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Sample records for fibroblastic cell lines

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

    PubMed Central

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

    2015-01-01

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

  2. Fibroblast cell line establishment, cryopreservation and interspecies embryos reconstruction in red panda ( Ailurus fulgens).

    PubMed

    Tao, Yong; Liu, Jianming; Zhang, Yunhai; Zhang, Meiling; Fang, Junshun; Han, Wei; Zhang, Zhizhong; Liu, Ya; Ding, Jianping; Zhang, Xiaorong

    2009-05-01

    In evolution, the red panda (Ailurus fulgens) plays a pivotal role in the higher level phylogeny of arctoides carnivore mammals. The red panda inhabits certain Asian countries only and its numbers are decreasing. Therefore, the development of feasible ways to preserve this species is necessary. Genetic resource cryopreservation and somatic cell nuclear transfer (SCNT) have been used extensively to rescue this endangered species. The present study describes the establishment, for the first time, of a red panda ear fibroblast cell line, which was then cryopreserved, thawed and cultured. Through micromanipulation, interspecies embryos were reconstructed using the cryopreserved-thawed fibroblasts of the red panda as the donor and rabbit oocytes as recipients. A total of 194 enucleated rabbit oocytes were reconstructed with red panda ear fibroblasts; enucleated oocytes were activated without fusion as the control. The results show that the fibroblast cell line was established successfully by tissue culture and then cryopreserved in liquid nitrogen. Supplementation with 20% fetal bovine serum and 8% dimethyl sulphoxide in basic medium facilitated the cryopreservation. The interspecies embryos were successfully reconstructed. The cleavage, morulae and blastocyst rates after in vitro culture were 71, 47 and 23% (31/194), respectively. This study indicated that a somatic cell line could be established and cryopreserved from red panda and that rabbit cytoplast supports mitotic cleavage of the red panda karyoplasts and is capable of reprogramming the nucleus to achieve blastocysts.

  3. Episomal-based generation of an iPS cell line from human fetal foreskin fibroblasts.

    PubMed

    Matz, Peggy; Adjaye, James

    2016-01-01

    Human fetal foreskin fibroblasts (HFF1) were used to generate the iPSC line epiHFF1-B1 employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC, and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHFF1-B1 and the human embryonic stem cell line-H1 have a pearson correlation of 0.936.

  4. Isolation and characterization of flat revertant cell lines from A-MuLV-transformed fibroblasts.

    PubMed

    Glass, D J; Rees-Jones, R W; Goff, S P

    1990-01-01

    Transformation of lymphoid and fibroblastic cells by Abelson murine leukemia virus (A-MuLV) is mediated by the viral tyrosine protein kinase. We do not yet know the important target proteins in the cell, the host proteins that modulate the kinase activity, or the host proteins involved in the signal-transduction pathway ultimately leading to altered patterns of cell growth. As a first step toward identifying these host proteins, we have isolated and characterized several flat revertant cell lines from transformed lines carrying v-abl. Clonal transformed cell lines used as parental strains were prepared by infecting Rat-2 fibroblasts with A-MuLV, using M-MuLV as helper. A rhodamine dye screening procedure was used to obtain three clones of morphologically flat revertant cells. Each of the three lines was non-refractile and contact inhibited. All the lines retained a transformation-competent copy of A-MuLV; all released high titers of virus capable of inducing foci on previously uninfected Rat-2 cells. Analyses of the revertant lines suggest that diverse mechanisms can lead to loss of transformed morphology.

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

    PubMed

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

    2015-10-01

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

  6. Prevalent expression of fibroblast growth factor (FGF) receptors and FGF2 in human tumor cell lines.

    PubMed

    Chandler, L A; Sosnowski, B A; Greenlees, L; Aukerman, S L; Baird, A; Pierce, G F

    1999-05-05

    Basic fibroblast growth factor (FGF2) has potent mitogenic and angiogenic activities that have been implicated in tumor development and malignant progression. The biological effects of FGF2 and other members of the FGF ligand family are mediated by 4 transmembrane tyrosine kinase receptors (FGFRs). To better understand the roles of FGFRs in cancer, the expression of FGF2 and each of the 4 FGFRs was assessed by RNase protection analysis of 60 human tumor cell lines, representing 9 tumor types. Expression of at least one FGFR isoform was detected in 90% and FGF2 mRNA in 35% of the cell lines. Our comprehensive analysis of FGF2 and FGFR expression in human tumor cell lines provides evidence that FGF signaling pathways are active in a majority of human tumor cell lines, and lends support to the development of anti-tumor strategies that target FGFRs.

  7. In vitro study for laser gene transfer in BHK-21 fibroblast cell line

    NASA Astrophysics Data System (ADS)

    Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

    2009-02-01

    Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated

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

    PubMed

    Singh, Mahipal; Sharma, Anil K

    2011-02-01

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

  9. Generation of human iPS cell line SKiPSc1 from healthy Human Neonatal Foreskin Fibroblast cells.

    PubMed

    Alawad, Abdullah; Alhazzaa, Othman; Altuwaijri, Saleh; Alkhrayef, Mohammad; Alagrafi, Faisal; Alhamdan, Ziyad; Alenazi, Abdullah; Alharbi, Sultan; Hammad, Mohamed

    2016-06-25

    The SKiPSc1 induced pluripotent stem (iPS) cell line was generated from Human Neonatal Foreskin Fibroblasts (HNFFs) obtained from a healthy donor infant that were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

  10. Establishment and cryopreservation of a fibroblast cell line derived from Bengal tiger (Panthera tigris tigris).

    PubMed

    Guan, W J; Liu, C Q; Li, C Y; Liu, D; Zhang, W X; Ma, Y H

    2010-01-01

    The Bengal tiger ear marginal tissue fibroblasts cell line (BTF22), containing 157 tubes of frozen cells, was successfully established by using primary explants technique and cell cryoconservation technology. Biological analysis showed that the population doubling time (PDT) for revival cells was approximately 28 h. Measurement of LDH and MDH isoenzymes showed no cross-contamination among the cells. Karyotyping showed that the frequency of cells with chromosome number 2n = 38 was 90.6-92.2%. Tests for bacteria, fungi, viruses and mycoplasma were negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 4.4% and 31.9%. Every index of the BTF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). Not only has the germline of this important Bengal tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform would provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2015-04-24

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

  13. In vitro myogenic and adipogenic differentiation model of genetically engineered bovine embryonic fibroblast cell lines.

    PubMed

    Yin, Jinlong; Jin, Xun; Beck, Samuel; Kang, Dong Ho; Hong, Zhongshan; Li, Zhehu; Jin, Yongcheng; Zhang, Qiankun; Choi, Yun-Jaie; Kim, Sung-Chan; Kim, Hyunggee

    2010-02-01

    Our current understanding of muscle and adipose tissue development has been largely restricted to the study of murine myogenic and adipogenic cell lines, since attempts to establish these cell lines from other species have met with only limited success. Here we report that a spontaneously immortalized bovine embryonic fibroblast cell line (BEFS) undergoes differentiation into adipogenic or myogenic lineages when ectopically transduced with PPARgamma2 (an adipogenic lineage determinant) or MyoD (a myogenic lineage determinant) and grown in adipogenic and myogenic differentiation culture media (ADCM and MDCM, respectively). We also found that PPARgamma2-overexpressing BEFS cells (BEFS-PPARgamma2) grown in ADCM with or without the PPARgamma2 ligand, troglitazone, preferentially differentiate into adipogenic cells in the presence of ectopic MyoD expression. Ectopic expression of PPARgamma2 in the inducible MyoD-overepxressing BEFS cells (BEFS-TetOn-MyoD) completely suppresses myogenic differentiation and leads to a significant increase in adipogenic differentiation, suggesting that the adipogenic differentiation program might be dominant. Therefore, BEFS, BEFS-PPARgamma2, and BEFS-TetOn-MyoD would be a valuable biological model for understanding a fundamental principle underlying myogenic and adipogenic development, and for isolating various genetic and chemical factors that enable muscle and adipocyte differentiation.

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

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

    PubMed

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

    2013-05-01

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

  16. Increased liposome-mediated gene transfer into haematopoietic cells grown in adhesion to stromal or fibroblast cell line monolayers.

    PubMed

    Marit, G; Cao, Y; Froussard, P; Ripoche, J; Dupouy, M; Elandaloussi, A; Lacombe, F; Mahon, F X; Keller, H; Pla, M; Reiffers, J; Theze, J

    2000-01-01

    We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.

  17. Differential regulation of papilloma virus early gene expression in transformed fibroblasts and carcinoma cell lines.

    PubMed Central

    Kleiner, E; Dietrich, W; Pfister, H

    1986-01-01

    Treatment of bovine papilloma virus (BPV) 1-transformed mouse fibroblasts with cycloheximide led to a 10-fold increase in the amount of viral transcripts, after as little as 1 h of protein synthesis inhibition. Northern blots revealed no qualitative changes in the RNA pattern. Nuclear run-on experiments showed about a 7-fold increase in specific transcriptional activity after cycloheximide treatment. The half-life of BPV1 mRNA was twice as long as in untreated controls. These results indicate that both RNA synthesis and degradation of viral RNA are controlled by labile proteins. Cycloheximide stimulation turned out to be independent of the BPV1 E2 gene activity which enhances viral transcription. Cycloheximide treatment had no effect on the amount of human papilloma virus (HPV) 18 transcripts in cervical carcinoma derived HeLa and C4-1 cells. Transcription of HPV16 in the cervical carcinoma line SiHa was likewise unaffected. The differential regulation of transcription in transformed fibroblasts and cancer-derived cells, and the significance for malignant conversion are discussed. Images Fig. 1. Fig. 3. Fig. 4. Fig. 6. PMID:3019673

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

    PubMed

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

    2012-12-01

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

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

    PubMed

    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 < 0.05). After transferring cloned 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.

  20. Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line

    PubMed Central

    1985-01-01

    The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle- specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary- derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place. PMID:3988800

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols.

    PubMed

    Mei, Xin; Wu, Yuan-Yuan; Mao, Xiao; Tu, You-Ying

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene.

  3. Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models.

    PubMed

    Samluk, Anna; Zakrzewska, Karolina Ewa; Pluta, Krzysztof Dariusz

    2013-07-01

    Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.

  4. Genome-wide expression analysis in fibroblast cell lines from probands with Pallister Killian syndrome.

    PubMed

    Kaur, Maninder; Izumi, Kosuke; Wilkens, Alisha B; Chatfield, Kathryn C; Spinner, Nancy B; Conlin, Laura K; Zhang, Zhe; Krantz, Ian D

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2010-01-01

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

  7. A comparative evaluation of photo-toxic effect of fractionated melanin and chlorpromazine hydrochloride on human (dermal fibroblasts and epidermal keratinocytes) and mouse cell line/s (fibroblast Balb/c 3T3).

    PubMed

    Rai, V; Dayan, N; Michniak-Kohn, B

    2011-03-01

    Fractionated melanin (Mel-HEV), a bleached version of natural melanin, offers protection against the high energy visible (HEV/UVA) and ultraviolet (specifically UVA) irradiation making it a potential compound to be added to skin care and sunscreen formulations and other cosmetic and personal care products. Chlorpromazine (CPZ) has been shown to exhibit photosensitivity and phototoxicity reaction in vitro and in vivo. Comparative evaluation of chemotoxicity and phototoxicity using Mel-HEV and CPZ (as positive control) was performed on mouse fibroblast cell line 'Balb/c 3T3'. This is the recommended method for evaluating the phototoxic potential of compounds under the European Center of Validation of Alternative Methods (ECVAM) guidelines (OECD, 2004). This study was expanded from a mouse cell line - Balb 3T3/c to two human cell lines - HDF and HEKn for two reasons: to compare the difference between the sensitivity and behavior of two fibroblast cell lines (Balb/c 3T3 vs. HDF) and to compare the differences between two fibroblast cell lines with the keratinocyte cell line (HDF & Balb/c 3T3 vs. HEKn). It was found that Balb/c 3T3 and HEKn were both sensitive to the phototoxic potential of CPZ. However, HDF showed insensitivity to phototoxic evaluation. The test compound, Mel-HEV, was found to be non-phototoxic. The mean toxic concentration (MTC) for CPZ during HEV and UVA exposure conditions was found to be similar using Balb/c 3T3 (36.25 μg/ml) and HEKn (39.99 μg/ml) showing that cells exhibit similar responses at HEV/UVA- conditions. However, Balb/c 3T3 showed more sensitivity to CPZ at HEV/UVA+ condition (MTC=0.87 μg/ml; mean PIF=55.33; MPE=0.395) than HEKn (MTC=5.35 μg/ml; PIF=7.61; MPE=0.276) making it the preferred cell line for phototoxicity evaluations.

  8. Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

    PubMed

    Wu, Shuai Shuai; Li, Dong Jiang; Lv, Chun Rong; Yang, Hong Yuan; Zhu, Lan; Li, Wei Juan; Quan, Guo Bo; Hong, Qiong Hua

    2013-01-01

    In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P < 0.05). In conclusion, the skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in

  9. Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture

    PubMed Central

    2012-01-01

    Introduction The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available. Methods We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs. Results NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank. Conclusions The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine. PMID:22472092

  10. 12p microRNA expression in fibroblast cell lines from probands with Pallister-Killian syndrome.

    PubMed

    Izumi, Kosuke; Zhang, Zhe; Kaur, Maninder; Krantz, Ian D

    2014-12-01

    Pallister-Killian syndrome is a multisystem sporadic genetic diagnosis characterized by facial dysmorphia, variable developmental delay and intellectual impairment, hypotonia, seizures, diaphragmatic hernia, and other systemic abnormalities. Pallister-Killian syndrome is typically caused by the presence of a supernumerary isochromosome that is always present in a tissue limited mosaic pattern, resulting in tetrasomy 12p due to the two extra copies of 12p. We evaluated the potential contribution of microRNAs located on 12p to the pathogenesis of Pallister-Killian syndrome phenotype. Using skin fibroblast cell lines from 13 probands with Pallister-Killian syndrome and 5 normal matched controls, the expression level of 5 microRNAs located on 12p and their target gene mRNA levels were measured. All measured micro RNAs located on 12p were overexpressed in Pallister-Killian syndrome fibroblasts, although the fold difference of the expression level was lower than copy number differences. Among the five microRNAs, miR-1244 had the highest fold difference. Many of computer-predicted target genes of miR-1244 were downregulated in Pallister-Killian syndrome skin fibroblasts. In particular, expression levels of MEIS2 and UQCRB were significantly decreased in Pallister-Killian syndrome samples, and an inverse linear correlation was seen between the level of miR-1244 and MEIS2 and UQCRB expression levels. Since many of computer-predicted miR-1244 target genes play roles in transcriptional regulation, overexpression of miR-1244 due to tetrasomy 12p may contribute to the pleiotropic phenotype of Pallister-Killian syndrome by modulating its downstream target genes including MEIS2 and UQCRB.

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

    PubMed

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

    1990-01-01

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

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

    PubMed

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

    2011-01-01

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

  13. Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids

    SciTech Connect

    Yuan, Z.Q.; Gault, E.A.; Gobeil, P.; Nixon, C.; Campo, M.S.; Nasir, L.

    2008-04-10

    It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independently of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.

  14. Mouse fibroblast L929 cells are less permissive to infection by Nelson Bay orthoreovirus compared to other mammalian cell lines.

    PubMed

    Mok, Lawrence; Wynne, James W; Grimley, Samantha; Shiell, Brian; Green, Diane; Monaghan, Paul; Pallister, Jackie; Bacic, Antony; Michalski, Wojtek P

    2015-07-01

    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.

  15. Synthesis and secretion of transforming growth factor-beta1 by human desmoid fibroblast cell line and its modulation by toremifene.

    PubMed

    Locci, P; Bellocchio, S; Lilli, C; Marinucci, L; Cagini, L; Baroni, T; Giustozzi, G; Balducci, C; Becchetti, E

    2001-11-01

    The present study provides evidence that the in vitro cultured fibroblast cell line from desmoid tumors differs from normal fibrobasts in its extracellular matrix (ECM) macromolecule composition and is modulated by treatment with toremifene, an antiestrogen that reduces tumor mass by an unknown mechanism. The results showed increased transforming growth factor-beta 1 (TGF-beta1) production, TGF-beta1 mRNA expression, and TGF-beta1 receptor number in desmoid fibroblasts compared with normal cells. As desmoid fibroblasts did not produce tumor necrosis factor-alpha (TNF-alpha) but were sensitive to it, which enhanced glycosaminoglycans (GAG) accumulation, we assessed the TGF-beta1 effects on TNF-alpha production by human monocytes. Our results showed TGF-beta1 significantly increased TNF-alpha secretion by monocytes. Toremifene mediated its effects in desmoid fibroblasts via an estrogen receptor-independent pathway. It inhibited GAG accumulation and the secretion of both latent and active forms of TGF-beta1 and had an inhibitory effect on TNF-alpha production by monocytes. Our results suggest that in reducing TGF-beta1 production by desmoid fibroblasts and TNF-alpha production by monocytes, toremifene may restore the balance between the two growth factors.

  16. Novel real-time cell analysis platform for the dynamic monitoring of ionizing radiation effects on human tumor cell lines and primary fibroblasts.

    PubMed

    Mán, Imola; Szebeni, Gábor J; Plangár, Imola; Szabó, Emilia R; Tőkés, Tünde; Szabó, Zoltán; Nagy, Zoltán; Fekete, Gábor; Fajka-Boja, Roberta; Puskás, László G; Hideghéty, Katalin; Hackler, László

    2015-09-01

    Translational research in radiation oncology is important for the detection of adverse radiation effects, cellular responses, and radiation modifications, and may help to improve the outcome of radiation therapy in patients with cancer. The present study aimed to optimize and validate a real‑time label‑free assay for the dynamic monitoring of cellular responses to ionizing radiation. The xCELLigence system is an impedance‑based platform that provides continuous information on alterations in cell size, shape, adhesion, proliferation, and survival. In the present study, various malignant human primary fibroblast cells (U251, GBM2, MCF7, A549, HT‑29) were exposed to 0, 5 and 10 Gy of Cobalt60 radiation. As well as the xCELLigence system, cell survival and proliferation was evaluated using the following conventional end‑point cell‑based methods: Clonogenic, MTS, and lactate dehydrogenase assays, and apoptosis was detected by fluorescence‑activated cell sorting. The effects of ionizing radiation were detected for each cell line using impedance monitoring. The real‑time data correlated with the colony forming assay results. At low cell densities (1,000‑2,000 cells/well) the impedance‑based method was more accurate at monitoring dose‑dependent changes in the malignant human primary fibroblast cell lines, as compared with the end‑point assays. The results of the present study demonstrated that the xCELLigence system may be a reliable and rapid diagnostic method for the monitoring of dynamic cell behavior following radiation. In addition, the xCELLigence system may be used to investigate the cellular mechanisms underlying the radiation response, as well as the time‑dependent effects of radiation on cell proliferation and viability.

  17. Cytotoxicity evaluation of a new fast set highly viscous conventional glass ionomer cement with L929 fibroblast cell line

    PubMed Central

    Ahmed, Hany Mohamed Aly; Omar, Nor Shamsuria; Luddin, Norhayati; Saini, Rajan; Saini, Deepti

    2011-01-01

    Aim: This study aims to evaluate the cytotoxicity of a new fast set highly viscous conventional glass ionomer cement (GIC) with L929 fibroblasts. Materials and Methods: The cement capsule was mixed and introduced into a paraffin wax mould. After setting, the cement was incubated in Dulbecco's Modified Eagle's Medium. Six replicates of the material extract were added to the culture medium in 96-well plates. L929 mouse fibroblast cells were added into the wells and then incubated for 48 h. Dimethylthiazol diphenyltetrazolium bromide test was performed for cytotoxicity evaluation. Results: The results showed that this GIC brand did not yield a half-maximal inhibitory concentration value, IC50, as the cell viability was above 50% at all concentrations. Cell viability over 90% was observed at the concentrations of 3.125 and 1.5625 mg/ml. Maximum concentration of the material showed cell viability of 59.4%. Conclusions: This new fast set highly viscous conventional GIC showed low cytotoxicity to mouse fibroblast cells, and it can be suggested as a substitute for dental cements exhibiting a long setting time. PMID:22144813

  18. Probing pyruvate metabolism in normal and mutant fibroblast cell lines using 13C-labeled mass isotopomer analysis and mass spectrometry.

    PubMed

    Riazi, Roya; Khairallah, Maya; Cameron, Jessie M; Pencharz, Paul B; Des Rosiers, Christine; Robinson, Brian H

    2009-12-01

    Fibroblast cell lines are frequently used to diagnose genetic mitochondrial defects in children. The effect of enzyme deficiency on overall flux rate through metabolic pathways is, however, not generally considered. We have transposed an experimental paradigm that was developed for isolated perfused organs using (13)C-labeled substrates and (13)C-isotopomer analysis to probe pyruvate mitochondrial metabolism in cultured human fibroblast cell lines with normal or genetically mutant pyruvate decarboxylation (PDC) or carboxylation (PC) activity. Cells were incubated with 1mM [U-(13)C]pyruvate, and the (13)C-molar percent enrichment (MPE) of intracellular pyruvate, citrate, malate (as a surrogate of oxaloacetate) and aspartate was assessed by mass spectrometry. We estimated various flux ratios relevant to metabolic pathways involved in energy production, namely pyruvate formation, PDC, PC, and citrate recycling in the citric acid cycle (CAC). In all cell lines, exogenous pyruvate was predominately decarboxylated (PC/PDC ratios 0.01-0.3). PC-deficient cell lines displayed an expected negligible contribution of PC flux to oxaloacetate formation for citrate synthesis (PC/CS), which was associated with a greater contribution of PDC to acetyl-CoA formation (PDC/CS), and greater recycling of (13)C-labeled citrate into the CAC. In PDH-deficient cell lines, metabolic flux alterations were most apparent in cells with more than 50% reduction in enzyme activity. This led to an unexpected lower PC/CS flux ratio, while the PDC/CS flux ratio was unchanged. These data illustrate the usefulness of this approach in identifying unexpected metabolic consequences of genetic defects related to pyruvate metabolism.

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

    PubMed

    Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

    2010-06-01

    Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

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

    PubMed Central

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

    2006-01-01

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

  1. Evaluation of apoptosis induction using PARP cleavage on gastric adenocarcinoma and fibroblast cell lines by different strains of Helicobacter pylori.

    PubMed

    Mojtahedi, Ali; Salehi, Rasoul; Navabakbar, Farahtaj; Tamizifar, Hasan; Tavakkoli, Hamid; Duronio, Vincent

    2007-11-15

    Helicobacter pylori is one of the most common pathogens affecting humans and is the major environmental factor in the development of gastric cancer increasing from 4 to 6 folds the risk of its development. Variations in cancer risk among H. pylori infected individuals may correlate to difference in H. pylori strains, variable host characteristics and specific interactions between host and microbial determinants. To determine the effect of different strains of H. pylori on cellular apoptosis this study was designed an in vitro model using AGS and HEF cell lines. After specified time intervals total cell proteins was extracted and subjected to SDS-PAGE and immunoblotting using anti poly ADP-ribose polymerase (PARP) antibody. Decrease in densitometric value of PARP was indicative of higher level of apoptosis. The ability of apoptosis induction in AGS and HEF cell lines by wild type (cagA+/vacA+), cagA-/vacA+, vacA-/cagA+ and double negative (cagA-/vacA-) strains were significantly different. The assessed apoptosis in AGS cell line co-cultured with wild type strain was 3.22 +/- 0.2 in 24 h, 2.8 +/- 0.1 in 48 and 2.1 +/- 0.09 in 72 h of incubation time. Similar assessment with cagA-/vacA+ strains in AGS cells was 4.17 +/- 1.49 in 24 h, 3.32 +/- 0.45 in 48 h and 2.32 +/- 0.61 in 72 h incubation. A variation in apoptotic potential between the H. pylori strains on two cells (AGS and HEF) was observed. Based on present results, it is concluded that H. pylori strains as well as target cell types are important in pathogenesis and induction of apoptosis during a specified time interval.

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

    PubMed Central

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

    2016-01-01

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

  3. Increasing the susceptibility of the rat 208F fibroblast cell line to radiation-induced apoptosis does not alter its clonogenic survival dose-response.

    PubMed Central

    Aldridge, D. R.; Arends, M. J.; Radford, I. R.

    1995-01-01

    Recent studies have suggested a correlation between the rate and incidence of apoptosis and the radiation response of particular cell lines. However, we found that increasing the rate of induction of apoptosis in the fibroblast line 208F, by transfecting it with human c-myc, did not lead to a change in its clonogenic survival dose-response for either gamma-irradiation or 125I-induced DNA damage. It was also found that expression of mutant (T24) Ha-ras in the 208F line appeared to decrease the level of apoptosis per mitosis after irradiation and inhibited the formation of nucleosomal ladders, but did not affect either the onset of the morphological features of apoptosis or the clonogenic survival dose-response of the cells to either gamma-irradiation or 125I-induced DNA damage. Our findings suggest that it may be incorrect to make predictions about the radiosensitivity of cells based only on knowledge of their mode of death. Images Figure 3 Figure 4 PMID:7880740

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

    PubMed

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

    2016-08-01

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

  5. Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production.

    PubMed

    Hisaoka, Kazue; Tsuchioka, Mami; Yano, Ryoya; Maeda, Natsuko; Kajitani, Naoto; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2011-06-17

    Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway

  6. Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line.

    PubMed Central

    Thomson, T M; Green, S H; Trotta, R J; Burstein, D E; Pellicer, A

    1990-01-01

    A cell line was generated from U7 cells (a subline of PC12 rat pheochromocytoma cells) that contains a stably integrated transforming mouse N-ras (Lys-61) gene under the control of the long terminal repeat from mouse mammary tumor virus. Such cells, designated UR61, undergo neuronal differentiation upon exposure to nanomolar concentrations of dexamethasone, as a consequence of expression of the activated N-ras gene (I. Guerrero, A. Pellicer, and D.E. Burstein, Biochem, Biophys. Res. Commun. 150:1185-1192, 1988). Exposure of UR61 cells to either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) results in a marked induction of c-fos RNA, with kinetics paralleling those of NGF- or bFGF-induced expression of c-fos RNA in PC12 cells. Dexamethasone-induced expression of activated N-ras p21 results in blocking of c-fos RNA induction by NGF or bFGF in a time-dependent manner. Activated N-ras p21-mediated inhibition of c-fos RNA induction in UR61 cells is selective for NGF and bFGF and is not due to selective degradation of c-fos RNA. Normal and transforming N-ras can trans activate the chloramphenicol acetyltransferase gene linked to mouse c-fos regulatory sequences when transient expression assays are performed. Our observations suggest that N-ras p21 selectively interacts with pathways involved in induction of c-fos expression which initiate at the receptors for NGF and bFGF. Images PMID:2108319

  7. Pyropia yezoensis peptide promotes collagen synthesis by activating the TGF-β/Smad signaling pathway in the human dermal fibroblast cell line Hs27

    PubMed Central

    Kim, Cho-Rong; Kim, Young-Min; Lee, Min-Kyeong; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-01-01

    Pyropia yezoensis (P. yezoensis) is a marine algae that exhibits antioxidant, anti-inflammatory, antitumor and anti-aging activities. In this study, we investigated the effects of the P. yezoensis peptide, PYP1-5, on collagen synthesis in the human dermal fibroblast cell line Hs27. Skin aging is related to reduced collagen production and the activities of multiple enzymes, including matrix metalloproteinases (MMPs), which degrade collagen structure in the dermis, and tissue inhibitor of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the action of MMPs. While collagen synthesis is associated with a number of signaling pathways, we examined the increased collagen synthesis via the upregulation of the transforming growth factor-β (TGF-β)/Smad signaling pathway. Using MTS assay, we found that PYP1-5 did not affect cell viability. Moreover, we confirmed that PYP1-5 increased type 1 collagen expression using enzyme-linked immunosorbent assay (ELISA), western blot analysis and quantitative PCR. In addition, we identified changes in various enzymes, as well as the mechanisms behind the PYP1-5-induced collagen synthesis. PYP1-5 decreased the MMP-1 protein and mRNA levels, and increased the TIMP-1 and TIMP-2 protein and mRNA levels. In addition, PYP1-5 activated the TGF-β/Smad signaling pathway, which increased TGF-β1, p-Smad2 and p-Smad3 expression, while inhibiting Smad7, an inhibitor of the TGF-β/Smad pathway. Furthermore, PYP1-5 upregulated transcription factor specificity protein 1 (Sp1) expression, which is reportedly involved in type 1 collagen expression. These findings indicate that PYP1-5 activates the TGF-β/Smad signaling pathway, which subsequently induces collagen synthesis in Hs27 cells. PMID:27878236

  8. Cell motility and local viscoelasticity of fibroblasts.

    PubMed

    Park, S; Koch, D; Cardenas, R; Käs, J; Shih, C K

    2005-12-01

    Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48 +/- 0.51 kPa) and of H-ras transformed fibroblasts (0.42 +/- 0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01 +/- 0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1 +/- 4.5 microm/h for BALB 3T3 fibroblasts, 13.1 +/- 5.2 microm/h for SV-T2 fibroblasts, and 26.2 +/- 11.5 microm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension.

