture polystyrene (TCPS). Superhydrophobic FEP-Teflon was prepared by ion etching followed by oxygen glow-discharge. Hy

  19. Development of a full-thickness human gingiva equivalent constructed from immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    J.K. Buskermolen; C.M.A. Reijnders; S.W. Spiekstra; T. Steinberg; C.J. Kleverlaan; A.J. Feilzer; A.D. Bakker; S. Gibbs

    2016-01-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines

  20. Assembly of fibronectin into the extracellular matrix of early and late passage human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mann, D.M.

    1987-01-01

    The specific binding of soluble /sup 125/I-human plasma fibronectin (/sup 125/I-HFN-P) to confluent cultures of early and late passage human skin fibroblasts was investigated. Previous studies HFN-P bound to fibroblast cell layers indicated that HNF-P was present in the cultures in two separate pools, distinguishable on the basis of their solubility in 1% deoxycholate. Examination of the kinetics of /sup 125/I-HFN-P binding to Pool I of early and late passage cultures revealed that both cultures required 2-4 h to approach steady-state conditions. Other kinetic studies showed that the rates of low of /sup 125/I-HFN-P from either Pool I or Pool II were similar for both cultures. Further, Scatchard analysis revealed a single class of Pool I binding sites with apparent dissociation constants (K/sub d/) of 5.3 x 10/sup -8/M (early passage) and 4.2 x 10/sup -8/M (late passage). These results indicate that early and late passage cultures of human fibroblasts exhibit differences in the number of cell surface biding sites for soluble fibronectin, and in the extent to which they incorporate soluble fibronectin into the extracellular matrix. Parameters which affect the fibronectin matrix assembly system of human skin fibroblasts were also examined. In addition, several monoclonal anti-fibronectin antibodies were characterized and developed as experimental probes for fibronectin structure and function.

  1. ADHESION AND SPREADING OF HUMAN SKIN FIBROBLASTS ON PHYSICOCHEMICALLY CHARACTERIZED GRADIENT SURFACES

    NARCIS (Netherlands)

    RUARDY, TG; SCHAKENRAAD, JM; VANDERMEI, HC; BUSSCHER, HJ

    1995-01-01

    In this study, adhesion and spreading of human skin fibroblasts on gradient surfaces of dichlorodimethylsilane (DDS) coupled to glass was investigated. Gradient surfaces were prepared by the diffusion technique and characterized by the Wilhelmy plate technique for their wettability and by scanning x

  2. SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells.

    Science.gov (United States)

    Pan, Chuanying; Hicks, Amy; Guan, Xuan; Chen, Hong; Bishop, Colin E

    2010-04-01

    Induced pluripotent stem (iPS) cells can be derived from human somatic cells by cellular reprogramming. This technology provides a potential source of non-controversial therapeutic cells for tissue repair, drug discovery, and opportunities for studying the molecular basis of human disease. Normally, mouse embryonic fibroblasts (MEFs) are used as feeder layers in the initial derivation of iPS lines. The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cells reprogrammed from somatic cells using lentiviral expressed reprogramming factors. In our study, iPS cells expressed common pluripotency markers, displayed human embryonic stem cells (hESCs) morphology and unmethylated promoters of NANOG and OCT4. These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    Directory of Open Access Journals (Sweden)

    Ruvinov Emil

    2008-11-01

    Full Text Available Abstract Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO would improve tissue repair in rat after myocardial infarction (MI. Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat.

  5. 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.

  6. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Jun [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Xiang, Jun-Jian, E-mail: txjj@jnu.edu.cn [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China); Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Li, Dan [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Deng, Ning; Wang, Hong; Gong, Yi-Ping [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China)

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  7. Human chorionic gonadotropin (hCG) concentrations during the late first trimester are associated with fetal growth in a fetal sex-specific manner.

    Science.gov (United States)

    Barjaktarovic, Mirjana; Korevaar, Tim I M; Jaddoe, Vincent W V; de Rijke, Yolanda B; Visser, Theo J; Peeters, Robin P; Steegers, Eric A P

    2017-02-01

    Human chorionic gonadotropin (hCG) is a pregnancy-specific hormone that regulates placental development. hCG concentrations vary widely throughout gestation and differ based on fetal sex. Abnormal hCG concentrations are associated with adverse pregnancy outcomes including fetal growth restriction. We studied the association of hCG concentrations with fetal growth and birth weight. In addition, we investigated effect modification by gestational age of hCG measurement and fetal sex. Total serum hCG (median 14.4 weeks, 95 % range 10.1-26.2), estimated fetal weight (measured by ultrasound during 18-25th weeks and >25th weeks) and birth weight were measured in 7987 mother-child pairs from the Generation R cohort and used to establish fetal growth. Small for gestational age (SGA) was defined as a standardized birth weight lower than the 10th percentile of the study population. There was a non-linear association of hCG with birth weight (P = 0.009). However, only low hCG concentrations measured during the late first trimester (11th and 12th week) were associated with birth weight and SGA. Low hCG concentrations measured in the late first trimester were also associated with decreased fetal growth (P = 0.0002). This was the case for both male and female fetuses. In contrast, high hCG concentrations during the late first trimester were associated with increased fetal growth amongst female, but not male fetuses. Low hCG in the late first trimester is associated with lower birth weight due to a decrease in fetal growth. Fetal sex differences exist in the association of hCG concentrations with fetal growth.

  8. Influence of maternal folate status on human fetal growth parameters

    NARCIS (Netherlands)

    van Uitert, Evelyne M.; Steegers-Theunissen, Regine P. M.

    2013-01-01

    Worldwide periconceptional folic acid supplement use is recommended to prevent neural tube defects. This also stimulated research on maternal folate status in association with fetal growth, an important predictor of perinatal and future development and health. We provide an overview of literature on

  9. Effect of low-power red light laser irradiation on the viability of human skin fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Bednarska, K.; Rozga, B.; Leyko, W.; Bryszewska, M. [Institute of Biophysics, University of Lodz (Poland); Kolodziejczyk, K.; Szosland, D. [Diabetological Clinic, Medical Academy of Lodz (Poland)

    1998-10-01

    Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm{sup 2}). The energy densities were 2 J/cm{sup 2} and 12 J/cm{sup 2}, respectively, and the irradiation was carried out at a temperature of 22 C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 C, in the presence of 5% CO{sub 2}, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation. (orig.) With 4 figs., 2 tabs., 13 refs.

  10. 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.

  11. 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

  12. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

    NARCIS (Netherlands)

    Hartmann-Fritsch, Fabienne; Hosper, Nynke; Luginbuehl, Joachim; Biedermann, Thomas; Reichmann, Ernst; Meuli, Martin

    Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis

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

    Directory of Open Access Journals (Sweden)

    Paola Brun

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

  14. Imatinib mesylate inhibits proliferation and exerts an antifibrotic effect in human breast stroma fibroblasts.

    Science.gov (United States)

    Gioni, Vassiliki; Karampinas, Theodoros; Voutsinas, Gerassimos; Roussidis, Andreas E; Papadopoulos, Savvas; Karamanos, Nikos K; Kletsas, Dimitris

    2008-05-01

    Tumor stroma plays an important role in cancer development. In a variety of tumors, such as breast carcinomas, a desmoplastic response, characterized by stromal fibroblast and collagen accumulation, is observed having synergistic effects on tumor progression. However, the effect of known anticancer drugs on stromal cells has not been thoroughly investigated. Imatinib mesylate is a selective inhibitor of several protein tyrosine kinases, including the receptor of platelet-derived growth factor, an important mediator of desmoplasia. Recently, we have shown that imatinib inhibits the growth and invasiveness of human epithelial breast cancer cells. Here, we studied the effect of imatinib on the proliferation and collagen accumulation in breast stromal fibroblasts. We have shown that it blocks the activation of the extracellular signal-regulated kinase and Akt signaling pathways and up-regulates cyclin-dependent kinase inhibitor p21(WAF1), leading to the inhibition of fibroblast proliferation, by arresting them at the G(0)/G(1) phase of the cell cycle. Imatinib inhibits more potently the platelet-derived growth factor-mediated stimulation of breast fibroblast proliferation. By using specific inhibitors, we have found that this is due to the inhibition of the Akt pathway. In addition, imatinib inhibits fibroblast-mediated collagen accumulation. Conventional and quantitative PCR analysis, as well as gelatin zymography, indicates that this is due to the down-regulation of mRNA synthesis of collagen I and collagen III-the main collagen types in breast stroma-and not to the up-regulation or activation of collagenases matrix metalloproteinase 2 and matrix metalloproteinase 9. These data indicate that imatinib has an antifibrotic effect on human breast stromal fibroblasts that may inhibit desmoplastic reaction and thus tumor progression.

  15. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  16. MAOA and GYG2 are submitted to X chromosome inactivation in human fibroblasts.

    Science.gov (United States)

    Stabellini, Raquel; Vasques, Luciana R; de Mello, Joana Carvalho Moreira; Hernandes, Lys Molina; Pereira, Lygia V

    2009-08-16

    X chromosome inactivation (XCI) is a comprehensively studied phenomenon that helped to highlight the heritable nature of epigenetic modifications. Although it consists of the transcriptional inactivation of a whole X chromosome in females, some genes escape this process and present bi-allelic expression. Using human fibroblasts with skewed inactivation, we determined allele-specific expression of two X-linked genes previously described to escape XCI in rodent/human somatic cell hybrids, MAOA and GYG2, and the pattern of DNA methylation of their 5' end. Results from these complementary methodologies let us to conclude that both genes are subjected to X inactivation in normal human fibroblasts, indicating that hybrid cells are not an adequate system for studying epigenotypes. We emphasize the need of an analysis of XCI in normal human cell lines, helping us to determine more precisely which X-linked genes contribute to differences among genders and to the phenotypes associated with sex chromosomes aneuploidies.

  17. Enumeration of the colony-forming units–fibroblast from mouse and human bone marrow in normal and pathological conditions

    OpenAIRE

    Kuznetsov, Sergei A; Mankani, Mahesh H.; Bianco, Paolo; Robey, Pamela G.

    2008-01-01

    Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units–fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum hea...

  18. Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1 Downregulates ELOVL1 Gene Expression and Fatty Acid Synthesis in Goat Fetal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Weipeng Wang

    2015-07-01

    Full Text Available Elongation of very-long-chain fatty acids 1 (ELOVL1 is a ubiquitously expressed gene that belongs to the ELOVL family and regulates the synthesis of very-long-chain fatty acids (VLCFAs and sphingolipids, from yeast to mammals. Mammalian target of rapamycin complex 1 (mTORC1 is a central regulator of cell metabolism and is associated with fatty acids synthesis. In this study, we cloned the cDNA that encodes Cashmere goat (Capra hircus ELOVL1 (GenBank Accession number KF549985 and investigated its expression in 10 tissues. ELOVL1 cDNA was 840 bp, encoding a deduced protein of 279 amino acids, and ELOVL1 mRNA was expressed in a wide range of tissues. Inhibition of mTORC1 by rapamycin decreased ELOVL1 expression and fatty acids synthesis in Cashmere goat fetal fibroblasts. These data show that ELOVL1 expression is regulated by mTORC1 and that mTORC1 has significant function in fatty acids synthesis in Cashmere goat.

  19. 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.

  20. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    OpenAIRE

    Shan-Shan Liu; Hao-Yan Wang; Jun-Ming Tang; Xiu-Mei Zhou

    2013-01-01

    The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II) on collagen synthesis in hypoxic human lung fibroblast (HLF) cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression levels in human ...

  1. Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor.

    OpenAIRE

    Francki, A.; Uciechowski, P.; Floege, J; von der Ohe, J.; Resch, K.; Radeke, H. H.

    1995-01-01

    For various forms of human glomerulonephritis a close relationship between inflammatory injury and a local mesangial proliferative response has been described. Herein, we used primary cultures of human glomerular mesangial cells (HMCs) from five different donors to determine the autocrine growth-inducing capacity of their supernatants after stimulation with different cytokines and lipopolysaccharide (LPS) to determine whether this effect is due to basic fibroblast growth factor (bFGF). The ba...

  2. The expression pattern of two novel cytokines (IL-24 and IL-29) in human fetal membranes.

    Science.gov (United States)

    Nace, Judith; Fortunato, Stephen J; Maul, Holger; Menon, Ramkumar

    2010-11-01

    interleukin (IL)-24 and -29 are novel cytokines, produced by immune cells in response to microbial antigens. The functions of these cytokines in the reproductive system are unknown. We examined the expression pattern of IL-24 and IL-29 in human fetal membranes from preterm and term births and in in vitro in response to microbial antigens. fetal membranes collected from cesarean sections at term (normal, not in labor) were placed in culture for 48 h. These membranes were then stimulated with bacterial lipopolysaccharide (LPS) or viral antigen poly-inosinic and cytidylic acid (polyIC) for an additional 24 h. Amniotic fluids (AF) and fetal membranes were also collected from preterm and term deliveries. IL-24 and IL-29 expressions were studied by RT-PCR. ELISA documented culture media and AF cytokine concentrations. IL-24 and IL-29 expressions were seen in cultured fetal membranes regardless of stimulation. Expressions were also found in preterm and term labor membranes, but not in non-labor tissues at term. IL-24 concentrations were higher after LPS stimulation whereas IL-29 concentrations were higher after polyIC-stimulation. AF analysis did not detect either of the cytokines either preterm or term. this is the first study to report IL-24 and IL-29 expressions in human fetal membranes. Higher concentrations of these cytokines in response to distinct infectious stimuli suggest different pathways for fetal immune response during infection.

  3. Ethical issues surrounding the transplantation of human fetal tissues.

    Science.gov (United States)

    Hurd, R E

    1992-12-01

    Organ transplants have been one of the greatest advances in medicine. However, organs from living relatives or cadavers are in short supply, and many people die awaiting a donor organ. Increasing the donor pool by using organs from aborted fetuses has been proposed to increase the supply. In addition, there are benefits of using fetal tissue including its particular usefulness in children, the fact that it is not readily rejected, and its potential for growth. Guidelines for fetal research were issued in 1975, but a research moratorium was imposed in 1988 to allow study of ethical and legal issues. While the federal government delays in lifting the ban, several states have written laws governing experimentation with fetuses. Ethical arguments against using fetal tissue for organ transplant include a concern that this would create a branch of biomedicine which depends on the continuation of induced abortions. This could lead to neglect of research for other therapies. The timing and type of abortion should continue to benefit the mother, rather than the organ recipient. Ethicists debate whether or not use of aborted tissue implies complicity in the abortion process beyond that which exists for all members of a society which permits abortion. They also wonder whether knowing that some good could come of an abortion would influence a woman's decision to have one. Proposals to keep the use of fetal tissue ethical include banning the commercial use of sale of tissues, forbidding designation of the tissue recipient (to prevent harvesting fetal tissue for a relative), separating abortion counseling and management from harvesting of the tissue, and obtaining informed consent (perhaps from a proxy surrogate rather than from the mother) for the use of fetal tissue. When the medical and ethical communities have reached some consensus on these issues, crafted safeguards, and precluded conflicts of interest, then restrictions on government funding should be lifted. Whereas it

  4. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells.

    Science.gov (United States)

    Yang, Chao; Ji, Lei; Yue, Wen; Shi, Shuang-Shuang; Wang, Ruo-Yong; Li, Yan-Hua; Xie, Xiao-Yan; Xi, Jia-Fei; He, Li-Juan; Nan, Xue; Pei, Xue-Tao

    2012-02-01

    Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

  5. KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas

    NARCIS (Netherlands)

    Roost, M.S.; Iperen, L. van; Ariyurek, Y.; Buermans, H.P.; Arindrarto, W.; Devalla, H.D.; Passier, R.; Mummery, C.L.; Carlotti, F.; Koning, E.J. de; Zwet, E.W. van; Goeman, J.J.; Chuva de Sousa Lopes, S.M.

    2015-01-01

    Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and s

  6. KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas

    NARCIS (Netherlands)

    Roost, Matthias S; van Iperen, Liesbeth; Ariyurek, Yavuz; Buermans, Henk P; Arindrarto, Wibowo; Devalla, Harsha D; Passier, Robert; Mummery, Christine L; Carlotti, Françoise; de Koning, Eelco J P; van Zwet, Erik W; Goeman, Jelle J; Chuva de Sousa Lopes, Susana M

    2015-01-01

    Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and

  7. Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

    Science.gov (United States)

    Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

    2016-01-01

    This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

  8. Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

    Directory of Open Access Journals (Sweden)

    Humidah Alanazi

    2014-01-01

    Full Text Available The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P<0.01 sensitive to oxidation but significantly (P<0.01 resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P<0.01 slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

  9. Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

    Science.gov (United States)

    Alanazi, Humidah; Semlali, Abdelhabib; Perraud, Laura; Chmielewski, Witold; Zakrzewski, Andrew

    2014-01-01

    The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers. PMID:25302312

  10. The effectiveness of potent dental adhesives on the viability of LPS challenged human gingival fibroblasts.

    Science.gov (United States)

    Garner, Angelia D; Tucci, Michelle A; Benghuzzi, Hamed A

    2014-01-01

    Dental adhesives are necessary for the retention of specific dental restorations utilized to repair the anatomy of the tooth after dental decay is removed. Adhesives come into contact with healthy and diseased periodontal tissues. Porphyromonas gingivalis is a gram negative bacterial pathogen, and lipopolysaccharide (LPS-PG) is an endotoxin found in gingival connective tissues of patients who suffer from periodontal disease. The presence of the endotoxin causes inflammation. This study aims to evaluate the effectiveness of potent dental adhesives when human gingival fibroblasts are challenged with LPS-PG. The fibroblasts were exposed to the dental adhesives polymethly methacrylate (PMMA), OptiBond®, and Prime & Bond® which were purchased from Patterson Dental, a national dental materials supplier. The human gingival fibroblasts (HGF-1, ATCC® CRL-2014™) were purchased from American Type Culture Collection (ATCC). The porphyromonas gingival lipopolysaccharide (LPS-PG) was purchased from Fisher Scientific (Pittsburg, PA). No significant differences in metabolic behavior was detected among the groups (padhesives and LPS-PG at 48 hour intervals (p<0.003). No significant changes were noted in cellular morphology at any phases, and all cells demonstrated typical fibroblast spindle shape.

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

    Directory of Open Access Journals (Sweden)

    Takayuki Ueno

    2015-01-01

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

  12. The beta(2)-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts

    NARCIS (Netherlands)

    Lamyel, F.; Warnken-Uhlich, M.; Seemann, W. K.; Mohr, K.; Kostenis, E.; Ahmedat, A. S.; Smit, M.; Gosens, R.; Meurs, H.; Miller-Larsson, A.; Racke, Kurt

    2011-01-01

    Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by beta-adrenoceptor activation. We aimed, first, to characterise the expression pattern of beta-adrenoceptor subtypes in human lung fibroblasts and, second, to p

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

    Science.gov (United States)

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

    2016-01-15

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

  14. Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Kegel Magdalena

    2011-10-01

    Full Text Available Abstract Background The kynurenine pathway (KP is the main route of tryptophan degradation in the human body and generates several neuroactive and immunomodulatory metabolites. Altered levels of KP-metabolites have been observed in neuropsychiatric and neurodegenerative disorders as well as in patients with affective disorders. The purpose of the present study was to investigate if skin derived human fibroblasts are useful for studies of expression of enzymes in the KP. Methods Fibroblast cultures were established from cutaneous biopsies taken from the arm of consenting volunteers. Such cultures were subsequently treated with interferon (IFN-γ 200 U/ml and/or tumor necrosis factor (TNF-α, 100 U/ml for 48 hours in serum-free medium. Levels of transcripts encoding different enzymes were determined by real-time PCR and levels of kynurenic acid (KYNA were determined by HPLC. Results At base-line all cultures harbored detectable levels of transcripts encoding KP enzymes, albeit with considerable variation across individuals. Following cytokine treatment, considerable changes in many of the transcripts investigated were observed. For example, increases in the abundance of transcripts encoding indoleamine 2,3-dioxygenase, kynureninase or 3-hydroxyanthranilic acid oxygenase and decreases in the levels of transcripts encoding tryptophan 2,3-dioxygenase, kynurenine aminotransferases or quinolinic acid phosphoribosyltransferase were observed following IFN-γ and TNF-α treatment. Finally, the fibroblast cultures released detectable levels of KYNA in the cell culture medium at base-line conditions, which were increased after IFN-γ, but not TNF-α, treatments. Conclusions All of the investigated genes encoding KP enzymes were expressed in human fibroblasts. Expression of many of these appeared to be regulated in response to cytokine treatment as previously reported for other cell types. Fibroblast cultures, thus, appear to be useful for studies of disease

  15. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids.

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    Takamasa Tobita

    Full Text Available There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes.

  16. The period length of fibroblast circadian gene expression varies widely among human individuals.

    Directory of Open Access Journals (Sweden)

    Steven A Brown

    2005-10-01

    Full Text Available Mammalian circadian behavior is governed by a central clock in the suprachiasmatic nucleus of the brain hypothalamus, and its intrinsic period length is believed to affect the phase of daily activities. Measurement of this period length, normally accomplished by prolonged subject observation, is difficult and costly in humans. Because a circadian clock similar to that of the suprachiasmatic nucleus is present in most cell types, we were able to engineer a lentiviral circadian reporter that permits characterization of circadian rhythms in single skin biopsies. Using it, we have determined the period lengths of 19 human individuals. The average value from all subjects, 24.5 h, closely matches average values for human circadian physiology obtained in studies in which circadian period was assessed in the absence of the confounding effects of light input and sleep-wake cycle feedback. Nevertheless, the distribution of period lengths measured from biopsies from different individuals was wider than those reported for circadian physiology. A similar trend was observed when comparing wheel-running behavior with fibroblast period length in mouse strains containing circadian gene disruptions. In mice, inter-individual differences in fibroblast period length correlated with the period of running-wheel activity; in humans, fibroblasts from different individuals showed widely variant circadian periods. Given its robustness, the presented procedure should permit quantitative trait mapping of human period length.

  17. Dimethylarsenic acid damages cellular DNA and inhibits gap junctional intercellular communication between human skin fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    GuoXB; DengFR

    2002-01-01

    Although arsenic is identified as a human carcinogen,there is currently no accepted mechanism for its action or an established animal model for evaluating the carcinogenic activity of arsenic.To elucidate the mechanism of arsenic arcinogenesis,we investigated the effect of dimethylarsenic acid(DMAA),the main metabolite of inorganic arsenic in humans,on the cellular DNA and gap junctional intercellular communication (GJIC) between human skin fibroblast cells.Single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage in human skin fibroblast cells exposed to DMAA,and the GJIC between cells was detected by the scrape loading/dye transfer assay.DMAA at concentrations of 0.01-1.0 mmol·L-1 induced DNA damage in a dose-dependent manner,and GJIC between human skin fibroblast cells was significantly inhibited by DMAA at 1.0 mmol·L-1.Our results suggest that both genotoxic and nongenotoxic mechanism are involved in the mechanism of DMAA-induced cellular toxicity.

  18. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

    OpenAIRE

    2013-01-01

    PURPOSE: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. METHODS: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thicknes...

  19. Pluripotent stem cells exhibiting similar characteristics can be isolated from human fetal bone marrow,heart,liver,muscle,lung,derma,kidney,and fat

    Institute of Scientific and Technical Information of China (English)

    FANG Baijun; SONG Yongping; ZHAO Chunhua; SHI Mingxia; LIN Quande

    2007-01-01

    Previously,we reported that a cell population derived from human fetal bone marrow fBM),termed here Flk1+CD34-postembryonic pluripotent stem cells(PPSCs)that have the characteristics of mesenchymal stem cells (MSCs),could difierentiate into ectodermal,endodermal and mesodermal celI types at the single cell level in vitro,and that these cells could also difierentiate into the epithelium of liver,lung,gut,as well as the hematopoietic and endothelial lineages after transplantion into irradiated non-obese diabetic/severe combined immunodeficient(NOD/SCID) mice.In this study,we further isolated pluripotent stem cells from human fetal heart,liver,muscle,lung,derma,kidney,and fat and then analyzed the characteristics and function of these stem cells.It was found that the phenotype of the culture-expanded pluripotent stem cells from different fetal tissues was similar to BM-derived Flk1+CD34-PPSCs.i.e.Flk1 and CD44 positive,GlyA,CD34,CD45,class I-HLA and HLA-DR negative.Morphologically,these cells were fibroblast-like and the doubling time was about 30 h.More importantly,culture-expanded pluripotent stem cells from all these fetal tissues were able to differentiate into cells with morphologic and phenotypic characteristics of adipocytes,osteocytes,neurons,gilal cells and hepatocytes.These pluripotent stem cells with characteristics similar to fetal BM-derived Flk1+CD34-PPSCs can be selected and cultured from tissues other than the BM.This phenomenon may help explain the"stem cell plasticity"found in multiple human tissues.In addition,as fetal BM-derived Flk1+CD34-PPSCs,these pluripotent stem cells from different fetal tissues had the capacity for self-renewal and multi-lineage difierentiation even after being expanded for more than 40 population doublings in vitro.Thus,they may be an ideal source of stem cells for treatment of inherited or degenerative diseases.

  20. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products.

    Science.gov (United States)

    El Ghalbzouri, Abdoelwaheb; Commandeur, Suzan; Rietveld, Marion H; Mulder, Aat A; Willemze, Rein

    2009-01-01

    Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.

  1. Binding of furosemide to albumin isolated from human fetal and adult serum.

    Science.gov (United States)

    Viani, A; Cappiello, M; Silvestri, D; Pacifici, G M

    1991-01-01

    Albumin was isolated from pooled fetal serum from 58 placentas obtained at normal delivery at term and from pooled adult plasma from 8 individuals. Albumin isolation was carried out by means of PEG precipitation followed by ion-exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The electrophoresis on SDS-polyacrylamide gels showed only one spot that comigrated with commercial human albumin. Binding to albumin was measured by equilibrium dialysis of an aliquot of albumin solution (0.7 ml) against the same volume of 0.13 M sodium orthophosphate buffer (pH 7.4). At a total concentration of 2 micrograms/ml (therapeutic range), the unbound fraction of furosemide was 2.71% (fetal albumin) and 2.51% (adult albumin). Two classes of binding sites for furosemide were observed in fetal and adult albumin. The number of binding sites (moles of furosemide per mole of albumin) was 1.22 (fetal albumin) and 1.58 (adult albumin) for the high-affinity site and 2.97 (fetal albumin) and 3.25 (adult albumin) for the low-affinity site. The association constants (M-1) were 3.1 X 10(4) (fetal albumin) and 2.6 X 10(4) (adult albumin) for the high-affinity set of sites and 0.83 X 10(4) (fetal albumin) and 1.0 X 10(4) (adult albumin) low-affinity site. The displacement of furosemide from albumin was studied with therapeutic concentrations of several drugs. Valproic acid, salicylic acid, azapropazone and tolbutamide had the highest displacing effects which were significantly higher with fetal than with adult albumin.

  2. 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.

  3. Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains.

    Directory of Open Access Journals (Sweden)

    Ching-Wen Tsai

    Full Text Available Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS entered senescence after 55-60 population doublings (PDs, and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB, and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3 to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70-75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.

  4. Enhanced Biological Behavior of In Vitro Human Gingival Fibroblasts on Cold Plasma-Treated Zirconia.

    Directory of Open Access Journals (Sweden)

    Miao Zheng

    Full Text Available To evaluate whether atmospheric-pressure dielectric-barrier-discharge plasma treatment of zirconia enhances its biocompatibility with human gingival fibroblasts.The zirconia disks were divided into four groups and treated using helium atmospheric-pressure dielectric-barrier-discharge plasmas for 30, 60 or 90 s or left untreated. The surface morphology, wettability and chemical elements were analyzed. Fibroblasts density, morphology, morphometry and attachment-related genes expression were measured at different time points from 3 to 72 h.After plasma treatment, the surface morphology and roughness remained the same, while the contact angle decreased from 78.31° to 43.71°, and the surface C/O ratio decreased from 3.17 to 0.89. The surficial areas and perimeters of HGFs were increased two-fold in the treated groups at 3 h. Fibroblasts density increased on treated disks at all time points, especially the ones treated for 60 s. Attachment-related genes in the groups treated for 30 and 60 s were significantly higher at 3 and 24 h.The helium atmospheric-pressure dielectric-barrier-discharge plasma treatment enhances the biological behavior of fibroblasts on zirconia by increasing the expression of attachment-related genes within 24 h and promoting the cell density during longer culture times. Wettability of zirconia, an important physicochemical property, has a vital influence on the cell behaviors.

  5. Protective Effect of Strawberry Extract against Inflammatory Stress Induced in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Massimiliano Gasparrini

    2017-01-01

    Full Text Available A protracted pro-inflammatory state is a major contributing factor in the development, progression and complication of the most common chronic pathologies. Fruit and vegetables represent the main sources of dietary antioxidants and their consumption can be considered an efficient tool to counteract inflammatory states. In this context an evaluation of the protective effects of strawberry extracts on inflammatory stress induced by E. coli LPS on human dermal fibroblast cells was performed in terms of viability assays, ROS and nitrite production and biomarkers of oxidative damage of the main biological macromolecules. The results demonstrated that strawberry extracts exerted an anti-inflammatory effect on LPS-treated cells, through an increase in cell viability, and the reduction of ROS and nitrite levels, and lipid, protein and DNA damage. This work showed for the first time the potential health benefits of strawberry extract against inflammatory and oxidative stress in LPS-treated human dermal fibroblast cells.

  6. Proteome oxidative carbonylation during oxidative stress-induced premature senescence of WI-38 human fibroblasts

    DEFF Research Database (Denmark)

    Le Boulch, Marine; Ahmed, Emad K; Rogowska-Wrzesinska, Adelina

    2017-01-01

    Accumulation of oxidatively damaged proteins is a hallmark of cellular and organismal ageing, and is also a phenotypic feature shared by both replicative senescence and stress-induced premature senescence of human fibroblasts. Moreover, proteins that are building up as oxidized (i.e. the "Oxi......-proteome") during ageing and age-related diseases represent a restricted set of cellular proteins, indicating that certain proteins are more prone to oxidative carbonylation and subsequent intracellular accumulation. The occurrence of specific carbonylated proteins upon oxidative stress induced premature senescence...... of WI-38 human fibroblasts and their follow-up identification have been addressed in this study. Indeed, it was expected that the identification of these proteins would give insights into the mechanisms by which oxidatively damaged proteins could affect cellular function. Among these proteins, some...

  7. MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes.

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    Damon Polioudakis

    Full Text Available miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.

  8. MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes.

    Science.gov (United States)

    Polioudakis, Damon; Abell, Nathan S; Iyer, Vishwanath R

    2015-01-01

    miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.

  9. Maternal endotoxemia, fetal anomalies, and central nervous system damage: a rat model of a human problem.

    Science.gov (United States)

    Ornoy, A; Altshuler, G

    1976-01-15

    Endotoxemia is a common consequence of the gram-negative urinary tract infections that complicate human pregnancies. Only rarely, however, have the effects of maternal endotoxemia been evaluated by animal experiments or by human investigations. Data of the Collaborative Perinatal Study suggest an association between maternal endotoxemia and fetal central nervous system damage. For these reasons we performed controlled studies of the fetal effects of treatment of pregnant rats, at appropriate gestational ages, with E. coli endotoxin. We found a maximum 7 per cent incidence of fetal anomalies in the treated animals but no anomalies in controls. Placental light microscopy examinations indicated the mechanism to include Shwartzman-lixemia produces periventricular leukomalacia. We obtained an incidence of neuronal necrosis in treated fetuses that was 10 times greater than in control fetuses. It is therefore of importance that additional studies of the pathologic effects of endotoxin be performed.

  10. Stability and biological activity evaluations of PEGylated human basic fibroblast growth factor

    OpenAIRE

    Hadadian, Shahin; Shamassebi, Dariush Norouzian; Mirzahoseini, Hasan; Shokrgozar, Mohamad Ali; Bouzari, Saeid; Sepahi, Mina

    2015-01-01

    Background: Human basic fibroblast growth factor (hBFGF) is a heparin-binding growth factor and stimulates the proliferation of a wide variety of cells and tissues causing survival properties and its stability and biological activity improvements have received much attention. Materials and Methods: In the present work, hBFGF produced by engineered Escherichia coli and purified by cation exchange and heparin affinity chromatography, was PEGylated under appropriate condition employing 10 kD pol...

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

    OpenAIRE

    Patwardhan, Juilee; Bhatt, Purvi

    2015-01-01

    Background: The exposure of skin to ultraviolet-B (UV-B) radiations leads to deoxyribonucleic acid (DNA) damage and can induce production of free radicals which imbalance the redox status of the cell and lead to increased oxidative stress. Clove has been traditionally used for its analgesic, anti-inflammatory, anti-microbial, anti-viral, and antiseptic effects. Objective: To evaluate the UV-B protective activity of flavonoids from Eugenia caryophylata (clove) buds on human dermal fibroblast c...

  12. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

    Directory of Open Access Journals (Sweden)

    Jordan R Plews

    Full Text Available BACKGROUND: Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. CONCLUSION/SIGNIFICANCE: Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  13. Gelam Honey Protects against Gamma-Irradiation Damage to Antioxidant Enzymes in Human Diploid Fibroblasts

    OpenAIRE

    Suzana Makpol; Tengku Ahbrizal Farizal Tengku Ahmad; Yasmin Anum Mohd Yusof; Nor Fadilah Rajab; Khairuddin Abdul Rahim; Zakiah Jubri

    2013-01-01

    The present study was designed to determine the radioprotective effects of Malaysian Gelam honey on gene expression and enzyme activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of human diploid fibroblasts (HDFs) subjected to gamma-irradiation. Six groups of HDFs were studied: untreated control, irradiated HDFs, Gelam honey-treated HDFs and HDF treated with Gelam honey pre-, during- and post-irradiation. HDFs were treated with 6 mg/mL of sterilized Gelam ...

  14. DNA damage in human skin fibroblasts exposed to UVA light used in clinical PUVA treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bredberg, A.

    1981-06-01

    Human skin fibroblasts were irradiated with a clinically used UVA light source. The doses (1.1 and 3 J/cm2) were similar to those reaching the dermis during clinical PUVA treatment of psoriasis. DNA strand breaks, as determined by alkaline elution, were formed in a dose-dependent way and disappeared within 1 hr of postincubation at 37 degrees C. These findings have clinical implications since UVA-induced DNA damage may be accompanied by mutagenic and tumor promoting effects.

  15. The Role of Human Adult Peripheral and Umbilical Cord Blood Platelet-Rich Plasma on Proliferation and Migration of Human Skin Fibroblasts.

    Science.gov (United States)

    Hashemi, Seyedeh-Sara; Mahmoodi, Mahdokht; Rafati, Ali Reza; Manafi, Farzad; Mehrabani, Davood

    2017-05-01

    Wound healing is a complex and dynamic process following damage in tissue structures. Due to extensive skin damage caused by burn injuries, this study determined the role of human adult peripheral and umbilical cord blood platelet-rich plasma on proliferation and migration in human skin fibroblasts. Platelet-rich plasma (5, 10, 15, 20 and 50% PRP) from human umbilical cord blood and adult peripheral blood were provided and added to fibroblasts cultured from a human skin sample. Migration and proliferation of fibroblasts were assessed in comparison to 10% FBS and by the fibroblast responses to a concentration gradient. All components of the umbilical cord blood PRP significantly stimulated the growth of fibroblasts when compared to the negative control. Fibroblast growth was enhanced in a dose dependent manner. All fibroblast cultures retained normal morphology. No significant difference was noted between umbilical cord blood and adult peripheral blood PRP preparations regarding cell proliferation and migration, but the difference to 10% FBS was significant. 1% and 50% PRP reduced cellular proliferation. The 20% umbilical cord blood PRP and 10% adult peripheral blood PRP had a significant stimulatory effect on the migration of the skin fibroblast cells in comparison with 10% FBS. As PRP could promote the migration and proliferation of dermal fibroblasts, it can be safely added in cultures when treatment of chronic wounds without triggering the immune response is needed.

  16. Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Zhongqiu Li

    2017-01-01

    Full Text Available Purpose. The functions of vascular endothelial growth factor (VEGF in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

  17. Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA.

    Science.gov (United States)

    Li, Zhongqiu; Hua, Wen; Li, Xuedong; Wang, Wei

    2017-01-01

    Purpose. The functions of vascular endothelial growth factor (VEGF) in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF) cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF) were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

  18. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

    Science.gov (United States)

    Saini, Natalie; Chan, Kin; Grimm, Sara A.; Dai, Shuangshuang; Fargo, David C.; Kaufmann, William K.; Taylor, Jack A.; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J.; Schurman, Shepherd H.; Malc, Ewa P.; Mieczkowski, Piotr A.

    2016-01-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307 PMID:27788131

  19. Resinous perforation-repair materials inhibit the growth, attachment, and proliferation of human gingival fibroblasts.

    Science.gov (United States)

    Huang, Fu-Mei; Tai, Kuo-Wei; Chou, Ming-Yung; Chang, Yu-Chao

    2002-04-01

    The choice of repair material is one of the important factors in the prognosis of the endodontically treated tooth with a perforation defect. The cytotoxicity of perforation-repair materials must be investigated to ensure a safe biological response. The aim of this in vitro study was to evaluate the effect of resin-modified, glass-ionomer cement, compomer, and resin on human-gingival fibroblasts. Human gingival fibroblasts from crown lengthening surgery were cultured by using an explant technique with the consent of the patient. Cytotoxicity was judged by using an assay of tetrazolium bromide reduction. The results showed that resin-modified, glass-ionomer cement Fuji II LC, compomer Compoglass, and resin SpectrumTPH (TPH) were cytotoxic to primary human gingival fibroblast cultures by inhibiting cell growth and proliferation. TPH alone had an effect on cell attachment. It was found that TPH was the most cytotoxic repair material among those tested in all cultures. The toxicity decreased in the order of TPH>FLC>CG.