  9. Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

    PubMed Central

    Brown, P C; Beverley, S M; Schimke, R T

    1981-01-01

    Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally. Images PMID:6287217

  10. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    PubMed

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  11. Active compound of Zingiber cassumunar Roxb. down-regulates the expression of genes involved in joint erosion in a human synovial fibroblast cell line.

    PubMed

    Chaiwongsa, Rujirek; Ongchai, Siriwan; Boonsing, Phorani; Kongtawelert, Prachya; Panthong, Ampai; Reutrakul, Vichai

    2012-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovium. It is involved in up-regulation of pro-inflammatory cytokines and matrix metalloproteinases (MMPs), resulting in joint inflammation and erosion. Zingiber cassumunar Roxb. has long been used to reduce joint pain and inflammation. This study aimed to investigate the inhibitory activities of an active compound of Z. cassumunar, (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D), against cytokine-induced up-regulation of catabolic genes involved in cartilage degradation in RA. Synovial fibroblast cell line, SW982, was cultured in media containing interleukin-1β (IL-1β), in the presence or absence of compound D at the concentration range of 1 to 100 µM. After 24 hours, the cells were analyzed for the expressions of MMPs, IL-1β and interleukin-1β-converting enzyme (ICE) by RT-PCR. MMPs activities in the culture media were analyzed by zymographic techniques. Dexamethasone was used as the positive control. It was found that compound D at the concentration of 10 - 100 µM significantly decreased the mRNA expressions of MMP-1, -2, -3, and -13 which was induced by IL-1β (P<0.05) concomitantly with a decrease in activities of these MMPs in the culture media. An increase in the mRNA expression of IL-1β and ICE was also suppressed by compound D. The results suggest that the potent activities of this compound may be involved in the reduction of IL-1β protein synthesis in both pro-form and active form which played an important role in up-regulation of MMPs. This study first revealed the chondroprotective activity of Z. cassumunar in the transcriptional level by suppressing cytokine-induced catabolic genes which caused cartilage erosion in RA.

  12. Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

    PubMed Central

    Gatzka, Martina; Treiber, Nicolai; Schneider, Lars A.; Mulaw, Medhanie A.; Lucas, Tanja; Kochanek, Stefan; Dummer, Reinhard; Levesque, Mitchell P.; Wlaschek, Meinhard; Scharffetter-Kochanek, Karin

    2016-01-01

    Aging is associated with a rising incidence of cutaneous squamous cell carcinoma (cSCC), an aggressive skin cancer with the potential for local invasion and metastasis. Acquisition of a senescence-associated secretory phenotype (SASP) in dermal fibroblasts has been postulated to promote skin cancer progression in elderly individuals. The underlying molecular mechanisms are largely unexplored. We show that Chemerin, a previously unreported SASP factor released from senescent human dermal fibroblasts, promotes cSCC cell migration, a key feature driving tumor progression. Whereas the Chemerin abundance is downregulated in malignant cSCC cells, increased Chemerin transcripts and protein concentrations are detected in replicative senescent fibroblasts in vitro and in the fibroblast of skin sections from old donors, indicating that a Chemerin gradient is built up in the dermis of elderly. Using Transwell® migration assays, we show that Chemerin enhances the chemotaxis of different cSCC cell lines. Notably, the Chemerin receptor CCRL2 is remarkably upregulated in cSCC cell lines and human patient biopsies. Silencing Chemerin in senescent fibroblasts or the CCRL2 and GPR1 receptors in the SCL-1 cSCC cell line abrogates the Chemerin-mediated chemotaxis. Chemerin triggers the MAPK cascade via JNK and ERK1 activation, whereby the inhibition impairs the SASP- or Chemerin-mediated cSCC cell migration. Taken together, we uncover a key role for Chemerin, as a major factor in the secretome of senescent fibroblasts, promoting cSCC cell migration and possibly progression, relaying its signals through CCRL2 and GPR1 receptors with subsequent MAPK activation. These findings might have implications for targeted therapeutic interventions in elderly patients. PMID:27907906

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

    PubMed

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

    2016-12-01

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

  14. Smooth muscle differentiation in scleroderma fibroblastic cells.

    PubMed Central

    Sappino, A. P.; Masouyé, I.; Saurat, J. H.; Gabbiani, G.

    1990-01-01

    Using antibodies to alpha-smooth muscle actin and desmin on paraffin-embedded formalin-fixed tissue sections, the authors demonstrate that fibroblastic cells of localized and systemic scleroderma lesions express features of smooth muscle differentiation. Eleven of eleven skin specimens of systemic sclerosis patients and two of four skin specimens of localized scleroderma displayed the presence of fibroblasts expressing alpha-smooth muscle actin, a cell population that predominated in areas of prominent collagen deposition. A similar fibroblastic phenotype was found in the esophagus, the liver, and the lung specimens obtained from four patients who died of progressive systemic sclerosis. Immunostaining for desmin, performed on adjacent tissue sections, demonstrated that a minority of these fibroblastic cells present in skin and visceral lesions contained this protein. The authors' observations indicate that scleroderma fibroblasts are phenotypically related to the stromal cells previously identified in hypertrophic scars, fibromatoses, and desmoplasia; they might provide novel criteria for the characterization of scleroderma lesions and help to identify the factors responsible for phenotypic modulations in fibroblastic cells. Images Figure 1 Figure 2 Figure 3 PMID:1698026

  15. Assembly and cell surface expression of TAP-independent, chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin.

    PubMed

    Fromm, Sharon Vigodman; Duady-Ben Yaakov, Shirly; Schechter, Chana; Ehrlich, Rachel

    2002-02-01

    Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.

  16. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

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

    PubMed

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

    2016-05-24

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

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

    SciTech Connect

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

    2015-03-06

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

  19. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    PubMed

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  20. Inducible responses to DNA damage in the mouse embryo fibroblasts cell line C3H/10T1/2 and its transformed counterpart C3H/MCA

    SciTech Connect

    Miller, L.S.

    1987-01-01

    Early passage mouse embryo fibroblasts cells (C3H/10T1/2) were treated with ultraviolet (UV) radiation of 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to determine whether such treatment induced DNA repair processes, measured as increased survival and mutagenesis of Herpes simplex (HSV-1). No enhanced host cell reactivation of UV-irradiated virus was observed following treatment of cells with UV-irradiation or TPA. Replication of undamaged virus in untreated C3H cells resulted in an increase over the background mutation frequency. When the cells were UV-irradiated and infected with unirradiated virus, a decrease in mutagenesis was observed. Decreased untargeted mutagenesis was shown to be dose- and time-dependent, reaching a minimum at a fluence of 5-7 Jm/sup /minus/2/ for 24 hours between irradiation and infection of cells. There was no change in mutagenesis of UV-irradiated virus grown in UV-irradiated cells compared to untreated cells. The repair capacity of methylcholanthrene-transformed C3H cells (MCA cells) was compared with untransformed C3H cells. The cell lines demonstrated similar cell survival curves following UV-irradiation but differed markedly in their ability to repair damaged HSV-1.

  1. Stable release of BDNF from the fibroblast cell line NIH3T3 grown on silicone elastomers enhances survival of spiral ganglion cells in vitro and in vivo.

    PubMed

    Warnecke, Athanasia; Sasse, Susanne; Wenzel, Gentiana I; Hoffmann, Andrea; Gross, Gerhard; Paasche, Gerrit; Scheper, Verena; Reich, Uta; Esser, Karl-Heinz; Lenarz, Thomas; Stöver, Timo; Wissel, Kirsten

    2012-07-01

    The treatment of choice for profound sensorineural hearing loss (SNHL) is direct electrical stimulation of spiral ganglion cells (SGC) via a cochlear implant (CI). The number and excitability of SGC seem to be critical for the success that can be achieved via CI treatment. However, SNHL is associated with degeneration of SGC. Long-term drug delivery to the inner ear for improving SGC survival may be achieved by functionalisation of CI electrodes with cells providing growth factors. Therefore, the capacity of brain-derived neurotrophic factor (BDNF)-secreting NIH3T3 cells grown on cylindrically shaped silicone elastomers (SE) to exert local and sustained neuroprotective effects was assessed in vitro and in vivo. An in vitro model to investigate adhesion and cell growth of lentivirally modified NIH3T3 cells synthesising BDNF on SE was established. The bioactivity of BDNF was characterised by co-cultivation of SGC with cell-coated SE. In addition, cell-coated SE were implanted into deafened guinea pigs. The recombinant NIH3T3 cells proliferated on silicone surfaces during 14 days of cultivation and expressed significantly increasing BDNF levels. Enhanced survival rates and neurite outgrowth of SGC demonstrated the bioactivity of BDNF in vitro. Implantation of SE with adhering BDNF-secreting NIH3T3 cells into the cochleae of systemically deafened guinea pigs induced a significant increase in SGC survival in comparison to SE without cell coating. Our data demonstrate a novel approach of cell-based long-term drug delivery to support SGC survival in vitro and in vivo. This therapeutic strategy--once transferred to cells suitable for clinical application--may improve CI performance.

  2. Electrically excitable normal rat kidney fibroblasts: A new model system for cell-semiconductor hybrids.

    PubMed Central

    Parak, W J; Domke, J; George, M; Kardinal, A; Radmacher, M; Gaub, H E; de Roos, A D; Theuvenet, A P; Wiegand, G; Sackmann, E; Behrends, J C

    1999-01-01

    In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters. PMID:10049346

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

    SciTech Connect

    Mayer, D.; Branscheid, D. )

    1992-04-01

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

  4. Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

    PubMed Central

    Jiménez Pérez, Zuly Elizabeth; Mathiyalagan, Ramya; Markus, Josua; Kim, Yeon-Ju; Kang, Hyun Mi; Abbai, Ragavendran; Seo, Kwang Hoon; Wang, Dandan; Soshnikova, Veronika; Yang, Deok Chun

    2017-01-01

    There has been a growing interest in the design of environmentally affable and biocompatible nanoparticles among scientists to find novel and safe biomaterials. Panax ginseng Meyer berries have unique phytochemical profile and exhibit beneficial pharmacological activities such as antihyperglycemic, antiobesity, antiaging, and antioxidant properties. A comprehensive study of the biologically active compounds in ginseng berry extract (GBE) and the ability of ginseng berry (GB) as novel material for the biosynthesis of gold nanoparticles (GBAuNPs) and silver nanoparticles (GBAgNPs) was conducted. In addition, the effects of GBAuNPs and GBAgNPs on skin cell lines for further potential biological applications are highlighted. GBAuNPs and GBAgNPs were synthesized using aqueous GBE as a reducing and capping agent. The synthesized nanoparticles were characterized for their size, morphology, and crystallinity. The nanoparticles were evaluated for antioxidant, anti-tyrosinase, antibacterial, and cytotoxicity activities and for morphological changes in human dermal fibroblast and murine melanoma skin cell lines. The phytochemicals contained in GBE effectively reduced and capped gold and silver ions to form GBAuNPs and GBAgNPs. The optimal synthesis conditions (ie, temperature and v/v % of GBE) and kinetics were investigated. Polysaccharides and phenolic compounds present in GBE were suggested to be responsible for stabilization and functionalization of nanoparticles. GBAuNPs and GBAgNPs showed increased scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radicals compared to GBE. GBAuNPs and GBAgNPs effectively inhibited mushroom tyrosinase, while GBAgNPs showed antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, GBAuNPs were nontoxic to human dermal fibroblast and murine melanoma cell lines, and GBAgNPs showed cytotoxic effect on murine melanoma cell lines. The current results evidently suggest that GBAgNPs can act as potential

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

    SciTech Connect

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

    2014-09-05

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

  6. Copper Redistribution in Atox1-deficient Mouse Fibroblast Cells

    PubMed Central

    McRae, Reagan; Lai, Barry

    2010-01-01

    Quantitative SXRF imaging of adherent mouse fibroblast cells deficient in Atox1, a metallochaperone protein responsible for delivering copper (Cu) to cuproenzymes in the trans-Golgi network, revealed striking differences in the subcellular Cu distribution compared to wildtype cells. While the latter showed a pronounced perinculear localization of Cu, the Atox1-deficient cells displayed a mostly unstructured and diffuse distribution throughout the entire cell body. Comparison of the SXRF elemental maps for Zn and Fe of the same samples showed no marked differences between the two cell lines. The data underscore the importance of Atox1, not only as a metallochaperone for delivering Cu to cuproenzymes, but also as a key player in maintaining the proper distribution and organization of Cu at the cellular level. PMID:19865834

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

    PubMed

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

    2016-03-01

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

  8. Mesenchymal stem cells induce dermal fibroblast responses to injury

    SciTech Connect

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

    2010-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2012-04-01

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

  12. Fibrogenic Lung Injury Induces Non-Cell-Autonomous Fibroblast Invasion.

    PubMed

    Ahluwalia, Neil; Grasberger, Paula E; Mugo, Brian M; Feghali-Bostwick, Carol; Pardo, Annie; Selman, Moisés; Lagares, David; Tager, Andrew M

    2016-06-01

    Pathologic accumulation of fibroblasts in pulmonary fibrosis appears to depend on their invasion through basement membranes and extracellular matrices. Fibroblasts from the fibrotic lungs of patients with idiopathic pulmonary fibrosis (IPF) have been demonstrated to acquire a phenotype characterized by increased cell-autonomous invasion. Here, we investigated whether fibroblast invasion is further stimulated by soluble mediators induced by lung injury. We found that bronchoalveolar lavage fluids from bleomycin-challenged mice or patients with IPF contain mediators that dramatically increase the matrix invasion of primary lung fibroblasts. Further characterization of this non-cell-autonomous fibroblast invasion suggested that the mediators driving this process are produced locally after lung injury and are preferentially produced by fibrogenic (e.g., bleomycin-induced) rather than nonfibrogenic (e.g., LPS-induced) lung injury. Comparison of invasion and migration induced by a series of fibroblast-active mediators indicated that these two forms of fibroblast movement are directed by distinct sets of stimuli. Finally, knockdown of multiple different membrane receptors, including platelet-derived growth factor receptor-β, lysophosphatidic acid 1, epidermal growth factor receptor, and fibroblast growth factor receptor 2, mitigated the non-cell-autonomous fibroblast invasion induced by bronchoalveolar lavage from bleomycin-injured mice, suggesting that multiple different mediators drive fibroblast invasion in pulmonary fibrosis. The magnitude of this mediator-driven fibroblast invasion suggests that its inhibition could be a novel therapeutic strategy for pulmonary fibrosis. Further elaboration of the molecular mechanisms that drive non-cell-autonomous fibroblast invasion consequently may provide a rich set of novel drug targets for the treatment of IPF and other fibrotic lung diseases.

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

    PubMed

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

    2013-02-01

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

  14. Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

    PubMed Central

    Wang, Yao-wen; Ren, Ji-hao; Xia, Kun; Wang, Shu-hui; Yin, Tuan-fang; Xie, Ding-hua; Li, Li-hua

    2012-01-01

    Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF. Conclusions: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In additon, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin’s side effect when it is applied clinically. PMID

  15. Cellular metabolic rates in cultured primary dermal fibroblasts and myoblast cells from fast-growing and control Coturnix quail.

    PubMed

    Jimenez, Ana Gabriela; Cooper-Mullin, Clara; Anthony, Nicholas B; Williams, Joseph B

    2014-05-01

    Fibroblast cells have been extensively used in research, including in medicine, physiology, physiological-ecology, and conservation biology. However, whether the physiology of fibroblasts reflects the physiology of other cell types in the same animal is unknown. Dermal fibroblasts are responsible for generating connective tissue and involved in wound healing, but generally, this cell type is thought to be metabolically inactive until it is required at the site of tissue damage. Thus, one might question whether fibroblasts are a representative model system to portray the metabolic profile of the whole organism, as compared with cells isolated from other tissues, like muscle, brain or kidneys. To explore whether fibroblasts have the same metabolic profile as do myoblast cells, we cultured cells from day-old chicks of quail (Coturnix coturnix japonica) selected for fast-growth or normal growth (our control group). Our results suggest that isolated primary fibroblasts and myoblast cells had higher rates of glycolysis, oxygen consumption and more mitochondria in the fast-growing line than in the control line. Our findings lend support for the idea that fibroblasts are a representative cell system to characterize the whole organism metabolic signature at the cellular-level. These data are striking, however, because fibroblasts had higher rates of metabolism for every parameter measured than myoblast cells isolated from the same individuals.

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

    PubMed Central

    2014-01-01

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

  17. Fibroblast cell interactions with human melanoma cells affect tumor cell growth as a function of tumor progression.

    PubMed Central

    Cornil, I; Theodorescu, D; Man, S; Herlyn, M; Jambrosic, J; Kerbel, R S

    1991-01-01

    It is known from a variety of experimental systems that the ability of tumor cells to grow locally and metastasize can be affected by the presence of adjacent normal tissues and cells, particularly mesenchymally derived stromal cells such as fibroblasts. However, the comparative influence of such normal cell-tumor cell interactions on tumor behavior has not been thoroughly investigated from the perspective of different stages of tumor progression. To address this question we assessed the influence of normal dermal fibroblasts on the growth of human melanoma cells obtained from different stages of tumor progression. We found that the in vitro growth of most (4 out of 5) melanoma cell lines derived from early-stage radial growth phase or vertical growth phase metastatically incompetent primary lesions is repressed by coculture with normal dermal fibroblasts, suggesting that negative homeostatic growth controls are still operative on melanoma cells from early stages of disease. On the other hand, 9 out of 11 melanoma cell lines derived from advanced metastatically competent vertical growth phase primary lesions, or from distant metastases, were found to be consistently stimulated to grow in the presence of dermal fibroblasts. Evidence was obtained to show that this discriminatory fibroblastic influence is mediated by soluble inhibitory and stimulatory growth factor(s). Taken together, these results indicate that fibroblast-derived signals can have antithetical growth effects on metastatic versus metastatically incompetent tumor subpopulations. This resultant conversion in responsiveness to host tissue environmental factors may confer upon small numbers of metastatically competent cells a growth advantage, allowing them to escape local growth constraints both in the primary tumor site and at distant ectopic tissue sites. PMID:2068080

  18. Fibroblast PER2 Circadian Rhythmicity Depends on Cell Density

    PubMed Central

    Noguchi, Takako; Wang, Lexie L.; Welsh, David K.

    2013-01-01

    Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high and low density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these two possibilities, we mixed PER2::LUC wild type (WT) cells with non-luminescent, non-rhythmic Bmal1−/− cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high density cultures. We also mixed PER2::LUC WT cells with non-luminescent, long period Cry2−/− cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or non-rhythmic Bmal1−/− cells. In previous work, we found that low K+ suppresses fibroblast rhythmicity, and we and others have found that either low K+ or low Ca2+ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low density fibroblasts with high K+ (21 mM), high Ca2+ (3.6 mM), or conditioned medium. Conditioned medium from high density fibroblast cultures rescued rhythmicity of low density cultures, whereas high K+ or Ca2+ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity

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

    PubMed

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

    2014-09-05

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

  20. Interaction between head and neck squamous cell carcinoma cells and fibroblasts in the biosynthesis of PGE2

    PubMed Central

    Alcolea, Sonia; Antón, Rosa; Camacho, Mercedes; Soler, Marta; Alfranca, Arantzazu; Avilés-Jurado, Francesc-Xavier; Redondo, Juan-Miguel; Quer, Miquel; León, Xavier; Vila, Luis

    2012-01-01

    Prostaglandin (PG)E2 is relevant in tumor biology, and interactions between tumor and stroma cells dramatically influence tumor progression. We tested the hypothesis that cross-talk between head and neck squamous cell carcinoma (HNSCC) cells and fibroblasts could substantially enhance PGE2 biosynthesis. We observed an enhanced production of PGE2 in cocultures of HNSCC cell lines and fibroblasts, which was consistent with an upregulation of COX-2 and microsomal PGE-synthase-1 (mPGES-1) in fibroblasts. In cultured endothelial cells, medium from fibroblasts treated with tumor cell-conditioned medium induced in vitro angiogenesis, and in tumor cell induced migration and proliferation, these effects were sensitive to PGs inhibition. Proteomic analysis shows that tumor cells released IL-1, and tumor cell-induced COX-2 and mPGES-1 were suppressed by the IL-1-receptor antagonist. IL-1α levels were higher than those of IL-1β in the tumor cell-conditioning medium and in the secretion from samples obtained from 20 patients with HNSCC. Fractionation of tumor cell-conditioning media indicated that tumor cells secreted mature and unprocessed forms of IL-1. Our results support the concept that tumor-associated fibroblasts are a relevant source of PGE2 in the tumor mass. Because mPGES-1 seems to be essential for a substantial biosynthesis of PGE2, these findings also strengthen the concept that mPGES-1 may be \\a target for therapeutic intervention in patients with HNSCC. PMID:22308510

  1. Fibroblast Cell-Based Therapy for Experimental Autoimmune Diabetes

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Fibroblast nemosis induces angiogenic responses of endothelial cells

    SciTech Connect

    Enzerink, Anna; Rantanen, Ville; Vaheri, Antti

    2010-03-10

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

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

    PubMed Central

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

    2016-01-01

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

  5. New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally

    PubMed Central

    1989-01-01

    We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM- 2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation. PMID:2466037

  6. Melanoma Cells Block PEDF Production in Fibroblasts to Induce the Tumor-Promoting Phenotype of Cancer-Associated Fibroblasts.

    PubMed

    Nwani, Nkechiyere G; Deguiz, Maria L; Jimenez, Benilde; Vinokour, Elena; Dubrovskyi, Oleksii; Ugolkov, Andrey; Mazar, Andrew P; Volpert, Olga V

    2016-04-15

    Loss of pigment epithelium-derived factor (PEDF, SERPINF1) in cancer cells is associated with poor prognosis and metastasis, but the contribution of stromal PEDF to cancer evolution is poorly understood. Therefore, we investigated the role of fibroblast-derived PEDF in melanoma progression. We demonstrate that normal dermal fibroblasts expressing high PEDF levels attenuated melanoma growth and angiogenesis in vivo, whereas PEDF-depleted fibroblasts exerted tumor-promoting effects. Accordingly, mice with global PEDF knockout were more susceptible to melanoma metastasis. We also demonstrate that normal fibroblasts in close contact with PEDF-null melanoma cells lost PEDF expression and tumor-suppressive properties. Further mechanistic investigations underlying the crosstalk between tumor and stromal cells revealed that melanoma cells produced PDGF-BB and TGFβ, which blocked PEDF production in fibroblasts. Notably, cancer-associated fibroblasts (CAF) isolated from patient-derived tumors expressed markedly low levels of PEDF. Treatment of patient CAF and TGFβ-treated normal fibroblasts with exogenous PEDF decreased the expression of CAF markers and restored PEDF expression. Finally, expression profiling of PEDF-depleted fibroblasts revealed induction of IL8, SERPINB2, hyaluronan synthase-2, and other genes associated with tumor promotion and metastasis. Collectively, our results demonstrate that PEDF maintains tumor-suppressive functions in fibroblasts to prevent CAF conversion and illustrate the mechanisms by which melanoma cells silence stromal PEDF to promote malignancy. Cancer Res; 76(8); 2265-76. ©2016 AACR.

  7. Analysis of primary cilia in directional cell migration in fibroblasts.

    PubMed

    Christensen, Søren T; Veland, Iben R; Schwab, Albrecht; Cammer, Michael; Satir, Peter

    2013-01-01

    Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus and point toward the leading edge. Recent work has shown that primary cilia coordinate a series of signaling pathways critical to fibroblast cell migration during development and in wound healing. In particular, platelet-derived growth factor receptor alpha (PDGFRα) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by micropipette addition of PDGFRα ligands to cultures of fibroblasts. These methods should be useful not only in studying cell migration but also more generally in delineating response pathways in cells with primary cilia.

  8. Cell line provenance.

    PubMed

    Freshney, R Ian

    2002-07-01

    Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology. However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and contamination, given the will to apply it. Combined with proper records of the origin and history of the cell line, assays for authentication and contamination contribute to the provenance of the cell line. Detailed records should start from the initiation or receipt of the cell line, and should incorporate data on the donor as well as the tissue from which the cell line was derived, should continue with details of maintenance, and include any accidental as well as deliberate deviations from normal maintenance. Records should also contain details of authentication and regular checks for contamination. With this information, preferably stored in a database, and suitable backed up, the provenance of the cell line so created makes the cell line a much more valuable resource, fit for validation in industrial applications and more likely to provide reproducible experimental results when disseminated for research in other laboratories.

  9. SPARC regulates collagen interaction with cardiac fibroblast cell surfaces.

    PubMed

    Harris, Brett S; Zhang, Yuhua; Card, Lauren; Rivera, Lee B; Brekken, Rolf A; Bradshaw, Amy D

    2011-09-01

    Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

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

    SciTech Connect

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

    2010-08-20

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

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

    SciTech Connect

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

    1990-01-01

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

  12. Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability.

    PubMed

    Perlman, H; Georganas, C; Pagliari, L J; Koch, A E; Haines, K; Pope, R M

    2000-05-15

    The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.