  20. Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

    Science.gov (United States)

    Gupta, S L; Carlin, J M; Pyati, P; Dai, W; Pfefferkorn, E R; Murphy, M J

    1994-01-01

    Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts. Images PMID:8188349

  1. Treatment of postoperative lower extremity wounds using human fibroblast-derived dermis: a retrospective analysis.

    Science.gov (United States)

    Carlson, Russell M; Smith, Nicholas C; Dux, Katherine; Stuck, Rodney M

    2014-04-01

    Human fibroblast-derived dermis skin substitute is a well-studied treatment for diabetic foot ulcers; however, no case series currently exist for its use in healing postoperative wounds of the lower extremity. A retrospective analysis was conducted on 32 lower extremity postoperative wounds treated weekly with human fibroblast-derived dermis skin substitute. Postoperative wounds were defined as a wound resulting from an open partial foot amputation, surgical wound dehiscence, or nonhealing surgical wound of the lower extremity. Wound surface area was calculated at 4 and 12 weeks or until wound closure if prior to 12 weeks. Postoperative wounds treated with weekly applications showed mean improvement in surface area reduction of 63.6% at 4 weeks and 96.1% at 12 weeks. More than 56% of all wounds healed prior to the 12-week endpoint. Additionally, only one adverse event was noted in this group. This retrospective review supports the use of human fibroblast-derived dermis skin substitute in the treatment of postoperative lower extremity wounds. This advanced wound care therapy aids in decreased total healing time and increased rate of healing for not only diabetic foot wounds but also postoperative wounds of the lower extremity, as demonstrated by this retrospective review.

  2. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Natalie Saini

    2016-10-01

    Full Text Available Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

  3. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

    Science.gov (United States)

    Saini, Natalie; Roberts, Steven A; Klimczak, Leszek J; Chan, Kin; Grimm, Sara A; Dai, Shuangshuang; Fargo, David C; Boyer, Jayne C; Kaufmann, William K; Taylor, Jack A; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J; Schurman, Shepherd H; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

    2016-10-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

  4. Complex epithelial remodeling underlie the fusion event in early fetal development of the human penile urethra.

    Science.gov (United States)

    Shen, Joel; Overland, Maya; Sinclair, Adriane; Cao, Mei; Yue, Xuan; Cunha, Gerald; Baskin, Laurence

    We recently described a two-step process of urethral plate canalization and urethral fold fusion to form the human penile urethra. Canalization ("opening zipper") opens the solid urethral plate into a groove, and fusion ("closing zipper") closes the urethral groove to form the penile urethra. We hypothesize that failure of canalization and/or fusion during human urethral formation can lead to hypospadias. Herein, we use scanning electron microscopy (SEM) and analysis of transverse serial sections to better characterize development of the human fetal penile urethra as contrasted to the development of the human fetal clitoris. Eighteen 7-13 week human fetal external genitalia specimens were analyzed by SEM, and fifteen additional human fetal specimens were sectioned for histologic analysis. SEM images demonstrate canalization of the urethral/vestibular plate in the developing male and female external genitalia, respectively, followed by proximal to distal fusion of the urethral folds in males only. The fusion process during penile development occurs sequentially in multiple layers and through the interlacing of epidermal "cords". Complex epithelial organization is also noted at the site of active canalization. The demarcation between the epidermis of the shaft and the glans becomes distinct during development, and the epithelial tag at the distal tip of the penile and clitoral glans regresses as development progresses. In summary, SEM analysis of human fetal specimens supports the two-zipper hypothesis of formation of the penile urethra. The opening zipper progresses from proximal to distal along the shaft of the penis and clitoris into the glans in identical fashion in both sexes. The closing zipper mechanism is active only in males and is not a single process but rather a series of layered fusion events, uniquely different from the simple fusion of two epithelial surfaces as occurs in formation of the palate and neural tube. Copyright © 2016 International Society

  5. Complex epithelial remodeling underlie the fusion event in early fetal development of the human penile urethra

    Science.gov (United States)

    Sinclair, Adriane; Cao, Mei; Yue, Xuan; Cunha, Gerald; Baskin, Laurence

    2016-01-01

    We recently described a two-step process of urethral plate canalization and urethral fold fusion to form the human penile urethra. Canalization (“opening zipper”) opens the solid urethral plate into a groove, and fusion (“closing zipper”) closes the urethral groove to form the penile urethra. We hypothesize that failure of canalization and/or fusion during human urethral formation can lead to hypospadias. Herein, we use scanning electron microscopy (SEM) and analysis of transverse serial sections to better characterize development of the human fetal penile urethra as contrasted to the development of the human fetal clitoris. Eighteen 7-13 week human fetal external genitalia specimens were analyzed by SEM, and fifteen additional human fetal specimens were sectioned for histologic analysis. SEM images demonstrate canalization of the urethral/vestibular plate in the developing male and female external genitalia, respectively, followed by proximal to distal fusion of the urethral folds in males only. The fusion process during penile development occurs sequentially in multiple layers and through the interlacing of epidermal “cords”. Complex epithelial organization is also noted at the site of active canalization. The demarcation between the epidermis of the shaft and the glans becomes distinct during development, and the epithelial tag at the distal tip of the penile and clitoral glans regresses as development progresses. In summary, SEM analysis of human fetal specimens supports the two-zipper hypothesis of formation of the penile urethra. The opening zipper progresses from proximal to distal along the shaft of the penis and clitoris into the glans in identical fashion in both sexes. The closing zipper mechanism is active only in males and is not a single process but rather a series of layered fusion events, uniquely different from the simple fusion of two epithelial surfaces as occurs in formation of the palate and neural tube. PMID:27397682

  6. Expression of human acidic fibroblast growth factor in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    YU Ying; CAI Shaoxi; Harald G. WERIRICH; XIA Yuxian

    2003-01-01

    Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoriS KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mg.L-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 1 7 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.

  7. Expression and function of connexin 43 in human gingival wound healing and fibroblasts.

    Directory of Open Access Journals (Sweden)

    Rana Tarzemany

    Full Text Available Connexins (C×s are a family of transmembrane proteins that form hemichannels and gap junctions (GJs on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of

  8. In vitro exposure of human fibroblasts to local anaesthetics impairs cell growth

    Science.gov (United States)

    Fedder, C; Beck-Schimmer, B; Aguirre, J; Hasler, M; Roth-Z'graggen, B; Urner, M; Kalberer, S; Schlicker, A; Votta-Velis, G; Bonvini, J M; Graetz, K; Borgeat, A

    2010-01-01

    Lidocaine, bupivacaine or ropivacaine are used routinely to manage perioperative pain. Sparse data exist evaluating the effects of local anaesthetics (LA) on fibroblasts, which are involved actively in wound healing. Therefore, we investigated the effects of the three LA to assess the survival, viability and proliferation rate of fibroblasts. Human fibroblasts were exposed to 0·3 mg/ml and 0·6 mg/ml of each LA for 2 days, followed by incubation with normal medium for another 1, 4 or 7 days (group 1). Alternatively, cells were incubated permanently with LA for 3, 6 or 9 days (group 2). Live cell count was assessed using trypan blue staining. Viability was measured by the tetrazolium bromide assay. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine assay. Production of reactive oxygen species (ROS) was determined, measuring the oxidation of non-fluorescent-2,7′-dichlorofluorescin. Treatment of cells with the three LA showed a concentration-dependent decrease of live cells, mitochondrial activity and proliferation rate. Group arrangement played a significant role for cell count and proliferation, while exposure time influenced viability. Among the analysed LA, bupivacaine showed the most severe cytotoxic effects. Increased production of ROS correlated with decreased viability of fibroblasts in lidocaine- and bupivacaine-exposed cells, but not upon stimulation with ropivacaine. This study shows a concentration-dependent cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts in vitro, with more pronounced effects after continuous incubation. A possible mechanism of cell impairment could be triggered by production of ROS upon stimulation with lidocaine and bupivacaine. PMID:20819090

  9. HEMA but not TEGDMA induces autophagy in human gingival fibroblasts

    Science.gov (United States)

    Teti, Gabriella; Orsini, Giovanna; Salvatore, Viviana; Focaroli, Stefano; Mazzotti, Maria C.; Ruggeri, Alessandra; Mattioli-Belmonte, Monica; Falconi, Mirella

    2015-01-01

    Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3 mmol/L of HEMA or 3 mmol/L of TEGDMA for 24, 48, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase – 3 and PARP) and autophagy (beclin – 1 and LC3B I/II) were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis. PMID:26483703

  10. HEMA but not TEGDMA Induces Autophagy in Human Gingival Fibroblasts.

    Directory of Open Access Journals (Sweden)

    gabriella eteti

    2015-10-01

    Full Text Available Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA and triethylene glycol dimethacrylate (TEGDMA to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3mmol/L of HEMA or 3mmol/L of TEGDMA for 24 h, 48h, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase – 3 and PARP and autophagy (beclin – 1 and LC3B I/II were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis.

  11. Effects of conditioned medium from LL-37 treated adipose stem cells on human fibroblast migration.

    Science.gov (United States)

    Yang, Eun-Jung; Bang, Sa-Ik

    2017-07-01

    Adipose stem cell-conditioned medium may promote human dermal fibroblast (HDF) proliferation and migration by activating paracrine peptides during the re-epithelization phase of wound healing. Human antimicrobial peptide LL-37 is upregulated in the skin epithelium as part of the normal response to injury. The effects of conditioned medium (CM) from LL-37 treated adipose stem cells (ASCs) on cutaneous wound healing, including the mediation of fibroblast migration, remain to be elucidated, therefore the aim of the present study was to determine how ASCs would react to an LL-37-rich microenvironment and if CM from LL-37 treated ASCs may influence the migration of HDFs. The present study conducted migration assays with HDFs treated with CM from LL-37 treated ASCs. Expression of CXC chemokine receptor 4 (CXCR4), which controls the recruitment of HDFs, was analyzed at the mRNA and protein levels. To further characterize the stimulatory effects of LL-37 on ASCs, the expression of stromal cell-derived factor-1α (SDF-1α), a CXC chemokine, was investigated. CM from LL-37-treated ASCs induced migration of HDFs in a time- and dose-dependent manner, with a maximum difference in migration observed 24 h following stimulation with LL-37 at a concentration of 10 µg/ml. The HDF migration and the expression of CXCR4 in fibroblasts was markedly increased upon treatment with CM from LL-37-treated ASCs compared with CM from untreated ASCs. SDF-1α expression was markedly increased in CM from LL-37 treated ASCs. It was additionally observed that SDF-1α blockade significantly reduced HDF migration. These findings suggest the feasibility of CM from LL-37-treated ASCs as a potential therapeutic for human dermal fibroblast migration.

  12. Preventive effects of tamarind seed coat extract on UVA-induced alterations in human skin fibroblasts.

    Science.gov (United States)

    Phetdee, Khemjira; Rakchai, Racharat; Rattanamanee, Kwanchai; Teaktong, Thanasak; Viyoch, Jarupa

    2014-01-01

    One of the most damaging actions on skin is from solar radiation, particularly from its ultraviolet (UV) component, through the formation of oxidative species. Thus, an antioxidant strategy that prevents the formation of these oxidants could form the basis of an efficacious cutaneous protectant. Many herbal materials contain antioxidant polyphenols, and this study assessed the possibility that tamarind seed coat extract could fulfill this role. An alcoholic extract of the tamarind (Tamarindus indica L.) seed coat showed stronger antioxidant activity (2,2-diphenyl-1-picrylhydrazyl inhibition, EC(50) = 12.9 μg/ml) than L-ascorbic acid (EC(50) = 22.9 μg/ml) and α-tocopherol (EC(50) = 29.3 μg/ml). In cultured fibroblasts taken from human skin, hydrogen peroxide (100-1000 μM) damaged 62-92% of the cells compared to only 35-47% when the cells were preincubated in extract (200 μg/ml) for 24 h. UVA (40 J/cm2) irradiation of human fibroblasts damaged 25% of the cells but the death rate was reduced to 10% with extract. UV irradiation increased the proportion of cells arrest in G(0)/G(1) phase (from 59% to 78%) but this was largely prevented by the extract (64%), according to flow cytometry. Intracellular total glutathione of UVA-irradiated cells pretreated with the extract increased to 10-25% compared to the non-pretreated group at 24-72 h after irradiation. Fibroblasts typically increased matrix metalloproteinase-1 secretion after photodamage, and this is prevented by the extract. This is the first report showing that tamarind seed coat extract is an antioxidant and can protect human skin fibroblasts from cellular damage produced by UVA and thus may form the foundation for an antiaging cosmetic.

  13. Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation.

    Science.gov (United States)

    Tahir, Ammar; Bileck, Andrea; Muqaku, Besnik; Niederstaetter, Laura; Kreutz, Dominique; Mayer, Rupert L; Wolrab, Denise; Meier, Samuel M; Slany, Astrid; Gerner, Christopher

    2017-02-07

    During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

  14. Diagnosis of the human fetal age based on the development of the normal kidney

    Directory of Open Access Journals (Sweden)

    Lizardo-Daudt Helena Maria

    2002-01-01

    Full Text Available Background and aims: The diagnosis of human fetal age is usually estimated based on the measurement of crown-rump length or crown-heel length and the weight of the fetus. However, this estimate is not totally accurate and sometimes is necessary to combine other data to determine the fetal age. An analysis of the normal embryological development of the kidney may assist in this determination. The histology of this process, although well described, lacks photographic documentation. We intend to fill this gap by providing histologists and pathologists, especially inexperienced ones, with information about the staging of the renal development through microphotography. The objective of the present study was to achieve greater accuracy for the diagnosis of human fetal age through the proposed classification and the photographic documentation presented. Material and methods: Normal embryological development of the human kidney was studied by light microscopy. The fetal period from 6 to 40 weeks of gestation was observed according the stage of maturity of glomeruli and tubules; localization of glomeruli, occurrence of nephrogenic tissue and cortico-medullary differentiation. At least 5 different exams were observed from each week of development. Two hundred four exams were analyzed in the whole study. The histological characteristics were quantified and the process was documented by microphotography. Results and final considerations: The fetal development of the kidney was divided into 8 stages, which was documented through microphotography. Nephron structural formation occurred until the 34th week of prenatal development. From the 35th week on, tubules and glomeruli continued to mature without the formation of new nephrons. The proposed classification intends to improve the accuracy of the fetal age diagnosis.

  15. Blood flow in the human fetal descending aorta : a pulsed Doppler study

    NARCIS (Netherlands)

    L. Pijpers (Leendert)

    1985-01-01

    textabstractIn 1628 William Harvey introduced his concept ofthe human circulation. Although a lot of studies concerning the fetal circulation were done before it was not until the 1930s that Barcroft (1934, 1939) and associates performed radiograpbic studies on the feta! goal and lamb to establish t

  16. Towards a New Study on Associative Learning in Human Fetuses: Fetal Associative Learning in Primates

    Science.gov (United States)

    Kawai, Nobuyuki

    2010-01-01

    Research has revealed that fetuses can learn from events in their environment. The most convincing evidence for fetal learning is habituation to vibroacoustic stimulation (VAS) in human fetuses and classical conditioning in rat fetuses. However, these two research areas have been independent of each other. There have been few attempts at classical…

  17. Reduced cell number in the neocortical part of the human fetal brain in Down syndrome

    DEFF Research Database (Denmark)

    Larsen, K.B.; Laursen, H.; Graem, N.

    2008-01-01

    Mental retardation is seen in all individuals with Down syndrome (DS) and different brain abnormalities are reported. The aim of this study was to investigate if mental retardation at least in part is a result of a lower cell number in the neocortical part of the human fetal forebrain. We therefore...

  18. Dissecting human cerebral organoids and fetal neocortex using single-cell RNAseq

    Science.gov (United States)

    Treutlein, Barbara

    Cerebral organoids - three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells - have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

  19. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

    NARCIS (Netherlands)

    Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

    2013-01-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three h

  20. Resveratrol inhibits steroidogenesis in human fetal adrenocortical cells at the end of first trimester

    DEFF Research Database (Denmark)

    Savchuk, Iuliia; Morvan, Marie-Line; Søeborg, Tue

    2017-01-01

    steroidogenesis at gestational weeks (GW) 9-12. METHODS AND RESULTS: Adrenals from aborted fetuses (GW10-12) were used to prepare primary cultures of human fetal adrenocortical cells (HFAC). HFAC were treated in the presence or absence of ACTH (10 ng/ml) with or without resveratrol (10 μM) for 24 hours...

  1. Towards a New Study on Associative Learning in Human Fetuses: Fetal Associative Learning in Primates

    Science.gov (United States)

    Kawai, Nobuyuki

    2010-01-01

    Research has revealed that fetuses can learn from events in their environment. The most convincing evidence for fetal learning is habituation to vibroacoustic stimulation (VAS) in human fetuses and classical conditioning in rat fetuses. However, these two research areas have been independent of each other. There have been few attempts at classical…

  2. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.

    Science.gov (United States)

    Camp, J Gray; Badsha, Farhath; Florio, Marta; Kanton, Sabina; Gerber, Tobias; Wilsch-Bräuninger, Michaela; Lewitus, Eric; Sykes, Alex; Hevers, Wulf; Lancaster, Madeline; Knoblich, Juergen A; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B; Treutlein, Barbara

    2015-12-22

    Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

  3. Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations.

    Science.gov (United States)

    Fowler, Paul A; Abramovich, David R; Haites, Neva E; Cash, Phillip; Groome, Nigel P; Al-Qahtani, Ahmed; Murray, Tessa J; Lea, Richard G

    2007-11-01

    Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis. Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10-1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated. Exposure to dieldrin reduced LH-induced testosterone secretion (P Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals. Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.

  4. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

    Science.gov (United States)

    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  5. Production of transgenic embryos through nuclear transfer using ovine fetal fibroblasts transferred with foreign genes%体细胞核移植生产绵羊转hALR基因囊胚

    Institute of Scientific and Technical Information of China (English)

    马玉珍; 任宇; 周雪原; 刘东军; 旭日干

    2011-01-01

    扩增人肝细胞再生增强因子(human augmenter of liver regeneration,ALR)基因,利用质粒pIRES2-EGFP 构建新霉素(Neo)、增强绿色荧光蛋白(enhanced green fluorescence protein,EGFP)双标记基因且EGFP和ALR基因为双顺反子的真核表达载体.LipofectAMINETM介导其转染体外培养的绵羊胎儿成纤维细胞(sheep fetal fibroblast cells,sFFCs);经G418筛选转基因细胞;激光共聚焦显微镜挑选绿色荧光单克隆细胞.PCR、RT-PCR和免疫组织化学方法进一步检测ALR基因及其表达;稳定表达外源基因的sFFCs作供体,移入去核的绵羊卵母细胞中,进行体细胞核移植.通过激光共聚焦显微镜和ALR抗体检测EGFP、ALR基因在胚胎水平上的表达,其结果表明:由IRES连接的EGFP和ALR基因可在绵羊胎儿成纤维细胞内同时表达,由此细胞核移植产生的转基因胚胎在发育的各阶段均可见绿色荧光;囊胚中所有细胞表达EGFP基因;发绿色荧光的胚胎中ALR基因同时存在.因此,由IRES连接标记基因和目的基因,以标记基因指示目的基因的表达,可简化检测目的基因的繁琐手段;用筛选的转基因早期胚胎进行移植,可提高制备转基因动物的效率.%Human ALR gene sequence was amplified by PCR from human total DNA and inserted into PIRES2-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/AZJ? By the induction of lipofectAMINE?. The positive cell clones were selected with medium containing G418 (800 ug/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and

  6. Knockdown of CDK2AP1 in primary human fibroblasts induces p53 dependent senescence.

    Directory of Open Access Journals (Sweden)

    Khaled N Alsayegh

    Full Text Available Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1 is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1 in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a no increase in senescence associated beta-galactosidase activity, (b decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1

  7. Developmental origins of health and disease: experimental and human evidence of fetal programming for metabolic syndrome.