  13. Capsaicin partially mimics heat in mouse fibroblast cells in vitro.

    PubMed

    Sugimoto, Naotoshi; Katakura, Masanori; Matsuzaki, Kentaro; Nakamura, Hiroyuki; Yachie, Akihiro; Shido, Osamu

    2017-03-01

    Capsaicin activates transient receptor potential vanilloid 1 (TRPV1), a cation channel in the transient receptor potential family, resulting in the transient entry of Ca(2+) and Mg(2+) and a warm sensation. However, the effects of capsaicin on cells have not fully elucidated in fibroblasts. In this study, we investigated whether capsaicin could induce signal transduction in mouse fibroblast cells and compared the effect with that of heat-induced signal transduction. The activation of the mitogen-activated protein kinases (MAPKs) ERK and p38 MAPK, expression levels of heat shock protein 70 (HSP70) and HSP90, actin assembly, and cell proliferation were analyzed in NIH3T3 mouse fibroblast cells. A 15-min stimulation with capsaicin (∼100 μM) phosphorylated ERK and p38 MAPK and induced actin assembly. A 2-day stimulation with capsaicin increased the level of HSP70, but not HSP90, and the 2-day stimulation with capsaicin (∼100 μM) did not affect cell proliferation. A 15-min exposure to moderate heat (39.5 °C) phosphorylated both ERK and p38 MAPK and induced actin assembly to similar degrees as stimulation with capsaicin. A 2-day exposure to moderate heat increased the levels of both HSP70 and HSP90 and prevented cell proliferation. However, the 2-day stimulation with capsaicin (100 μM) failed to prevent heat shock-induced cell death. Thus, our results suggest that the effects of capsaicin on fibroblast cells partially differ from those of heat. Notably, the 2-day stimulation with capsaicin was not sufficient to develop heat tolerance in fibroblast cells.

  14. Generation of iPSC line HEL24.3 from human neonatal foreskin fibroblasts.

    PubMed

    Trokovic, Ras; Weltner, Jere; Otonkoski, Timo

    2015-07-01

    Human iPSC line HEL24.3 was generated from healthy human foreskin fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses.

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

    SciTech Connect

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

    1994-02-01

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

  16. Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells.

    PubMed

    Wei, Quan-Xiang; van der Hoeven, Franciscus; Hollstein, Monica; Odell, Adam F

    2012-05-17

    This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.

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

    PubMed Central

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

    2011-01-01

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

  18. Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells.

    PubMed Central

    Lüke, H J; Prehm, P

    1999-01-01

    The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others. PMID:10493913

  19. Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent

    PubMed Central

    1994-01-01

    Directional cellular locomotion is thought to involve localized intracellular calcium changes and the lateral transport of cell surface molecules. We have examined the roles of both calcium and cell surface glycoprotein redistribution in the directional migration of two murine fibroblastic cell lines, NIH 3T3 and SV101. These cell types exhibit persistent, cathode directed motility when exposed to direct current electric fields. Using time lapse phase contrast microscopy and image analysis, we have determined that electric field-directed locomotion in each cell type is a calcium independent process. Both exhibit cathode directed motility in the absence of extracellular calcium, and electric fields cause no detectable elevations or gradients of cytosolic free calcium. We find evidence suggesting that galvanotaxis in these cells involves the lateral redistribution of plasma membrane glycoproteins. Electric fields cause the lateral migration of plasma membrane concanavalin A receptors toward the cathode in both NIH 3T3 and SV101 fibroblasts. Exposure of directionally migrating cells to Con A inhibits the normal change of cell direction following a reversal of electric field polarity. Additionally, when cells are plated on Con A- coated substrata so that Con A receptors mediate cell-substratum adhesion, cathode-directed locomotion and a cathodal accumulation of Con A receptors are observed. Immunofluorescent labeling of the fibronectin receptor in NIH 3T3 fibroblasts suggests the recruitment of integrins from large clusters to form a more diffuse distribution toward the cathode in field-treated cells. Our results indicate that the mechanism of electric field directed locomotion in NIH 3T3 and SV101 fibroblasts involves the lateral redistribution of plasma membrane glycoproteins involved in cell-substratum adhesion. PMID:7929557

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

    PubMed Central

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

    1986-01-01

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

  1. Autophagy in cancer associated fibroblasts promotes tumor cell survival

    PubMed Central

    Martinez-Outschoorn, Ubaldo E; Trimmer, Casey; Lin, Zhao; Whitaker-Menezes, Diana; Chiavarina, Barbara; Zhou, Jie; Wang, Chengwang; Pavlides, Stephanos; Martinez-Cantarin, Maria P; Capozza, Franco; Witkiewicz, Agnieszka K; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Caro, Jaime

    2010-01-01

    Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB—another inducer of autophagy—prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer

  2. Pituitary follicular cells produce basic fibroblast growth factor

    SciTech Connect

    Ferrara, N.; Schweigerer, L.; Neufeld, G.; Mitchell, R.; Gospodarowicz, D.

    1987-08-01

    Cultured monolayers of bovine pituitary follicular cells, which transport ions, contain high amounts of mitogenic activity for endothelial cells which, on the basis of gene expression analysis, heparin-Sepharose elution profile, bioassay, immunoblotting, radioimmunoassay, and radioreceptor assay, has been identified as basic fibroblast growth factor (bFGF). These data indicate that follicular cells may be a major source of bFGF in the pituitary gland. Considering that bFGF has been proposed to play a role in paracrine regulation of pituitary hormone secretion, the data also suggest that these cells may exert important local regulatory functions.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    1989-12-01

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

  5. Identification of cell lines permissive for human coronavirus NL63.

    PubMed

    Schildgen, Oliver; Jebbink, Maarten F; de Vries, Michel; Pyrc, Krzysztov; Dijkman, Ronald; Simon, Arne; Müller, Andreas; Kupfer, Bernd; van der Hoek, Lia

    2006-12-01

    Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

  6. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

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

    PubMed Central

    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. PMID:28168047

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

    PubMed

    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.

  9. CLO: The cell line ontology

    PubMed Central

    2014-01-01

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

  10. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  11. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. Fibroblast-Derived Exosomes Contribute to Chemoresistance through Priming Cancer Stem Cells in Colorectal Cancer

    PubMed Central

    Hu, Yibing; Yan, Chang; Mu, Lei; Huang, Kaiyu; Li, Xiaolan; Tao, Deding; Wu, Yaqun; Qin, Jichao

    2015-01-01

    Chemotherapy resistance observed in patients with colorectal cancer (CRC) may be related to the presence of cancer stem cells (CSCs), but the underlying mechanism(s) remain unclear. Carcinoma-associated fibroblasts (CAFs) are intimately involved in tumor recurrence, and targeting them increases chemo-sensitivity. We investigated whether fibroblasts might increase CSCs thus mediating chemotherapy resistance. CSCs were isolated from either patient-derived xenografts or CRC cell lines based on expression of CD133. First, CSCs were found to be inherently resistant to cell death induced by chemotherapy. In addition, fibroblast-derived conditioned medium (CM) promoted percentage, clonogenicity and tumor growth of CSCs (i.e., CD133+ and TOP-GFP+) upon treatment with 5-fluorouracil (5-Fu) or oxaliplatin (OXA). Further investigations exhibited that exosomes, isolated from CM, similarly took the above effects. Inhibition of exosome secretion decreased the percentage, clonogenicity and tumor growth of CSCs. Altogether, our findings suggest that, besides targeting CSCs, new therapeutic strategies blocking CAFs secretion even before chemotherapy shall be developed to gain better clinical benefits in advanced CRCs. PMID:25938772

  14. Fibroblast morphogenesis on 3D collagen matrices: the balance between cell clustering and cell migration.

    PubMed

    da Rocha-Azevedo, Bruno; Grinnell, Frederick

    2013-10-01

    Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell-cell interactions. In vitro studies have shown that increasing the potential for cell-cell adhesion relative to cell-substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.

  15. Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras.

    PubMed

    Kruglova, A A; Kizilova, E A; Zhelezova, A I; Gridina, M M; Golubitsa, A N; Serov, O L

    2008-12-01

    Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.

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

    SciTech Connect

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

    1991-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  19. Regulation of skeletal muscle stem cells by fibroblast growth factors.

    PubMed

    Pawlikowski, Bradley; Vogler, Thomas Orion; Gadek, Katherine; Olwin, Bradley B

    2017-03-01

    Fibroblast growth factors (FGFs) are essential for self-renewal of skeletal muscle stem cells (satellite cells) and required for maintenance and repair of skeletal muscle. Satellite cells express high levels of FGF receptors 1 and 4, low levels of FGF receptor 3, and little or no detectable FGF receptor 2. Of the multiple FGFs that influence satellite cell function in culture, FGF2 and FGF6 are the only members that regulate satellite cell function in vivo by activating ERK MAPK, p38α/β MAPKs, PI3 kinase, PLCγ and STATs. Regulation of FGF signaling is complex in satellite cells, requiring Syndecan-4, a heparan sulfate proteoglycan, as well as ß1-integrin and fibronectin. During aging, reduced responsiveness to FGF diminishes satellite cell self-renewal, leading to impaired skeletal muscle regeneration and depletion of satellite cells. Mislocalization of ß1-integrin, reductions in fibronectin, and alterations in heparan sulfate content all contribute to reduced FGF responsiveness in satellite cells. How these cell surface proteins regulate satellite cell self-renewal is incompletely understood. Here we summarize the current knowledge, highlighting the role(s) for FGF signaling in skeletal muscle regeneration, satellite cell behavior, and age-induced muscle wasting. Developmental Dynamics, 2017. © 2017 Wiley Periodicals, Inc.

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

    PubMed

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

    2015-01-01

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

  1. Exploring the effects of low-level laser therapy on fibroblasts and tumor cells following gamma radiation exposure.

    PubMed

    Ramos Silva, Camila; Cabral, Fernanda Viana; de Camargo, Claudinei Francisco Morais; Núñez, Silvia Cristina; Mateus Yoshimura, Tania; de Lima Luna, Arthur Cássio; Maria, Durvanei Augusto; Ribeiro, Martha Simões

    2016-12-01

    Ionizing radiation (IR) induces DNA damage and low-level laser therapy (LLLT) has been investigated to prevent or repair detrimental outcomes resulting from IR exposure. Few in vitro studies, however, explore the biological mechanisms underlying those LLLT benefits. Thus, in this work, fibroblasts and tumor cells are submitted to IR with doses of 2.5 Gy and 10 Gy. After twenty-four-h, the cells are exposed to LLLT with fluences of 30 J cm(-2) , 90 J cm(-2) , and 150 J cm(-2) . Cellular viability, cell cycle phases, cell proliferation index and senescence are evaluated on days 1 and 4 after LLLT irradiation. For fibroblasts, LLLT promotes - in a fluence-dependent manner - increments in cell viability and proliferation, while a reduction in the senescence was observed. Regarding tumor cells, no influences of LLLT on cell viability are noticed. Whereas LLLT enhances cell populations in S and G2 /M cell cycle phases for both cellular lines, a decrease in proliferation and increase in senescence was verified only for tumor cells. Putting together, the results suggest that fibroblasts and tumor cells present different responses to LLLT following exposure to gamma-radiation, and these promising results should stimulate further investigations. Senescence of tumor cells and fibroblasts on the 4(th) day after ionizing radiation (IR) and low-level laser therapy (LLLT) exposures. The number of senescent cells increased significantly for tumor cells (a) while for fibroblasts no increment was observed (b). The blue collor indicates senescence activity.

  2. Halomethane-induced cytotoxicity and cell proliferation in human lung MRC-5 fibroblasts and NL20-TA epithelial cells.

    PubMed

    Nájera-Martínez, Minerva; García-Latorre, Ethel A; Reyes-Maldonado, Elba; Domínguez-López, M Lilia; Vega-López, Armando

    2012-09-01

    Halomethanes (HMs) can be formed during the chlorination process to obtain drinking water. In liver cells, HMs had been shown to be mutagenic and carcinogenic; however, their bioactivation by CYP 2E1 and GSTT1 is required. Although inhalation is the most common pathway of exposure, reports on the toxic effects induced by HMs in human lung are contradictory. The aim of this study was therefore to evaluate in vitro cytotoxicity and cell proliferation induced by CH(2)Cl(2), CHCl(3) and BrCHCl(2) in human lung NL20-TA epithelial cells and MRC-5 fibroblasts, and their relationship with CYP 2E1 and GSTT1 activity. High concentrations of these HMs induced cytotoxicity, particularly in cells treated with BrCHCl(2). Low concentrations of BrCHCl(2) stimulated hyperproliferation of fibroblasts, the most probable consequence of which is regenerative proliferation related to collagen induction. Fibroblasts exposed to BrCHCl(2) exhibited low levels of CYP 2E1 activity suggesting that released bromine is able to alter this activity by affecting the active site or auto regulating the activity itself. GSTT1 was up to ten times more active than CYP 2E1 in both cell lines, indicating that potential lung damage is due to formation of pro-carcinogens such as formaldehyde.

  3. Engineered Hypopharynx from Coculture of Epithelial Cells and Fibroblasts Using Poly(ester urethane) as Substratum

    PubMed Central

    Shen, Zhisen; Chen, Jingjing; Kang, Cheng; Gong, Changfeng

    2013-01-01

    Porous polymeric scaffolds have been much investigated and applied in the field of tissue engineering research. Poly(ester urethane) (PEU) scaffolds, comprising pores of 1–20 μm in diameter on one surface and ≥200 μm on the opposite surface and in bulk, were fabricated using phase separation method for hypopharyngeal tissue engineering. The scaffolds were grafted with silk fibroin (SF) generated from natural silkworm cocoon to enhance the scaffold's hydrophilicity and further improve cytocompatibility to both primary epithelial cells (ECs) and fibroblasts of human hypopharynx tissue. Coculture of ECs and fibroblasts was conducted on the SF-grafted PEU scaffold (PEU-SF) to evaluate its in vitro cytocompatibility. After co-culture for 14 days, ECs were lined on the scaffold surface while fibroblasts were distributed in scaffold bulk. The results of in vivo investigation showed that PEU porous scaffold possessed good biocompatibility after it was grafted by silk fibroin. SF grafting improved the cell/tissue infiltration into scaffold bulk. Thus, PEU-SF porous scaffold is expected to be a good candidate to support the hypopharynx regeneration. PMID:24455669

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

    PubMed

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

    2014-12-01

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

  5. Ethanol and Cancer Induce Similar Changes on Protein Expression Pattern of Human Fibroblast Cell

    PubMed Central

    Zamanian–Azodi, Mona; Rezaei-Tavirani, Mostafa; Rahmati-Rad, Sara; Rezaei Tavirani, Majid

    2016-01-01

    Ethanol has a vast consumption around the world. Many researches confirmed some adverse effect of this component on human health. In addition, recent studies showed significant alteration in both cellular population, and protein profile of human foreskin fibroblast cell line (HFFF2) in the specific dosage of ethanol. Here, the role and interaction of some proteins (characterized by significant alteration in expression due to ethanol effect) analyzed by proteomics and evaluated by considering cancerous case. 2D-electrophoresis findings of comparison of normal fibroblast cells and treated fibroblast with 270 mM dosage of ethanol analyzed by using SameSpots software, R software, and Cytoscape for protein-protein interaction (PPI) investigation. Six proteins with significantly altered expression associated with fundamental properties in a cell identified in ethanol-treated sample. These include AnnexinA5, Heterogeneous nuclear ribonucleoprotein A1, Rho-GDP dissociation inhibitor, Cathepsin L, Cu/Zn-SOD, Rho-GDP dissociation inhibitor, and Serpin peptidase inhibitor. Surprisingly, all these proteins were down-regulated and this pattern is similar to nasopharyngeal carcinoma-associated stromal fibroblast sample. Additionally, protein-protein interaction (PPI) indicates that HNRNPA1, SERPINE1 are hub proteins. Once their expression alters, it can impose vast changes on other molecular function. Based on this approach, ethanol may target same pathways that are related to cancer onset. In addition, some epidemiologic studies proved that ethanol consumption is related to increment of cancer risk. Therefore, more investigation is required in this regard to elicit the feasible relationship. PMID:28228815

  6. Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth.

    PubMed

    Di Francesco, P; Testa, E P; Testa, U; Liboi, E

    1989-06-01

    Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Lymphoblastoid lines and skin fibroblasts from patients with tuberous sclerosis are abnormally sensitive to ionizing radiation and to a radiomimetic chemical

    SciTech Connect

    Scudiero, D.A.; Moshell, A.N.; Scarpinato, R.G.; Meyer, S.A.; Clatterbuck, B.E.; Tarone, R.E.; Robbins, J.H.

    1982-03-01

    Lymphoblastoid lines, derived by transforming peripheral blood lymphocytes with Epstein-Barr virus, and skin fibroblast lines were established from two patients with tuberous sclerosis. The number of viable lymphoblastoid cells was determined by their ability to exclude the vital dye trypan blue after their irradiation with x-rays or 254 nm ultraviolet light. The growth of fibroblasts was determined by their ability to form colonies after treatment with the radiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine. The tuberous sclerosis lymphoblastoid lines were hypersensitive to x-rays but had normal sensitivity to the ultraviolet radiation. The tuberous sclerosis fibroblast lines were hypersensitive to the N-methyl-N'-nitro-N-nitrosoguanidine. The hypersensitivity of tuberous sclerosis cells to x-rays and to N-methyl-N'-nitro-N-nitrosoguanidine is believed to reflect defective repair of DNA damaged by these agents and may provide the basis for in vitro, including prenatal, diagnostic tests for tuberous sclerosis.

  8. Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts.

    PubMed

    Gonçalves, N J N; Bressan, F F; Roballo, K C S; Meirelles, F V; Xavier, P L P; Fukumasu, H; Williams, C; Breen, M; Koh, S; Sper, R; Piedrahita, J; Ambrósio, C E

    2017-04-01

    Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.

  9. Growth of Murine Cytomegalovirus in Various Cell Lines

    PubMed Central

    Kim, Kwang Soo; Carp, Richard I.

    1971-01-01

    Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV. PMID:4327583

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

    PubMed Central

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

    2014-01-01

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

  11. The elemental analysis of normal and Menkes' fibroblast cells with the SPMP

    NASA Astrophysics Data System (ADS)

    Allan, G. L.; Camakaris, J.; Legge, G. J. F.

    1991-03-01

    A scanning proton microprobe has been used to study the elemental distributions, including trace elements, in human skin fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. Cells grown in a copper-rich medium exhibited higher levels of intracellular copper but were found to be susceptible to the toxic effect of the elevated copper level. In normal maintenance medium the Menkes' cells recorded an average intracellular copper level six times higher than normal fibroblasts.

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

    SciTech Connect

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

    1990-09-15

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

  13. Pediatric brain tumor cell lines.

    PubMed

    Xu, Jingying; Margol, Ashley; Asgharzadeh, Shahab; Erdreich-Epstein, Anat

    2015-02-01

    Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research.

  14. Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen.

    PubMed

    Griffin, M; Bhandari, R; Hamilton, G; Chan, Y C; Powell, J T

    1993-06-01

    During alveolar development and alveolar repair close contacts are established between fibroblasts and lung epithelial cells through gaps in the basement membrane. Using co-culture systems we have investigated whether these close contacts influence synthesis and secretion of the principal surfactant apoprotein (SP-A) by cultured rat lung alveolar type II cells and the synthesis and secretion of type I collagen by fibroblasts. The alveolar type II cells remained cuboidal and grew in colonies on fibroblast feeder layers and on Matrigel-coated cell culture inserts but were progressively more flattened on fixed fibroblast monolayers and plastic. Alveolar type II cells cultured on plastic released almost all their SP-A into the medium by 4 days. Alveolar type II cells cultured on viable fibroblasts or Matrigel-coated inserts above fibroblasts accumulated SP-A in the medium at a constant rate for the first 4 days, and probably recycle SP-A by endocytosis. The amount of mRNA for SP-A was very low after 4 days of culture of alveolar type II cells on plastic, Matrigel-coated inserts or fixed fibroblast monolayers: relatively, the amount of mRNA for SP-A was increased 4-fold after culture of alveolar type II cells on viable fibroblasts. Co-culture of alveolar type II cells with confluent human dermal fibroblasts stimulated by 2- to 3-fold the secretion of collagen type I into the culture medium, even after the fibroblasts' growth had been arrested with mitomycin C. Collagen secretion, by fibroblasts, also was stimulated 2-fold by conditioned medium from alveolar type II cells cultured on Matrigel. The amount of mRNA for type I collagen increased only modestly when fibroblasts were cultured in this conditioned medium. This stimulation of type I collagen secretion diminished as the conditioned medium was diluted out, but at high dilutions further stimulation occurred, indicating that a factor that inhibited collagen secretion also was being diluted out. The conditioned medium

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  16. Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

    PubMed

    Daneshvar Amoli, Abdolreza; Mohebali, Nazanin; Farzaneh, Parvaneh; Shahzadeh Fazeli, Seyed Abolhassan; Nikfarjam, Laleh; Ashouri Movasagh, Sepideh; Moradmand, Zahra; Ganjibakhsh, Meysam; Nasimian, Ahmad; Izadpanah, Mehrnaz; Vakhshiteh, Faezeh; Gohari, Neda Sadat; Masoudi, Najmeh Sadat; Farghadan, Maryam; Mohamadi Moghanjoghi, Shiva; Khalili, Masoud; Khaledi, Kourosh J

    2016-12-30

    Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

  17. Hypoxia Enhances Direct Reprogramming of Mouse Fibroblasts to Cardiomyocyte-Like Cells.

    PubMed

    Wang, Yanyan; Shi, Shujun; Liu, Huiwen; Meng, Li

    2016-02-01

    Recent work has shown that mouse and human fibroblasts can be reprogrammed to cardiomyocyte-like cells with a combination of transcription factors. Current research has focused on improving the efficiency and mechanisms for fibroblast reprogramming. Previously, it has been reported that hypoxia enhances fibroblast cell reprogramming to pluripotent stem cells. In this study, we observed that 6 h of hypoxic conditions (2% oxygen) on newborn mouse dermal fibroblasts can improve the efficiency of reprogramming to cardiomyocyte-like cells. Expression of cardiac-related genes and proteins increased at 4 weeks after transfer of three transcription factors (Gata4/Mef2c/Tbx5 [GMT]). However, beating cardiomyocyte cells were not detected. The epigenetic mechanism of hypoxia-induced fibroblast reprogramming to cardiomyocyte cells requires further study.

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

    SciTech Connect

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

    2004-07-14

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

  19. Ex vivo culture of circulating tumor cells using magnetic force-based coculture on a fibroblast feeder layer.

    PubMed

    Yamamoto, Shuhei; Shimizu, Kazunori; Fei, Jiahui; Iwata, Hiroji; Okochi, Mina; Nakanishi, Hayao; Honda, Hiroyuki

    2016-11-01

    Phenotype-based analysis of circulating tumor cells (CTCs) is a promising approach to identification of new therapeutic targets and to elucidation of the biological properties. Nonetheless, ex vivo culturing of CTCs is still a technical challenge. Here, we develop a novel ex vivo culture method for CTCs using a fibroblast feeder layer and a magnetic coculture protocol. CTCs in the blood of a mouse metastasis model are labeled magnetically with magnetite nanoparticles. The labeled CTCs are isolated by a magnetic capture column and a size-selective capture filter. The isolated CTCs are positioned on a fibroblast feeder layer by the magnetic force. As a result, we observe adhesion and proliferation of the CTCs under the conditions of the fibroblast feeder layer and the magnetic force, whereas no adhesion or proliferation is observed without the feeder layer. After that, we culture the CTCs and obtain three CTC-derived cell lines. Using these cell lines, we perform phenotype-based analyses of invasiveness and drug resistance and find that the CTC-derived cell lines are more malignant than the original cells. Thus, the proposed method would be a promising approach to ex vivo culture of CTCs for phenotype-based analysis, and possibly used in cancer treatment.

  20. TGF beta induces a sustained c-fos expression associated with stimulation or inhibition of cell growth in EL2 or NIH 3T3 fibroblasts.

    PubMed

    Liboi, E; Di Francesco, P; Gallinari, P; Testa, U; Rossi, G B; Peschle, C

    1988-02-29

    We have previously indicated that epidermal growth factor (EGF) plays a fundamental role in the proliferation control of EL2 rat fibroblast line. It is shown here that transforming growth factor beta (TGF beta) stimulates both DNA synthesis and proliferation of EL2 cells, while exerting an inhibitory effect on the growth of murine NIH-3T3 fibroblasts. We also report the effect of TGF beta and EGF on c-fos expression in EL2 cells, as compared to that of TGF beta in NIH-3T3 fibroblasts. In EL2 cells EGF induces a transient c-fos expression at both mRNA and protein level, as previously observed in NIH-3T3 fibroblasts treated with platelet-derived or fibroblast growth factor (PDGF, FGF). Conversely, TGF beta induces in EL2 cells a sustained expression of fos mRNA and protein, which are still detectable at least 24 and 7 hr after treatment respectively. In NIH-3T3 fibroblasts TGF beta causes a sustained fos RNA expression, which is not associated, however, with detectable fos protein. We conclude that in fibroblasts stimulated by mitogens c-fos expression may be differentially modulated, depending of the growth factor and the cell line. This is seemingly due to differential regulation of fos gene expression, not only at the transcriptional and/or post-transcriptional level (transient or sustained fos RNA induction by EGF or TGF beta in EL2 cells), but also at the translational level (fos protein(s) induction by TGF beta in EL2 but not NIH-3T3 fibroblasts, possibly related to the stimulatory vs inhibitory effect of this factor on the growth of the former vs the latter line).

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

    PubMed Central

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

    2015-01-01

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

  2. Ultrasound Gene Transfer into Fibroblast Cells using Microbubbles

    NASA Astrophysics Data System (ADS)

    Nakamura, Yoji; Hirayama, Kota; Yoshinaka, Kiyoshi; Tei, Yuichi; Takagi, Shu; Matsumoto, Yoichiro

    2009-04-01

    Ultrasound is widely applied in the medical field and offers the strong advantages of non-invasiveness and high-selectivity. Gene transfer using ultrasound, which is called sonoporation, is one application. Ultrasound has the potential to deliver therapeutic materials such as genes, drugs or proteins into cells. Microbubbles are known to be able to improve delivery efficiency. This is attributed to therapeutic materials passing through the cell membrane after permeability is increased by destruction or oscillation of microbubbles. The present study tried to deliver the GFP plasmids into fibroblast cells. Cells were cultured in 6-well culture plates and exposed to ultrasound (frequency, 2.1 MHz; wave pattern, duty cycle 10%; intensity, 0-26 W/cm2; time, 0-200 s) transmitted through medium containing microbubbles (Levovist® (void fraction, 8×10-5) or Sonazoid® (void fraction, 0-24×10-4)) and GFP plasmids at a concentration of 15 μg/mL. Density of microbubbles after ultrasound irradiation was measured. When ultrasound intensity was increased with Levovist® 8×10-4, transfection efficiency increased, cell viability decreased and microbubbles disappeared. With Sonazoid®, transfection efficiency and cell viability were basically unchanged and microbubbles decreased, but did not disappear. Transfection efficiency also improved with increased ultrasound irradiation time or microbubble density. Microbubble destruction appeared to have the main effect on gene transfection under Levovist® and microbubble oscillation had the main effect under Sonazoid®.

  3. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    PubMed

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

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

    NASA Astrophysics Data System (ADS)

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

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

  5. Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts

    PubMed Central

    Capetian, Philipp; Azmitia, Luis; Pauly, Martje G.; Krajka, Victor; Stengel, Felix; Bernhardi, Eva-Maria; Klett, Mariana; Meier, Britta; Seibler, Philip; Stanslowsky, Nancy; Moser, Andreas; Knopp, Andreas; Gillessen-Kaesbach, Gabriele; Nikkhah, Guido; Wegner, Florian; Döbrössy, Máté; Klein, Christine

    2016-01-01

    Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at

  6. Tumour-associated fibroblasts and mesenchymal stem cells: more similarities than differences.

    PubMed

    Paunescu, Virgil; Bojin, Florina M; Tatu, Calin A; Gavriliuc, Oana I; Rosca, Adriana; Gruia, Alexandra T; Tanasie, Gabriela; Bunu, Carmen; Crisnic, Daniela; Gherghiceanu, Mihaela; Tatu, Fabian R; Tatu, Carmen S; Vermesan, Simona

    2011-03-01

    Tumour-associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF-like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs' definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen-DR subregion (HLA-DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α-smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor-β1, interleukins (IL-4, IL-10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of 'specialized' MSCs.