    Science.gov (United States)

    de Gusmão Correia, M L; Volpato, A M; Águila, M B; Mandarim-de-Lacerda, C A

    2012-07-01

    The concept of developmental origins of health and disease has been defined as the process through which the environment encountered before birth, or in infancy, shapes the long-term control of tissue physiology and homeostasis. The evidence for programming derives from a large number of experimental and epidemiological observations. Several nutritional interventions during diverse phases of pregnancy and lactation in rodents are associated with fetal and neonatal programming for metabolic syndrome. In this paper, recent experimental models and human epidemiological studies providing evidence for the fetal programming associated with the development of metabolic syndrome and related diseases are revisited.

  8. Induced pluripotent stem (iPS) cells from human fetal stem cells.

    Science.gov (United States)

    Guillot, Pascale V

    2016-02-01

    Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications.

  9. Proteolytic processing of anti-Müllerian hormone differs between human fetal testes and adult ovaries

    DEFF Research Database (Denmark)

    Mamsen, Linn; Petersen, TS; Jeppesen, JV

    2015-01-01

    and specificity of a panel of five novel high-affinity AMH monoclonal antibodies. Two recognize the mature C-terminal form of AMH, whereas three recognize the active pro-mature form of AMH in human tissue. The antibodies were tested on fetal male testicular and mesonephric tissue aged 8-19 weeks post conception...... of AMH was hardly detected in Sertoli cells, but was readily detected in GCs. This particular form was also located to the nucleus in GCs, whereas the other investigated AMH forms remained in the cytoplasm. Interestingly, the distribution of the AMH forms in the fetal serum of boys showed...

  10. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  11. Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Sivapragasam Gothai

    2016-03-01

    Full Text Available Background/Aim: Wounds are the outcome of injuries to the skin that interrupt the soft tissue. Healing of a wound is a complex and long-drawn-out process of tissue repair and remodeling in response to injury. A large number of plants are used by folklore traditions for treatment of cuts, wounds and burns. Moringa oleifera is an herb used as traditional folk medicine for the treatment of various skin wounds and associated diseases. The underlying mechanisms of wound healing activity of ethyl acetate fraction of M. oleifera leaves extract are completely unknown. Methods: In the current study, ethyl acetate fraction of Moringa oleifera leaves was investigated for its efficacy on cell viability, proliferation and migration (wound closure rate in human normal dermal fibroblast cells. Results: Results revealed that lower concentration (12.5 and micro;g/ml, 25 and micro;g/ml, and 50 and micro;g/ml of ethyl acetate fraction of M. oleifera leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. Conclusion: The present study suggested that ethyl acetate fraction of M. oleifera leaves might be a potential therapeutic agent for skin wound healing by promoting fibroblast proliferation and migration through increasing the wound closure rate corroborating its traditional use. [J Intercult Ethnopharmacol 2016; 5(1.000: 1-6

  12. IFN-Dependent and -Independent Reduction in West Nile Virus Infectivity in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Lisa I. Hoover

    2014-03-01

    Full Text Available Although dermal fibroblasts are one of the first cell types exposed to West Nile virus (WNV during a blood meal by an infected mosquito, little is known about WNV replication within this cell type. Here, we demonstrate that neuroinvasive, WNV-New York (WNV-NY, and nonneuroinvasive, WNV-Australia (WNV-AUS60 strains are able to infect and replicate in primary human dermal fibroblasts (HDFs. However, WNV-AUS60 replication and spread within HDFs was reduced compared to that of WNV-NY due to an interferon (IFN-independent reduction in viral infectivity early in infection. Additionally, replication of both strains was constrained late in infection by an IFN-β-dependent reduction in particle infectivity. Overall, our data indicates that human dermal fibroblasts are capable of supporting WNV replication; however, the low infectivity of particles produced from HDFs late in infection suggests that this cell type likely plays a limited role as a viral reservoir in vivo.

  13. Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts

    Science.gov (United States)

    Huh, Christine J; Zhang, Bo; Victor, Matheus B; Dahiya, Sonika; Batista, Luis FZ; Horvath, Steve; Yoo, Andrew S

    2016-01-01

    Aging is a major risk factor in many forms of late-onset neurodegenerative disorders. The ability to recapitulate age-related characteristics of human neurons in culture will offer unprecedented opportunities to study the biological processes underlying neuronal aging. Here, we show that using a recently demonstrated microRNA-based cellular reprogramming approach, human fibroblasts from postnatal to near centenarian donors can be efficiently converted into neurons that maintain multiple age-associated signatures. Application of an epigenetic biomarker of aging (referred to as epigenetic clock) to DNA methylation data revealed that the epigenetic ages of fibroblasts were highly correlated with corresponding age estimates of reprogrammed neurons. Transcriptome and microRNA profiles reveal genes differentially expressed between young and old neurons. Further analyses of oxidative stress, DNA damage and telomere length exhibit the retention of age-associated cellular properties in converted neurons from corresponding fibroblasts. Our results collectively demonstrate the maintenance of age after neuronal conversion. DOI: http://dx.doi.org/10.7554/eLife.18648.001 PMID:27644593

  14. Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    Science.gov (United States)

    Sklirou, Aimilia D.; Ralli, Marianna; Dominguez, Maria; Papassideri, Issidora; Skaltsounis, Alexios-Leandros; Trougakos, Ioannis P.

    2015-01-01

    Despite the fact that several natural products (e.g. crude extracts or purified compounds) have been found to activate cell antioxidant responses and/or delay cellular senescence the effect(s) of small peptides on cell viability and/or modulation of protective mechanisms (e.g. the proteostasis network) remain largely elusive. We have thus studied a hexapeptide (Hexapeptide-11) of structure Phe–Val–Ala–Pro–Phe–Pro (FVAPFP) originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity. We show herein that Hexapeptide-11 exhibits no significant toxicity in normal human diploid lung or skin fibroblasts. Exposure of fibroblasts to Hexapeptide-11 promoted dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. Moreover, it promoted increased nuclear accumulation of Nrf2; higher expression levels of proteasomal protein subunits and increased proteasome peptidase activities. In line with these findings we noted that Hexapeptide-11 conferred significant protection in fibroblasts against oxidative-stress-mediated premature cellular senescence, while at in vivo skin deformation assays in human subjects it improved skin elasticity. Finally, Hexapeptide-11 was found to induce the activity of extracellular MMPs and it also suppressed cell migration. Our presented findings indicate that Hexapeptide-11 is a promising anti-ageing agent. PMID:25974626

  15. Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    Directory of Open Access Journals (Sweden)

    Aimilia D. Sklirou

    2015-08-01

    Full Text Available Despite the fact that several natural products (e.g. crude extracts or purified compounds have been found to activate cell antioxidant responses and/or delay cellular senescence the effect(s of small peptides on cell viability and/or modulation of protective mechanisms (e.g. the proteostasis network remain largely elusive. We have thus studied a hexapeptide (Hexapeptide-11 of structure Phe–Val–Ala–Pro–Phe–Pro (FVAPFP originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity. We show herein that Hexapeptide-11 exhibits no significant toxicity in normal human diploid lung or skin fibroblasts. Exposure of fibroblasts to Hexapeptide-11 promoted dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. Moreover, it promoted increased nuclear accumulation of Nrf2; higher expression levels of proteasomal protein subunits and increased proteasome peptidase activities. In line with these findings we noted that Hexapeptide-11 conferred significant protection in fibroblasts against oxidative-stress-mediated premature cellular senescence, while at in vivo skin deformation assays in human subjects it improved skin elasticity. Finally, Hexapeptide-11 was found to induce the activity of extracellular MMPs and it also suppressed cell migration. Our presented findings indicate that Hexapeptide-11 is a promising anti-ageing agent.

  16. Effects of Panax ginseng extract on human dermal fibroblast proliferation and collagen synthesis.

    Science.gov (United States)

    Lee, Geum-Young; Park, Kang-Gyun; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2016-03-01

    Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  17. 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

  18. 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.

  19. 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.

  20. In vitro toxicity of grey MTA in comparison to white MTA on human periodontal ligament fibroblasts.

    Science.gov (United States)

    Al-Haj Ali, S N; Al-Jundi, S H; Ditto, D J

    2014-12-01

    This was to define and compare the in vitro toxicity of grey MTA with that of white MTA on cultured human periodontal ligament (PDL) fibroblasts. PDL cells were obtained from sound first permanent molars and cultured in Dulbecco's Modified Eagle's Medium. Cultures were subjected to different concentrations of grey and white MTA (0.5, 5, 50 and 500 µg/ml) for 24 h at 37 °C. Cells that were not exposed to grey or white MTA served as the negative control. In vitro toxicity was assessed using MTT assay. The results were compared using ANOVA and Tukey statistical tests (p MTA presented higher in vitro toxicity than grey MTA. However, the differences were almost insignificant (p > 0.05). Both colours of MTA are biocompatible since they were both able to preserve PDL fibroblasts for up to 24 h. MTA is as a promising alternative in pulpotomy of primary teeth.

  1. Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts.

    Science.gov (United States)

    Chiu, Po Yee; Lam, Philip Y; Yan, Chung Wai; Ko, Kam Ming

    2011-06-01

    The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging.

  2. Recent advances in the prenatal interrogation of the human fetal genome.

    Science.gov (United States)

    Hui, Lisa; Bianchi, Diana W

    2013-02-01

    The amount of genetic and genomic information obtainable from the human fetus during pregnancy is accelerating at an unprecedented rate. Two themes have dominated recent technological advances in prenatal diagnosis: interrogation of the fetal genome in increasingly high resolution and the development of non-invasive methods of fetal testing using cell-free DNA in maternal plasma. These two areas of advancement have now converged with several recent reports of non-invasive assessment of the entire fetal genome from maternal blood. However, technological progress is outpacing the ability of the healthcare providers and patients to incorporate these new tests into existing clinical care, and further complicates many of the economic and ethical dilemmas in prenatal diagnosis. This review summarizes recent work in this field and discusses the integration of these new technologies into the clinic and society.

  3. GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation.

    Science.gov (United States)

    Larijani, Bagher; Aghayan, Hamid-Reza; Goodarzi, Parisa; Arjmand, Babak

    2015-01-01

    Stem cell therapy seems a promising avenue in regenerative medicine. Within various stem cells, mesenchymal stem cells have progressively used for cellular therapy. Because of the age-related decreasing in the frequency and differentiating capacity of adult MSCs, fetal tissues such as fetal liver, lung, pancreas, spleen, etc. have been introduced as an alternative source of MSCs for cellular therapy. On the other hand, using stem cells as advanced therapy medicinal products, must be performed in compliance with cGMP as a quality assurance system to ensure the safety, quality, and identity of cell products during translation from the basic stem cell sciences into clinical cell transplantation. In this chapter the authors have demonstrated the manufacturing of GMP-grade human fetal liver-derived mesenchymal stem cells.

  4. The Role of Placental Homeobox Genes in Human Fetal Growth Restriction

    Directory of Open Access Journals (Sweden)

    Padma Murthi

    2011-01-01

    Full Text Available Fetal growth restriction (FGR is an adverse pregnancy outcome associated with significant perinatal and paediatric morbidity and mortality, and an increased risk of chronic disease later in adult life. One of the key causes of adverse pregnancy outcome is fetal growth restriction (FGR. While a number of maternal, fetal, and environmental factors are known causes of FGR, the majority of FGR cases remain idiopathic. These idiopathic FGR pregnancies are frequently associated with placental insufficiency, possibly as a result of placental maldevelopment. Understanding the molecular mechanisms of abnormal placental development in idiopathic FGR is, therefore, of increasing importance. Here, we review our understanding of transcriptional control of normal placental development and abnormal placental development associated with human idiopathic FGR. We also assess the potential for understanding transcriptional control as a means for revealing new molecular targets for the detection, diagnosis, and clinical management of idiopathic FGR.

  5. [Morphogenesis of Human Fetal Thymus during Weeks 22-27 of Development].

    Science.gov (United States)

    Kulida, L V; Peretyatko, L P; Nazarov, S B

    2015-01-01

    Distinctive features of human fetal thymus morphogenesis in early ontogeny in the case of uncomplicated pregnancy have been characterized. A steady increase of thymus dimensions and weight occurred concomitantly to differentiation of morphofunctional zones within the organ. Cell differentiation in the subcapsular and inner cortical zones of the thymus lobes was manifested as changes in parameters of expression of T-lymphocyte antigens CD1, CD2, and CD3 and ultrastructural features of reticuloepithelial cells (REC) type I and II forming a microenvironment for lymphocytes. RECs of the medullar zone formed a glomerular syncytium with desmosomal interepithelial contacts by week 22 of fetal development. Small lymphocytes predominated among thymocytes (66%). Hassall's corpuscles, the structural correlates of morphological and functional maturity, predominated in the fetal thymuses during developmental weeks 25-27.

  6. Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts.

    Science.gov (United States)

    Opländer, Christian; Wetzel, Wiebke; Cortese, Miriam M; Pallua, Norbert; Suschek, Christoph V

    2008-05-01

    Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

  7. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  8. Discovery and Characterization of piRNAs in the Human Fetal Ovary

    Directory of Open Access Journals (Sweden)

    Zev Williams

    2015-10-01

    Full Text Available Piwi-interacting RNAs (piRNAs, a class of 26- to 32-nt non-coding RNAs (ncRNAs, function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis—developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.

  9. Expression and activation of caspase-6 in human fetal and adult tissues.

    Directory of Open Access Journals (Sweden)

    Nelly Godefroy

    Full Text Available Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.

  10. Anti-inflammatory effects of budesonide in human lung fibroblasts are independent of histone deacetylase 2

    Directory of Open Access Journals (Sweden)

    Wang X

    2013-08-01

    Full Text Available Xingqi Wang,1 Amy Nelson,1 Zachary M Weiler,1 Amol Patil,1 Tadashi Sato,1 Nobuhiro Kanaji,1 Masanori Nakanishi,1 Joel Michalski,1 Maha Farid,1 Hesham Basma,1 Tricia D LeVan,1 Anna Miller-Larsson,2 Elisabet Wieslander,2 Kai-Christian Muller,3 Olaf Holz,3 Helgo Magnussen,3 Klaus F Rabe,3 Xiangde Liu,1 Stephen I Rennard1 1Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA; 2AstraZeneca R&D Molndal, Molndal, Sweden; 3Hospital Grosshansdorf, Center for Pneumology and Thoracic Surgery, Grosshansdorf, Germany Objective and design: Reduced expression of histone deacetylase 2 (HDAC2 in alveolar macrophages and epithelial cells may account for reduced response of chronic obstructive pulmonary disease (COPD patients to glucocorticoids. HDAC2 expression and its role in mediating glucocorticoid effects on fibroblast functions, however, has not been fully studied. This study was designed to investigate whether HDAC2 mediates glucocorticoid effects on release of inflammatory cytokines and matrix metalloproteinases (MMPs from human lung fibroblasts. Methods: Human lung fibroblasts (HFL-1 cells were stimulated with interleukin (IL-1β plus tumor necrosis factor (TNF-α in the presence or absence of the glucocorticoid budesonide. Cytokines (IL-6 and IL-8 were quantified by enzyme linked immunosorbent assay (ELISA and MMPs (MMP-1 and MMP-3 by immunoblotting in culture medium. The role of HDAC2 was investigated using a pharmacologic inhibitor as well as a small interfering ribonucleic acid (siRNA targeting HDAC2. Results: We have demonstrated that budesonide concentration-dependently (10-10–10-7 M inhibited IL-6, IL-8, MMP-1, and MMP-3 release by HFL-1 cells in response to IL-1β plus TNF-a. While an HDAC inhibitor significantly blocked the inhibitory effect of budesonide on human bronchial epithelial cells (HBECs and monocytes (THP-1 cells, it did not block the inhibitory

  11. Insights in spatio-temporal characterization of human fetal neural stem cells.

    Science.gov (United States)

    Martín-Ibáñez, Raquel; Guardia, Inés; Pardo, Mónica; Herranz, Cristina; Zietlow, Rike; Vinh, Ngoc-Nga; Rosser, Anne; Canals, Josep M

    2017-05-01

    Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4-5, 6-7 and 8-9weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3-4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were

  12. Cloning, Expression and Functional Characterization of In-House Prepared Human Basic Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    Hassan Rassouli

    2013-01-01

    Full Text Available Objective: Human basic fibroblast growth factor (bFGF plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs.Materials and Methods: In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability.Results: This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo.Conclusion: The results show no significant difference in function between our in-house produced and commercialized bFGF.

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

    Directory of Open Access Journals (Sweden)

    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

  14. Gene-specific mitochondria dysfunctions in human TARDBP and C9ORF72 fibroblasts.

    Science.gov (United States)

    Onesto, Elisa; Colombrita, Claudia; Gumina, Valentina; Borghi, Maria Orietta; Dusi, Sabrina; Doretti, Alberto; Fagiolari, Gigliola; Invernizzi, Federica; Moggio, Maurizio; Tiranti, Valeria; Silani, Vincenzo; Ratti, Antonia

    2016-05-05

    Dysregulation of RNA metabolism represents an important pathogenetic mechanism in both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) due to the involvement of the DNA/RNA-binding proteins TDP-43 and FUS and, more recently, of C9ORF72. A potential link between dysregulation of RNA metabolism and mitochondrial dysfunction is recently emerged in TDP-43 disease models. To further investigate the possible relationship between these two pathogenetic mechanisms in ALS/FTD, we studied mitochondria functionality in human mutant TARDBP(p.A382T) and C9ORF72 fibroblasts grown in galactose medium to induce a switch from a glycolytic to an oxidative metabolism. In this condition we observed significant changes in mitochondria morphology and ultrastructure in both mutant cells with a fragmented mitochondria network particularly evident in TARDBP(p.A382T) fibroblasts. From analysis of the mitochondrial functionality, a decrease of mitochondria membrane potential with no alterations in oxygen consumption rate emerged in TARDBP fibroblasts. Conversely, an increased oxygen consumption and mitochondria hyperpolarization were observed in C9ORF72 fibroblasts in association to increased ROS and ATP content. We found evidence of autophagy/mitophagy in dynamic equilibrium with the biogenesis of novel mitochondria, particularly in mutant C9ORF72 fibroblasts where an increase of mitochondrial DNA content and mass, and of PGC1-α protein was observed. Our imaging and biochemical data show that wild-type and mutant TDP-43 proteins do not localize at mitochondria so that the molecular mechanisms responsible for such mitochondria impairment remain to be further elucidated. For the first time our findings assess a link between C9ORF72 and mitochondria dysfunction and indicate that mitochondria functionality is affected in TARDBP and C9ORF72 fibroblasts with gene-specific features in oxidative conditions. As in neuronal metabolism mitochondria are actively used for ATP

  15. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Buskermolen, Jeroen K; Reijnders, Christianne M A; Spiekstra, Sander W; Steinberg, Thorsten; Kleverlaan, Cornelis J; Feilzer, Albert J; Bakker, Astrid D; Gibbs, Susan

    2016-08-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  16. CYCLOSPORIN A AFFECTS THE PROLIFERATION PROCESS IN NORMAL HUMAN DERMAL FIBROBLASTS.

    Science.gov (United States)

    Janikowska, Grazyna; Janikowsk, Tomasz; Pyka, Alina; Wilczok, Adam; Mazurek, Urszula

    2016-01-01

    Cyclosporin A is an immunosuppressant drug that is used not only in solid transplant rejection, but also in moderate and severe forms of psoriasis, pyoderma, lupus or arthritis. Serious side effects of the drug such as skin cancer or gingival hyperplasia probably start with the latent proliferation process. Little is known about the influence of cyclosporin A on molecular signaling in epidermal tissue. Thus, the aim of this study was to estimate the influence of cyclosporin A on the process of proliferation in normal human dermal fibroblasts. Fibroblasts were cultured in a liquid growth medium in standard conditions. Cyclosporin A was added to the culture after the confluence state. Survival and proliferation tests on human dermal fibroblast cells were performed. Total RNA was extracted from fibroblasts, based on which cDNA and cRNA were synthesized. The obtained cRNA was hybridized with the expression microarray HGU-133A_2.0. Statistical analysis of 2734 mRNAs was performed by the use of GeneSpring 13.0 software and only results with p cyclosporin A) was performed to lower the number of statistically significant results from 679 to 66, and less. Between statistically and biologically significant mRNAs down-regulated were EGRJ, BUBIB, MKI67, CDK1, TTK, E2F8, TPX2, however, the INSIG1, FOSL1, HMOX1 were up-regulated. The experiment data revealed that cyclosporin A up-regulated FOSL1 in the first 24 h, afterwards down-regulating its expression. The HMOX1 gene was up-regulated in the first stage of the experiment (CsA 8 h), however, after the next 16 h of culture time its expression was down-regulated (CsA 24 h), to finally increased in the later time period. The results indicate that cyclosporin A had a significant effect on proliferation in normal human dermal fibroblasts through the changes in the expression of genes related to the cell cycle and transcription regulation process.

  17. 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....

  18. Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect.

    Science.gov (United States)

    Kovářová, Nikola; Pecina, Petr; Nůsková, Hana; Vrbacký, Marek; Zeviani, Massimo; Mráček, Tomáš; Viscomi, Carlo; Houštěk, Josef

    2016-06-01

    This paper describes data related to a research article entitled "Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects" [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1 (-/-) ) and control (SURF1 (+/+) ) mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX), to reversible inhibition of mitochondrial translation in SURF1 (-/-) mouse and SURF1 patient fibroblast cell lines.

  19. Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

    Science.gov (United States)

    Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

    2013-01-01

    Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID

  20. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yulianti

    2015-08-01

    Full Text Available Asiatiocoside, a saponin component isolated from Centella asiatica can improve wound healing by promoting the proliferation of human dermal fibroblasts (HDF and synthesis of collagen. The skin-renewing cells and type I and III collagen synthesis decrease with aging, resulting in the reduction of skin elasticity and delayed wound healing. Usage of natural active compounds from plants in wound healing should be evaluated and compared to retinoic acid as an active agent that regulates wound healing. The aim of this study was to compare and evaluate the effect of asiaticoside and retinoic acid to induce greater cell proliferation and type I and III collagen synthesis in human dermal fibroblast. Methods Laboratory experiments were conducted using human dermal fibroblasts (HDF isolated from human foreskin explants. Seven passages of HDF were treated with asiaticoside and retinoic acid at several doses and incubated for 24 and 48 hours. Cell viability in all groups was tested with the MTT assay to assess HDF proliferation. Type I and III collagen synthesis was examined using the respective ELISA kits. Analysis of variance was performed to compare the treatment groups. Results Asiaticoside had significantly stronger effects on HDF proliferation than retinoic acid (p<0.05. The type III collagen production was significantly greater induction with asiaticoside compared to retinoic acid (p<0.05. Conclusion Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

  1. Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

    Science.gov (United States)

    Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

    2015-06-01

    Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic

  2. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition

    Directory of Open Access Journals (Sweden)

    Ramkumar Menon

    2017-08-01

    Full Text Available Human parturition is an inflammatory process that involves both fetal and maternal compartments. The precise immune cell interactions have not been well delineated in human uterine tissues during parturition, but insights into human labor initiation have been informed by studies in animal models. Unfortunately, the timing of parturition relative to fetal maturation varies among viviparous species—indicative of different phylogenetic clocks and alarms—but what is clear is that important common pathways must converge to control the birth process. Herein, we hypothesize a novel signaling mechanism initiated by human fetal membrane aging and senescence-associated inflammation. Programmed events of fetal membrane aging coincide with fetal growth and organ maturation. Mechanistically, senescence involves in telomere shortening and activation of p38 mitogen-activated signaling kinase resulting in aging-associated phenotypic transition. Senescent tissues release inflammatory signals that are propagated via exosomes to cause functional changes in maternal uterine tissues. In vitro, oxidative stress causes increased release of inflammatory mediators (senescence-associated secretory phenotype and damage-associated molecular pattern markers that can be packaged inside the exosomes. These exosomes traverse through tissues layers, reach maternal tissues to increase overall inflammatory load transitioning them from a quiescent to active state. Animal model studies have shown that fetal exosomes can travel from fetal to the maternal side. Thus, aging fetal membranes and membrane-derived exosomes cargo fetal signals to the uterus and cervix and may trigger parturition. This review highlights a novel hypothesis in human parturition research based on data from ongoing research using human fetal membrane model system.

  3. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition.

    Science.gov (United States)

    Menon, Ramkumar; Mesiano, Sam; Taylor, Robert N

    2017-01-01

    Human parturition is an inflammatory process that involves both fetal and maternal compartments. The precise immune cell interactions have not been well delineated in human uterine tissues during parturition, but insights into human labor initiation have been informed by studies in animal models. Unfortunately, the timing of parturition relative to fetal maturation varies among viviparous species-indicative of different phylogenetic clocks and alarms-but what is clear is that important common pathways must converge to control the birth process. Herein, we hypothesize a novel signaling mechanism initiated by human fetal membrane aging and senescence-associated inflammation. Programmed events of fetal membrane aging coincide with fetal growth and organ maturation. Mechanistically, senescence involves in telomere shortening and activation of p38 mitogen-activated signaling kinase resulting in aging-associated phenotypic transition. Senescent tissues release inflammatory signals that are propagated via exosomes to cause functional changes in maternal uterine tissues. In vitro, oxidative stress causes increased release of inflammatory mediators (senescence-associated secretory phenotype and damage-associated molecular pattern markers) that can be packaged inside the exosomes. These exosomes traverse through tissues layers, reach maternal tissues to increase overall inflammatory load transitioning them from a quiescent to active state. Animal model studies have shown that fetal exosomes can travel from fetal to the maternal side. Thus, aging fetal membranes and membrane-derived exosomes cargo fetal signals to the uterus and cervix and may trigger parturition. This review highlights a novel hypothesis in human parturition research based on data from ongoing research using human fetal membrane model system.

  4. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition

    Science.gov (United States)

    Menon, Ramkumar; Mesiano, Sam; Taylor, Robert N.

    2017-01-01

    Human parturition is an inflammatory process that involves both fetal and maternal compartments. The precise immune cell interactions have not been well delineated in human uterine tissues during parturition, but insights into human labor initiation have been informed by studies in animal models. Unfortunately, the timing of parturition relative to fetal maturation varies among viviparous species—indicative of different phylogenetic clocks and alarms—but what is clear is that important common pathways must converge to control the birth process. Herein, we hypothesize a novel signaling mechanism initiated by human fetal membrane aging and senescence-associated inflammation. Programmed events of fetal membrane aging coincide with fetal growth and organ maturation. Mechanistically, senescence involves in telomere shortening and activation of p38 mitogen-activated signaling kinase resulting in aging-associated phenotypic transition. Senescent tissues release inflammatory signals that are propagated via exosomes to cause functional changes in maternal uterine tissues. In vitro, oxidative stress causes increased release of inflammatory mediators (senescence-associated secretory phenotype and damage-associated molecular pattern markers) that can be packaged inside the exosomes. These exosomes traverse through tissues layers, reach maternal tissues to increase overall inflammatory load transitioning them from a quiescent to active state. Animal model studies have shown that fetal exosomes can travel from fetal to the maternal side. Thus, aging fetal membranes and membrane-derived exosomes cargo fetal signals to the uterus and cervix and may trigger parturition. This review highlights a novel hypothesis in human parturition research based on data from ongoing research using human fetal membrane model system. PMID:28861041

  5. The upregulation of heat shock protein 47 expression in human buccal fibroblasts stimulated with arecoline.

    Science.gov (United States)

    Yang, Shun-Fa; Tsai, Chung-Hung; Chang, Yu-Chao

    2008-04-01

    Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. HSP47 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare HSP47 expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further to explore the potential mechanisms that may lead to induce HSP47 expression. The mRNA levels of HSP47 from fibroblasts cultured from 20 OSF and 10 normal buccal mucosal fibroblasts (BMFs) were evaluated by reverse transcription polymerase chain reaction. The effect of arecoline, the major areca nut alkaloid, was added to explore the potential mechanisms that may lead to induce HSP47 expression. Furthermore, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, p38 inhibitor SB203580, cyclo-oxygenase-2 (COX-2) inhibitor NS-398, and glutathione precursor N-acetyl-l-cysteine were added to find the possible mechanisms. OSF demonstrated significantly higher HSP47 mRNA expression than BMFs (P Arecoline was also found to elevate HSP47 mRNA expression in a dose-dependent manner (P arecoline when compared with control (P arecoline-induced HSP47 mRNA expression (P arecoline in fibroblasts may be mediated by MEK, PI3K, and COX-2 signal transduction pathways.

  6. Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches

    Energy Technology Data Exchange (ETDEWEB)

    Muczynski, V. [Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unité 967, F-92265, Fontenay aux Roses (France); Cravedi, J.P. [INRA, INP, Université de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France); Lehraiki, A.; Levacher, C.; Moison, D.; Lecureuil, C.; Messiaen, S. [Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unité 967, F-92265, Fontenay aux Roses (France); Perdu, E. [INRA, INP, Université de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France); Frydman, R. [Service de Gynécologie-Obstétrique, Hôpital A. Béclère, Université Paris Sud F-92141 Clamart (France); Habert, R. [Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unité 967, F-92265, Fontenay aux Roses (France); and others

    2012-05-15

    The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10{sup −5} M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7–12 weeks) of gestation and cultured in the presence or not of 10{sup −5} M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with {sup 14}C-MEHP. A 10{sup −5} M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10{sup −5} M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells. -- Highlights: ► 10{sup −5} M of MEHP impairs germ cell development in the human fetal testis. ► Organotypic culture is a suitable approach to investigate phthalate effects in human. ► MEHP is not metabolized in the human fetal testis. ► In mice, MEHP triggers similar effects both in vivo and in vitro.

  7. Comparative studies of different cryopreservation methods for mesenchymal stem cells derived from human fetal liver.

    Science.gov (United States)

    Todorov, Plamen; Hristova, Elena; Konakchieva, Rossitza; Michova, Antoaneta; Dimitrov, Josif

    2010-03-29

    Fetal stem cells possess some intriguing characteristics, which delineate them as promising cellular therapeutics. They are less immunogenic, at lower stage of differentiation and have higher potential for repopulation and migration. Furthermore, the fetal stem cells secrete a set of cytokines and growth factors, which stimulate the regeneration of the recipient tissue. The present study indicated that the adhesive fraction of human fetal liver cells possessed the morphological characteristics of mesenchymal stem cells, as well as potential to differentiate into adipocyte and osteoblast lineages. The immunophenotypic analysis showed that the cells expressed CD13, CD73, CD90 and CD105 (typical for mesenchymal stem cells) and lacked the haematopoietic lineage markers CD34 and CD45. Addressing the issue of the low-temperature storage of the human fetal liver cells, four different methods for cryopreservation were assessed: conventional slow freezing, program freezing and two vitrification protocols. The obtained results demonstrated that the cells were cryotolerant and maintained their properties and differentiation potential after thawing. Program freezing showed to be the most efficient method for cryopreservation of the investigated cells.

  8. Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells.

    Science.gov (United States)

    Park, Tae Sub; Galic, Zoran; Conway, Anne E; Lindgren, Anne; van Handel, Benjamin J; Magnusson, Mattias; Richter, Laura; Teitell, Michael A; Mikkola, Hanna K A; Lowry, William E; Plath, Kathrin; Clark, Amander T

    2009-04-01

    The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real-time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure.

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

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2017-02-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37°C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37°C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

  10. Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2017-02-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37 °C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37 °C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

  11. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst, E-mail: e.wolvetang@uq.edu.au

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.

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

    Science.gov (United States)

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

    2016-08-01

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

  13. Cytotoxicity and Proliferation Studies with Arsenic in Established Human Cell Lines: Keratinocytes, Melanocytes, Dendritic Cells, Dermal Fibroblasts, Microvascular Endothelial Cells, Monocytes and T-Cells

    Directory of Open Access Journals (Sweden)

    Hari H. P. Cohly

    2003-01-01

    Full Text Available Abstract: Based on the hypothesis that arsenic exposure results in toxicity and mitogenecity, this study examined the dose-response of arsenic in established human cell lines of keratinocytes (HaCaT, melanocytes (1675, dendritic cells (THP-1/A23187, dermal fibroblasts (CRL1904, microvascular endothelial cells (HMEC, monocytes (THP-1, and T cells (Jurkat. Cytotoxicity was determined by incubating THP-1, THP-1+ A23187 and JKT cells in RPMI 1640, 1675 in Vitacell, HMEC in EBM, and dermal fibroblasts and HaCaT in DMEM with 10% fetal bovine serum, 1% streptomycin and penicillin for 72 hrs in 96-well microtiter plates, at 37oC in a 5% CO2 incubator with different concentrations of arsenic using fluorescein diacetate (FDA. Cell proliferation in 96-well plates was determined in cultured cells starved by prior incubation for 24 hrs in 1% FBS and exposed for 72 hours, using the 96 cell titer proliferation solution (Promega assay. Cytotoxicity assays yielded LD50s of 9 μg/mL for HaCaT, 1.5 μg/mL for CRL 1675, 1.5 μg/mL for dendritic cells, 37 μg/mL for dermal fibroblasts, 0.48 μg/mL for HMEC, 50 μg/mL for THP-1 cells and 50 μg/mL for JKT-T cells. The peak proliferation was observed at 6 μg/mL for HaCaT and THP-1 cells, 0.19 μg/mL for CRL 1675, dendritic cells, and HMEC, and 1.5 μg/mL for dermal fibroblasts and Jurkat T cells. These results show that arsenic is toxic at high doses to keratinocytes, fibroblasts, monocytes and T cells, and toxic at lower doses to melanocytes, microvascular endothelial cells and dendritic cells. Proliferation studies showed sub-lethal doses of arsenic to be mitogenic.

  14. Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue.

    Science.gov (United States)

    Ardehali, Reza; Ali, Shah R; Inlay, Matthew A; Abilez, Oscar J; Chen, Michael Q; Blauwkamp, Timothy A; Yazawa, Masayuki; Gong, Yongquan; Nusse, Roeland; Drukker, Micha; Weissman, Irving L

    2013-02-26

    A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

  15. Intraarticular Sprifermin (Recombinant Human Fibroblast Growth Factor 18) in Knee Osteoarthritis

    DEFF Research Database (Denmark)

    Lohmander, L. S.; Hellot, S.; Dreher, D.

    2014-01-01

    Objective. To evaluate the efficacy and safety of intraarticular sprifermin (recombinant human fibroblast growth factor 18) in the treatment of symptomatic knee osteoarthritis (OA). Methods. The study was a randomized, double-blind, placebo-controlled, proof-of-concept trial. Intraarticular...... in joint space width (JSW) seen on radiographs, and pain scores on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Results. One hundred ninety-two patients were randomized and evaluated for safety, 180 completed the trial, and 168 were evaluated for the primary efficacy end...

  16. Comparison of the Efficiency of Viral Transduction and Episomal Transfection in Human Fibroblast Reprogramming.

    Science.gov (United States)

    Vdovin, A S; Lupatov, A Yu; Kholodenko, I V; Yarygin, K N

    2015-11-01

    Induced pluripotent cells were derived from adult human skin fibroblast by using two methods of reprogramming. Episomal transfection with vectors containing oriP/EBNA-1 sequence for delivery of reprogramming genes Oct4, Sox2, Klf4, L-Myc, and Lin28 proved to be more effective than viral transduction with Sendai virus-based vector: ~200 and 8 colonies per 10(5) cells were found on day 21 of culturing, respectively. Colonies of induced pluripotent cells obtained by these two methods expressed pluripotency marker Tra1-60.

  17. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

    OpenAIRE

    Soldatov, N M

    1992-01-01

    The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of thes...

  18. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts

    OpenAIRE

    1984-01-01

    The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to th...

  19. 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

  20. STAT6-dependent collagen synthesis in human fibroblasts is induced by bovine milk

    OpenAIRE

    Stefan Kippenberger; Nadja Zöller; Johannes Kleemann; Jutta Müller; Roland Kaufmann; Matthias Hofmann; August Bernd; Markus Meissner; Eva Valesky

    2015-01-01

    Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold,...

  1. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Directory of Open Access Journals (Sweden)

    Donejko M

    2017-03-01

    Full Text Available Magdalena Donejko,1 Edyta Rysiak,2 Elżbieta Galicka,1 Robert Terlikowski,3 Edyta Katarzyna Głażewska,1 Andrzej Przylipiak1 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, 3Department of Health Restoration, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK, and the influence of HA on those processes. Materials and methods: Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results: Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion: This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. Keywords: apoptosis, skin fibroblast, focal adhesion kinase, hyaluronic acid, ethanol

  2. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3.

    Science.gov (United States)

    Rauchenberger, Robert; Borges, Eric; Thomassen-Wolf, Elisabeth; Rom, Eran; Adar, Rivka; Yaniv, Yael; Malka, Michael; Chumakov, Irina; Kotzer, Sarit; Resnitzky, Dalia; Knappik, Achim; Reiffert, Silke; Prassler, Josef; Jury, Karin; Waldherr, Dirk; Bauer, Susanne; Kretzschmar, Titus; Yayon, Avner; Rothe, Christine

    2003-10-03

    The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., and Virnekäs, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

  3. Combined transcriptome analysis of fetal human and mouse cerebral cortex exposed to alcohol.

    Science.gov (United States)

    Hashimoto-Torii, Kazue; Kawasawa, Yuka Imamura; Kuhn, Alexandre; Rakic, Pasko

    2011-03-08

    Fetal exposure to environmental insults increases the susceptibility to late-onset neuropsychiatric disorders. Alcohol is listed as one of such prenatal environmental risk factors and known to exert devastating teratogenetic effects on the developing brain, leading to complex neurological and psychiatric symptoms observed in fetal alcohol spectrum disorder (FASD). Here, we performed a coordinated transcriptome analysis of human and mouse fetal cerebral cortices exposed to ethanol in vitro and in vivo, respectively. Up- and down-regulated genes conserved in the human and mouse models and the biological annotation of their expression profiles included many genes/terms related to neural development, such as cell proliferation, neuronal migration and differentiation, providing a reliable connection between the two species. Our data indicate that use of the combined rodent and human model systems provides an effective strategy to reveal and analyze gene expression changes inflicted by various physical and chemical environmental exposures during prenatal development. It also can potentially provide insight into the pathogenesis of environmentally caused brain disorders in humans.

  4. Human cytomegalovirus induces a distinct innate immune response in the maternal-fetal interface.

    Science.gov (United States)

    Weisblum, Yiska; Panet, Amos; Zakay-Rones, Zichria; Vitenshtein, Alon; Haimov-Kochman, Ronit; Goldman-Wohl, Debra; Oiknine-Djian, Esther; Yamin, Rachel; Meir, Karen; Amsalem, Hagai; Imbar, Tal; Mandelboim, Ofer; Yagel, Simcha; Wolf, Dana G

    2015-11-01

    The initial interplay between human cytomegalovirus (HCMV) and innate tissue response in the human maternal-fetal interface, though crucial for determining the outcome of congenital HCMV infection, has remained unknown. We studied the innate response to HCMV within the milieu of the human decidua, the maternal aspect of the maternal-fetal interface, maintained ex vivo as an integral tissue. HCMV infection triggered a rapid and robust decidual-tissue innate immune response predominated by interferon (IFN)γ and IP-10 induction, dysregulating the decidual cytokine/chemokine environment in a distinctive fashion. The decidual-tissue response was already elicited during viral-tissue contact, and was not affected by neutralizing HCMV antibodies. Of note, IFNγ induction, reflecting immune-cell activation, was distinctive to the maternal decidua, and was not observed in concomitantly-infected placental (fetal) villi. Our studies in a clinically-relevant surrogate human model, provide a novel insight into the first-line decidual tissue response which could affect the outcome of congenital infection.