  7. Schwann cell migration and neurite outgrowth are influenced by media conditioned by epineurial fibroblasts.

    PubMed

    van Neerven, S G A; Pannaye, P; Bozkurt, A; Van Nieuwenhoven, F; Joosten, E; Hermans, E; Taccola, G; Deumens, R

    2013-11-12

    The regenerative capacity of the peripheral nervous system is largely related to Schwann cells undergoing proliferation and migration after injury and forming growth-supporting substrates for severed axons. Novel data show that fibroblasts to a certain extent regulate the pro-regenerative behavior of Schwann cells. In the setting of peripheral nerve injury, the fibroblasts that form the epineurium come into close contact with both Schwann cells and peripheral axons, but the potential influence on these latter two cell types has not been studied yet. In the present study we explored whether culture media, conditioned by epineurial fibroblasts can influence Schwann cells and/or neurite outgrowth from dorsal root ganglia neurons in vitro. Our data indicate that epineurial fibroblast-conditioned culture media substantially increase Schwann cell migration and the outgrowth of neurites. Schwann cell proliferation remained largely unaffected. These same read-out parameters were assayed in a condition where epineurial fibroblasts were subjected to stretch-cell-stress, a mechanical stressor that plays an important role in traumatic peripheral nerve injuries. Stretch-cell-stress of epineurial fibroblasts did not further change the positive effects of conditioned media on Schwann cell migration and neurite outgrowth. From these data we conclude that an as yet unknown pro-regenerative role can be attributed to epineurial fibroblasts, implying that such cells may affect the outcome of severe peripheral nerve injury.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  10. CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

    PubMed Central

    Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

  11. Characterization of human fibroblastic reticular cells as potential immunotherapeutic tools.

    PubMed

    Valencia, Jaris; Jiménez, Eva; Martínez, Víctor G; Del Amo, Beatriz G; Hidalgo, Laura; Entrena, Ana; Fernández-Sevilla, Lidia M; Del Río, Francisco; Varas, Alberto; Vicente, Ángeles; Sacedón, Rosa

    2017-05-01

    Fibroblastic reticular cells (FRCs) are essential players during adaptive immune responses not only as a structural support for the encounter of antigen-presenting cells and naive T lymphocytes but also as a source of modulatory signals. However, little is known about this cell population in humans. To address the phenotypical and functional analysis of human FRCs here we established splenic (SP) and mesenteric lymph node (LN) CD45(-)CD31(-)CD90(+)podoplanin(+) myofibroblastic cell cultures. They shared the phenotypical characteristics distinctive of FRCs, including the expression of immunomodulatory factors and peripheral tissue antigens. Nevertheless, human FRCs also showed particular features, some differing from mouse FRCs, like the lack of nitric oxide synthase (NOS2) expression after interferon (IFN)γstimulation. Interestingly, SP-FRCs expressed higher levels of interleukin (IL)-6, BMP4, CCL2, CXCL12 and Notch molecules, and strongly adapted their functional profile to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly I:C) and IFNγ stimulation. In contrast, we found higher expression of transforming growth factor (TGF)β and Activin A in LN-FRCs that barely responded via Toll-Like Receptor (TLR)3 and constitutively expressed retinaldehyde dehydrogenase 1 enzyme, absent in SP-FRCs. This study reveals human FRCs can be valuable models to increase our knowledge about the physiology of human secondary lymphoid organs in health and disease and to explore the therapeutic options of FRCs.

  12. Susceptibilities of 14 cell lines to bluetongue virus infection.

    PubMed Central

    Wechsler, S J; McHolland, L E

    1988-01-01

    The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations. PMID:2853175

  13. Intestinal smooth muscle cell maintenance by basic fibroblast growth factor.

    PubMed

    Lee, Min; Wu, Benjamin M; Stelzner, Matthias; Reichardt, Holger M; Dunn, James C Y

    2008-08-01

    Intestinal tissue engineering is a potential therapy for patients with short bowel syndrome. Tissue engineering scaffolds that promote smooth muscle cell proliferation and angiogenesis are essential toward the regeneration of functional smooth muscles for peristalsis and motility. Since basic fibroblast growth factor (bFGF) can stimulate smooth muscle proliferation and angiogenesis, the delivery of bFGF was employed to stimulate proliferation and survival of primary intestinal smooth muscle cells. Two methods of local bFGF delivery were examined: the incorporation of bFGF into the collagen coating and the encapsulation of bFGF into poly(D,L-lactic-co-glycolic acid) microspheres. Cell-seeded scaffolds were implanted into the omentum and were retrieved after 4, 14, and 28 days. The seeded cells proliferated from day 4 to day 14 in all implants; however, at 28 days, significantly higher density of implanted cells and blood vessels was observed, when 10 microg of bFGF was incorporated into the collagen coating of scaffolds as compared to scaffolds with either no bFGF or 1 microg of bFGF in collagen. Microsphere encapsulation of 1 microg of bFGF produced similar effects as 10 microg of bFGF mixed in collagen and was more effective than the delivery of 1 microg of bFGF by collagen incorporation. The majority of the implanted cells also expressed alpha-smooth muscle actin. Scaffolds coated with microsphere-encapsulated bFGF and seeded with smooth muscle cells may be a useful platform for the regeneration of the intestinal smooth muscle.

  14. Blockade of the chemokine receptor, CCR5, reduces the growth of orthotopically injected colon cancer cells via limiting cancerassociated fibroblast accumulation

    PubMed Central

    Tanabe, Yamato; Sasaki, Soichiro; Mukaida, Naofumi; Baba, Tomohisa

    2016-01-01

    We previously demonstrated that cancer-associated fibroblasts (CAFs) accumulate at tumor sites through the interaction between a chemokine, CCL3, and its receptor, CCR5, in the late phase of colitis-associated colon carcinogenesis. Here we examined the effect of a CCR5 antagonist, maraviroc, on tumor growth arising from the orthotopic injection of mouse or human colon cancer cell lines into the cecal wall by focusing on CAFs. Orthotopic injection of either cell line caused tumor formation together with leukocyte infiltration and fibroblast accumulation. Concomitant oral administration of maraviroc reduced tumor formation with few effects on leukocyte infiltration. In contrast, maraviroc reduced the intratumor number of α-smooth muscle actin-positive fibroblasts, which express epidermal growth factor, a crucial growth factor for colon cancer cell growth. These observations suggest that maraviroc or other CCR5 antagonists might act as novel anti-CRC drugs to dampen CAFs, an essential cell component for tumor progression. PMID:27340784

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

    PubMed Central

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

    2016-01-01

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

  16. Exposure to Varying Strain Magnitudes Influences the Conversion of Normal Skin Fibroblasts Into Hypertrophic Scar Cells.

    PubMed

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Cai, Xia; Liu, Tao

    2016-04-01

    Mechanical strain is a key contributor in the pathogenesis of hypertrophic scarring, whose optimal stretch magnitudes to initiate the differentiation of normal skin fibroblasts into aberrant fibroblasts phenotype remains largely unresolved. Influence of varying cyclic strain magnitudes on cultured human normal skin fibroblasts and its transformation into hypertrophic scar fibroblast-like phenotype is investigated in this study. Cultured fibroblasts isolated from hypertrophic scar and normal skin tissue were subjected to cyclic mechanical stretching under individual 10%, 15%, and 20% strain magnitudes at a frequency of 0.1 Hz for 24 hours. Stretched normal skin fibroblasts demonstrated significantly increased rates of cell proliferation, and also apparently oriented away nearly perpendicular to the applied stretching direction. Interestingly, the applied 10% strains magnitude resulted in a markedly enhanced cell proliferative ability compared with that of 20% strain magnitude. Parameters involving the mechanotransduction signaling, such as integrin β1 and P130Cas, were significantly improved at both mRNA and protein levels in the stretched normal skin fibroblasts, which was demonstrated in a negative magnitude-dependent manner. In addition, 10% strains magnitude triggered the highest expression levels of growth factor TGF-β1 and collagen matrix in stretched normal skin fibroblasts. Collectively, these results indicate that the 10% stretching magnitude, of the 3 strain magnitudes studied, is most effective for triggering the optimal mechanotransduction effects and biological responses inside cultured skin fibroblasts. The demonstrable conversion of normal skin fibroblasts into hypertrophic scar fibroblasts was also observed when 10% stretching magnitude was applied to cultured fibroblasts in vitro.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. The characterization of fibrocyte-like cells: a novel fibroblastic cell of the placenta.

    PubMed

    Riddell, M R; Winkler-Lowen, B; Chakrabarti, S; Dunk, C; Davidge, S T; Guilbert, L J

    2012-03-01

    The placenta is a highly vascularized organ thus angiogenesis is a key process in placental development. The contribution that different cells in the villous stroma play in placental angiogenesis is largely unknown. In this study we identified a novel stromal cell type in sections of term placenta which is morphologically fibroblastic and expressing the fibroblast marker TE-7 but also positive for the monocytic markers CD115 and CD14 and designated these cells as fibrocyte-like cells. Populations of fibrocyte-like cells from the placenta were isolated by two methods: culture of adherence-selected placental cells and, for higher purity, by CD45 fluorescence activated cell sorting (FACS). Fibrocyte-like cell conditioned medium increased endothelial tubule-like structure formation 2-fold versus control medium. Both pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and the anti-angiogenic factor soluble-Flt were found in the conditioned medium. Neutralizing antibodies against VEGF and b-FGF reduced endothelial cell tubule-like structures to control levels. These data suggests that fibrocyte-like cells, a previously unidentified cell of the villous stroma, may play an important role in the regulation of placental angiogenesis.

  20. Integral membrane protease fibroblast activation protein sensitizes fibrosarcoma to chemotherapy and alters cell death mechanisms.

    PubMed

    Baird, Sarah K; Rigopoulos, Angela; Cao, Diana; Allan, Laura; Renner, Christoph; Scott, Fiona E; Scott, Andrew M

    2015-11-01

    Fibroblast activation protein (FAP), an integral membrane serine protease, is found on fibro- and osteo-sarcoma and on myofibroblasts in epithelial carcinoma, but rarely on other adult tissue. FAP has been demonstrated to be an excellent target for tumor imaging in clinical trials, and antibodies and other FAP-targeting drugs are in development. Here we have shown that FAP overexpression increased the growth of HT1080 fibrosarcoma cells in vitro and in vivo, and found that the expression of FAP affects response to chemotherapy. When treated with doxorubicin, expression of FAP increased susceptibility to the drug. In spite of this, FAP-HT1080 cells had fewer markers of classical apoptosis than HT1080 cells and neither necrosis nor necroptosis were enhanced. However, levels of early mitochondrial and lysosomal membrane permeability markers were increased, and autophagy switched from a protective function in HT1080 cells to part of the cell death mechanism with FAP expression. Therefore, FAP may affect how the tumor responds to chemotherapeutic drugs overall, which should be considered in targeted drug development. The overexpression of FAP also alters cell signaling and responses to the environment in this cell line. This includes cell death mechanisms, changing the response of HT1080 cells to doxorubicin from classical apoptosis to an organelle membrane permeability-dependent form of cell death.

  1. A functional role for the 'fibroblast-like cells' in gastrointestinal smooth muscles.

    PubMed

    Kurahashi, Masaaki; Zheng, Haifeng; Dwyer, Laura; Ward, Sean M; Koh, Sang Don; Sanders, Kenton M

    2011-02-01

    Smooth muscles, as in the gastrointestinal tract, are composed of several types of cells. Gastrointestinal muscles contain smooth muscle cells, enteric neurons, glial cells, immune cells, and various classes of interstitial cells. One type of interstitial cell, referred to as 'fibroblast-like cells' by morphologists, are common, but their function is unknown. These cells are found near the terminals of enteric motor neurons, suggesting they could have a role in generating neural responses that help control gastrointestinal movements. We used a novel mouse with bright green fluorescent protein expressed specifically in the fibroblast-like cells to help us identify these cells in the mixture of cells obtained when whole muscles are dispersed with enzymes. We isolated these cells and found they respond to a major class of inhibitory neurotransmitters - purines. We characterized these responses, and our results provide a new hypothesis about the role of fibroblast-like cells in smooth muscle tissues.

  2. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    PubMed

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  3. Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer.

    PubMed

    Koo, Ok Jae; Hossein, Mohammad Shamim; Hong, So Gun; Martinez-Conejero, Jose A; Lee, Byeong Chun

    2009-02-01

    Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents - dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) - on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

  4. Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells

    PubMed Central

    Liu, Jiewei; Xu, Kun; Chase, Maya; Ji, Yuxin; Logan, Jennifer K.; Buchsbaum, Rachel J.

    2012-01-01

    The tumor microenvironment undergoes changes concurrent with neoplastic progression. Cancer incidence increases with aging and is associated with tissue accumulation of senescent cells. Senescent fibroblasts are thought to contribute to tumor development in aging tissues. We have shown that fibroblasts deficient in the Rac exchange factor Tiam1 promote invasion and metastasis of associated epithelial tumor cells. Here, we use a three-dimensional culture model of cellular invasiveness to outline several steps underlying this effect. We find that stress-induced senescence induces decreased fibroblast Tiam1 protein levels and increased osteopontin levels, and that senescent fibroblast lysates induce Tiam1 protein degradation in a calcium- and calpain-dependent fashion. Changes in fibroblast Tiam1 protein levels induce converse changes in osteopontin mRNA and protein. Senescent fibroblasts induce increased invasion and migration in co-cultured mammary epithelial cells. These effects in epithelial cells are ameliorated by either increasing fibroblast Tiam1 or decreasing fibroblast osteopontin. Finally, in seeded cell migration assays we find that either senescent or Tiam1-deficient fibroblasts induce increased epithelial cell migration that is dependent on fibroblast secretion of osteopontin. These findings indicate that one mechanism by which senescent fibroblasts promote neoplastic progression in associated tumors is through degradation of fibroblast Tiam1 protein and the consequent increase in secretion of osteopontin by fibroblasts. PMID:22302986

  5. Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung

    PubMed Central

    Lugo, Roberto; Gabasa, Marta; Andriani, Francesca; Puig, Marta; Facchinetti, Federica; Ramírez, Josep; Gómez-Caro, Abel; Pastorino, Ugo; Fuster, Gemma; Almendros, Isaac; Gascón, Pere; Davalos, Albert; Reguart, Noemí; Roz, Luca; Alcaraz, Jordi

    2016-01-01

    Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors. PMID:27384989

  6. Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung.

    PubMed

    Lugo, Roberto; Gabasa, Marta; Andriani, Francesca; Puig, Marta; Facchinetti, Federica; Ramírez, Josep; Gómez-Caro, Abel; Pastorino, Ugo; Fuster, Gemma; Almendros, Isaac; Gascón, Pere; Davalos, Albert; Reguart, Noemí; Roz, Luca; Alcaraz, Jordi

    2016-12-13

    Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors.

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

    SciTech Connect

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

    2011-10-07

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

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

    PubMed Central

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

    2015-01-01

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

  9. Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch.

    PubMed

    Dicker, Anthony J; Serewko, Magdalena M; Russell, Terry; Rothnagel, Joseph A; Strutton, Geoff M; Dahler, Alison L; Saunders, Nicholas A

    2002-05-01

    In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2. These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2).

  10. Human foreskin fibroblast-like stromal cells can differentiate into functional hepatocytic cells.

    PubMed

    Huang, Hsing-I; Chen, Shao-Kuan; Wang, Robert Y-L; Shen, Chia-Rui; Cheng, Yu-Che

    2013-12-01

    Foreskin fibroblast-like stromal cells (FDSCs) are progenitors isolated from human tissue that can differentiate into diverse cell types. Many types of stem cells can differentiate into hepatocyte-like cells, which could be used for drug testing or in liver regeneration therapy, but whether FDSCs can be converted into functional hepatocytes is unknown. FDSCs show divergent properties when cultured in distinct media, forming spheres in Dulbecco's modified Eagle's medium (DMEM) containing F12, epidermal growth factor (EGF), and basic fibroblast growth factor (b-FGF), but have fibroblast-like morphology when cultured in DMEM-based growth medium. Both cell populations express the typical mesenchymal stem cell markers CD90, CD105, and CD73, but the p75 neurotrophin receptor (p75NTR) was detected only in FDSC spheres. Both types of FDSCs can differentiate into hepatocyte-like cells, which express typical liver markers, including albumin and hepatocyte paraffin 1 (Hep Par1), along with liver-specific biological activities. When plasmids containing the human hepatitis B virus (HBV) genome were transfected transiently into FDSCs, differentiated hepatocyte-like cells secrete large amounts of HBe and HBs antigens. FDSCs could be used for clinical hepatic therapy and/or serve as a model of HBV.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons.

    PubMed

    Talluri, Thirumala R; Kumar, Dharmendra; Glage, Silke; Garrels, Wiebke; Ivics, Zoltan; Debowski, Katharina; Behr, Rüdiger; Kues, Wilfried A

    2014-07-18

    The generation of induced pluripotent stem (iPS) cells represents a promising approach for innovative cell therapies. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity. Transposition of reprogramming cassettes represents a recent alternative to viral approaches. Since binary transposons can be produced as common plasmids they provide a safe and cost-efficient alternative to viral delivery methods. Here, we compared the efficiency of two different transposon systems, Sleeping Beauty (SB) and piggyBac (PB), for the generation of murine iPS. Murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, and fibroblasts derived from outbred NMRI mice were employed for reprogramming. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-Myc was compatible with iPS cell line derivation, albeit with reduced reprogramming efficiencies. The transposon-derived iPS cells featured typical hallmarks of pluripotency, including teratoma growth in immunodeficient mice. Thus SB and PB transposons represent a promising non-viral approach for iPS cell derivation.

  14. Inhibition of Breast Cancer Progression by Blocking Heterocellular Contact between Epithelial Cells and Fibroblasts

    DTIC Science & Technology

    2012-04-01

    between MCF-DCIS cells and human mammary fibroblasts (HMFs) in breast cancer progression by employing a microfluidic -based compartmentalized 3D co-culture platform enabling both contact-free and contact-associated co-cultures.

  15. Quantitative methods to characterize morphological properties of cell lines.

    PubMed

    Mancia, Annalaura; Elliott, John T; Halter, Michael; Bhadriraju, Kiran; Tona, Alessandro; Spurlin, Tighe A; Middlebrooks, Bobby L; Baatz, John E; Warr, Gregory W; Plant, Anne L

    2012-07-01

    Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

  16. Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages.

    PubMed

    Wang, Qing-Hua; Peng, Yun; Cai, Xin-Yong; Wan, Meng; Liu, Yu; Wei, Hong

    2015-08-01

    Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

  17. Mast cell differentiation depends on T cells and granule synthesis on fibroblasts.

    PubMed Central

    Davidson, S; Mansour, A; Gallily, R; Smolarski, M; Rofolovitch, M; Ginsburg, H

    1983-01-01

    Mast cell differentiation was generated in the following three experimental situations: (i) infection of mice with Schistosoma Mansoni or with Nippostrongylus brasiliensis and growth of the lymph node cells in the presence of the corresponding helminth antigen; (ii) immunization with horse serum and growth of blood and lymph node cells in the presence of the horse serum; (iii) exposure of T-cell-depleted suspensions of lymph node cells from unimmunized mice to T-cell factor (TCF) released into medium of the young cultures of (i) and (ii). This differentiation was also obtained when lymph node cells from athymic nude mice were exposed to TCF. The cell suspensions were plated on X-irradiated fibroblast monolayers prepared from embryonic mouse skin. Screening of the suspensions before plating on the fibroblasts in culture revealed no young forms of mast cells, and none were present in culture of nude mice lymph node cells maintained without TCF. Primordial appearance of metachromatic granules generally in the golgi zone was first seen in many 'large lymphoid cells' as early as 18 hr after plating. This was followed by increase in the cytoplasm volume, increase in granule number and mitosis, ending at 10-18 days with homogeneous populations of mature mast cells. When the mesenteric lymph node cells from mice infected with the helminths were grown in the absence of fibroblasts but in the presence of the antigen, homogeneous populations of cells with extended cytoplasm, filled with unstained vacuoles developed during days 7-13. These cells did not contain histamine (or at most 0.2 microgram per 10(6) vacuolated cells). When these cells were plated on fibroblast monolayers clear granule formation in all the vacuoles was seen 2 days later. It increased progressively in size and staining intensity, until the vacuoles transformed into typical mast cell granules. By the fourth day the vacuolated cells attained the typical mast cell morphology and the histamine content greatly

  18. Thyroid cell lines in research on goitrogenesis.

    PubMed

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

    1991-12-01

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

  19. In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells.

    PubMed

    Martinez, Elizabeth F; Donato, Tatiani A G; Arana-Chavez, Victor E

    2012-10-01

    Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.

  20. Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3).

    PubMed

    Bisht, Gunjan; Rayamajhi, Sagar; Kc, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G

    2016-12-01

    Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe3O4-ZnO magnetic composite particles (MCPs).

  1. Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3)

    NASA Astrophysics Data System (ADS)

    Bisht, Gunjan; Rayamajhi, Sagar; KC, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G.

    2016-12-01

    Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles.

  2. Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts

    SciTech Connect

    Malaise, E.P.; Fertil, B.; Deschavanne, P.J.; Chavaudra, N.; Brock, W.A.

    1987-08-01

    The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.

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

    SciTech Connect

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

    2014-04-01

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

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

    PubMed

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

    2015-01-01

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

  5. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility

    PubMed Central

    Dzik, Luciana M.; Iglesia, Rebeca P.; Cruz, Mário C.; Zelanis, André; de Siqueira, Adriane S.; Serrano, Solange M.T.; Goldberg, Gary S.; Jaeger, Ruy G.; Freitas, Vanessa M.

    2016-01-01

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment. PMID:27374178

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

    PubMed

    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. Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation.

    PubMed

    Agley, Chibeza C; Rowlerson, Anthea M; Velloso, Cristiana P; Lazarus, Norman R; Harridge, Stephen D R

    2013-12-15

    We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells. The CD56(-) fraction obtained from the first sort was highly enriched in TE-7(+) fibroblasts. Using quantitative analysis of immunofluorescent staining for lipid content, lineage markers and transcription factors, we tested if the purified cell populations could differentiate into adipocytes in response to treatment with either fatty acids or adipocyte-inducing medium. Both treatments caused the fibroblasts to differentiate into adipocytes, as shown by loss of intracellular TE-7, upregulation of the adipogenic transcription factors PPARγ and C/EBPα, and adoption of a lipid-laden adipocyte morphology. By contrast, myogenic cells did not undergo adipogenesis and showed differential regulation of PPARγ and C/EBPα in response to these adipogenic treatments. Our results show that human skeletal muscle fibroblasts are at least bipotent progenitors that can remain as extracellular-matrix-producing cells or differentiate into adipocytes.

  8. Cell imaging and DNA delivery in fibroblastic cells by conjugated polyelectrolytes.

    PubMed

    Shin, Yong Cheol; Lee, Jong Ho; Jeong, Ji-Eun; Kim, Boram; Lee, Eun Ji; Jin, Oh Seong; Jung, Tae Gon; Lee, Jun Jae; Woo, Han Young; Han, Dong-Wook

    2013-01-01

    This study concentrates on the potential application of conjugated polyelectrolytes (CPEs) to cell imaging and DNA delivery. Four different types of polyfluorene copolymers, namely, PAHFP-Br, PAEFP-Br, PAHFbT-Br, and PSBFP-Na, which have the same π-conjugated backbone but different side chains, were synthesized. For cytotoxicity testing, L-929 fibroblastic cells were treated with increasing concentrations (0-50 µM) of each CPE and then cell viability was determined by WST-8 assay. Cellular uptake of CPEs into cultured L-929 cells was observed by fluorescence microscopy. To examine DNA delivery by CPEs, the cells were incubated for 1 H with PAHFP-Br/fluorescein (Fl)-labeled single-stranded DNA (ssDNA-Fl) complex and then visualized by fluorescence microscopy. Cytotoxicity of CPEs was increased in a dose-dependent manner but at lower than 10 µM, PAHFP-Br, PAEFP-Br, and PSBFP-Na did not show any cytotoxic effects on the cells. When added to cell cultures at 1 µM, PAHFP-Br/ssDNA-Fl complex was delivered and then dissociated into PAHFP-Br and ssDNA-Fl within the cells. This result implies that PAHFP-Br can enable cell imaging and DNA delivery into fibroblastic cells. Therefore, it is suggested that PAHFP-Br with various advantages such as low cytotoxicity and high fluorescence efficiency can be extensively used as a potential agent for cell imaging and gene delivery.

  9. Differences of cell surface marker expression between bone marrow- and kidney-derived murine mesenchymal stromal cells and fibroblasts.

    PubMed

    Cakiroglu, F; Osbahr, J W; Kramer, J; Rohwedel, J

    2016-10-31

    Mesenchymal stromal cells (MSC) are undifferentiated, multipotent adult cells with regenerative properties. They are particularly relevant for therapeutic approaches due to the simplicity of their isolation and cultivation. Since MSC show an expression pattern of cell surface marker, which is almost identical to fibroblasts, many attempts have been made to address the similarities and differences between MSC and fibroblasts. In this study we aimed to isolate murine MSC from bone marrow (BM) and kidney to characterize them in comparison to fibroblasts. Cells were isolated from murine kidney, BM and abdominal skin by plastic adherence and subsequently characterized by analysing their capability to build colony-forming unit-fibroblasts (CFU-F), their morphology, their proliferation, expression of telomerase activity and cell surface antigens as well as their differentiation capacity. Plastic adherent cells from the 3 mouse tissues showed similar morphology, proliferation profiles and CFU-F building capacities. However, while MSC from BM and kidney differentiated into the adipogenic, chondrogenic and osteogenic direction, fibroblasts were not able to do so efficiently. In addition, a tendency for lower expression of telomerase was found in the fibroblast population. Proliferating cells from kidney and BM expressed the MSC-specific cell surface markers CD105 and Sca-1 on a significantly higher and CD117 on a significantly lower level compared to fibroblasts and were thereby distinguishable from fibroblasts. Furthermore, we found that certain CD markers were specifically expressed on a higher level, either in BM-derived cells or fibroblasts. This study demonstrates that murine MSC isolated from different organs express certain specific markers, which enable their discrimination.