  5. Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Min Lin; Qun Chen; Li-Ye Yang; Wen-Yu Li; Xi-Biao Cao; Jiao-Ren Wu; You-Peng Peng; Mo-Rui Chen

    2007-01-01

    AIM:To investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODS:The human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTS:A stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSION:HBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.

  6. Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih [Institute of Biomedical Sciences, Taiwan (China)] [and others

    1995-08-01

    Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.

  7. Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus.

    Science.gov (United States)

    Chen, Daxiang; Long, Mingjian; Xiao, Bin; Xiong, Yufeng; Chen, Huiqin; Chen, Yu; Kuang, Zhenzhan; Li, Ming; Wu, Yingsong; Rock, Daniel L; Gong, Daoyuan; Wang, Yong; He, Haijian; Liu, Fang; Luo, Shuhong; Hao, Wenbo

    2017-08-29

    Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

  8. Metabolic gene profile in early human fetal heart development

    National Research Council Canada - National Science Library

    Iruretagoyena, J I; Davis, W; Bird, C; Olsen, J; Radue, R; Teo Broman, A; Kendziorski, C; Splinter BonDurant, S; Golos, T; Bird, I; Shah, D

    2014-01-01

    .... In order to describe normal cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and created a corresponding 'normal' database...

  9. Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders.

    Science.gov (United States)

    Hattori, Yuka; Mukaide, Takahiro; Jiang, Li; Kotani, Tomomi; Tsuda, Hiroyuki; Mano, Yukio; Sumigama, Seiji; Hirayama, Tasuku; Nagasawa, Hideko; Kikkawa, Fumitaka; Toyokuni, Shinya

    2015-01-01

    Amniotic fluid contains numerous biomolecules derived from fetus and mother, thus providing precious information on pregnancy. Here, we evaluated oxidative stress of human amniotic fluid and measured the concentration of catalytic Fe(II). Amniotic fluid samples were collected with consent from a total of 89 subjects in Nagoya University Hospital, under necessary medical interventions: normal pregnancy at term, normal pregnancy at the 2nd trimester, preterm delivery with maternal disorders but without fetal disorders, congenital diaphragmatic hernia, fetal growth restriction, pregnancy-induced hypertension, gestational diabetes mellitus, Down syndrome and trisomy 18. Catalytic Fe(II) and oxidative stress markers (8-hydroxy-2'-deoxyguanosine, 8-OHdG; dityrosine) were determined with RhoNox-1 and specific antibodies, respectively, using plate assays. Levels of 8-OHdG and dityrosine were higher in the 3rd trimester compared with the 2nd trimester in normal subjects, and the abnormal groups generally showed lower levels than the controls, thus suggesting that they represent fetal metabolic activities. In contrast, catalytic Fe(II) was higher in the 2nd trimester than the 3rd trimester in the normal subjects, and overall the abnormal groups showed higher levels than the controls, suggesting that high catalytic Fe(II) at late gestation reflects fetal pathologic alterations. Notably, products of H2O2 and catalytic Fe(II) remained almost constant in amniotic fluid.

  10. A high-resolution MRI study of linear growth of the human fetal skull base

    Energy Technology Data Exchange (ETDEWEB)

    Jeffery, N. [University Coll., London (United Kingdom). Dept. of Anatomy and Development Biology

    2002-04-01

    The skull base, otherwise referred to as the basicranium or cranial base, plays a key role in the process of skull development, providing both support for the brain and an architectural component of the craniofacial complex. Consequently, the fetal skull base has been the focus of numerous studies employing various methods, including sectioning, plain radiography and CT. This paper investigates high-resolution (hr) MRI as an alternative method for looking at and quantifying the fetal skull base. The evaluation tests two basic hypotheses drawn from previous studies. These suggest that the anterior segment of the midline skull base grows more rapidly than the posterior segment and that the width of the posterior cranial fossa increases disproportionately in relation to its length. I imaged 42 formalin preserved human fetuses from museum collections with hrMRI. The T2-weighted image voxels were significantly smaller than those acquired with conventional clinical MRI. Landmarks of the fetal skull base were identified on reformatted axial and sagittal images. Bivariate plots revealed that the growth rate of the anterior skull base is almost twice that of the posterior skull base and that increases in the width of the posterior cranial fossa exceed those in its length. These findings confirm those of previous investigations and show that hrMRI offers a way forward in noninvasive quantification of fetal morphology. ----------------------------------------------------------------------------.

  11. Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta.

    Science.gov (United States)

    Sardesai, Varda S; Shafiee, Abbas; Fisk, Nicholas M; Pelekanos, Rebecca A

    2017-04-01

    Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender-discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi-derived MSC (CV-MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV-MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070-1084.

  12. Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker

    Institute of Scientific and Technical Information of China (English)

    Parveen Nyamath; Ayesha AM; Aejaz Habeeb; Sanjeev Khosla; Aleem A Khan; CM Habibullah

    2007-01-01

    AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker.METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation.RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture.CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.

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

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

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

  14. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. The chromene sargachromanol E inhibits ultraviolet A-induced ageing of skin in human dermal fibroblasts.

    Science.gov (United States)

    Kim, J-A; Ahn, B-N; Kong, C-S; Kim, S-K

    2013-05-01

    Skin ageing is influenced by environmental factors such as ultraviolet (UV) radiation. The effects of UV radiation on skin functions should be investigated using human in vitro models to understand the mechanisms of skin ageing. Additionally, marine algae provide a valuable source for identifying and extracting biologically active substances. In this study, sargachromanol E was isolated from a marine brown alga, Sargassum horneri, and its inhibitory effect on skin ageing was investigated using UVA-irradiated dermal fibroblasts. Formation of intracellular reactive oxygen species (ROS), lipid peroxidation and protein oxidation induced by UVA irradiation were investigated in UVA-irradiated human dermal fibroblasts. The levels of matrix metalloproteinases (MMPs) were determined by reverse-transcriptase polymerase chain reaction and Western blot analysis. Sargachromanol E did not exhibit any significant cytotoxicity or phototoxicity in UVA-exposed dermal fibroblasts. Additionally, sargachromanol E suppressed intracellular formation of ROS, membrane protein oxidation, lipid peroxidation and expression of collagenases such as MMP-1, MMP-2 and MMP-9, all of which are caused by UVA exposure. It was further found that these inhibitions were related to an increase in the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP2. Moreover, we have shown that the transcriptional activation of activator protein 1 (AP-1) signalling caused by UVA irradiation was inhibited by treatment with sargachromanol E. This study suggests that UVA irradiation modulates MMP expression via the transcriptional activation of AP-1 signalling, whereas treatment with sargachromanol E protected cell damage caused by UVA irradiation. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  17. Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

    Science.gov (United States)

    Meyer-Ter-Vehn, Tobias; Katzenberger, Barbara; Han, Hong; Grehn, Franz; Schlunck, Günther

    2008-09-01

    The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-beta, Rho-dependent contractility, and cell-matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-beta-mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-beta1. alpha-Smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. alpha-SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. Lovastatin inhibited TGF-beta-induced CTGF transcription, alpha-SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-beta-induced alpha-SMA expression. Lovastatin decreased TGF-beta-induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery.

  18. Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation

    Directory of Open Access Journals (Sweden)

    Y Boza

    2010-12-01

    Full Text Available The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM, primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001. HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05. The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

  19. A three-dimensional study of human fetal endocervix with special reference to its epithelium

    OpenAIRE

    Barberini, F.; Makabe, S.; Motta, P M

    1998-01-01

    The development of human fetal cervix has been systematically studied by SEM, obtaining a detailed map of its fine structure, particularly concerning the differentiation and maturation of the endocervical epithelium, including its "eversion" and "squamous metaplasia", normally occurring in postnatal life, but not yet observed in detail by electron microscopy in the fetus. Cervices from spontaneous abortion at 12, 15, 18, 20, 21 and 22 weeks and from intrauterin...

  20. MCT8 expression in human fetal cerebral cortex is reduced in severe intrauterine growth restriction

    OpenAIRE

    Shiao Y Chan; Hancox, Laura A; Martín-Santos, Azucena; Loubière, Laurence S; Walter, Merlin N.M.; González, Ana-Maria; Cox, Phillip M; Logan, Ann; Christopher J. McCabe; Franklyn, Jayne A; Kilby, Mark D

    2014-01-01

    The importance of the thyroid hormone (TH) transporter, monocarboxylate transporter 8 (MCT8), to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with MCT8 (SLC16A2) mutations. Intrauterine growth restriction (IUGR), usually due to uteroplacental failure, is associated with milder neurodevelopmental deficits, which have been partly attributed to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and de...

  1. Expression and localization of VEGF receptors in human fetal skeletal tissues.

    Science.gov (United States)

    Marini, M; Sarchielli, E; Toce, M; Acocella, A; Bertolai, R; Ciulli, C; Orlando, C; Sgambati, E; Vannelli, G B

    2012-12-01

    During development the vertebrate skeleton is the product of deriving cells from distinct embryonic lineages. The craniofacial skeleton is formed by migrating cranial neural crest cells, whereas the axial and limb skeletons are derived from mesodermal cells. The Vascular Endothelial Growth Factors (VEGFs) / receptors (VEGFRs) system plays an important role in angiogenesis, as well as osteogenesis, during bone development, growth, and remodeling, attracting endothelial cells and osteoclasts and stimulating osteoblast differentiation. Recent evidence has shown that during development VEGFR-3 is also expressed in neural and glial precursors of forebrain and cerebellum, as well as in the eye. In this study, we found that VEGFR-1, VEGFR-2 and VEGFR-3 are expressed in human bone both in fetal and adult life. The gene expression levels were significantly higher in fetal samples especially in mandibles. In addition, higher levels of VEGFR-3 in orofacial district were confirmed by western blotting analysis. We also observed that in fetal mandibular samples VEGFRs colocalized in several osteoblasts, osteoclasts and osteoprogenitor cells. Furthermore, some cells coexpressed VEGFR-3 and ET-1, a marker of neural crest cells. The results demonstrated different expression of VEGFRs in human mandibular and femoral bones which could be correlated to their different structure, function and development during organogenesis. VEGFR-3 might represent a specific signal for ectomesenchymal lineage differentiation during early human development.

  2. Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging.

    Directory of Open Access Journals (Sweden)

    Solène Mine

    Full Text Available Understanding the contribution of the dermis in skin aging is a key question, since this tissue is particularly important for skin integrity, and because its properties can affect the epidermis. Characteristics of matched pairs of dermal papillary and reticular fibroblasts (Fp and Fr were investigated throughout aging, comparing morphology, secretion of cytokines, MMPs/TIMPs, growth potential, and interaction with epidermal keratinocytes. We observed that Fp populations were characterized by a higher proportion of small cells with low granularity and a higher growth potential than Fr populations. However, these differences became less marked with increasing age of donors. Aging was also associated with changes in the secretion activity of both Fp and Fr. Using a reconstructed skin model, we evidenced that Fp and Fr cells do not possess equivalent capacities to sustain keratinopoiesis. Comparing Fp and Fr from young donors, we noticed that dermal equivalents containing Fp were more potent to promote epidermal morphogenesis than those containing Fr. These data emphasize the complexity of dermal fibroblast biology and document the specific functional properties of Fp and Fr. Our results suggest a new model of skin aging in which marked alterations of Fp may affect the histological characteristics of skin.

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

    Science.gov (United States)

    Ohshima, Susumu; Seyama, Atsushi

    2012-09-01

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

  4. Effects of differently polarized microwave radiation on the microscopic structure of the nuclei in human fibroblasts.

    Science.gov (United States)

    Shckorbatov, Yuriy G; Pasiuga, Vladimir N; Goncharuk, Elena I; Petrenko, Tatiana Ph; Grabina, Valentin A; Kolchigin, Nicolay N; Ivanchenko, Dmitry D; Bykov, Victor N; Dumin, Oleksandr M

    2010-10-01

    To investigate the influence of microwave radiation on the human fibroblast nuclei, the effects of three variants of electromagnetic wave polarization, linear and left-handed and right-handed elliptically polarized, were examined. Experimental conditions were: frequency (f) 36.65 GHz, power density (P) at the surface of exposed object 1, 10, 30, and 100 µW/cm(2), exposure time 10 s. Human fibroblasts growing in a monolayer on a cover slide were exposed to microwave electromagnetic radiation. The layer of medium that covered cells during microwave exposure was about 1 mm thick. Cells were stained immediately after irradiation by 2% (w/v) orcein solution in 45% (w/v) acetic acid. Experiments were made at room temperature (25 °C), and control cell samples were processed in the same conditions. We assessed heterochromatin granule quantity (HGQ) at 600× magnification. Microwave irradiation at the intensity of 1 µW/cm(2) produced no effect, and irradiation at the intensities of 10 and 100 µW/cm(2) induced an increase in HGQ. More intense irradiation induced more chromatin condensation. The right-handed elliptically polarized radiation revealed more biological activity than the left-handed polarized one.

  5. 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.

  6. Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Masatoshi Suzuki

    2012-01-01

    Full Text Available Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

  7. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

    Science.gov (United States)

    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  8. Scopoletin has a potential activity for anti-aging via autophagy in human lung fibroblasts.

    Science.gov (United States)

    Nam, Hyang; Kim, Moon-Moo

    2015-03-15

    Autophagy was known to be associated with aging in addition to cancer and neurodegeneration. The effects of scopoletin on autophagy and anti-aging were investigated in human lung fibroblast cell line, IMR 90. Here we show that scopoletin induces autophagy. It is also identified that the modulation of p53 by scopoletin are related to the induction of autophagy. Moreover, the level of SA-β-Gal staining, an aging marker, is reduced by scopoletin. In addition, while the expression levels of histone deacetylases such as HDAC1, SIRT1 and SIRT6 are increased in IMR 90 cells in the presence of scopoletin, the expression levels of histone acetyltransferases are decreased. Furthermore, scopoletin enhances the level of transcription factors such as Nrf-2and p-FoxO1 related to anti-aging. In addition, scopoletin modulates the reprogramming proteins. Therefore, these findings suggest that scopoletin could exert a positive effect on anti-aging related to autophagy through modulation of p53 in human lung fibroblasts. Copyright © 2015 Elsevier GmbH. All rights reserved.

  9. Arecoline stimulated early growth response-1 production in human buccal fibroblasts: suppression by epigallocatechin-3-gallate.

    Science.gov (United States)

    Hsieh, Yu-Ping; Chen, Hsin-Ming; Chang, Jenny Zwei-Chieng; Chiang, Chun-Pin; Deng, Yi-Ting; Kuo, Mark Yen-Ping

    2015-04-01

    Early growth response-1 (Egr-1) protein plays an important role in many human fibrotic diseases. Areca nut chewing is the most important risk factor of oral submucous fibrosis (OSF). Egr-1 protein expression in OSF was examined using antibody to Egr-1. Arecoline-induced Egr-1 expression and its signaling pathways were assessed by Western blot analyses in human buccal mucosal fibroblasts (BMFs). Elevated Egr-1 staining was observed in epithelial cells, fibroblast, and inflammatory cells in 7 of 10 OSF cases. Arecoline, a main alkaloid found in the areca nut, stimulated Egr-1 synthesis in BMFs. Pretreatment with antioxidant N-acetyl-L-cysteine, c-Jun NH2-terminal kinase inhibitor SP600125, and extracellular signal-regulated kinase inhibitor PD98059 significantly reduced arecoline-induced Egr-1 synthesis. Epigallocatechin-3-gallate (EGCG) inhibited arecoline-induced Egr-1 synthesis and collagen gel contraction in a dose-responsive manner. Constitutive Egr-1 expression during areca nut chewing may play a role in the pathogenesis of OSF. EGCG could be a good candidate for prevention or treatment of OSF. © 2014 Wiley Periodicals, Inc.

  10. Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

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    Layman, D.L.; Diedrich, D.L.

    1987-06-01

    Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by /sup 3/H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in /sup 3/H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin.

  11. In Vitro Cytotoxicity Evaluation of Three Root-End Filling Materials in Human Periodontal Ligament Fibroblasts.

    Science.gov (United States)

    Coaguila-Llerena, Hernán; Vaisberg, Abraham; Velásquez-Huamán, Zulema

    2016-01-01

    The aim of this study was to evaluate in vitro the cytotoxicity on human periodontal ligament fibroblasts of three root-end filling materials: MTA Angelus®, EndoSequence Root Repair Material Putty® and Super EBA®. A primary culture of human periodontal ligament fibroblasts was previously obtained in order to evaluate the cytotoxicity of the three extracts from the root-end filling materials after 2 and 7 days of setting. Serial dilutions of these extracts (1:1, 1:2, 1:4 and 1:8) were evaluated at 1, 3 and 7 days using the methyl-thiazol-tetrazolium (MTT) colorimetric assay. Cell viability was evaluated as percentage of the negative control group, which represented 100% cell viability. Statistical analyses were done with t-test, ANOVA and Kruskal-Wallis test at a significance level of 5%. It was found that the main difference among root-end filling materials was in the higher dilutions (p0.05). Cell viability of MTA Angelus® was superior for 2-day setting (pMaterial Putty®. Super EBA® showed the lowest percentage of cell viability at higher dilutions (pMaterial Putty® were less cytotoxic in the highest dilution (1:1) compared with Super EBA®.

  12. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts

    Science.gov (United States)

    Budiyanto, Arief; Soebono, Hardyanto

    2016-01-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm-2) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined. PMID:27401663

  13. Transmission of HIV-1 by primary human uterine epithelial cells and stromal fibroblasts.

    Science.gov (United States)

    Asin, Susana N; Fanger, Michael W; Wildt-Perinic, Dunja; Ware, Patricia L; Wira, Charles R; Howell, Alexandra L

    2004-07-15

    Women can become infected with human immunodeficiency virus type 1 (HIV-1) after the heterosexual transmission of virus from an infected male partner. To understand the events that result in transmission of HIV-1 across the female reproductive tract, we characterized the life-cycle events of HIV-1 in primary cultures of human uterine epithelial cells and stromal fibroblasts. Epithelial cells and stromal fibroblasts released virus particles after exposure to either X4- or R5-tropic strains of HIV-1. Virus released by these cells was able to infect CD4(+) T cells. When exposed to an X4-tropic strain of HIV-1, these cells supported HIV-1 reverse transcription, integration, and viral DNA transcription. When exposed to an R5-tropic strain, however, these cells released unmodified virus. These data suggest that uterine cells are targets for productive infection with X4-tropic strains and release unmodified R5-tropic viruses that would then be able to infect submucosal target cells, including T cells and macrophages.