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

    PubMed Central

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

    2015-01-01

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

  11. Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity

    PubMed Central

    Balsamo, Mirna; Scordamaglia, Francesca; Pietra, Gabriella; Manzini, Claudia; Cantoni, Claudia; Boitano, Monica; Queirolo, Paola; Vermi, William; Facchetti, Fabio; Moretta, Alessandro; Moretta, Lorenzo; Mingari, Maria Cristina; Vitale, Massimo

    2009-01-01

    Although the role of the tumor microenvironment in the process of cancer progression has been extensively investigated, the contribution of different stromal components to tumor growth and/or evasion from immune surveillance is still only partially defined. In this study we analyzed fibroblasts derived from metastatic melanomas and provide evidence for their strong immunosuppressive activity. In coculture experiments, melanoma-derived fibroblasts sharply interfered with NK cell functions including cytotoxicity and cytokine production. Thus, both the IL-2-induced up-regulation of the surface expression of NKp44, NKp30, and DNAM-1 triggering receptors and the acquisition of cytolytic granules were inhibited in NK cells. This resulted in an impairment of the NK cell-mediated killing of melanoma target cells. Transwell cocultures and the use of specific inhibitors suggested that cell-to-cell contact was required for inducing DNAM-1 modulation. In contrast, modulation of NKp44 and NKp30 was due to PGE2 released by fibroblasts during coculture. Normal skin fibroblasts could also partially affect NK cell phenotype and function. However, the inhibitory effect of tumor-derived fibroblasts was far stronger and directly correlated with their ability to produce PGE2 either constitutively or upon induction by NK cells. PMID:19934056

  12. Interaction between human lung fibroblasts and T-lymphocytes prevents activation of CD4+ cells

    PubMed Central

    Vancheri, Carlo; Mastruzzo, Claudio; Trovato-Salinaro, Elisa; Gili, Elisa; Lo Furno, Debora; Pistorio, Maria P; Caruso, Massimo; La Rosa, Cristina; Crimi, Claudia; Failla, Marco; Crimi, Nunzio

    2005-01-01

    Background T lymphocytes are demonstrated to play an important role in several chronic pulmonary inflammatory diseases. In this study we provide evidence that human lung fibroblasts are capable of mutually interacting with T-lymphocytes leading to functionally significant responses by T-cells and fibroblasts. Methods Human lung fibroblast were co-cultured with PMA-ionomycin activated T-CD4 lymphocytes for 36 hours. Surface as well as intracellular proteins expression, relevant to fibroblasts and lymphocytes activation, were evaluated by means of flow cytometry and RT-PCR. Proliferative responses of T lymphocytes to concanavalin A were evaluated by the MTT assay. Results In lung fibroblasts, activated lymphocytes promote an increase of expression of cyclooxygenase-2 and ICAM-1, expressed as mean fluorescence intensity (MFI), from 5.4 ± 0.9 and 0.7 ± 0.15 to 9.1 ± 1.5 and 38.6 ± 7.8, respectively. Fibroblasts, in turn, induce a significant reduction of transcription and protein expression of CD69, LFA-1 and CD28 in activated lymphocytes and CD3 in resting lymphocytes. In activated T lymphocytes, LFA-1, CD28 and CD69 expression was 16.6 ± 0.7, 18.9 ± 1.9 and 6.6 ± 1.3, respectively, and was significantly reduced by fibroblasts to 9.4 ± 0.7, 9.4 ± 1.4 and 3.5 ± 1.0. CD3 expression in resting lymphocytes was 11.9 ± 1.4 and was significantly reduced by fibroblasts to 6.4 ± 1.1. Intracellular cytokines, TNF-alpha and IL-10, were evaluated in T lymphocytes. Co-incubation with fibroblasts reduced the number of TNF-alpha positive lymphocytes from 54,4% ± 6.12 to 30.8 ± 2.8, while IL-10 positive cells were unaffected. Finally, co-culture with fibroblasts significantly reduced Con A proliferative response of T lymphocytes, measured as MTT absorbance, from 0.24 ± 0.02 nm to 0.16 ± 0.02 nm. Interestingly, while the activation of fibroblasts is mediated by a soluble factor, a cognate interaction ICAM-1 mediated was demonstrated to be responsible for the modulation

  13. Excision repair in xeroderma pigmentosum group C cells is regulated differently in transformed cells and primary fibroblasts

    SciTech Connect

    Cleaver, J.E.

    1988-10-14

    Excision repair in xeroderma pigmentosum group C cells occurs at about 20-30% of normal levels. In confluent fibroblasts a unique characteristic of this low repair is that it is clustered, representing very efficient repair in a small region of the genome. In SV40-transformed fibroblasts and Epstein-Barr virus-transformed lymphocytes of complementation group C, however, excision repair is randomly distributed. This may be a consequence of the high rate of proliferation of both of these cell types, because random repair is also observed in rapidly proliferating group C fibroblasts. The distribution of sites that can be mended in group C cells, therefore, varies according to the transformed and proliferative state of the cells, demonstrating that transformed cells do not always exhibit repair characteristics identical to those of primary fibroblasts.

  14. Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

    PubMed Central

    Lai, Pei-Lun; Lin, Hsuan; Chen, Shang-Fu; Yang, Shang-Chih; Hung, Kuo-Hsuan; Chang, Ching-Fang; Chang, Hsiang-Yi; Lu, Frank Leigh; Lee, Yi-Hsuan; Liu, Yu-Chuan; Huang, Hsiao-Chun; Lu, Jean

    2017-01-01

    Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine. PMID:28303927

  15. Priming cancer cells for drug resistance: role of the fibroblast niche

    PubMed Central

    FANG, Wei Bin; YAO, Min; CHENG, Nikki

    2014-01-01

    Conventional and targeted chemotherapies remain integral strategies to treat solid tumors. Despite the large number of anti-cancer drugs available, chemotherapy does not completely eradicate disease. Disease recurrence and the growth of drug resistant tumors remain significant problems in anti-cancer treatment. To develop more effective treatment strategies, it is important to understand the underlying cellular and molecular mechanisms of drug resistance. It is generally accepted that cancer cells do not function alone, but evolve through interactions with the surrounding tumor microenvironment. As key cellular components of the tumor microenvironment, fibroblasts regulate the growth and progression of many solid tumors. Emerging studies demonstrate that fibroblasts secrete a multitude of factors that enable cancer cells to become drug resistant. This review will explore how fibroblast secretion of soluble factors act on cancer cells to enhance cancer cell survival and cancer stem cell renewal, contributing to the development of drug resistant cancer. PMID:25045348

  16. Highly efficient direct conversion of human fibroblasts to neuronal cells by chemical compounds.

    PubMed

    Dai, Ping; Harada, Yoshinori; Takamatsu, Tetsuro

    2015-05-01

    Direct conversion of mammalian fibroblasts into induced neuronal (iN) cells has been attained by forced expression of pro-neural transcriptional factors, or by combining defined factors with either microRNAs or small molecules. Here, we show that neuronal cells can be converted from postnatal human fibroblasts into cell populations with neuronal purities of up to >80% using a combination of six chemical compounds. The chemical compound-induced neuronal cells (CiNCs) express neuron-specific proteins and functional neuron markers. The efficiency of CiNCs is unaffected by either the donor's age or cellular senescence (passage number). We propose this chemical direct converting strategy as a potential approach for highly efficient generation of neuronal cells from human fibroblasts for such uses as in neural disease modeling and regenerative medicine.

  17. Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast

    PubMed Central

    1986-01-01

    NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found

  18. B cell homeostasis and follicle confines are governed by fibroblastic reticular cells.

    PubMed

    Cremasco, Viviana; Woodruff, Matthew C; Onder, Lucas; Cupovic, Jovana; Nieves-Bonilla, Janice M; Schildberg, Frank A; Chang, Jonathan; Cremasco, Floriana; Harvey, Christopher J; Wucherpfennig, Kai; Ludewig, Burkhard; Carroll, Michael C; Turley, Shannon J

    2014-10-01

    Fibroblastic reticular cells (FRCs) are known to inhabit T cell-rich areas of lymphoid organs, where they function to facilitate interactions between T cells and dendritic cells. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools that target this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrated their indispensable role in antiviral T cell responses. Unexpectedly, loss of FRCs also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and identifies a subset of FRCs that control B cell homeostasis and follicle identity.

  19. Cell cycle synchronization of skin fibroblast cells in four species of family Felidae.

    PubMed

    Wittayarat, M; Thongphakdee, A; Saikhun, K; Chatdarong, K; Otoi, T; Techakumphu, M

    2013-04-01

    This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 μm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 μm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.

  20. Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

    PubMed

    Bikfalvi, A; Cramer, E M; Tenza, D; Tobelem, G

    1991-01-01

    Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Chapter 6. available lepidopteran insect cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. More than 320 lines from 65 species are listed. The official designation is given for each cell line as well as the species, tissue source, and, when kno...

  5. 46,XY/45,X mosaicism in an amniotic fluid cell culture: suppression of abnormal cell line after subcultivation.

    PubMed Central

    Hasholt, L

    1979-01-01

    An abnormal cell population, 45,X, appeared in 3 of 4 cell lines established from an amniotic fluid specimen obtained from a normal mid-trimester pregnancy. Two of the cell lines were subjected to repeated chromosome analyses until VII passage. The abnormal cells were suppressed after repeated trypsinisations; simultaneously, fibroblast-like cells outgrew the cultures, which were previously predominated by epithelial-like cells. Polyploidy was found in 0 to 12% of the cells, the highest level existing in the early passages. The question of whether chromosomally abnormal cells present in primary cultures and the early subcultures reflect the karyotype of the fetus is discussed. PMID:573801

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

    SciTech Connect

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

    2009-05-01

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

  7. Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7

    PubMed Central

    Czerwinska, Areta M.; Nowacka, Joanna; Aszer, Magdalena; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Grabowska, Iwona

    2016-01-01

    ABSTRACT The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E. PMID:27610933

  8. Temporal variations in cell migration and traction during fibroblast-mediated gel compaction.

    PubMed

    Shreiber, David I; Barocas, Victor H; Tranquillo, Robert T

    2003-06-01

    Current models used in our laboratory to assess the migration and traction of a population of cells within biopolymer gels are extended to investigate temporal changes in these parameters during compaction of mechanically constrained gels. The random cell migration coefficient, micro (t) is calculated using a windowing technique by regressing the mean-squared displacement of cells tracked at high magnification in three dimensions with a generalized least squares algorithm for a subset of experimental time intervals, and then shifting the window interval-by-interval until all time points are analyzed. The cell traction parameter, tau(0)(t), is determined by optimizing the solution of our anisotropic biphasic theory to tissue equivalent compaction. The windowing technique captured simulated sinusoidal and step changes in cell migration superposed on a persistent random walk in simulated cell movement. The optimization software captured simulated time dependence of compaction on cell spreading. Employment of these techniques on experimental data using rat dermal fibroblasts (RDFs) and human foreskin fibroblasts (HFFs) demonstrated that these cells exhibit different migration-traction relationships. Rat dermal fibroblast migration was negatively correlated to traction, suggesting migration was not the driving force for compaction with these cells, whereas human foreskin fibroblast migration was positively correlated to traction.

  9. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

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

    PubMed Central

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

    2012-01-01

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

  11. Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Caulfield, J.P.; Hein, A.; Bloes, W.F.; Stevens, R.L.

    1985-11-01

    Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The /sup 35/S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1988-05-01

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

  15. Eosinophil activation of fibroblasts from chronic allergen-induced disease utilizes stem cell factor for phenotypic changes.

    PubMed

    Dolgachev, Vladislav; Berlin, Aaron A; Lukacs, Nicholas W

    2008-01-01

    In the present studies the role of stem cell factor (SCF) in mediating eosinophil and fibroblast activation during their interaction was investigated. SCF was significantly higher in fibroblasts grown from lungs of chronic allergen-challenged mice compared to fibroblasts grown from normal mice. When eosinophils were layered onto fibroblasts from allergic mice, a significant increase in SCF was detected compared to fibroblasts from nonallergic mice. The interaction of fibroblasts with eosinophils also increased the production of asthma-associated chemokines, CCL5 and CCL6, was dependent on cell-to-cell interaction, and was observed only with fibroblasts derived from lungs of chronic allergen-challenged mice and not from those derived from unchallenged normal mice. Chemokine production was significantly decreased when anti-SCF antibodies were added during eosinophil-fibroblast interaction. The interaction of fibroblasts from chronic allergen-challenged mice with eosinophils also increased alpha-smooth muscle cell actin and procollagen I expression as well as induced transforming growth factor-beta. The changes in myofibroblast activation were dependent on SCF-mediated pathways because anti-SCF antibody treatment reduced the expression of all three of these latter fibrosis-associated markers. Thus, our data suggest that SCF mediates an important activation pathway for fibroblasts during chronic allergic responses on interaction with recruited eosinophils and suggest a potential mechanism of airway remodeling during chronic disease.

  16. Generation of KCL038 clinical grade human embryonic stem cell line

    PubMed Central

    Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345799

  17. Generation of KCL031 clinical grade human embryonic stem cell line

    PubMed Central

    Jacquet, Laureen; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL031 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27345813

  18. Generation of KCL039 clinical grade human embryonic stem cell line

    PubMed Central

    Devito, Liani; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL039 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345806

  19. Generation of KCL037 clinical grade human embryonic stem cell line

    PubMed Central

    Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL037 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345800

  20. Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.

    PubMed Central

    Falciani, F; Terao, M; Goldwurm, S; Ronchi, A; Gatti, A; Minoia, C; Li Calzi, M; Salmona, M; Cazzaniga, G; Garattini, E

    1994-01-01

    The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor. Images Figure 1 Figure 2 Figure 4 PMID:8129733

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

    PubMed

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

    2014-01-01

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

  2. Mechanism of enhanced fibroblast arachidonic acid metabolism by mononuclear cell factor.

    PubMed Central

    Whiteley, P J; Needleman, P

    1984-01-01

    Chronic inflammation is associated with an infiltration of mononuclear cells, fibroblast proliferation, and elevated levels of prostaglandin (PG) E2. Mononuclear cell conditioned factor (MNCF) medium (5%) stimulated a 100-fold increase in basal human dermal fibroblast PGE2 release over 48 h as compared with fibroblasts that were incubated with control medium (conditioned medium prepared without cells). The MNCF-induced PGE2 production was suppressed by protein synthesis inhibitors. Fibroblasts pretreated with control medium released PGE2 only modestly in response to 1 nM bradykinin for 1 h (basal, 50 +/- 7 pg PGE2/micrograms protein; stimulated, 104 +/- 12 pg PGE2/micrograms protein), whereas cells that had been pretreated with MNCF showed a greatly facilitated bradykinin-induced release of PGE2. (basal, 297 +/- 59 pg PGE2/micrograms protein; stimulated, 866 +/- 85 pg PGE2/micrograms protein). The exaggerated agonist response is not specific for bradykinin because platelet-derived growth factor elicits a similar response. Exogenous arachidonic acid conversion to PGE2 was also facilitated (two- to threefold) by MNCF pretreatment as compared with control. Both the enhanced agonist-stimulated and exogenous arachidonic acid-induced PGE2 release from the MNCF pretreated cells were inhibited by actinomyin D or cycloheximide. A kinetic study of microsomal cyclooxygenase prepared from fibroblasts pretreated with MNCF showed a threefold increase in the maximum velocity (Vmax) but the same Michaelis constant (Km) as control-treated cells. This augmented arachidonic acid metabolism and subsequent enhanced PGE2 production may play an important role in macrophage-fibroblast interactions at sites of inflammation. PMID:6439745

  3. Helium Generated Cold Plasma Finely Regulates Activation of Human Fibroblast-Like Primary Cells

    PubMed Central

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

    2014-01-01

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

  4. Fibroblastic reticular cells: organization and regulation of the T lymphocyte life cycle.

    PubMed

    Brown, Flavian D; Turley, Shannon J

    2015-02-15

    The connective tissue of any organ in the body is generally referred to as stroma. This complex network is commonly composed of leukocytes, extracellular matrix components, mesenchymal cells, and a collection of nerves, blood, and lymphoid vessels. Once viewed primarily as a structural entity, stromal cells of mesenchymal origin are now being intensely examined for their ability to directly regulate various components of immune cell function. There is particular interest in the ability of stromal cells to influence the homeostasis, activation, and proliferation of T lymphocytes. One example of this regulation occurs in the lymph node, where fibroblastic reticular cells support the maintenance of naive T cells, induce Ag-specific tolerance, and restrict the expansion of newly activated T cells. In an effort to highlight the varied immunoregulatory properties of fibroblastic reticular cells, we reviewed the most recent advances in this field and provide some insights into potential future directions.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    1982-01-01

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

  7. Sister chromatid exchange response of human diploid fibroblasts and Chinese hamster ovary cells to dimethylnitrosamine and benzo(a)pyrene

    SciTech Connect

    Tomkins, D.J.; Kwok, S.E.; Douglas, G.R.; Biggs, D.

    1982-01-01

    In the search for relevant assays for mutagenicity testing, considerable attention has been given to the use of mammalian cells in vitro and the incorporation of metabolic activation in the protocol. Chinese hamster ovary (CHO) cells are commonly chosen as the target cells for cytogenetic tests because of their excellent growth characteristics and long lifespan in culture. However, there may be cellular factors affecting the uptake, metabolism, and repair of damage which are not the same in cell lines. The response of CHO cells and three human diploid fibroblast strains (1MR-90, WI-38, S-3299) to benzo(a)pyrene (BP) and dimethylnitrosamine (DMN) were compared using sister chromatid exchange (SCE) analysis as a measure of genetic damage. For both BP and DMN the human cells and the CHO cells showed dose-response slopes that were significantly different from zero, except CHO cells treated with BP for 1 hr and S-3299 cells treated with DMN. Whereas human and CHO cells showed similar dose-response to BP and the three human cell strains had similar dose-responses to BP and DMN, the dose-response of the human cells to DMN was statistically less significant than that of CHO cells. Reducing the duration of chemical treatment in CHO cells had no effect on the slope of the dose-response curves for BP or DMN. The observed differences between human and CHO cells may reflect differences in the fate of metabolic intermediates of DMN.

  8. The relationship of fibroblast translocations to cell morphology and stress fibre density.

    PubMed

    Lewis, L; Verna, J M; Levinstone, D; Sher, S; Marek, L; Bell, E

    1982-02-01

    Translocation of human fibroblasts in culture was studied using techniques of time-lapse cinemicrography, indirect immunofluorescence, and computer analysis. An inverse relationship between the velocity of cells during the last hour of life and the density of stress fibers seen by immune staining was demonstrated. Translocating cells generally assumed one of two interconvertible morphologies: a triangular tailed shape or tailed fibroblast (TF), and a tailless form that resembled a half-moon, which we call a half-moon fibroblast (HMF). The tail of TFs formed only on regions of substrate that had been previously traversed by cells. The half-moon morphology developed either on previously used or on virgin substrate. Cells adopted the HMF rather than the TF morphology with a four-fold greater frequency. HMFs translocated slightly faster than TFs. The foregoing observation suggest that the fibroblast tail is not an organelle essential for translocation. Since our technique allowed us to distinguish between cells which were cycling and those which had left cycle, we compared their velocities and found them to be similar. Also the average velocities of cells of different population-doubling levels (10th, 30th, 40th) were approximately equal.

  9. Proliferation of the synovial lining cell layer in suggested metal hypersensitivity.

    PubMed

    Burkandt, Andreas; Katzer, Alexander; Thaler, Karlheinz; Von Baehr, Volker; Friedrich, Reinhard E; Rüther, Wolfgang; Amling, Michael; Zustin, Jozef

    2011-01-01

    Synovial tissues in joints with prostheses display characteristic morphological changes in cases with aseptic failure, particularly macrophage infiltration. Since proliferation of the synovial lining cell layer represents a feature characteristic of autoimmune joint diseases, the possibility of morphological changes of the synovial lining cell layer in periprosthetic tissues was investigated. Synovial biopsies from five groups of morphologically well-defined lesions (osteoarthritis, rheumatoid arthritis, aseptic loosened metal-on-polyethylene and metal-on-metal arthroplasty and suggested metal hypersensitivity) were compared using a conventional staining method and immunohistochemistry. The synovial lining cell layer was substantially enlarged in both rheumatoid arthritis and cases suggestive of metal hypersensitivity. Macrophage infiltrates were apparent in rheumatoid arthritis and all specimens from retrieved hip arthroplasties. Although both synovial and subsynovial macrophages were positive for CD163 (indicating synovial M2 macrophages), the remaining fibroblast-like synoviocytes and scattered stromal fibroblasts showed a positive reaction with the D2-40 antibody (indicating fibroblast-like synoviocytes). Furthermore, in contrast to CD163-positive macrophages, the enlarged D2-40-positive fibroblast-like synoviocytes displayed cytoplasmatic tubular projections. Proliferation of the periprosthetic synovial lining cell layer occurred in cases with unexplained groin pain following metal-on-metal hip resurfacing arthroplasty, suggestive of hypersensitivity. Despite some important study limitations, the present observation adds to the evidence that metal hypersensitivity shares characteristic morphological features with autoimmune diseases of the joints.

  10. Effects of Hypoxia, Surrounding Fibroblasts, and p16 Expression on Breast Cancer Cell Migration and Invasion.

    PubMed

    Zhang, Jun; Li, Liyuan; Lu, Yi

    2015-01-01

    Cancer cell migration and invasion play essential roles in the metastatic cascade that transforms the local, noninvasive confined tumor cells to the motile, metastatic cancer cells moving through the extracellular matrix and basement into the circulation. Accumulated evidences suggest that intratumoral hypoxia, a characteristic of fast-growing solid tumors, promotes cancer cell motile and invasive abilities. In this study, we investigated the effects of hypoxia, surrounding fibroblasts, and p16 expression on the migration and invasion of breast cancer cells. We found that hypoxia promoted breast cancer cell migration and invasion, and cocultured fibroblasts stimulated invasiveness of breast cancer cells. Moreover, by using a Tet-on inducible system, we found that p16 is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer cells. These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia-mediated cancer cell migration and invasion. This study may provide important validation for p16-mediated cancer therapy either by gene therapy or pharmacological activation of internal p16 gene that is usually inactive due to hypermethylation in the tumor cells.

  11. Radiation-induced division delay in Chinese hamster ovary fibroblast and carcinoma cells: dose effect and ploidy. [X-ray

    SciTech Connect

    Kimler, B.F.; Leeper, D.B.; Schneiderman, M.H.

    1981-02-01

    The mitotic selection procedure for cell cycle analysis was utilized to investigate the G/sub 2/ transition point for and the duration of radiation-induced division delay in diploid and tetraploid Chinese hamster ovary (CHO) fibroblasts and in Chinese hamster ovarian carcinoma cells. The location of the radiation-induced division delay transition point was dose independent at high doses and located approximately 42 min before division. At lower doses only an estimate of the point of blockade was possible; but the G/sub 2/ transition point appeared to be earlier in the cell cycle. The duration of radiation-induced division delay was dose dependent. This response is consistent with a sensitive population of cells in late G/sub 2/ that define the location of the transition point and the length of division delay. There was no difference observed in the dose response for radiation-induced division delay between the pseudotetraploid cell line of CHO and the pseudodiploid parent strain. However, in the cell line derived from a spontaneous Chinese hamster ovarian carcinoma the division delay was 39 +- 4 min/Gy. Therefore, radiation-induced division delay is independent of chromosome ploidy, but can show intraspecies cell line specificity.

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

    PubMed

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

    2016-06-02

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

  13. Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells

    PubMed Central

    Kohgadai, Nargis; Müller, Janis A.; Laustsen, Anders; Thavachelvam, Karthiga; Stürzel, Christina M.; Jones, Jennifer J.; Somsouk, Ma; Garcia, Maurice M.; Smith, James F.; Greenblatt, Ruth M.; Münch, Jan; Jakobsen, Martin R.; Giudice, Linda C.; Greene, Warner C.; Roan, Nadia R.

    2017-01-01

    Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells–by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV–without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy. PMID:28207890

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-15

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

  16. Biological effects of cobalt-chromium nanoparticles and ions on dural fibroblasts and dural epithelial cells.

    PubMed

    Behl, Bharat; Papageorgiou, Iraklis; Brown, Christopher; Hall, Richard; Tipper, Joanne L; Fisher, John; Ingham, Eileen

    2013-05-01

    The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121 μm(3)per cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm(3) (epithelial cells) and 121 μm(3) (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell types at any dose. Reactive oxygen species were induced in both cell types at 50 μm(3) per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of the particles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses.

  17. Tumor cells derived exosomes contain hTERT mRNA and transform nonmalignant fibroblasts into telomerase positive cells

    PubMed Central

    Beery, Einat; Nordenberg, Jardena; Pinchasi, Maria; Goldvaser, Hadar; Henick, Steven; Goldberg, Michal; Lahav, Meir

    2016-01-01

    Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter-cell communicators and affecting biological processes in “recipient” cells upon their uptake. The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from cancer cells via exosomes into telomerase negative fibroblasts, where it is translated into a fully active enzyme and transforms these cells into telomerase positive, thus creating a novel type of cells; non malignant cells with telomerase activity. All tested telomerase positive cells, including cancer cells and non malignant cells with overexpressed telomerase secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. Similarly exosomes isolated from sera of patients with pancreatic and lung cancer contained hTERT mRNA as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including increased proliferation, extension of life span and postponement of senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by phleomycin and from apoptosis, indicating that also telomerase “extracurricular” activities are manifested in the recipient cells. The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the alterations of cancer microenvironment and its role in cancer. The described process has an obvious therapeutic potential which will be explored in further studies. PMID:27385095

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

    PubMed Central

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

    2016-01-01

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

  19. Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia

    PubMed Central

    Hirakawa, Toshiki; Yashiro, Masakazu; Doi, Yosuke; Kinoshita, Haruhito; Morisaki, Tamami; Fukuoka, Tatsunari; Hasegawa, Tsuyoshi; Kimura, Kenjiro; Amano, Ryosuke; Hirakawa, Kosei

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs), the expression of insulin-like growth factor-1 (IGF1) and IGF1 receptor (IGF1R) was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9) was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O2) and hypoxia (1% O2). IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC. PMID:27487118

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

    PubMed

    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; You, Seungkwon

    2012-01-31

    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.

  1. Acidic fibroblast growth factor modulates Staphylococcus aureus adherence to human endothelial cells.

    PubMed Central

    Blumberg, E A; Hatcher, V B; Lowy, F D

    1988-01-01

    Alteration of human endothelial cells may increase their susceptibility to staphylococcal invasion and thus may contribute to the development of intravascular staphylococcal disease. Acidic fibroblast growth factor, a potent regulator of endothelial cell function, had a significant effect on Staphylococcus aureus infection of cultured human endothelial cells. Three of four S. aureus strains had diminished adherence to endothelial cells when the latter were grown in the presence of acidic fibroblast growth factor (P less than 0.05). The diminished adherence was time dependent, maximal at 72 h, and independent of the initial bacterial inoculum. A twofold enhancement of S. aureus adherence was observed when endothelial cells were pretreated with heparitinase. Adherence was unaffected by endothelial cell activation by interleukin-1 or endotoxin. Thus, acidic fibroblast growth factor exerted a protective effect, deterring S. aureus adherence to cultured endothelial cells. Endothelial cell heparan sulfate was also directly involved in the adherence process. Subtle modulations of endothelial cells can significantly affect the ability of S. aureus to adhere to and then infect these cells. Similar alterations may contribute to the ability of S. aureus to infect endovascular tissue in vivo. PMID:3259546

  2. Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

    PubMed

    Meng, Mingyao; Wang, Wenju; Yan, Jun; Tan, Jing; Liao, Liwei; Shi, Jianlin; Wei, Chuanyu; Xie, Yanhua; Jin, Xingfang; Yang, Li; Jin, Qing; Zhu, Huirong; Tan, Weiwei; Yang, Fang; Hou, Zongliu

    2016-08-01

    Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

  3. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  4. Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts.

    PubMed

    French, Andrew J; Adams, Catharine A; Anderson, Linda S; Kitchen, John R; Hughes, Marcus R; Wood, Samuel H

    2008-02-01

    Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20-24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.

  5. Stromal fibroblasts facilitate cancer cell invasion by a novel invadopodia-independent matrix degradation process

    PubMed Central

    Krueger, Eugene W.; Chen, Jing; Qiang, Li; McNiven, Mark A.