  14. Mitochondrial proteomics on human fibroblasts for identification of metabolic imbalance and cellular stress

    Directory of Open Access Journals (Sweden)

    Bross Peter

    2009-05-01

    Full Text Available Abstract Background Mitochondrial proteins are central to various metabolic activities and are key regulators of apoptosis. Disturbance of mitochondrial proteins is therefore often associated with disease. Large scale protein data are required to capture the mitochondrial protein levels and mass spectrometry based proteomics is suitable for generating such data. To study the relative quantities of mitochondrial proteins in cells from cultivated human skin fibroblasts we applied a proteomic method based on nanoLC-MS/MS analysis of iTRAQ-labeled peptides. Results When fibroblast cultures were exposed to mild metabolic stress – by cultivation in galactose medium- the amount of mitochondria appeared to be maintained whereas the levels of individual proteins were altered. Proteins of respiratory chain complex I and IV were increased together with NAD+-dependent isocitrate dehydrogenase of the citric acid cycle illustrating cellular strategies to cope with altered energy metabolism. Furthermore, quantitative protein data, with a median standard error below 6%, were obtained for the following mitochondrial pathways: fatty acid oxidation, citric acid cycle, respiratory chain, antioxidant systems, amino acid metabolism, mitochondrial translation, protein quality control, mitochondrial morphology and apoptosis. Conclusion The robust analytical platform in combination with a well-defined compendium of mitochondrial proteins allowed quantification of single proteins as well as mapping of entire pathways. This enabled characterization of the interplay between metabolism and stress response in human cells exposed to mild stress.

  15. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts.

    Science.gov (United States)

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure,membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans.

  16. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    Science.gov (United States)

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  17. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

    NARCIS (Netherlands)

    Hartmann-Fritsch, Fabienne; Hosper, Nynke; Luginbuehl, Joachim; Biedermann, Thomas; Reichmann, Ernst; Meuli, Martin

    2013-01-01

    Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formati

  18. Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    C.M.A. Reijnders; A. van Lier; S. Roffel; D. Kramer; R.J. Scheper; S. Gibbs

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  19. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

    NARCIS (Netherlands)

    Reijnders, Christianne M. A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.; Gibbs, Susan

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  20. Intracellular Immunization of Human Fetal Cord Blood Stem/Progenitor Cells with a Ribozyme Against Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Yu, Mang; Leavitt, Mark C.; Maruyama, Midori; Yamada, Osamu; Young, Dennis; Ho, Anthony D.; Wong-Staal, Flossie

    1995-01-01

    Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34^+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

  1. Chikungunya virus exploits miR-146a to regulate NF-κB pathway in human synovial fibroblasts.

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    Sakthi Priya Selvamani

    Full Text Available OBJECTIVES: Chikungunya virus causes chronic infection with manifestations of joint pain. Human synovial fibroblasts get infected with CHIKV and could lead to pro-inflammatory responses. MicroRNAs have potentials to regulate the gene expression of various anti-viral and pro-inflammatory genes. The study aims to investigate the role of miR-146a in modulation of inflammatory responses of human synovial fibroblasts by Chikungunya virus. METHODS: To study the role of miR-146a in CHIKV pathogenesis in human synovial cells and underlying inflammatory manifestations, we performed CHIKV infection in primary human synovial fibroblasts. Western blotting, real-time PCR, luciferase reporter assay, overexpression and knockdown of cellular miR-146a strategies have been employed to validate the role of miR-146a in regulation of pro-inflammatory NF-κB pathway. RESULTS: CHIKV infection induced the expression of cellular miR-146a, which resulted into down-regulation of TRAF6, IRAK1, IRAK2 and increased replication of CHIKV in human synovial fibroblasts. Exogenous expression of miR-146a in human synovial fibroblasts led to decreased expression of TRAF6, IRAK1, IRAK2 and decreased replication of CHIKV. Inhibition of cellular miR-146a by anti-miR-146a restored the expression levels of TRAF6, IRAK1 and IRAK2. Downregulation of TRAF6, IRAK1 and IRAK2 led to downstream decreased NF-κB activation through negative feedback loop. CONCLUSION: This study demonstrated the mechanism of exploitation of cellular miR-146a by CHIKV in modulating the host antiviral immune response in primary human synovial fibroblasts.

  2. Characterization of the autocrine/paracrine function of vitamin D in human gingival fibroblasts and periodontal ligament cells.

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    Kaining Liu

    Full Text Available BACKGROUND: We previously demonstrated that 25-hydroxyvitamin D(3, the precursor of 1α,25-dihydroxyvitamin D(3, is abundant around periodontal soft tissues. Here we investigate whether 25-hydroxyvitamin D(3 is converted to 1α,25-dihydroxyvitamin D(3 in periodontal soft tissue cells and explore the possibility of an autocrine/paracrine function of 1α,25-dihydroxyvitamin D(3 in periodontal soft tissue cells. METHODOLOGY/PRINCIPAL FINDINGS: We established primary cultures of human gingival fibroblasts and human periodontal ligament cells from 5 individual donors. We demonstrated that 1α-hydroxylase was expressed in human gingival fibroblasts and periodontal ligament cells, as was cubilin. After incubation with the 1α-hydroxylase substrate 25-hydroxyvitamin D(3, human gingival fibroblasts and periodontal ligament cells generated detectable 1α,25-dihydroxyvitamin D(3 that resulted in an up-regulation of CYP24A1 and RANKL mRNA. A specific knockdown of 1α-hydroxylase in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 1α,25-dihydroxyvitamin D(3 production and mRNA expression of CYP24A1 and RANKL. The classical renal regulators of 1α-hydroxylase (parathyroid hormone, calcium and 1α,25-dihydroxyvitamin D(3 and Porphyromonas gingivalis lipopolysaccharide did not influence 1α-hydroxylase expression significantly, however, interleukin-1β and sodium butyrate strongly induced 1α-hydroxylase expression in human gingival fibroblasts and periodontal ligament cells. CONCLUSIONS/SIGNIFICANCE: In this study, the expression, activity and functionality of 1α-hydroxylase were detected in human gingival fibroblasts and periodontal ligament cells, raising the possibility that vitamin D acts in an autocrine/paracrine manner in these cells.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    Objective:To estimate electroporation (EP) influence on malignant and normal cells.Methods:Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following:250,1000,1750,2500 V/cm;50 μs by5 impulses for every case. The viability of cells after EP was estimated byMTT assay. The ultrastructural analysis was observed by transmission electron microscope (ZeissEM900). Results:In the current study we observed the intracellular effect followingEP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated byEP. Conversely, we showed thatEP in some conditions can stimulate cells to proliferation. Some changes induced byEP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters ofEP (250 and1000 V/cm). After applying higher electric field intensities (2500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications afterEP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters ofEP.Conclusions:We can claim thatEP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude thatEP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.

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

    Science.gov (United States)

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

    2011-04-01

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

  5. Neuropeptide Y in the Adult and Fetal Human Pineal Gland

    Directory of Open Access Journals (Sweden)

    Morten Møller

    2014-01-01

    Full Text Available Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally.

  6. Neuropeptide Y in the adult and fetal human pineal gland.

    Science.gov (United States)

    Møller, Morten; Phansuwan-Pujito, Pansiri; Badiu, Corin

    2014-01-01

    Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally.

  7. Prominent periventricular fiber system related to ganglionic eminence and striatum in the human fetal cerebrum.

    Science.gov (United States)

    Vasung, L; Jovanov-Milošević, N; Pletikos, M; Mori, S; Judaš, M; Kostović, Ivica

    2011-01-01

    Periventricular pathway (PVP) system of the developing human cerebrum is situated medial to the intermediate zone in the close proximity to proliferative cell compartments. In order to elucidate chemical properties and developing trajectories of the PVP we used DTI in combination with acetylcholinesterase histochemistry, SNAP-25 immunocytochemistry and axonal cytoskeletal markers (SMI312, MAP1b) immunocytochemistry on postmortem paraformaldehyde-fixed brains of 30 human fetuses ranging in age from 10 to 38 postconceptional weeks (PCW), 2 infants (age 1-3 months) and 1 adult brain. The PVP appears in the early fetal period (10-13 PCW) as two defined fibre bundles: the corpus callosum (CC) and the fetal fronto-occipital fascicle (FOF). In the midfetal period (15-18 PCW), all four components of the PVP can be identified: (1) the CC, which at rostral levels forms a voluminous callosal plate; (2) the FOF, with SNAP-25-positive fibers; (3) the fronto-pontine pathway (FPP) which for a short distance runs within the PVP; and (4) the subcallosal fascicle of Muratoff (SFM) which contains cortico-caudate projections. The PVPs are situated medial to the internal capsule at the level of the cortico-striatal junction; they remain prominent during the late fetal and early preterm period (19-28 PCW) and represent a portion of the wider periventricular crossroad of growing associative, callosal and projection pathways. In the perinatal period, the PVPs change their topographical relationships, decrease in size and the FOF looses its SNAP-25-reactivity. In conclusion, the hitherto undescribed PVP of the human fetal cerebrum contains forerunners of adult associative and projection pathways. Its transient chemical properties and relative exuberance suggest that the PVP may exert influence on the development of cortical connectivity (intermediate targeting) and other neurogenetic events such as neuronal proliferation. The PVP's topographical position also indicates that it is a major

  8. Human placental lactogen and unconjugated estriol concentrations in twin pregnancy: monitoring of fetal development in intrauterine growth retardation and single intrauterine fetal death.

    Science.gov (United States)

    Trapp, M; Kato, K; Bohnet, H G; Gerhard, I; Weise, H C; Leidenberger, F

    1986-11-01

    Human placental lactogen and unconjugated estriol concentrations in maternal serum were evaluated in 100 uneventful twin pregnancies, and these values were compared with those observed in 16 twin pregnancies associated with intrauterine growth retardation or single intrauterine fetal death. In pregnancies associated with intrauterine growth retardation (n = 8), human placental lactogen levels were at the lower limit of normal range for singleton pregnancies, whereas estriol levels were normal in most cases. When one of the fetuses had died before week 33 of pregnancy (n = 5), both human placental lactogen and estriol levels were low and they were almost at the levels in singleton pregnancy. When intrauterine fetal death occurred after week 36 of pregnancy (n = 3), both hormone levels remained normal until term. Thus human placental lactogen rather than estriol is a good indicator of intrauterine growth retardation in twin pregnancy. Both human placental lactogen and estriol are useful for the monitoring of the surviving fetus in the case of single intrauterine fetal death.

  9. Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles

    Directory of Open Access Journals (Sweden)

    Tchou Julia

    2012-09-01

    Full Text Available Abstract Background Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2 of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. Methods To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC and six Her2+. Results We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer. Conclusions These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.

  10. Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

    Institute of Scientific and Technical Information of China (English)

    Juan Wang; Weibin Sun; Chun Lu; Guixia Tang

    2005-01-01

    Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2 (hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

  11. 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.

  12. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  13. The early fetal development of human neocortical GABAergic interneurons.

    Science.gov (United States)

    Al-Jaberi, Nahidh; Lindsay, Susan; Sarma, Subrot; Bayatti, Nadhim; Clowry, Gavin J

    2015-03-01

    GABAergic interneurons are crucial to controlling the excitability and responsiveness of cortical circuitry. Their developmental origin may differ between rodents and human. We have demonstrated the expression of 12 GABAergic interneuron-associated genes in samples from human neocortex by quantitative rtPCR from 8 to 12 postconceptional weeks (PCW) and shown a significant anterior to posterior expression gradient, confirmed by in situ hybridization or immunohistochemistry for GAD1 and 2, DLX1, 2, and 5, ASCL1, OLIG2, and CALB2. Following cortical plate (CP) formation from 8 to 9 PCW, a proportion of cells were strongly stained for all these markers in the CP and presubplate. ASCL1 and DLX2 maintained high expression in the proliferative zones and showed extensive immunofluorescent double-labeling with the cell division marker Ki-67. CALB2-positive cells increased steadily in the SVZ/VZ from 10 PCW but were not double-labeled with Ki-67. Expression of GABAergic genes was generally higher in the dorsal pallium than in the ganglionic eminences, with lower expression in the intervening ventral pallium. It is widely accepted that the cortical proliferative zones may generate CALB2-positive interneurons from mid-gestation; we now show that the anterior neocortical proliferative layers especially may be a rich source of interneurons in the early neocortex.

  14. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  15. Potential of human fetal chorionic stem cells for the treatment of osteogenesis imperfecta.

    Science.gov (United States)

    Jones, Gemma N; Moschidou, Dafni; Abdulrazzak, Hassan; Kalirai, Bhalraj Singh; Vanleene, Maximilien; Osatis, Suchaya; Shefelbine, Sandra J; Horwood, Nicole J; Marenzana, Massimo; De Coppi, Paolo; Bassett, J H Duncan; Williams, Graham R; Fisk, Nicholas M; Guillot, Pascale V

    2014-02-01

    Osteogenesis imperfecta (OI) is a genetic bone pathology with prenatal onset, characterized by brittle bones in response to abnormal collagen composition. There is presently no cure for OI. We previously showed that human first trimester fetal blood mesenchymal stem cells (MSCs) transplanted into a murine OI model (oim mice) improved the phenotype. However, the clinical use of fetal MSC is constrained by their limited number and low availability. In contrast, human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification. Exogenous cells preferentially homed to long bone epiphyses, expressed osteoblast genes, and produced collagen COL1A2. Together, our data suggest that exogenous cells decrease bone brittleness and BV by directly differentiating to osteoblasts and indirectly stimulating host chondrogenesis and osteogenesis. In conclusion, the placenta is a practical source of stem cells for the treatment of OI.

  16. Microarray-bioinformatics analysis of altered genomic expression profiles between human fetal and infant myocardium

    Institute of Scientific and Technical Information of China (English)

    KONG Bo; LIU Ying-long; L(U) Xiao-dong

    2008-01-01

    Background The physiological differences between fetal and postnatal heart have been well characterized at the cellular level. However, the genetic mechanisms governing and regulating these differences have only been partially elucidated. Elucidation of the differentially expressed genes profile before and after birth has never been systematically proposed and analyzed.Methods The human oligonuclectide microarray and bioinformatics analysis approaches were applied to isolate and classify the differentially expressed genes between fetal and infant cardiac tissue samples. Quantitative real-time PCR was used to confirm the results from the microarray.Results Two hundred and forty-two differentially expressed genes were discovered and classified into 13 categories, including genes related to energy metabolism, myocyte hyperplasia, development, muscle contraction, protein synthesis and degradation, extraceUular matrix components, transcription factors, apoptosis, signal pathway molecules, organelle organization and several other biological processes. Moreover, 95 genes were identified which had not previously been reported to be expressed in the heart.Conclusions The study systematically analyzed the alteration of the gene expression profile between the human fetal and infant myocardium. A number of genes were discovered which had not been reported to be expressed in the heart. The data provided insight into the physical development mechanisms of the heart before and after birth.KONG Bo and LU Xiao-dong contributed equally to this study.

  17. The Effect of Simvastatin on Infection-Induced Inflammatory Response of Human Fetal Membranes.

    Science.gov (United States)

    Basraon, Sanmaan K; Costantine, Maged M; Saade, George; Menon, Ramkumar

    2015-07-01

    Inflammatory cytokines and matrix metalloproteinases (MMPs) contribute to preterm labor pathophysiology. The objective of this study was to test anti-inflammatory properties of simvastatin in human fetal membranes exposed to lipopolysaccharide (LPS). Normal term human fetal membrane explants (n = 11) were allocated to one of the six study groups: control, LPS only (100 ng/mL), simvastatin only (125 ng/mL), simvastatin given 6 hrs prior to LPS (S-L), simvastatin given 6 hrs post-LPS (L-S), and simvastatin and LPS given simultaneously (L+S). Explants were incubated for 24 hrs. Multiplex ELISA for cytokines: IL-1β, IL-6, IL-10, and TNF-α; soluble cytokine receptors: sIL-1R2, sIL-6R, sTNFR1, and R2; MMPs (1, 2, 7, 9, and 10); and tissue inhibitor of metalloproteinase-2 (TIMP-2) was performed on tissue culture supernatants. Pairwise comparison between different groups was conducted by least square mean estimates. Compared with controls, LPS stimulation increased cytokine production and their tissue bioavailability (measured as the molar ratio of cytokine to its soluble receptor), thus confirming membrane immune reactivity (P fetal inflammatory response associated with infection-induced preterm birth. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. 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

  19. Trans—acting factors from the human fetal liver binding to the human ε—globin gene silencer

    Institute of Scientific and Technical Information of China (English)

    YANZHIJIANG; CHUJIANG; 等

    1997-01-01

    The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled,in part,by the silencer (-392bp- -177bp) upstream of this gene.In order to elucidate its role,the nuclear extract from the human fetal liver has been prepared and the interactions between trans-acting factors and this silencer element have been examined.By using DNaseI footprinting assay,a major protected region from -278bp to -235bp within this silencer element was identified.Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MV≈32,28,26 and 22kD) in the nuclear extract isolated from the human fetal liver,which could specifically bind to this region.Our results suggested that these trans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at the fetal stage through the interactions with this silencer.

  20. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

    Science.gov (United States)

    Mold, Jeff E; Venkatasubrahmanyam, Shivkumar; Burt, Trevor D; Michaëlsson, Jakob; Rivera, Jose M; Galkina, Sofiya A; Weinberg, Kenneth; Stoddart, Cheryl A; McCune, Joseph M

    2010-12-17

    Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

  1. Programmed cell death in developing human fetal CNS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The spatial and temporal distributions of programmed cell death (PCD) in developing central nervous system (CNS) of human fetuses ranging from 12 to 39 weeks of gestation were investigated using techniques of flow cytometry and terminal transferase-mediated nick end labeling (TUNEL). The results showed that PCD did occur in every representative brain region of all fetuses examined in different stages. It was found that there were two peaks of PCD appearing at the 12th and 39th weeks respectively, which suggested that the first peak of apoptosis may be involved in the selective elimination of neurons overproduced during the early development and the second may play an important role in establishing the correct neuronal circuitry.

  2. Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts

    Directory of Open Access Journals (Sweden)

    Dao Kim-Hien T

    2010-01-01

    Full Text Available Abstract Background The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. Methods Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. Results Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. Conclusion(s The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas

  3. Ultrastructural visualization of the Mesenchymal-to-Epithelial Transition during reprogramming of human fibroblasts to induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    M.K. Høffding

    2015-01-01

    Here, we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming, as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6–12 days after electroporation, in synchrony with the upregulation of early pluripotency markers, and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT which takes place during mammalian gastrulation.

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

    Directory of Open Access Journals (Sweden)

    María Questa

    2016-03-01

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

  5. Relocalization of cell adhesion molecules during neoplastic transformation of human fibroblasts.

    Science.gov (United States)

    Belgiovine, Cristina; Chiodi, Ilaria; Mondello, Chiara

    2011-11-01

    Studying neoplastic transformation of telomerase immortalized human fibroblasts (cen3tel), we found that the transition from normal to tumorigenic cells was associated with the loss of growth contact inhibition, the acquisition of an epithelial-like morphology and a change in actin organization, from stress fibers to cortical bundles. We show here that these variations were paralleled by an increase in N-cadherin expression and relocalization of different adhesion molecules, such as N-cadherin, α-catenin, p-120 and β-catenin. These proteins presented a clear membrane localization in tumorigenic cells compared to a more diffuse, cytoplasmic distribution in primary fibroblasts and non-tumorigenic immortalized cells, suggesting that tumorigenic cells could form strong cell-cell contacts and cell contacts did not induce growth inhibition. The epithelial-like appearance of tumorigenic cells did not reflect a mesenchymal-epithelial transition; in fact, cen3tel cells expressed vimentin and did not express cytokeratins at all transformation stages. Moreover, they did not express epithelial proteins such as occluding and claudin-1. In contrast, ZO-1 showed higher levels and a more defined membrane localization in tumorigenic cells compared to non-tumorigenic cells; this confirms its role in adherens junction formation in mesenchymal cells and is in agreement with the strong cell-cell contact formation by neoplastically transformed cells. Finally, we found α-catenin and ZO-1 nuclear localization in non-transformed cells, suggestive of possible additional roles of these proteins besides cell junction formation.