    2015-01-01

    Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process utilizes complex, actin-based, specializations of the plasma membrane termed invadopodia that act both to sequester and release matrix metalloproteinases. Here we report that cells of mesenchymal origin, including tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. These degradative sites lack the punctate shape of conventional invadopodia to spread along the cell base and are reticular and/or fibrous in character. In marked contrast to invadopodia, this degradation does not require the action of Src kinase, Cdc42, or Dyn2. Rather, inhibition of Dyn2 causes a dramatic upregulation of stromal matrix degradation. Further, expression and activity of matrix metalloproteinases are differentially regulated between tumor cells and stromal fibroblasts. This matrix remodeling by fibroblasts increases the invasive capacity of tumor cells, thereby illustrating how the tumor microenvironment can contribute to metastasis. These findings provide evidence for a novel matrix remodeling process conducted by stromal fibroblasts that is substantially more effective than conventional invadopodia, distinct in structural organization, and regulated by disparate molecular mechanisms. PMID:25982272

  6. Fibulin1C peptide induces cell attachment and extracellular matrix deposition in lung fibroblasts

    PubMed Central

    Ge, Qi; Chen, Ling; Jaffar, Jade; Argraves, William Scott; Twal, Waleed O.; Hansbro, Phil; Black, Judith L.; Burgess, Janette K.; Oliver, Brian

    2015-01-01

    Fibulin-1 is an extracellular matrix (ECM) protein, levels of which are elevated in serum and lung tissue from patients with idiopathic pulmonary fibrosis compared to healthy volunteers. Inhibition of fibulin-1C, one of four fibulin-1 isoforms, reduced proliferation and wound healing in human airway smooth muscle (ASM) cells. This study identified the bioactive region/s of fibulin-1C which promotes fibrosis. Seven fibulin-1C peptides were synthesized and used to pre-coat tissue culture plates before lung derived ASM cells and fibroblasts from patients with pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD) or neither disease (Control) were plated. Peptide effects on in vitro measures of fibrosis: cell attachment, proliferation and viability, and ECM deposition, were examined. Among these peptides, peptide 1C1 (FBLN1C1) enhanced ASM cell and fibroblast attachment. FBLN1C1 increased mitochondrial activity and proliferation in fibroblasts. In addition, FBLN1C1 stimulated fibulin1 deposition in PF and COPD fibroblasts, and augmented fibronectin and perlecan deposition in all three groups. Peptides FBLN1C2 to FBLN1C7 had no activity. The active fibulin-1C peptide identified in this study describes a useful tool for future studies. Ongoing investigation of the role of fibulin-1 may reveal the mechanisms underlying the pathphysiology of chronic lung diseases. PMID:25834989

  7. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    SciTech Connect

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-02-19

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor {gamma}2, CCAAT/enhancer-binding protein alpha (C/EBP{alpha}), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBP{beta} or C/EBP{delta}, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

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

    PubMed

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

    2016-11-28

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

  9. Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative

    SciTech Connect

    Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

    1982-08-01

    A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. The gaps may represent single-strand breaks. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or beta-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

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

    PubMed Central

    Persson, Henrik; Købler, Carsten; Mølhave, Kristian; Samuelson, Lars; Tegenfeldt, Jonas O; Oredsson, Stina; Prinz, Christelle N

    2013-01-01

    Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells. PMID:23813871

  11. Relocalization of cell adhesion molecules during neoplastic transformation of human fibroblasts.

    PubMed

    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.

  12. Focal adhesion kinase knockdown in carcinoma-associated fibroblasts inhibits oral squamous cell carcinoma metastasis via downregulating MCP-1/CCL2 expression.

    PubMed

    Min, Anjie; Zhu, Chao; Wang, Jingyi; Peng, Shuping; Shuai, Cijun; Gao, Shan; Tang, Zhangui; Su, Tong

    2015-02-01

    Carcinoma-associated fibroblasts (CAFs) have been demonstrated to play an important role in the occurrence and development of oral squamous cell carcinoma (OSCC). The aim of this study is to investigate the influence of CAFs on OSCC cells and to explore the role of focal adhesion kinase (FAK) in this process. The results showed that oral CAFs expressed a higher level of FAK than normal human gingival fibroblasts (HGFs), and the conditioned medium (CM) of CAFs could induce the invasion and migration of SCC-25, one oral squamous carcinoma cell line. However, knockdown of FAK by small interfering RNA (siRNA) resulted in inhibition of CAF-CM induced cell invasion and migration in SCC-25, probably by reducing the production of monocyte chemoattractant protein-1 (MCP-1/CCL2), one of downstream target chemokines. Therefore, our findings indicated that targeting FAK in CAFs might be a promising strategy for the treatment of OSCC in the future.

  13. PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

    PubMed Central

    Cheng, Ee-Chun; Kang, Dongwan; Wang, Zhong; Lin, Haifan

    2014-01-01

    PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts. PMID:25238487

  14. G1/S control of anchorage-independent growth in the fibroblast cell cycle

    PubMed Central

    1991-01-01

    We have developed methodology to identify the block to anchorage- independent growth and position it within the fibroblast cell cycle. Results with NRK fibroblasts show that mitogen stimulation of the G0/G1 transition and G1-associated increases in cell size are minimally affected by loss of cell anchorage. In contrast, the induction of G1/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked. Moreover, we demonstrate that the anchorage-dependent transition maps to late G1 and shortly before activation of the G1/S p34cdc2-like kinase. The G1/S block was also detectable in NIH-3T3 cells. Our results: (a) distinguish control of cell cycle progression by growth factors and anchorage; (b) indicate that anchorage mediates G1/S control in fibroblasts; and (c) identify a physiologic circumstance in which the phenotype of mammalian cell cycle arrest would closely resemble Saccharomyces cerevisiae START. The close correlation between anchorage independence in vitro and tumorigenicity in vivo emphasizes the key regulatory role for G1/S control in mammalian cells. PMID:1955482

  15. Rho-kinase inhibitor Y-27632 increases cellular proliferation and migration in human foreskin fibroblast cells.

    PubMed

    Piltti, Juha; Varjosalo, Markku; Qu, Chengjuan; Häyrinen, Jukka; Lammi, Mikko J

    2015-09-01

    The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho-kinase inhibitor Y-27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short-term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 μM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell-IQ time-lapse imaging analysis. Rho-kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin-associated focal adhesions were present in the ROCKi-treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte-specific genes, such as procollagen α1 (II) and aggrecan.

  16. Contribution of Fibroblast and Mast Cell (Afferent) and Tumor (Efferent) IL-6 Effects within the Tumor Microenvironment.

    PubMed

    Hugo, Honor J; Lebret, Stephanie; Tomaskovic-Crook, Eva; Ahmed, Nuzhat; Blick, Tony; Newgreen, Donald F; Thompson, Erik W; Ackland, M Leigh

    2012-04-01

    Hyperactive inflammatory responses following cancer initiation have led to cancer being described as a 'wound that never heals'. These inflammatory responses elicit signals via NFκB leading to IL-6 production, and IL-6 in turn has been shown to induce epithelial to mesenchymal transition in breast cancer cells in vitro, implicating a role for this cytokine in cancer cell invasion. We previously have shown that conditioned medium derived from cancer-associated fibroblasts induced an Epithelial to Mesenchymal transition (EMT) in PMC42-LA breast cancer cells and we have now identify IL-6 as present in this medium. We further show that IL-6 is expressed approximately 100 fold higher in a cancer-associated fibroblast line compared to normal fibroblasts. Comparison of mouse-specific (stroma) and human-specific (tumor) IL-6 mRNA expression from MCF-7, MDA MB 468 and MDA MB 231 xenografts also indicated the stroma rather than tumor as a significantly higher source of IL-6 expression. Mast cells (MCs) feature in inflammatory cancer-associated stroma, and activated MCs secrete IL-6. We observed a higher MC index (average number of mast cells per xenograft section/average tumor size) in MDA MB 468 compared to MDA MB 231 xenografts, where all MC were observed to be active (degranulating). This higher MC index correlated with greater mouse-specific IL-6 expression in the MDA MB 468 xenografts, implicating MC as an important source of stromal IL-6. Furthermore, immunohistochemistry on these xenografts for pSTAT3, which lies downstream of the IL-6 receptor indicated frequent correlations between pSTAT3 and mast cell positive cells. Analysis of publically available databases for IL-6 expression in patient tissue revealed higher IL-6 in laser capture microdissected stroma compared to adjacent tissue epithelium from patients with inflammatory breast cancer (IBC) and invasive non-inflammatory breast cancer (non-IBC) and we show that IL-6 expression was significantly higher in Basal

  17. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

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

    PubMed

    Ohshima, Susumu; Seyama, Atsushi

    2012-09-01

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

  19. Identification of beta-adrenergic receptors on cultured human fibroblast IMR-90 cells

    SciTech Connect

    Scarpace, P.J.

    1986-03-05

    Fibroblast cultures derived from normal human tissue undergo a finite number of population doublings when serial subcultivated in vitro. IMR-90 cells derived from human embryonic lung tissue undergo approximately 60 population doublings. Beta-adrenergic receptor (BAR) characteristics and isoproterenol-stimulated adenylate cyclase activity were assessed in IMR-90 cells at various population doublings (PDL). Scatchard analysis of /sup 125/I-Iodocyanopindolol (ICYP) binding yields a straight line consistent with a single class of antagonist binding sites with 452 +/- 35 sites/cell and a dissociation constant of 18.7 +/- 1.3 pM. There were no changes in BAR density between PDL 32 to PDL 46. Binding was both stereospecific and specific. Competition with epinephrine was 7.4 times more potent than with norepinephrine, suggesting predominate beta/sub 2/-type BAR. Competition with isoproterenol (Hill plots) indicated an apparent K/sub d/ of 1.3 +/- 0.1 x 10/sup -8/ M. Competition curves were resolved into high and low affinity binding sites yielding K/sub d/-high = 3.6 +/- 1.3 nM and k/sub d/-low = 1.6 +/- 0.7 x 10/sup -7/ M with 49.9 +/- 6.6% of the receptors in the high affinity state at PDL 31 to PDL 37. Cultures demonstrate isoproterenol-stimulated adenylate cyclase activity with a concentration of 1 x 10/sup -6/ M producing half-maximal stimulation and a maximum stimulation of 2 pmol cAMP/mg/min. Forskolin-stimulated was 10 pmol/cAMP/mg/min at PDL 46.

  20. Low-level laser therapy in 3D cell culture model using gingival fibroblasts.

    PubMed

    Basso, Fernanda G; Soares, Diana G; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-07-01

    Besides extensive data about the effects of low-level laser therapy (LLLT) on different cell types, so far, these results were obtained from monolayer cell culture models, which have limitations in terms of cell morphology and phenotype expression. Therefore, for better in vitro evaluation of the effects of LLLT, this study was performed with a 3D cell culture model, where gingival fibroblasts were seeded in collagen matrix. Cells isolated from a healthy patient were seeded in wells of 24-well plates with culture medium (DMEM) supplemented with 10 % fetal bovine serum and collagen type I solution. After 5 days, a serum-free DMEM was added to the matrices with cells that were subjected or not to three consecutive irradiations of LLLT by means of the LaserTABLE diode device (780 nm, 25 mW) at 0.5, 1.5, and 3 J/cm(2). Twenty-four hours after the last irradiation, cell viability and morphology as well as gene expression of growth factors were assessed. Histological evaluation of matrices demonstrated uniform distribution and morphology of gingival fibroblasts within the collagen matrix. LLLT at 3 J/cm(2) increased gingival fibroblast viability. Enhanced gene expression of hCOL-I and hEGF was observed for 0.5 J/cm(2), while no significant changes were detected for the other irradiation densities tested. In conclusion, LLLT promoted biostimulation of gingival fibroblasts seeded in a 3D cell culture model, demonstrating that this model can be applied for phototherapy studies and that LLLT could penetrate the collagen matrix to increase cell functions related to tissue repair.

  1. Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene

    PubMed Central

    Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

    2017-01-01

    This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells. PMID:28196103

  2. Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene.

    PubMed

    Wang, Kai; Jin, Song; Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

    2017-01-01

    This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells.

  3. Marrow stromal fibroblastic cell cultivation in vitro on decellularized bone marrow extracellular matrix.

    PubMed

    Dutra, Timothy F; French, Samuel W

    2010-02-01

    The in vitro biocompatibility of decellularized bone marrow extracellular matrix was evaluated. Following a freeze-thaw cycle, sectioned discs of fresh frozen rat metaphyseal bone were sequentially incubated in solutions of hypertonic, then hypotonic Ringer's solution, followed by deoxycholic acid, then DNAase I. The adequacy of decellularization of marrow stroma was examined by light microscopy. Marrow stromal fibroblastic cells were harvested by dispersion of rat long bone marrow, followed by concentration by discontinuous Ficoll-Paque gradient centrifugation. The fibroblastic cells were expanded by in vitro cultivation, and second passage cells were cryopreserved until needed. Cryopreserved marrow stromal cells were applied dropwise to sections of decellularized bone marrow extracellular matrix, and cultured in BJGb medium with 20% fetal bovine serum for ten days. Mature cultures were formalin fixed, decalcified, and embedded in paraffin. Light microscopy of hematoxylin and eosin stained sections showed individual spindle cells invading the upper portion of the decellularized extracellular matrix, and also a monolayer of spindle cells on the upper surfaces of exposed trabecular and cortical bone. This experiment showed that decellularized marrow extracellular matrix is a biocompatible three dimensional in vitro substrate for marrow stromal fibroblastic cells.

  4. Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells

    PubMed Central

    Mazzara, Pietro Giuseppe; Massimino, Luca; Pellegatta, Marta; Ronchi, Giulia; Ricca, Alessandra; Iannielli, Angelo; Giannelli, Serena Gea; Cursi, Marco; Cancellieri, Cinzia; Sessa, Alessandro; Del Carro, Ubaldo; Quattrini, Angelo; Geuna, Stefano; Gritti, Angela; Taveggia, Carla; Broccoli, Vania

    2017-01-01

    Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies. PMID:28169300

  5. Genetic polymorphism directs IL-6 expression in fibroblasts but not selected other cell types.

    PubMed

    Noss, Erika H; Nguyen, Hung N; Chang, Sook Kyung; Watts, Gerald F M; Brenner, Michael B

    2015-12-01

    Interleukin (IL)-6 blockade is an effective treatment for rheumatoid arthritis (RA), and synovial fibroblasts are a major IL-6 producer in the inflamed joint. We found that human RA and osteoarthritis (OA) synovial fibroblasts derived from independent donors reproducibly segregated into low, medium, and high IL-6 producers, independent of stimulus, cell passage, or disease state. IL-6 expression pattern correlated strongly with total mRNA expression, not mRNA stability, suggesting transcriptional rather than posttranscriptional regulation. High-fibroblast IL-6 expression was significantly associated with the IL-6 proximal promoter single nucleotide polymorphism (SNP) rs1800795 minor allele (CC) genotype. In contrast, no association between this SNP and IL-6 production was detected in CD14(+) monocytes, another major producer of synovial IL-6. Luciferase expression assays confirmed that this SNP was associated with differential IL-6 expression in fibroblasts. To date, several association studies examining rs1800795 allele frequency and disease risk have reported seemingly conflicting results ranging from no association to association with either the major or minor allele across a spectrum of conditions, including cancer and autoimmune, cardiovascular, infectious, and metabolic diseases. This study points to a prominent contribution from promoter genetic variation in fibroblast IL-6 regulation, but not in other IL-6-producing cell types. We propose that some of the heterogeneity in these clinical studies likely reflects the cellular source of IL-6 in specific diseases, much of which may be produced by nonhematopoietic cells. These results highlight that functional analysis of disease-associated SNPs on gene expression and pathologic processes must consider variation in diverse cell types.

  6. Altered Cellular Homeostasis in Murine MPS I Fibroblasts: Evidence of Cell-Specific Physiopathology.

    PubMed

    Viana, Gustavo Monteiro; do Nascimento, Cinthia Castro; Paredes-Gamero, Edgar Julian; D'Almeida, Vânia

    2017-02-21

    Mucopolysaccharidosis type I (MPS I), a rare autosomal recessive disease, is caused by a deficiency of the lysosomal enzyme alfa-L-iduronidase. Impaired enzyme activity promotes glycosaminoglycans accumulation in several tissues and organs, leading to complex multisystemic complications. Several studies using animal models indicated different intracellular pathways involving MPS I physiopathology; however, the exact mechanisms underlying this syndrome are still not understood. Previous results from our group showed alterations in ionic homeostasis and cell viability of splenocytes and macrophages in Idua-/- mice. In the present study, we found altered intracellular ionic homeostasis in a different cell type (fibroblasts) from the same murine model. Idua-/- fibroblasts from 3-month-old mice presented higher cytoplasmatic and endoplasmic reticulum Ca(2+) concentration, lower levels of mitochondrial Ca(2+) and mitochondrial membrane potential and higher cytoplasmatic pH when compared to Idua+/+ animals. Also, Idua-/- fibroblasts were more resistant to the apoptotic induction with staurosporine, indicating a possible resistance to apoptotic induction in those cells. In addition, despite the intracellular ionic imbalance, no significant alterations were found in apoptosis and autophagy in Idua-/- fibroblasts, which implies that the ionic alterations did not activate those pathways. The investigation of mechanisms underlying the cellular physiopathology of lysosomal diseases is crucial for a better understanding about the progression of these diseases. Since splenocytes, macrophages, and fibroblasts have different embryonic origins and distinct structural and functional features, potentially altered signaling pathways found in a cell-specific manner in an alfa-L-iduronidase-deficient environment provide additional understanding of the clinical multisystemic presentation of this disease and provide new basis for improved therapeutic approaches.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    PubMed Central

    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

  9. Cigarette smoke-exposed Candida albicans increased chitin production and modulated human fibroblast cell responses.

    PubMed

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

    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.

  10. Mononuclear cell modulation of connective tissue function: suppression of fibroblast growth by stimulation of endogenous prostaglandin production.

    PubMed Central

    Korn, J H; Halushka, P V; LeRoy, E C

    1980-01-01

    The role of immune cell products in modulating connective tissue metabolism was investigated. Supernates of both unstimulated and phytohemagglutinin-stimulated human mononuclear cell cultures suppressed fibroblast proliferation (up to 90%) and concomintantly stimulated fibroblast prostaglandin E(PGE) synthesis (20- to 70-fold). The growth suppression was, at least in part, a secondary result of the increased fibroblast PGE synthesis; growth suppression (a) paralled the increased fibroblast PGE synthesis, (b) was reversed by addition of inhibitors of prostaglandin synthesis (indomethacin, meclofenamate, and eicostaetraynoic acid), and (c) was reproduced by addition of exogenous PGE2 to fibroblast cultures. The prostaglandin-stimulatory, growth-suppressive activity was a product of non-T-lymphocyte, adherent cells and was present within 6 h of mononuclear cell culture. The activity was heat (56 degrees C) and trypsin sensitive, nondialyzable, and appeared in the 12,000-20,000 mol wt fractions by Sephadex G-100 chromatography. The activity in supernates of mononuclear cell cultures was removed by incubation with fibroblasts but not by similar incubation with peripheral blood mononuclear cells. Mononuclear cells release a factor(s) which modulates fibroblast proliferation by altering prostaglandin metabolism. PMID:7356693

  11. Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen.

    PubMed

    Chen, Wenshu; Xu, Xiuling; Bai, Lang; Padilla, Mabel T; Gott, Katherine M; Leng, Shuguang; Tellez, Carmen S; Wilder, Julie A; Belinsky, Steven A; Scott, Bobby R; Lin, Yong

    2012-07-01

    Despite decades of research in defining the health effects of low-dose (<100 mGy) ionizing photon radiation (LDR), the relationship between LDR and human cancer risk remains elusive. Because chemical carcinogens modify the tumor microenvironment, which is critical for cancer development, we investigated the role and mechanism of LDR in modulating the response of stromal cells to chemical carcinogen-induced lung cancer development. Secretion of proinflammatory cytokines such as interleukin-6 (IL-6), CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette-smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a single dose of LDR. The activation of NF-κB, which is important for BPDE-induced IL-6 secretion, was also effectively suppressed by LDR. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized normal human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody. Conditioned medium from LDR-primed and BPDE-treated fibroblast showed diminished capacity in activating STAT3. Furthermore, IL-6 enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke-induced lung carcinogenesis by suppressing secretion of cytokines such as IL-6 from fibroblasts in lung tumor-prone microenvironment.

  12. [Radioprotective effect of helium-neon laser radiation for fibroblast cells].

    PubMed

    Voskanian, K Sh; Mitsyn, G V; Gaevskiĭ, V N

    2007-01-01

    Effects of combined exposure to 633-nm laser waves and gamma-radiation, and laser waves and protons with the energy of 150 MeV on survivablilty of mice fibroblast cells C3H10T1/2 were compared. Cell suspension (1 - 5 x 10(5) cells/ml) was distributed in 2-ml plastic vials with 1 cm in diameter time interval between two exposures in a combination was no more than 60 s. immediately after exposure a required quantity of cells was inoculated in special vials for survivability assessment. Based on results of the experiment, preliminary and repeated laser treatment was favorable to survivability of fibroblast cells subjected to gamma- or proton irradiation (dose variation factor was within 1.3 to 2.2). Simultaneous exposure of C3H10T1/2 cells to the laser and proton beams also increased their survivability. The radioprotective effect of the helium-neon laser on fibroblasts earlier exposed to ionizing radiation is of chief interest, as most of the present-day radioprotectors are effective only if introduced into organism prior to exposure.

  13. Dual role of acetaminophen in promoting hepatoma cell apoptosis and kidney fibroblast proliferation

    PubMed Central

    YU, YUNG-LUEN; YIANG, GIOU-TENG; CHOU, PEI-LUN; TSENG, HSU-HUNG; WU, TSAI-KUN; HUNG, YU-TING; LIN, PEI-SHIUAN; LIN, SHU-YU; LIU, HSIAO-CHUN; CHANG, WEI-JUNG; WEI, CHYOU-WEI

    2014-01-01

    Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high-dose APAP-induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low-dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high-dose APAP treatment inhibited while therapeutic and low-dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase-9/-3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low-dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts. PMID:24682227

  14. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells.

    PubMed

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA1-LPA6) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA1 and LPA5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA1.

  15. Fibroblast growth factor signaling and inhibition in non-small cell lung cancer and their role in squamous cell tumors

    PubMed Central

    Salgia, Ravi

    2014-01-01

    With the introduction of targeted agents primarily applicable to non-small cell lung cancer (NSCLC) of adenocarcinoma histology, there is a heightened unmet need in the squamous cell carcinoma population. Targeting the angiogenic fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway is among the strategies being explored in squamous NSCLC; these efforts are supported by growth-promoting effects of FGF signaling in preclinical studies (including interactions with other pathways) and observations suggesting that FGF/FGFR-related aberrations may be more common in squamous versus adenocarcinoma and other histologies. A number of different anti-FGF/FGFR approaches have shown promise in preclinical studies. Clinical trials of two multitargeted tyrosine kinase inhibitors are restricting enrollment to patients with squamous NSCLC: a phase I/II trial of nintedanib added to first-line gemcitabine/cisplatin and a phase II trial of ponatinib for previously treated advanced disease, with the latter requiring not only squamous disease but also a confirmed FGFR kinase amplification or mutation. There are several ongoing clinical trials of multitargeted agents in general NSCLC populations, including but not limited to patients with squamous disease. Other FGF/FGFR-targeted agents are in earlier clinical development. While results are awaited from these clinical investigations in squamous NSCLC and other disease settings, additional research is needed to elucidate the role of FGF/FGFR signaling in the biology of NSCLC of different histologies. PMID:24711160

  16. Severe hypoxia and malnutrition collectively contribute to scar fibroblast inhibition and cell apoptosis.

    PubMed

    Lynam, Emily C; Xie, Yan; Dawson, Rebecca; Mcgovern, Jacqui; Upton, Zee; Wang, XiQiao

    2015-09-01

    This study aims to investigate whether severe hypoxia and malnutrition in scar tissue play key roles to induce hypertrophic scar regression. And scar-derived fibroblasts were treated with moderate/severe hypoxia and malnutrition to model condition of proliferative and regressive scar (5%O2 +5%FCS and 0.5%O2  + 0.5%FCS), and normoxia with well nutrition as control (10%O2  + 10%FCS). Our results demonstrated that severe hypoxia and malnutrition resulted in significantly reduced cell viability and collagen production, as well as HIF-1, VEGF, TGF-β1, and Bcl-2 protein expression when compared with control, and cell apoptosis occurred. Therefore, the severe hypoxia and malnutrition in scar tissue contribute to fibroblast inhibition and cell apoptosis, which is correlated with scar regression.

  17. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    PubMed

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  18. Increase in the radioresistance of normal skin fibroblasts but not tumor cells by mechanical injury.

    PubMed

    Chen, Zelin; Wang, Xin; Jin, Taotao; Wang, Yu; Hong, Christopher S; Tan, Li; Dai, Tingyu; Wu, Liao; Zhuang, Zhengping; Shi, Chunmeng

    2017-02-02

    The timing of radiation after mechanical injury such as in the case of surgery is considered a clinical challenge because radiation is assumed to impair wound healing. However, the physiological responses and underlying mechanisms of this healing impairment are still unclear. Here, we show that mechanical injury occurring before ionizing radiation decreases radiation-induced cell damage and increases cell repair in normal fibroblasts but not tumor cells in vitro and in vivo. At the molecular level, mechanical injury interrupts focal adhesion complexes and cell-cell cadherin interactions, transducing mechanical signals into intracellular chemical signals via activation of the phosphatidylinositol 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 beta (GSK-3β) pathways. We show that subsequent nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and β-catenin strengthen the stemness, antioxidant capabilities, and DNA double-strand break repair abilities of fibroblasts, ultimately contributing to increased radioresistance. Our findings demonstrate that mechanical injury to normal fibroblasts enhances radioresistance and may therefore question conventional wisdom surrounding the timing of radiation after surgery.

  19. Gravity spun polycaprolactone fibres for soft tissue engineering: interaction with fibroblasts and myoblasts in cell culture.

    PubMed

    Williamson, Matthew Richard; Adams, Eric F; Coombes, Allan G A

    2006-03-01

    Poly(epsilon-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. As-spun PCL fibres exhibited a mean strength and stiffness of 7.9 MPa and 0.1 GPa, respectively and a rough, porous surface morphology. Cold drawing to an extension of 500% resulted in increases in fibre strength (43 MPa) and stiffness (0.3 GPa) and development of an oriented, fibrillar surface texture. The proliferation rate of Swiss 3T3 mouse fibroblasts and C2C12 mouse myoblasts on as-spun, 500% cold-drawn and gelatin-modified PCL fibres was determined in cell culture to provide a basic measure of the biocompatibility of the fibres. Proliferation of both cell types was consistently higher on gelatin-coated fibres relative to as-spun fibres at time points below 7 days. Fibroblast growth rates on cold-drawn PCL fibres exceeded those on as-spun fibres but myoblast proliferation was similar on both substrates. After 1 day in culture, both cell types had spread and coalesced on the fibres to form a cell layer, which conformed closely to the underlying topography. The high fibre compliance combined with a potential for modifying the fibre surface chemistry with cell adhesion molecules and the surface architecture by cold drawing to enhance proliferation of fibroblasts and myoblasts, recommends further investigation of gravity-spun PCL fibres for 3-D scaffold production in soft tissue engineering.

  20. Skin deep: from dermal fibroblasts to pancreatic beta cells.

    PubMed

    Manzar, Gohar S; Kim, Eun-Mi; Rotti, Pavana; Zavazava, Nicholas

    2014-08-01

    Type I diabetes (T1D) is a chronic autoimmune disease caused by pancreatic β-cell destruction induced by autoantibodies and autoreactive T cells. After significant reduction of the β-cell mass, diabetes sets in and can cause significant complications. It is estimated that more than 3 million Americans have T1D, and its prevalence among young individuals is progressively rising; however, the reasons for this increase are not known. Islet transplantation is recognized as the ultimate cure for T1D, but unfortunately, the severe scarcity of available islets makes it necessary to establish alternative sources of β-cells. Our lab seeks to establish human-induced pluripotent stem cells as an unlimited, novel source of insulin-producing cells (IPCs) that are patient-specific, obviating the requirement for immunosuppression. Although several reports have emerged demonstrating successful derivation of IPCs from human pluripotent stem cells, the efficiencies of derivation are inadequate and these IPCs do not respond to glucose stimulation in vitro. We reasoned that the use of a growth factor sequestering bioscaffold and promotion of cell-cell signaling through 3D clustering would enhance the generation of functionally superior IPCs compared to those derived by 2D differentiation. Here, we discuss a novel 3D platform for the generation of highly efficient human IPCs.

  1. Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

    PubMed Central

    Agley, Chibeza C.; Rowlerson, Anthea M.; Velloso, Cristiana P.; Lazarus, Norman L.; Harridge, Stephen D. R.

    2015-01-01

    The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56– and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 106 ± 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56– cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package. PMID:25650991

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

    PubMed

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

    2013-10-01

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

  3. Lefty-1 alleviates TGF-β1-induced fibroblast-myofibroblast transdifferentiation in NRK-49F cells.

    PubMed

    Zhang, Lijun; Zhang, Jie; Xu, Changgeng; Zhou, Xiangjun; Wang, Wei; Zheng, Renping; Hu, Wei; Wu, Pin

    2015-01-01

    Fibroblast activation and proliferation are important for fibroblast-myofibroblast transdifferentiation, a crucial process in the pathological changes that define renal interstitial fibrosis. The left-right determination factor (Lefty) is an important cytokine of the transforming growth factor (TGF)-β family, with two variants, Lefty-1 and Lefty-2, in mice. Lefty has diverse functions, such as the regulation of embryonic development, the inhibition of TGF-β1 signaling, and the suppression of tumor activity. However, whether Lefty-1 influences fibroblast activation and proliferation, and consequently prevents fibroblast-myofibroblast transdifferentiation, remains unclear. This study aimed to investigate whether Lefty-1 can attenuate TGF-β1-induced fibroblast-myofibroblast transdifferentiation in normal rat kidney interstitial fibroblast cells (NRK-49F), as well as the mechanisms underlying any effects. Results showed that the typical fibroblast cell morphology of NRK-49F cells was altered after TGF-β1 treatment and that Lefty-1 significantly prevented this change in a dose-dependent manner. Further analyses demonstrated decreased proliferating cell nuclear antigen, cyclin D1, collagen I(A1), alpha-smooth muscle actin, and fibronectin expression. Lefty-1 further induced remarkable reductions in TGF-β1-induced Smad3 and mitogen-activated protein kinase-10/c-Jun N-terminal kinase (JNK-3) signaling, and enhanced expression of the antifibrotic factor bone morphogenetic protein (BMP)-5. However, without TGF-β1, Lefty-1 had no effect on Smad3, JNK-3, and BMP-5 activation and fibroblast-myofibroblast transdifferentiation. Taken together, these findings indicate that Lefty-1 can alleviate TGF-β1-mediated activation and the proliferation of fibroblasts. Furthermore, Lefty-1 may prevent fibroblast-myofibroblast transdifferentiation in part via modulations of Smad3, JNK-3, and BMP-5 activities in the TGF-β/BMP signaling pathway.

  4. Transition of Mesothelial Cell to Fibroblast in Peritoneal Dialysis: EMT, Stem Cell or Bystander?

    PubMed Central

    Liu, Yu; Dong, Zheng; Liu, Hong; Zhu, Jiefu; Liu, Fuyou; Chen, Guochun

    2015-01-01

    Long-term peritoneal dialysis (PD) can lead to fibrotic changes in the peritoneum, characterized by loss of mesothelial cells (MCs) and thickening of the submesothelial area with an accumulation of collagen and myofibroblasts. The origin of myofibroblasts is a central question in peritoneal fibrosis that remains unanswered at present. Numerous clinical and experimental studies have suggested that MCs, through epithelial-mesenchymal transition (EMT), contribute to the pool of peritoneal myofibroblasts. However, recent work has placed significant doubts on the paradigm of EMT in organ fibrogenesis (in the kidney particularly), highlighting the need to reconsider the role of EMT in the generation of myofibroblasts in peritoneal fibrosis. In particular, selective cell isolation and lineage-tracing experiments have suggested the existence of progenitor cells in the peritoneum, which are able to switch to fibroblast-like cells when stimulated by the local environment. These findings highlight the plastic nature of MCs and its contribution to peritoneal fibrogenesis. In this review, we summarize the key findings and caveats of EMT in organ fibrogenesis, with a focus on PD-related peritoneal fibrosis, and discuss the potential of peritoneal MCs as a source of myofibroblasts. PMID:25700459

  5. TP53inp1 Gene Is Implicated in Early Radiation Response in Human Fibroblast Cells.

    PubMed

    Sándor, Nikolett; Schilling-Tóth, Boglárka; Kis, Enikő; Fodor, Lili; Mucsányi, Fruzsina; Sáfrány, Géza; Hegyesi, Hargita

    2015-10-23

    Tumor protein 53-induced nuclear protein-1 (TP53inp1) is expressed by activation via p53 and p73. The purpose of our study was to investigate the role of TP53inp1 in response of fibroblasts to ionizing radiation. γ-Ray radiation dose-dependently induces the expression of TP53inp1 in human immortalized fibroblast (F11hT) cells. Stable silencing of TP53inp1 was done via lentiviral transfection of shRNA in F11hT cells. After irradiation the clonogenic survival of TP53inp1 knockdown (F11hT-shTP) cells was compared to cells transfected with non-targeting (NT) shRNA. Radiation-induced senescence was measured by SA-β-Gal staining and autophagy was detected by Acridine Orange dye and microtubule-associated protein-1 light chain 3 (LC3B) immunostaining. The expression of TP53inp1, GDF-15, and CDKN1A and alterations in radiation induced mitochondrial DNA deletions were evaluated by qPCR. TP53inp1 was required for radiation (IR) induced maximal elevation of CDKN1A and GDF-15 expressions. Mitochondrial DNA deletions were increased and autophagy was deregulated following irradiation in the absence of TP53inp1. Finally, we showed that silencing of TP53inp1 enhances the radiation sensitivity of fibroblast cells. These data suggest functional roles for TP53inp1 in radiation-induced autophagy and survival. Taken together, we suppose that silencing of TP53inp1 leads radiation induced autophagy impairment and induces accumulation of damaged mitochondria in primary human fibroblasts.

  6. Differentiation of human foreskin fibroblast-derived induced pluripotent stem cells into hepatocyte-like cells.

    PubMed

    Wang, Jianjun; Zhao, Ping; Wan, Zhihong; Jin, Xueyuan; Cheng, Yongqian; Yan, Tao; Qing, Song; Ding, Ning; Xin, Shaojie

    2016-10-01

    The aim of this study was to investigate the differentiation potential of induced pluripotent stem cells (iPSCs) derived from human foreskin fibroblasts (HFFs) into hepatocyte-like cells (HLCs). The iPSCs were firstly induced by transduction of OCT4, SOX2, KLF4, and c-MYC into HFFs using retrovirus. Afterwards, expressions of pluripotency factors were identified by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence staining, and karyotype, embryoid, and teratoma were observed by microscope. Then, iPSCs were gradually differentiated into endoderm cells, hepatic progenitor cells, and mature HLCs by special culture medium. During this process, differentiation efficiency into each kind of cells was evaluated by detecting SOX17, HNF4a, and ALB using flow cytometry, respectively. Besides, enzyme-linked immunosorbent assay was conducted to detect the secretion of ALB in iPSC-induced HLCs and quantitative reverse transcription-polymerase chain reaction was performed to detect the expression levels of hepatocyte-specific genes. The iPSCs were successfully induced by HFFs, which exhibited typical embryonic stem cells morphology, positive alkaline phosphatase staining, normal diploid karyotype, and positive expression of various pluripotency factors. Meanwhile, spherical embryoid and teratoma with 3 germ layers were formed by iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells and mature HLCs, and the differentiation efficiency was 55.7 ± 2.9%, 45.7 ± 4.8%, and 35.0 ± 3.9%, respectively. Besides, the secretion of ALB and expression of various hepatocyte-specific genes was highly detected in iPSC-induced HLCs. The iPSCs were successfully derived from HFFs and then differentiated into HLCs, which proved a new source for hepatocyte transplantation.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  8. Cellular metabolic rates from primary dermal fibroblast cells isolated from birds of different body masses.

    PubMed

    Jimenez, Ana Gabriela; Williams, Joseph B

    2014-10-01

    The rate of metabolism is the speed at which organisms use energy, an integration of energy transformations within the body; it governs biological processes that influence rates of growth and reproduction. Progress at understanding functional linkages between whole organism metabolic rate and underlying mechanisms that influence its magnitude has been slow despite the central role this issue plays in evolutionary and physiological ecology. Previous studies that have attempted to relate how cellular processes translate into whole-organism physiology have done so over a range of body masses of subjects. However, the data still remains controversial when observing metabolic rates at the cellular level. To bridge the gap between these ideas, we examined cellular metabolic rate of primary dermal fibroblasts isolated from 49 species of birds representing a 32,000-fold range in body masses to test the hypothesis that metabolic rate of cultured cells scales with body size. We used a Seahorse XF-96 Extracellular flux analyzer to measure cellular respiration in fibroblasts. Additionally, we measured fibroblast size and mitochondrial content. We found no significant correlation between cellular metabolic rate, cell size, or mitochondrial content and body mass. Additionally, there was a significant relationship between cellular basal metabolic rate and proton leak in these cells. We conclude that metabolic rate of cells isolated in culture does not scale with body mass, but cellular metabolic rate is correlated to growth rate in birds.

  9. UV-induced extracellular factor from human fibroblasts communicates the UV response to nonirradiated cells

    SciTech Connect

    Schorpp, M.; Mallick, U.; Rahmsdorf, H.J.; Herrlich, P.

    1984-07-01

    Ultraviolet light enhances the synthesis of at least eight abundant proteins in human fibroblasts within 2 hr. These proteins are identical with those induced by the tumor promoter TPA. The inducing signal is generated by DNA damage, as these proteins are induced by lower doses of UV in fibroblasts from patients with Cockayne's syndrome or Xeroderma pigmentosum. In the supernatant of UV-treated cells, a heat-labile ammonium sulfate precipitable factor of more than 10 kd (EPIF) was detected which, upon transfer to nonirradiated cells, mimicked UV in the UV-induced synthesis of gene products. The response to UV, TPA, or EPIF was inhibited by fluocinolone acetonide, but not by retinoic acid, protease inhibitors, or superoxide dismutase.

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

    NASA Astrophysics Data System (ADS)

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

    1991-08-01

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

  11. Defining the Diversity of Phenotypic Respecification Using Multiple Cell Lines and Reprogramming Regimens

    PubMed Central

    Alicea, Bradly; Murthy, Shashanka; Keaton, Sarah A.; Cobbett, Peter; Cibelli, Jose B.

    2013-01-01

    To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens–induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally “weak grip” on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines—even lines with similar origins—is likely the most direct means of improving reprogramming efficiency. PMID:23672680

  12. FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

    PubMed Central

    2011-01-01

    Background Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. Methods We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. Results We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Conclusion Cancer cell invasiveness can be affected by

  13. Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells.

    PubMed

    Aktug, Huseyin; Acikgoz, Eda; Uysal, Aysegul; Oltulu, Fatih; Oktem, Gulperi; Yigitturk, Gurkan; Demir, Kenan; Yavasoglu, Altug; Bozok Cetintas, Vildan

    2016-09-01

    Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and

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

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

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

  15. Raman spectroscopy: a powerful tool for the non-contact discrimination of bone marrow mesenchymal stem cells and fibroblasts

    NASA Astrophysics Data System (ADS)

    Pudlas, Marieke; Koch, Steffen; Bolwien, Carsten; Hirth, Thomas; Walles, Heike; Schenke-Layland, Katja

    2011-03-01

    Bone-marrow mesenchymal stem cells (BM-MSCs) are a promising cell source for regenerative medicine. However, it is challenging to determine whether isolated BM-MSC populations are free of fibroblasts. We employed traditional methods and Raman spectroscopy to distinguish BM-MSCs and human dermal fibroblasts (hDFs). Although in vitro differentiation assays revealed the multipotent character of BM-MSCs, long culture periods are a major disadvantage. Using Raman spectroscopy, we could quickly distinguish between BM-MSCs and hDFs. Therefore we conclude that this method is sufficient for the rapid detection of fibroblastic contaminations in BM-MSC cultures.

  16. Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts.

    PubMed

    Khammanit, R; Chantakru, S; Kitiyanant, Y; Saikhun, J

    2008-07-01

    The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.

  17. The many roads to Rome: induction of neural precursor cells from fibroblasts.

    PubMed

    Lujan, Ernesto; Wernig, Marius

    2012-10-01

    The experimental induction of specific cell fates in related or unrelated lineages has fascinated developmental biologists for decades. The evaluation of altered cell fates in response to ectopic expression during embryonic development has been a standard assay for interrogating gene function. However, until recently examples of cell lineage conversions were limited to closely related and primitive cell types. The induction of pluripotency in fibroblasts prominently highlighted that combinations of transcription factors can be extremely powerful and are much more effective than single genes. On the basis of this conclusion we previously identified transcription factor combinations that directly induce functional neuronal cells from mesodermal and endodermal cells. This work has evoked numerous additional studies demonstrating direct lineage conversion into neural and other lineages. Here, we review the generation of neural progenitor cells from fibroblasts, which is the newest addition to the arena of induced cell types. Surprisingly, two fundamentally different approaches have been taken to induce this cell type, one direct approach and another that involves the intermediate generation of a partially reprogrammed pluripotent state.

  18. Th17 cells favor inflammatory responses while inhibiting type I collagen deposition by dermal fibroblasts: differential effects in healthy and systemic sclerosis fibroblasts

    PubMed Central

    2013-01-01

    Introduction T helper (Th)-17 cells are increased in systemic sclerosis (SSc). We therefore assessed whether Th17 cells could modulate the inflammatory and fibrotic responses in dermal fibroblasts from healthy donors (HD) and SSc individuals. Methods Fibroblasts were obtained from 14 SSc and 8 HD skin biopsies. Th17 clones were generated from healthy peripheral blood upon enrichment of CC chemokine receptor (CCR)-4/CCR6/CD161 expressing cells. Their cytokine production was assessed by flow cytometry and multiplex beads immunoassay. Fibroblast production of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8, matrix metalloproteinase (MMP)-1, tissue inhibitor of metalloproteinase (TIMP)-1, MMP-2 and type-I collagen was quantified by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), and changes in their transcription levels assessed by real-time PCR. Intracellular signals were dissected by western blot and the use of pharmacological inhibitors. IL-17A, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) blocking reagents were used to assess the specificity of the observed effects. Results IL-17A increased MCP-1, IL-8 and MMP-1 production in a dose-dependent manner while having no effect on type I collagen in HD and SSc fibroblasts both at protein and mRNA levels. Nuclear factor-kappa B (NF-κB) and p38 were preferentially involved in the induction of MCP-1 and IL-8, while MMP-1 was most dependent on c-Jun N-terminal kinase (JNK). Supernatants of activated Th17 clones largely enhanced MCP-1, IL-8 and MMP-1 while strongly inhibiting collagen production. Of note, the production of MCP-1 and IL-8 was higher, while collagen inhibition was lower in SSc compared to HD fibroblasts. The Th17 clone supernatant effects were mostly dependent on additive/synergistic activities between IL-17A, TNF and in part IFN-γ. Importantly, the inhibition of type I collagen production induced by the Th17 clone supernatants was completely abrogated by

  19. Development and evaluation of topotecan loaded solid lipid nanoparticles: A study in cervical cancer cell lines.

    PubMed

    Chen, Zhao-Jie; Zhang, Zhen; Xie, Bei-Bei; Zhang, Hai-Yan

    2016-12-01

    The study aims at statistical development of solid lipid nanoparticles (SLNs) loaded with topotecan hydrochloride for avoiding the drawbacks of conventional drug therapies used in cervical cancer. Twenty SLN batches were prepared using organic solvent evaporation method to provide response surface curves. Thereafter, optimized SLNs were obtained using numeric method based on desirability functions providing maximum drug loading and appropriate particle size. Physical characterization of optimized TPH loaded SLNs was performed in terms of particle size, zeta potential, transmission and scanning electron microscopic evaluation. Cytotoxicity studies were performed against cervical cancer cell lines, including cervical squamous cell carcinoma cell line (HeLa) and human squamous cell carcinoma cell line (SiHa). Also, Swiss mouse embryo fibroblast cells (3T3-L1) and African green monkey kidney epithelial (Vero) cells were used to evaluate biocompatibility in normal cells. As pronounced from the results, optimized SLNs may provide an attractive alternative to conventional cervical cancer drug products.

  20. Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

    PubMed Central

    Malecka, Anna; Wang, Qunwei; Shah, Sabaria; Sutavani, Ruhcha V.; Spendlove, Ian; Ramage, Judith M.; Greensmith, Julie; Franks, Hester A.; Gough, Michael J.; Saalbach, Anja; Patel, Poulam M.; Jackson, Andrew M.

    2016-01-01

    Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-α/IL-1β from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk overcomes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radiation (1–6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-1β, -6, -10, and -27 or TNF-α and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Importantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-α/IL-1β and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would otherwise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity. PMID:27049023

  1. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    SciTech Connect

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  2. Interactions of ozone and antineoplastic drugs on rat lung fibroblasts and Walker rat carcinoma cells

    SciTech Connect

    Wenzel, D.G.; Morgan, D.L.

    1983-05-01

    Cultured rat lung fibroblasts (F-cells) and Walker rat carcinoma cells (WRC-cells) labeled with /sup 51/Cr were exposed to the following antitumor drugs alone or with O/sub 3/: carmustine (BCNU), doxorubicin (Dox), cisplatin (CPt), mitomycin C (Mit C) or vitamin K/sub 3/ (Vit K). Release of /sup 51/Cr (cell injury) was greater for F-cells than WRC-cells with any single treatment. Pretreatment with any drug (400 microM), except for Vit K with WRC-cells, did not significantly increase O/sub 3/-induced loss of /sup 51/Cr. Co-exposure of F-cells to drugs and O/sub 3/ resulted in a marked potentiation of O/sub 3/-induced injury with Vit K, and an inhibition with Dox.

  3. Overexpression of A-myb induces basic fibroblast growth factor-dependent proliferation of chicken neuroretina cells.

    PubMed Central

    Turque, N; Plaza, S; Klempnauer, K H; Saule, S

    1997-01-01

    A-Myb behaves similarly to c-Myb in chicken neuroretina cells in its ability to induce fibroblast-like differentiation, to promote growth in the presence of basic fibroblast growth factor (bFGF), and to induce Pax-6 and mim-1 expression. The one difference between c-Myb and A-Myb in these cells is that the former but not the latter protein causes colony formation in soft agar in the presence of bFGF. PMID:9371644

  4. miR-205 Hinders the Malignant Interplay Between Prostate Cancer Cells and Associated Fibroblasts

    PubMed Central

    Gandellini, Paolo; Giannoni, Elisa; Casamichele, Anna; Taddei, Maria Letizia; Callari, Maurizio; Piovan, Claudia; Valdagni, Riccardo; Pierotti, Marco Alessandro

    2014-01-01

    Abstract Aims: Tumor microenvironment is a strong determinant for the acquisition of metastatic potential of cancer cells. We have recently demonstrated that cancer-associated fibroblasts (CAFs) elicit a redox-dependent epithelial-mesenchymal transition (EMT) in prostate cancer (PCa) cells, driven by cycloxygenase-2/hypoxia-inducible factor-1 (HIF-1)/nuclear factor-κB pathway and enhancing tumor aggressiveness. Here, we investigated the involvement of microRNAs (miRNAs) in tumor-stroma interplay to identify possible tools to counteract oxidative stress and metastasis dissemination. Results: We found that miR-205 is the most downmodulated miRNA in PCa cells upon CAF stimulation, due to direct transcriptional repression by HIF-1, a known redox-sensitive transcription factor. Rescue experiments demonstrated that ectopic miR-205 overexpression in PCa cells counteracts CAF-induced EMT, thus impairing enhancement of cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. In addition, miR-205 blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion. Innovation: Overall, such findings suggest miR-205 as a brake against PCa metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts, thus interrupting the pro-oxidant and pro-inflammatory circuitries engaged by reactive stroma. Conclusion: The evidence that miR-205 replacement in PCa cells is able not only to prevent but also to revert the oxidative/pro-inflammatory axis leading to EMT induced by CAFs sets the rationale for developing miRNA-based approaches to prevent and treat metastatic disease. Antioxid. Redox Signal. 20, 1045–1059. PMID:23924028

  5. Cell line fingerprinting using retroelement insertion polymorphism.

    PubMed

    Ustyugova, Svetlana V; Amosova, Anna L; Lebedev, Yuri B; Sverdlov, Eugene D

    2005-04-01

    Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.

  6. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq

    PubMed Central

    Treutlein, Barbara; Lee, Qian Yi; Camp, J. Gray; Mall, Moritz; Koh, Winston; Shariati, Seyed Ali Mohammad; Sim, Sopheak; Neff, Norma F.; Skotheim, Jan M.; Wernig, Marius; Quake, Stephen R.

    2016-01-01

    Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation. PMID:27281220

  7. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.

    PubMed

    Treutlein, Barbara; Lee, Qian Yi; Camp, J Gray; Mall, Moritz; Koh, Winston; Shariati, Seyed Ali Mohammad; Sim, Sopheak; Neff, Norma F; Skotheim, Jan M; Wernig, Marius; Quake, Stephen R

    2016-06-16

    Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

  8. The scale of substratum topographic features modulates proliferation of corneal epithelial cells and corneal fibroblasts.

    PubMed

    Liliensiek, S J; Campbell, S; Nealey, P F; Murphy, C J

    2006-10-01

    The cornea is a complex tissue composed of different cell types, including corneal epithelial cells and keratocytes. Each of these cell types are directly exposed to rich nanoscale topography from the basement membrane or surrounding extracellular matrix. Nanoscale topography has been shown to influence cell behaviors, including orientation, alignment, differentiation, migration, and proliferation. We investigated whether proliferation of SV40-transformed human corneal epithelial cells (SV40-HCECs), primary human corneal epithelial cells (HCECs), and primary corneal fibroblasts is influenced by the scale of topographic features of the substratum. Using basement membrane feature sizes as our guide and the known dimensions of collagen fibrils of the corneal stroma (20-60 nm), we fabricated polyurethane molded substrates, which contain anisotropic feature sizes ranging from 200-2000 nm on pitches ranging from 400 to 4000 nm (pitch = ridge width + groove width). The planar regions separating each of the six patterned regions served as control surfaces. Primary corneal and SV40-HCEC proliferation decreased in direct response to decreasing nanoscale topographies down to 200 nm. In contrast to corneal epithelial cells, corneal fibroblasts did not exhibit significantly different response to any of the topographies when compared with planar controls at 5 days. However, decreased proliferation was observed on the smallest feature sizes after 14 days in culture. Results from these experiments are relevant in understanding the potential mechanisms involved in the control of proliferation and differentiation of cells within the cornea.

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

  10. Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

    2016-09-01

    The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

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

    SciTech Connect

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

    2015-02-27

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

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

    PubMed Central

    2012-01-01

    Background It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro. Results A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion. Conclusion The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT. PMID:22453032

  13. The propagation of avian viruses in a continuous cell line (QT35) of Japanese quail origin.

    PubMed

    Cowen, B S; Braune, M O

    1988-01-01

    Seven of nine avian virus families tested (Birnaviridae, Coronaviridae, Herpesviridae, Paramyxoviridae, Poxviridae, Reoviridae, and Retroviridae) were found to replicate in a quail fibroblast cell line, designated QT35, resulting in a cytopathic effect (CPE) visible with the naked eye or by low-power microscopy. In comparison, only one (Paramyxoviridae) of seven mammalian virus families tested produced an observable CPE. Cytopathic changes induced by examined viruses were round cell, syncytial, and focus formation. Trypsin did not promote cytopathic changes by selected CPE-negative avian and mammalian viruses in QT35 cells. Several avian viruses (infectious bursal disease virus, Newcastle disease virus, Canary pox virus, and reovirus) formed plaques under agar. Avian reovirus and infectious bursal disease virus produced similar titers in chicken embryo fibroblast (CEF) and QT35 cell cultures. Chicken-egg-yolk neutralizing-antibody titers to IBDV were comparable in CEF and QT35 cell-culture systems.

  14. Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts.

    PubMed

    Ozawa, Yumi; Hayashi, Shujiro; Hamasaki, Yoichiro; Hatamochi, Atsushi

    2016-12-01

    Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. This study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA) in the concentration range of 5 × 10(-7) to 5 × 10(-3) M. The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of (3)H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of (3)H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA in the concentration range of 5 × 10(-7) to 5 × 10(-4)M as compared to that in the GA-free control cultures; furthermore, the DNA-synthetic activity also rose in a concentration-dependent manner in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA at concentrations between 5 × 10(-7) and 5 × 10(-3) M. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect

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

    PubMed Central

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

    2016-01-01

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

  16. Toxic effects of various retrograde root filling materials on gingival fibroblasts and rat sarcoma cells.

    PubMed

    Peltola, M; Salo, T; Oikarinen, K

    1992-06-01

    The aim of this in vitro study was to evaluate the effect of amalgam, glass ionomer, composite and titanium on the growth of gingival fibroblasts (GF) and rat sarcoma cells (UMR) in vitro. The cells were either obtained from gingival biopsies taken during deliberation of unerupted canines (GF) or were of commercial origin (UMR). Equal numbers of cells were placed on culture dishes and incubated for a period of two weeks with the freshly prepared test materials. The cultures were photographed through a light microscope after 7 days incubation and finally counted after 14 days. It was shown that the proliferation of gingival fibroblasts was less disturbed by titanium, being approximately 96% of the control value (cell cultures without test particles), followed by composite, amalgam and glass ionomer (61%, 49% and 35% of the control value respectively). The number of UMR cells after 14 days incubation with the various materials was 76% of the control value with titanium, 12% with composite and 5% with both amalgam and glass ionomer. Inhibition of cell growth (UMR) around the test particles was most prominent around amalgam and glass ionomer, followed by composite and titanium. These effects were noted only with freshly prepared components however, so that the toxic reaction was less pronounced or minimal in a second incubation using the same particles sterilized in between. The results demonstrated that potential retrograde root filling materials have a variable toxic effect on gingival fibroblasts and rat sarcoma cells. The fact that the influence on proliferation disappeared when the test was performed with materials already tested once may be of clinical importance when estimating the biocompatibility in vivo.

  17. Repair of chromosome damage induced by X-irradiation during G/sub 2/ phase in a line of normal human fibroblasts and its malignant derivative

    SciTech Connect

    Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

    1982-08-01

    A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G/sub 2/ phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or ..beta..-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G/sub 2/ phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H/sub 2/O/sub 2/, or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G/sub 2/ phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

  18. Regulation of growth and gene activity in euploid hybrids between human neonatal fibroblasts and epithelioid amniotic fluid cells.