  6. 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.

  7. Donor's age and replicative senescence favour the in-vitro mineralization potential of human fibroblasts.

    Science.gov (United States)

    Boraldi, Federica; Bartolomeo, Angelica; Di Bari, Caterina; Cocconi, Andrea; Quaglino, Daniela

    2015-12-01

    Aberrant mineralization of soft connective tissues (ectopic calcification) may occur as a frequent age-related complication. Still, it remains unclear the role of mesenchymal cell donor's age and of replicative senescence on ectopic calcification. Therefore, the ability of cells to deposit in-vitro hydroxyapatite crystals and the expression of progressive ankylosis protein homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), tissue non specific alkaline phosphatase (TNAP) and osteopontin (OPN) have been evaluated in human dermal fibroblasts derived from neonatal (nHDF) and adult (aHDF) donors (ex-vivo ageing model) or at low and high cumulative population doublings (CPD) up to replicative senescence (in-vitro ageing model). This study demonstrates that: 1) replicative senescence favours hydroxyapatite formation in cultured fibroblasts; 2) donor's age acts as a major modulator of the mineralizing potential of HDF, since nHDF are less prone than aHDF to induce calcification; 3) donor's age and replicative senescence play in concert synergistically increasing the calcification process; 4) the ANKH+ENPP1/TNAP ratio, being crucial for pyrophosphate/inorganic phosphate balance, is greatly influenced by donor's age, as well as by replicative senescence, and regulates mineral deposition; 5) OPN is only modulated by replicative senescence.

  8. The effect of laser-treated titanium surface on human gingival fibroblast behavior.

    Science.gov (United States)

    Baltriukienė, D; Sabaliauskas, V; Balčiūnas, E; Melninkaitis, A; Liutkevičius, E; Bukelskienė, V; Rutkūnas, V

    2014-03-01

    Surface modification, as a means of enhancing soft tissue integration in titanium would have significant advantages including less marginal bone resorption, predictable esthetic outcome, improved soft tissue stability, and seal against bacterial leakage. The aim of this study was to evaluate the effects of laser-roughened titanium surfaces on human gingival fibroblast (HGF) viability, proliferation, and adhesion. Titanium discs were ablated with impulse laser in four different patterns. Polished and sand-blasted titanium discs were used as control groups. Specimen surface properties were determined using optical profilometry and scanning electron microscopy. HGF behavior on modified surfaces was analyzed using cell adhesion, viability, proliferation, and ELISA assays. Results suggested that modified Ti surfaces did not affect the viability of HGFs and improved adhesion was measured in laser treatment groups after 24 h. However, proliferation study showed that the adsorbance of fibroblast cells after 72 h cultured on polished titanium was higher and comparable with that of control cells. As for focal adhesion kinase (FAK), cells grown on laser modified surfaces had higher expression of FAK as compared with polished titanium. In conclusion, tested laser-treated surfaces seem to favor HGF adhesion. There were no significant differences between different laser treatment groups. Copyright © 2013 Society of Plastics Engineers.

  9. Deficient recovery from potentially lethal damage in some gamma-irradiated human fibroblast cell strains

    Energy Technology Data Exchange (ETDEWEB)

    Arlett, C.F.; Priestley, A. (Medical Research Council, Brighton (UK). Cell Mutation Unit)

    1984-01-01

    The repair of potentially lethal damage following treatment with gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0.5% foetal calf serum. Normal cells were found to be competent in the repair of PLD. Ataxia-telangiectasia cells were deficient as was a heterozygote suggesting that a failure to repair PLD may make it possible to detect such heterozygotes. Fibroblasts from Huntington's disease patients were either slightly or no more sensitive than cells from normal individuals. Cultures from two individuals in the former class showed limited capacity to repair PLD but cells from the latter class were as competent as normals. Thus assays of radiosensitivity where conditions allow for the repair of PLD may maximise small differences in sensitivity. Cells taken from three patients suffering from Basal Cell Naevus Syndrome were also shown to be defective in the repair of PLD. The existence of such a defect may be related to the increased frequency of basal cell cancer observed in exposed fields following irradiation of such individuals.

  10. Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Brydone, Alistair S; Dominic Meek, R M [Department of Orthopaedics, Southern General Hospital, 1345 Govan Road, Glasgow G51 4TF (United Kingdom); Dalby, Matthew J; Berry, Catherine C; McNamara, Laura E, E-mail: alibrydone@gmail.com [Centre for Cell Engineering, Joseph Black Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ (United Kingdom)

    2011-06-15

    Extracellular matrix (ECM) remodelling is an essential physiological process in which matrix-metalloproteinases (MMPs) have a key role. Manipulating the manner in which cells produce MMPs and ECMs may enable the creation of a desired tissue type, i.e. effect repair, or the prevention of tissue invasion (e.g. metastasis). The aim of this project was to determine if culturing fibroblasts on grooved topography altered collagen deposition or MMP production. Human fibroblasts were seeded on planar or grooved polycaprolactone substrates (grooves were 12.5 {mu}m wide with varying depths of 240 nm, 540 nm or 2300 nm). Cell behaviour and collagen production were studied using fluorescence microscopy and the spent culture medium was assessed using gel zymography to detect MMPs. Total collagen deposition was high on the 240 nm deep grooves, but decreased as the groove depth increased, i.e. as cell contact guidance decreased. There was an increase in gelatinase on the 2300 nm deep grooved topography and there was a difference in the temporal expression of MMP-3 observed on the planar surface compared to the 540 nm and 2300 nm topographies. These results show that topography can alter collagen and MMP production. A fuller understanding of these processes may permit the design of surfaces tailored to tissue regeneration e.g. tendon repair.

  11. [Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts].

    Science.gov (United States)

    Paco, Karen; Ponce-Soto, Luis Alberto; Lopez-Ilasaca, Marco; Aguilar, José L

    2016-01-01

    To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

  12. Attachment and growth behaviour of human gingival fibroblasts on titanium and zirconia ceramic surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Pae, Ahran; Kim, Hyeong-Seob; Woo, Yi-Hyung [Department of Prosthodontics, School of Dentistry, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Lee, Heesu [Department of Oral Anatomy, School of Dentistry, Kangnung National University, Gibyun-dong, Kangnung 210-702 (Korea, Republic of); Kwon, Yong-Dae, E-mail: ahranp@hotmail.co, E-mail: nightsu@kangnung.ac.k, E-mail: odontopia@khu.ac.k, E-mail: yongdae.kwon@gmail.co, E-mail: yhwoo@khu.ac.k [Department of Oral and Maxillofacial Surgery, School of Dentistry, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)

    2009-04-15

    The attachment, growth behaviour and the genetic effect of human gingival fibroblasts (HGF) cultured on titanium and different zirconia surfaces were investigated. HGF cells were cultured on (1) titanium discs with a machined surface, (2) yttrium-stabilized tetragonal zirconia polycrystals (Y-TZP) with a smooth surface and (3) Y-TZP with 100{mu}m grooves. The cell proliferation activity was evaluated through a MTT assay at 24 h and 48 h, and the cell morphology was examined by SEM. The mRNA expression of integrin-beta1, type I and III collagen, laminin and fibronectin in HGF were evaluated by RT-PCR after 24 h. From the MTT assay, the mean optical density values for the titanium and grooved zirconia surfaces after 48 h of HGF adhesion were greater than the values obtained for the smooth zirconia surfaces. SEM images showed that more cells were attached to the grooves, and the cells appeared to follow the direction of the grooves. The results of RT-PCR suggest that all groups showed comparable fibroblast-specific gene expression. A zirconia ceramic surface with grooves showed biological responses that were comparable to those obtained with HGF on a titanium surface.

  13. Regulation of hypoxia-inducible factor-1α in human buccal mucosal fibroblasts stimulated with arecoline

    Directory of Open Access Journals (Sweden)

    Yung-Chuan Ho

    2017-06-01

    Full Text Available Hypoxia-inducible factor (HIF-1α is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare HIF-1α expression from fibroblasts derived from human normal buccal mucosa and oral submucous fibrosis (OSF specimens and further to explore the potential mechanisms that may lead to induce HIF-1α expression. OSF buccal mucosal fibroblasts (BMFs demonstrated significantly higher HIF-1α mRNA expression than normal BMFs (p<0.005. Arecoline, the major areca nut alkaloid, was also found to elevate HIF-1α mRNA expression in a dose-dependent manner (p<0.05. Moreover, arecoline-induced HIF-1α expression was downregulated by mitogen-activated protein kinase inhibitor U0126, phosphatidylinositol 3-kinase inhibitor LY294002, p38 inhibitor SB203580, cyclooxygenase-2 inhibitor NS-398, and glutathione precursor N-acetyl-L-cysteine (p<0.05. Taken together, hypoxia plays an important role in the pathogenesis of areca quid chewing-associated OSF. These pharmacological agents may be further used as chemoprevention agents for OSF.

  14. Arecoline-stimulated connective tissue growth factor production in human buccal mucosal fibroblasts: Modulation by curcumin.

    Science.gov (United States)

    Deng, Yi-Ting; Chen, Hsin-Ming; Cheng, Shih-Jung; Chiang, Chun-Pin; Kuo, Mark Yen-Ping

    2009-09-01

    Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). We immunohistochemically examined the expression of CTGF protein in 20 cases of OSF and found positive CTGF staining in fibroblasts and endothelial cells in all cases. Western blot analysis showed that arecoline, a main alkaloid found in AN, stimulated CTGF synthesis in a dose- and time-dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of CTGF during AN chewing may enhance the fibrotic activity in OSF and play a role in the pathogenesis of OSF. Pretreatment with NF-kappaB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580 and antioxidant N-acetyl-l-cysteine, but not ERK inhibitor PD98059, significantly reduced arecoline-induced CTGF synthesis. Furthermore, curcumin completely inhibited arecoline-induced CTGF synthesis and the inhibition is dose-dependent. These results indicated that arecoline-induced CTGF synthesis was mediated by ROS, NF-kappaB, JNK, P38 MAPK pathways and curcumin could be a useful agent in controlling OSF.

  15. Photocytotoxicity in human dermal fibroblasts elicited by permanent makeup inks containing titanium dioxide.

    Science.gov (United States)

    Wamer, Wayne G; Yin, Jun-Jie

    2011-01-01

    Titanium dioxide (TiO2) is a pigment widely used in decorative tattoo and permanent makeup inks. However, little is known about the risks associated with its presence in these products. We have developed an in vitro assay to identify inks containing TiO2 that are cytotoxic and/or photocytotoxic. The presence of TiO2 in ten permanent makeup inks was established by X-ray fluorescence. Using X-ray diffraction, we found that seven inks contained predominately TiO2 (anatase), the more photocatalytically active crystalline form of TiO2. The remaining inks contained predominately TiO2 (rutile). To identify cytotoxic and/or photocytotoxic inks, human dermal fibroblasts were incubated for 18 h in media containing inks or pigments isolated from inks. Fibroblasts were then irradiated with 10 J/cm2 UVA radiation combined with 45 J/cm2 visible light for determining photocytotoxicity, or kept in the dark for determining cytotoxicity. Toxicity was assessed as inhibition of colony formation. No inks were cytotoxic. However eight inks, and the pigments isolated from these inks, were photocytotoxic. Using ESR, we found that most pigments from photocytotoxic inks generated hydroxyl radicals when photoexcited with UV radiation. Therefore, the possibility of photocytotoxicity should be considered when evaluating the safety of permanent makeup inks containing TiO2.

  16. Accelerated Telomere Shortening and Replicative Senescence in Human Fibroblasts Overexpressing Mutant and Wild Type Lamin A

    Science.gov (United States)

    Huang, Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectible WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. PMID:17870066

  17. Effect of Cyclosporin A and Angiotensin II on cytosolic calcium levels in primary human gingival fibroblasts

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    Ajitkumar Supraja

    2016-01-01

    Full Text Available Background: To evaluate the effect of Cyclosporin A (CsA and angiotensin II (Ang II on cytosolic calcium levels in cultured human gingival fibroblasts (HGFs. Materials and Methods: Healthy gingival samples from six volunteers were obtained, and primary HGFs were cultured. Cell viability and proliferation assay were performed to identify the ideal concentrations of CsA and Ang II. Cytosolic calcium levels in cultured gingival fibroblasts treated with CsA and Ang II were studied using colorimetric assay, confocal and fluorescence imaging. Statistical analyses were done using SPSS software and GraphPad Prism. Results: Higher levels of cytosolic levels were evident in cells treated with CsA and Ang II when compared to control group and was statistically significant (P < 0.05 in both colorimetric assay and confocal imaging. Fluorescent images of the cultured HGFs revealed the same. Conclusion: Thus calcium being a key player in major cellular functions, plays a major role in the pathogenesis of drug-induced gingival overgrowth.

  18. 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.

  19. Effects of PBM in different energy densities and irradiance on maintaining cell viability and proliferation of pulp fibroblasts from human primary teeth.

    Science.gov (United States)

    Marques, Nádia Carolina Teixeira; Neto, Natalino Lourenço; Prado, Mariel Tavares Oliveira; Vitor, Luciana Lourenço Ribeiro; Oliveira, Rodrigo Cardoso; Sakai, Vivien Thiemy; Santos, Carlos Ferreira; Machado, Maria Aparecida Andrade Moreira; Oliveira, Thais Marchini

    2017-08-11

    This study aimed to compare the effects of photobiomodulation (PBM) in different energy densities and irradiances on maintaining cell viability, and proliferation of pulp fibroblasts from human primary teeth (HPF) were cultured in DMEM and used between the fourth and eighth passages. Then, HPF were irradiated with the following different energy densities: 1.25 J/cm(2) (a), 2.50 J/cm(2) (b), 3.75 J/cm(2) (c), 5.00 J/cm(2) (d), and 6.25 J/cm(2) (e); but varying either the time of irradiation (groups 1a-1e) or the output power (groups 2a-2e). Positive (groups 1f and 2f) and negative controls (groups 1g and 2g), respectively, comprised non-irradiated cells grown in regular nutritional conditions (10% fetal bovine serum [FBS]) and under nutritional deficit (1% FBS). Cell viability and proliferation were respectively assessed through MTT and crystal violet (CV) assays at 24, 48, and 72 h after irradiation. Statistical analysis was performed by two-way ANOVA, followed by Tukey test (P < 0.05). The negative controls showed significantly lower viability in relation to most of the corresponding subgroups, both for MTT and CV assays. For both assays, the intragroup comparison showed that the periods of 24 h exhibited lower viability than the periods of 48 and 72 h for most of the subgroups, except the negative controls with lower viability. The different irradiation protocols (equal energy densities applied with different irradiances) showed no statistically significant differences on cell viability and proliferation at the evaluated periods. The proposed PBM in different energy densities and irradiance did not affect the viability and proliferation of pulp fibroblasts from human primary teeth.

  20. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

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    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  1. 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).

  2. Resveratrol Prevents High Fluence Red Light-Emitting Diode Reactive Oxygen Species-Mediated Photoinhibition of Human Skin Fibroblast Migration.

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    Andrew Mamalis

    Full Text Available Skin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease.The goal of our study was to investigate the how reactive oxygen species (ROS free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed.High fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR. For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2 on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2.High fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158.4% relative to matched

  3. Resveratrol Prevents High Fluence Red Light-Emitting Diode Reactive Oxygen Species-Mediated Photoinhibition of Human Skin Fibroblast Migration.

    Science.gov (United States)

    Mamalis, Andrew; Koo, Eugene; Isseroff, R Rivkah; Murphy, William; Jagdeo, Jared

    2015-01-01

    Skin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL) is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease. The goal of our study was to investigate the how reactive oxygen species (ROS) free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed. High fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR). For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2) on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2. High fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158.4% relative to matched controls

  4. Cytokeratin (CK5, CK8, CK14) expression and presence of progenitor stem cells in human fetal thymuses.

    Science.gov (United States)

    Gupta, Richa; Gupta, Tulika; Kaur, Harjeet; Sehgal, Shobha; Aggarwal, Anjali; Kapoor, Kanchan; Sharma, Anshu; Sahni, Daisy; Singla, Suhalika

    2016-09-01

    The aim of the current study was to observe the expression of cytokeratins in human fetal thymuses. Specific cytokeratin markers in adult humans and mice have been well described but there has been little similar work on human fetuses. We also aimed to see whether progenitor stem cells that could be harvested to treat various immunodeficiency disorders are present in fetal thymic tissue. Thymuses obtained from 30 aborted human fetuses (12 to 31 weeks) were examined immunohistochemically to investigate changes in cytokeratin expression in the epithelial cells (TEC) at various gestational ages. Before 16 weeks of gestation, cortical (cTEC) and medullary (mTEC) TEC exhibited homogenous staining for cytokeratins CK8 and CK5. After 16 weeks there was differential staining, with cTEC positive for CK8 and mTEC for CK5 and CK14. Interestingly, both CK5 + CK8+ progenitor stem cells were present in the fetal thymic cortex at all gestational ages, with a relatively high number from 12 to 16 weeks. Cytokeratin expression in fetal thymuses was quite different from that in the adult thymus owing to the presence of undifferentiated progenitor stem cells in fetal thymic stroma along with differentiated TEC. The best time to harvest these progenitor stem cells from fetal thymic stroma in order to treat various immune deficiency disorders appears to be 12-16 weeks. Clin. Anat. 29:711-717, 2016. © 2016 Wiley Periodicals, Inc.

  5. Inner ring deiodination of thyroxine and 3,5,3'-triiodothyronine by human fetal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Roti, E.; Fang, S.L.; Green, K.; Braverman, L.E.; Emerson, C.H.

    1983-12-01

    Indirect evidence, based on injection of thyroxine (T4) into the amniotic cavity of humans, and maternal thyroidectomy in the rat, suggests that fetal membranes might be capable of converting T4 to 3,3',5'-triiodothyronine (rT3) by virtue of inner ring iodothyronine deiodinase activity. The present study was undertaken to provide direct evidence that human fetal membranes contain inner ring iodothyronine deiodinase activity directed toward T4 and 3,5,3'-triiodothyronine (T3). Homogenates of human fetal membranes were incubated with 125I-labeled T4, rT3, and T3, and with stable T4. Conversion of 125(I)-T4 to 125(I)-rT3 was noted in chorion and amnion. 125I-T3 was converted to 125(I)-3,3'-diiodothyronine (T2) in chorion and amnion. 125(I)-rT3 was stable in fetal membranes under the incubation conditions employed. Time-, temperature-, pH-, and protein content-dependent conversion of stable T4 to rT3 was found in fetal membranes. Iodothyronine metabolism did not occur in the absence of dithiothreitol. These studies indicate that human fetal membranes contain an inner ring deiodinase enzyme. Because of its intimate contact with the amniotic cavity, this enzyme may generate a portion of the rT3 found in amniotic fluid.

  6. Ontological differences in first compared to third trimester human fetal placental chorionic stem cells.

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    Gemma N Jones

    Full Text Available Human mesenchymal stromal/stem cells (MSC isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC. We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.

  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 t