    PubMed Central

    Bryant, E M; Crouch, E; Bornstein, P; Martin, G M; Johnston, P; Hoehn, H

    1978-01-01

    Pure populations of proliferating synkaryons were obtained from polyethylene glycol-mediated crosses between diploid human foreskin fibroblasts and epithelioid amniotic fluid cells. These hybrids proved to be chromosomally stable tetraploids. They continuously produced heteropolymeric G6PD and showed strictly additive patterns of silver staining of both parental sets of nucleolar organizing chromosomes. Collagenous proteins characteristic of the fibroblast parent were synthesized, while fibronectin production appeared to be directed by the epithelioid portion of the genome. Even though these heterotypic hybrids proliferated at a reduced rate and achieved fewer population doublings relative to homotypic (fibroblast X fibroblast) crosses, they survived passage by trypsinization better than pure populations of epithelioid cells. These observations suggest a concerted action of both parental genomes with respect to proteins responsible for "household" functions, but complementation and possibly modulation of gene action with respect to "luxury" protein synthesis and cell growth. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:717401

  19. Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells.

    PubMed

    Hill, R P; MacNeil, S; Haycock, J W

    2006-02-01

    Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.

  20. Role of MEF feeder cells in direct reprogramming of mousetail-tip fibroblasts.

    PubMed

    Chen, Mengfei; Sun, Xuerong; Jiang, Ruzhang; Shen, Wenjuan; Zhong, Xiufeng; Liu, Bingqian; Qi, Ying; Huang, Bing; Xiang, Andy Peng; Ge, Jian

    2009-12-01

    Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c-Myc and Klf4 when co-cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder-free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine.

  1. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

  2. Mouse bone marrow-derived mast cells (BMMC) change their phenotype when cultured with fibroblasts

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Stevens, R.L.

    1986-03-05

    The heparin-containing mast cells (HP-MC) that reside in the connective tissues of the mouse, but not the chondroitin sulfate containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse BMMC (the presumptive in vitro counterpart of the in vivo differentiated mucosal mast cell) were cultured for 2-14 days with confluent skin-derived 3T3 fibroblasts in RPMI-1640 containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin-3 in the conditioned medium. The mast cells remained viable throughout the period of co-culture, since they failed to release LDG and because they increased their histamine content per cell approx.15-fold. After 8-9 days of co-culture, >50% of the BMMC changed histochemically becoming safranin positive. At this time, 30-50% of the (/sup 35/S)glycosaminoglycans on the proteoglycans synthesized by these co-cultured mass cells were heparin, whereas the initial BMMC synthesized proteoglycans containing only chondroitin sulfate E. That interleukin 3-dependent mouse BMMC can be induced to undergo a phenotypic change so as to express characteristics of a HP-MC suggests that the tissue microenvironment determines the differentiated characteristics of these cells.

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  4. AZD-4547 exerts potent cytostatic and cytotoxic activities against fibroblast growth factor receptor (FGFR)-expressing colorectal cancer cells.

    PubMed

    Yao, Ting-Jing; Zhu, Jin-Hai; Peng, De-Feng; Cui, Zhen; Zhang, Chao; Lu, Pei-hua

    2015-07-01

    Colorectal cancer (CRC) causes significant mortalities worldwide. Fibroblast growth factor (FGF) receptor (FGFR) signaling is frequently dysregulated and/or constitutively activated in CRCs, contributing to cancer carcinogenesis and progression. Here, we studied the activity of AZD-4547, a novel and potent FGFR kinase inhibitor, on CRC cells. AZD-4547 inhibited CRC cell growth in vitro, and its activity correlated with the FGFR-1/2 expression level. AZD-4547 was cytotoxic and pro-apoptotic in FGFR-1/2-expressed CRC cell lines (NCI-H716 and HCT-116), but not in FGFR-1/2 null HT-29 cells. Further, AZD-4547 inhibited cell cycle progression and attenuated the activation of FGFR1-FGFR substrate 2 (FRS-2), ERK/mitogen-activated protein kinase (MAPK), and AKT/mammalian target of rapamycin (AKT/mTOR) signalings in NCI-H716 and HCT-116 cells. In vivo, AZD-4547 oral administration at effective doses inhibited NCI-H716 (high FGFR-1/2 expression) xenograft growth in nude mice. Phosphorylation of FGFR-1, AKT, and ERK1/2 in xenograft specimens was also inhibited by AZD-4547 administration. Thus, our preclinical studies strongly support possible clinical investigations of AZD-4547 for the treatment of CRCs harboring deregulated FGFR signalings.

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

    PubMed

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

    2008-02-01

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

  6. Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells

    PubMed Central

    Santangelo, Kelly S; Johnson, Amanda L; Ruppert, Amy S; Bertone, Alicia L

    2007-01-01

    Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E2 (PGE2) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE2 production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE2, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE2, IL-6, or MMP3 production. Taken together, the anti-inflammatory and

  7. The Expression of Fibroblast Activation Protein in Clear Cell Renal Cell Carcinomas Is Associated with Synchronous Lymph Node Metastases

    PubMed Central

    Errarte, Peio; Guarch, Rosa; Pulido, Rafael; Blanco, Lorena; Nunes-Xavier, Caroline E.; Beitia, Maider; Gil, Javier; Angulo, Javier C.; López, José I.; Larrinaga, Gorka

    2016-01-01

    Clear cell renal cell carcinoma (CCRCC) is a heterogeneous and complex disease that frequently develops distant metastases. Fibroblast activation protein (FAP) is a serine peptidase the expression of which in cancer-associated fibroblasts has been associated with higher risk of metastases and poor survival. The objective of this study was to evaluate the role of FAP in metastatic CCRCC (mCCRCC). A series of 59 mCCRCC retrospectively collected was included in the study. Metastases developed either synchronous (n = 14) or metachronous to renal disease (n = 45). Tumor specimens were obtained from both primary lesion (n = 59) and metastases (n = 54) and FAP expression was immunohistochemically analyzed. FAP expression in fibroblasts from primary tumors correlated with FAP expression in the corresponding metastatic lesions. Also, primary and metastatic FAP expression was correlated with large tumor diameter (>7cm), high grade (G3/4), high stage (pT3/4), tumor necrosis and sarcomatoid transformation. The expression of FAP in primary tumors and in their metastases was associated both with synchronous metastases and also with metastases to the lymph nodes. FAP expression in the primary tumor was correlated with worse 10-year overall survival. Immunohistochemical detection of FAP in the stromal tumor fibroblasts could be a biomarker of early lymph node metastatic status and therefore could account for the poor prognosis of FAP positive CCRCC. PMID:28033421

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

    PubMed

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

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

  9. Fibroblast prostaglandin E2 synthesis. Persistence of an abnormal phenotype after short-term exposure to mononuclear cell products.

    PubMed

    Korn, J H

    1983-05-01

    Acquired abnormalities of connective tissue metabolism in inflammatory diseases often persist when lesional tissue is maintained in in vitro culture. Although connective tissue cells are exposed to inflammatory cell-derived mediators in vivo and such mediators have been shown to alter connective tissue cell behavior, it is unclear whether the persistence of metabolic defects in vitro could result from remote in vivo exposure to these mediators. An in vitro model was used to test whether transient exposure of normal fibroblasts to inflammatory mediators could lead to metabolic alterations that persist during in vitro culture. Short-term exposure of human foreskin fibroblasts in vitro to supernates of mitogen-activated peripheral blood mononuclear cells led to persistent abnormalities of prostaglandin E2 (PGE2) metabolism. Fibroblasts previously exposed to mononuclear cell products synthesized more than twice as much PGE2 when stimulated compared with similarly stimulated but previously unexposed control fibroblasts of the same strain. The enhanced PGE2 synthesis persisted for as long as 20 wk and 19 cell generations after the original exposure to mononuclear cell products. Exposure of fibroblast populations to mononuclear cell products may, thus, lead to metabolite alterations that are still evident after multiple cell generations.

  10. Cell surface abnormality in clones of skin fibroblasts from a carrier of Duchenne muscular dystrophy.

    PubMed Central

    Hillier, J; Jones, G E; Statham, H E; Witkowski, J A; Dubowitz, V

    1985-01-01

    We have previously reported that skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) have a lower intercellular adhesiveness than control cells, and that cells from carriers of DMD have normal adhesiveness instead of the expected intermediate value. We have now cloned skin fibroblasts from a carrier of DMD (subject AS) who is also heterozygous for G6PD B/G6PD Mediterranean and determined the intercellular adhesiveness and G6PD phenotypes of the clones. G6PD activity was determined using the 2d-G6P/G6P ratio method. Normal cells had a percentage utilisation of 7.31% and uncloned cells from AS a value of 25.16%. Of 16 clones, 15 had normal values (mean 8.72%) while one clone was G6PD Med with a value of 57.5%. Mean intercellular adhesiveness of normal and uncloned cells from AS were 2.95 and 2.90 respectively. Of 11 clones tested, nine had normal values of adhesiveness (mean 3.1) and all these clones were G6PD B. The single G6PD Med clone had a value of 0.88, compared with 1.39 for DMD cells. We have no explanation at present for the single clone that was G6PD B but DMD-like on aggregation. PMID:3989823

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

    PubMed

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

    2008-01-01

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

  12. GPER1 is regulated by insulin in cancer cells and cancer-associated fibroblasts.

    PubMed

    De Marco, Paola; Romeo, Enrica; Vivacqua, Adele; Malaguarnera, Roberta; Abonante, Sergio; Romeo, Francesco; Pezzi, Vincenzo; Belfiore, Antonino; Maggiolini, Marcello

    2014-10-01

    Elevated insulin levels have been associated with an increased cancer risk as well as with aggressive and metastatic cancer phenotypes characterized by a poor prognosis. Insulin stimulates the proliferation, migration, and invasiveness of cancer cells through diverse transduction pathways, including estrogen signaling. As G protein estrogen receptor 1 (GPER1) mediates rapid cell responses to estrogens, we evaluated the potential of insulin to regulate GPER1 expression and function in leiomyosarcoma cancer cells (SKUT-1) and breast cancer-associated fibroblasts (CAFs), which were used as a model system. We found that insulin transactivates the GPER1 promoter sequence and increases the mRNA and protein expression of GPER1 through the activation of the PRKCD/MAPK1/c-Fos/AP1 transduction pathway, as ascertained by means of specific pharmacological inhibitors and gene-silencing experiments. Moreover, cell migration triggered by insulin occurred through GPER1 and its main target gene CTGF, whereas the insulin-induced expression of GPER1 boosted cell-cycle progression and the glucose uptake stimulated by estrogens. Notably, a positive correlation between insulin serum levels and GPER1 expression was found in cancer fibroblasts obtained from breast cancer patients. Altogether, our data indicate that GPER1 may be included among the complex network of transduction signaling triggered by insulin that drives cells toward cancer progression.

  13. Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular Cell Network.

    PubMed

    Novkovic, Mario; Onder, Lucas; Bocharov, Gennady; Ludewig, Burkhard

    2017-01-01

    Secondary lymphoid organs have developed segregated niches that are able to initiate and maintain effective immune responses. Such global organization requires tight control of diverse cellular components, specifically those that regulate lymphocyte trafficking. Fibroblastic reticular cells (FRCs) form a densely interconnected network in lymph nodes and provide key factors necessary for T cell migration and retention, and foster subsequent interactions between T cells and dendritic cells. Development of integrative systems biology approaches has made it possible to elucidate this multilevel complexity of the immune system. Here, we present a graph theory-based analysis of the FRC network in murine lymph nodes, where generation of the network topology is performed using high-resolution confocal microscopy and 3D reconstruction. This approach facilitates the analysis of physical cell-to-cell connectivity, and estimation of topological robustness and global behavior of the network when it is subjected to perturbation in silico.

  14. HJURP regulates cellular senescence in human fibroblasts and endothelial cells via a p53-dependent pathway.

    PubMed

    Heo, Jong-Ik; Cho, Jung Hee; Kim, Jae-Ryong

    2013-08-01

    Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.

  15. Dynamic Assessment of Fibroblast Mechanical Activity during Rac-induced Cell Spreading in 3-D Culture

    PubMed Central

    Petroll, W. Matthew; Ma, Lisha; Kim, Areum; Ly, Linda; Vishwanath, Mridula

    2009-01-01

    The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 μm thick fibrillar collagen matrices and cultured for 1 to 2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent. PMID:18452153

  16. Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells. A methodological study.

    PubMed

    Hansen, T L; Andersen, H

    1983-01-01

    Through a methodological evaluation, reliable histochemical and biochemical methods for succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells were developed. The histochemical method includes a cleaning of the cultured cells in 1 mM malonate in 0.9% NaCl, air-drying and fixation in acetone (5 min at -20 degrees C), coating of cells with CoQ10 (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37 degrees C in 50 mM succinate and 0.5 mg/ml Nitro BT in 200 mM phosphate buffer, pH 7.6 PMS as an intermediate electron carrier was found inferior to exogenous CoQ10. Both types of cells exhibit equal activity. In the biochemical method homogenizing was performed in 50 mM Tris-HCl buffer, pH 7.5, and 200 mM sucrose. The standard incubation was 2.0 mM INT and 10 mM succinate in 10 mM Tris-HCl buffer, pH 7.5 for 1 h at 37 degrees C. The apparent Km values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I0.5 for malonate was 0.46 mM. Activity in amniotic fluid cells was 18.1 pkat/mg protein and in human skin fibroblasts 20.3 pkat/mg protein. Specificity of the methods was tested by use of a Chinese hamster fibroblast strain B9 known to be succinate dehydrogenase deficient in addition to various control experiments. Congruent results were obtained with the two methods.

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

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

    PubMed

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

    2014-07-01

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

  19. A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion.

    PubMed

    Coppe, Jean-Philippe; Boysen, Megan; Sun, Chung Ho; Wong, Brian J F; Kang, Mo K; Park, No-Hee; Desprez, Pierre-Yves; Campisi, Judith; Krtolica, Ana

    2008-07-01

    Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.

  20. Host cell reactivation of sunlamp-exposed adenovirus in fibroblasts from patients with Bloom's syndrome, ataxia telangiectasia, and Huntington's disease

    SciTech Connect

    Rainbow, A.J. )

    1991-01-01

    In this study, a sensitive host cell reactivation (HCR) technique was used to examine the repair capacity for DNA damaged by sunlamp exposure in fibroblast strains derived from 5 normal individuals and 8 patients representing three different diseases associated with DNA repair deficiencies. Adenovirus type 2 (Ad 2) was exposed to radiation from a GE 275 W sunlamp and subsequently used to infect fibroblast monolayers. At 48 hr after infection, cells were scored for the presence of viral structural antigens (Vag) using indirect immunofluorescent staining. Previous reports using this technique showed a substantial reduction in the HCR of sunlamp-exposed Ad 2 for infection of excision repair deficient fibroblasts from patients with xeroderma pigmentosum. In contrast, the HCR of Vag synthesis for sunlamp-exposed Ad 2 was in the normal range for the three ataxia telangiectasia, three Bloom's syndrome, and two Huntington's disease fibroblasts strains.

  1. Standards for Cell Line Authentication and Beyond

    PubMed Central

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  2. Anti-inflammatory effects of zinc in PMA-treated human gingival fibroblast cells

    PubMed Central

    Kim, Sangwoo; Jeon, Sangmi; Hui, Zheng; Kim, Young; Im, Yeonggwan; Lim, Wonbong; Kim, Changsu; Choi, Hongran; Kim, Okjoon

    2015-01-01

    Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)-treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis. Key words:Zinc, inflammatory response, cytokines, phorbol-12-myristate-13-acetate, gingival fibroblasts cells. PMID:25662537

  3. Fibroblast growth factor 10 represses premature cell differentiation during establishment of the intestinal progenitor niche.

    PubMed

    Nyeng, Pia; Bjerke, Maureen Ann; Norgaard, Gitte Anker; Qu, Xiaoling; Kobberup, Sune; Jensen, Jan

    2011-01-01

    Spatio-temporal regulation of the balance between cell renewal and cell differentiation is of vital importance for embryonic development and adult homeostasis. Fibroblast growth factor signaling relayed from the mesenchyme to the epithelium is necessary for progenitor maintenance during organogenesis of most endoderm-derived organs, but it is still ambiguous whether the signal is exclusively mitogenic. Furthermore, the downstream mechanisms are largely unknown. In order to elucidate these questions we performed a complementary analysis of fibroblast growth factor 10 (Fgf10), gain-of-function and loss-of-function in the embryonic mouse duodenum, where the progenitor niche is clearly defined and differentiation proceeds in a spatially organized manner. In agreement with a role in progenitor maintenance, FGF10 is expressed in the duodenal mesenchyme during early development while the cognate receptor FGFR2b is expressed in the epithelial progenitor niche. Fgf10 gain-of-function in the epithelium leads to spatial expansion of the progenitor niche and repression of cell differentiation, while loss-of-function results in premature cell differentiation and subsequent epithelial hypoplasia. We conclude that FGF10 mediated mesenchymal-to-epithelial signaling maintains the progenitor niche in the embryonic duodenum primarily by repressing cell differentiation, rather than through mitogenic signaling. Furthermore, we demonstrate that FGF10-signaling targets include ETS-family transcription factors, which have previously been shown to regulate epithelial maturation and tumor progression.

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

    SciTech Connect

    Tyrrell, R.M.; Pidoux, M.

    1986-06-01

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

  5. [Characteristics of quantitative karyotypic variability in cell line of kidney from rat kangaroo (Potorous tridactylis)].

    PubMed

    Polianskaia, G G; Samokish, V A

    1999-01-01

    The numerical regulations of karyotypic variability in cell line of rat kangaroo kidney, NBL-3-11, has been investigated. These regulations are similar with ones found for cell lines of the Indian muntjac skin fibroblasts (M, MT, M2). In particular the balanced karyotypic structure of cell population in vitro is determined by correlations of the structural variants of the karyotype (SVK). These correlations depend on the following regulations 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes with simultaneous numeral deviations some differences in the character of significant connections between the individual chromosomes. These connections have either single directed character, mainly (+) direction, or differently directed one by deviations of each chromosome mainly in one direction in cell line NBL-3-11. At the same time single directed character of simultaneous deviations is observed in cell lines of the Indian muntjac skin fibroblasts (M, MT, M2) either in (+) or (-) direction. Represented results confirm and extend considerably the known ideas of the regulations of karyotypic variability in cell populations in vitro.

  6. Immortalization of Werner syndrome and progeria fibroblasts

    SciTech Connect

    Saito, H.; Moses, R.E. )

    1991-02-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

  7. Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients

    PubMed Central

    Kim, Deok-Ho; Han, Karam; Gupta, Kshitiz; Kwon, Keon Woo

    2009-01-01

    In this report, we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell-substratum interactions. A single cell adhesion substratum with the constant ridge width (1 µm), and depth (400 nm) and variable groove widths (1 µm to 9.1 µm) allowed us to characterize the dependence of cellular responses, including cell shape, orientation, and migration, on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges, vs. those on the sparser areas, exhibiting a biphasic dependence of the migration speed on the pattern density. In addition, cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term). Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result, in addition to demonstrating a more sophisticated method to study aspects of wound healing processes, strongly suggests that even in the presence of considerable cell-cell interactions, the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell-matrix interactions, but also contribute to bioengineering of devices with the optimally structured design of cell-material interface. PMID:19595452

  8. Embryonic stem cell lines of nonhuman primates.

    PubMed

    Nakatsuji, Norio; Suemori, Hirofumi

    2002-06-26

    Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is

  9. Topological Small-World Organization of the Fibroblastic Reticular Cell Network Determines Lymph Node Functionality

    PubMed Central

    Abe, Jun; Bomze, David; Cremasco, Viviana; Scandella, Elke; Stein, Jens V.; Turley, Shannon J.; Ludewig, Burkhard

    2016-01-01

    Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses. PMID:27415420

  10. Topological Small-World Organization of the Fibroblastic Reticular Cell Network Determines Lymph Node Functionality.

    PubMed

    Novkovic, Mario; Onder, Lucas; Cupovic, Jovana; Abe, Jun; Bomze, David; Cremasco, Viviana; Scandella, Elke; Stein, Jens V; Bocharov, Gennady; Turley, Shannon J; Ludewig, Burkhard

    2016-07-01

    Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses.

  11. Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels

    NASA Technical Reports Server (NTRS)

    Goodwin, T. J.; Jessup, J. M.; Wolf, D. A.

    1992-01-01

    A new low shear stress microcarrier culture system has been developed at NASA's Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.

  12. Thyroid cancer cell lines: an overview

    PubMed Central

    Saiselet, Manuel; Floor, Sébastien; Tarabichi, Maxime; Dom, Geneviève; Hébrant, Aline; van Staveren, Wilma C. G.; Maenhaut, Carine

    2012-01-01

    Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during

  13. Fourier-transform infrared spectroscopic comparison of cultured human fibroblast and fibrosarcoma cells

    NASA Astrophysics Data System (ADS)

    Yang, Difei; Castro, Dan J.; El-Sayed, Ivan H.; El-Sayed, Mostafa A.; Saxton, Romaine E.; Zhang, Nancy Y.

    1995-05-01

    Infrared vibration spectroscopy appears to be a more powerful technique for diagnosis than visible or UV spectroscopy. Advantages of IR spectra include: 1) vibrational motion has a smaller tissue absorption coefficient than electronic motion, 2) scattering of infrared radiation has a lower cross section than visible or UV light, (these two facts allow deeper penetration of IR radiation) and 3) vibration spectra provide a better fingerprint of chemical groups present in cells than the unresolved broad electronic spectrum of biological molecules. In the present work, Fourier-transform IR spectroscopy was used to compare cultured human fibroblast and malignant fibrosarcoma cells. Significant differences were observed by comparing the spectra of the normal cells with that of the cancer cells. the PO2 symmetric stretching mode at 1082cm-1 in the cancer cell is reduced in intensity. These observations are similar to those reported previously by Wong et al in comparing the IR spectra of pairs of normal and cancerous cells from the colon and cervix. However, the observed increase in the relative intensity of the symmetric to antisymmetric CH3 bending mode are only found in fibrosarcoma and basal cell carcinoma. The decrease in intensity of the CH2 bending mode relative to that of CH3 mode was observed only for fibrosarcoma cells. This finding with paired human fibroblast and fibrosarcoma cells suggests that fatty acid chains or side chains of protein in the cancer cells are partially degraded leading to more terminal carbon. It is also possible that changes in the environment upon carcinogenesis induces a change in the relative absorption cross sections for the CH3 and CH2 bending vibrations.

  14. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or

  15. Screening of plants acting against Heterometrus laoticus scorpion venom activity on fibroblast cell lysis.

    PubMed

    Uawonggul, Nunthawun; Chaveerach, Arunrat; Thammasirirak, Sompong; Arkaravichien, Tarinee; Chuachan, Chattong; Daduang, Sakda

    2006-01-16

    The aqueous extracts of 64 plant species, listed as animal- or insect-bite antidotes in old Thai drug recipes were screened for their activity against fibroblast cell lysis after Heterometrus laoticus scorpion venom treatment. The venom was preincubated with plant extract for 30 min and furthered treated to confluent fibroblast cells for 30 min. More than 40% efficiency (test/control) was obtained from cell treatment with venom preincubated with extracts of Andrographis paniculata Nees (Acanthaceae), Barringtonia acutangula (L.) Gaertn. (Lecythidaceae), Calamus sp. (Palmae), Clinacanthus nutans Lindau (Acanthaceae), Euphorbia neriifolia L. (Euphorbiaceae), Ipomoea aquatica Forssk (Convolvulaceae), Mesua ferrea L. (Guttiferae), Passiflora laurifolia L. (Passifloraceae), Plectranthus amboinicus (Lour.) Spreng. (Labiatae), Ricinus communis L. (Euphorbiaceae), Rumex sp. (Polygonaceae) and Sapindus rarak DC. (Sapindaceae), indicating that they had a tendency to be scorpion venom antidotes. However, only Andrographis paniculata and Barringtonia acutangula extracts provided around 50% viable cells from extract treatments without venom preincubation. These two plant extracts are expected to be scorpion venom antidotes with low cytotoxicity.

  16. Fibroblast Growth Factor Receptor (FGFR): A New Target for Non-small Cell Lung Cancer Therapy.

    PubMed

    Biello, Federica; Burrafato, Giovanni; Rijavec, Erika; Genova, Carlo; Barletta, Giulia; Truini, Anna; Coco, Simona; Bello, Maria Giovanna Dal; Alama, Angela; Boccardo, Francesco; Grossi, Francesco

    2016-01-01

    Lung cancer is still the leading cause of cancer related death worldwide. Fibroblast growth factor receptor (FGFR) is a tirosine-kinase receptor that is seen to be amplified or mutated in non-small cell lung cancer (NSCLC) and it plays a crucial role in tumour development and maintenance. The authors analyzed the state of the art of FGFR by reviewing the current literature. Fibroblast growth factor (FGF)-FGFR pathway and their aberrations are described, with the evaluation of their possible prognostic role in NSCLC and in particular in squamous cell carcinomas, in which FGFR is more often amplified. New therapeutic agents targeting FGFR signaling have been developed and are now in clinical evaluation. Dysregulation of FGF signaling in tumour cells is related to FGFR gene amplification or mutation, although it is still uncertain which of these aberrations represents a real predictor of response to specific inhibitors. However, recent evidence has questioned whether FGFR is a real target in squamous cell histology. The effectiveness of FGFR inhibitors is also still unclear since there are no clinical data on selected patients. Moreover, the management of specific side effects related to inhibition of the physiological role of FGF should be more thorough.

  17. Cancer-Associated Fibroblasts from Hepatocellular Carcinoma Promote Malignant Cell Proliferation by HGF Secretion

    PubMed Central

    Fu, Bin-Sheng; Hua, XueFeng; Wang, Guo-Ying; Li, Tuan-Jie; Li, Xing; Wu, Xiang-Yuan; Tai, Yan; Zhou, Jie; Chen, Gui-Hua; Zhang, Qi

    2013-01-01

    Cancer-associated fibroblasts (CAFs) are reported to support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion in most solid tumors. However, the roles of CAFs in the liver cancer microenvironment have not been thoroughly studied. In our previous study, we successfully isolated CAFs from hepatocellular carcinoma (HCC) (H-CAFs) and proved that H-CAFs suppressed the activation of NK cells and thereby created favorable conditions for HCC progression. In our present study, we found that the proliferation of MHCC97L and Hep3B cells was significantly promoted by treatment with conditioned medium from H-CAFs. Pathological analysis also revealed that H-CAFs increased the proportion of Ki-67 (+) malignant cells and prevented them from undergoing necrosis. Moreover, the concentration of hepatocyte growth factor (HGF) cytokine in the conditioned medium of H-CAFs was higher than conditioned medium from normal skin fibroblasts (NSFs). Anti-HGF significantly reduced the proliferation-promoting capability of H-CAFs. In addition, we found that the abundance of H-CAFs correlated positively with tumor size. These results indicate that H-CAFs are an important factor for promoting the growth of HCC in vitro and in vivo, and that HGF plays a key role in HCC proliferation induced by H-CAFs. PMID:23667593

  18. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

    PubMed

    Abiko, Yoshimitsu; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Tsushima, Katsumasa; Ohta, Mitsuhiro; Sasahara, Hiroshige

    2004-03-01

    Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

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

    PubMed

    Wong, Wing Tak; Cooke, John P

    2016-01-01

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

  20. Triboelectric Nanogenerator Accelerates Highly Efficient Nonviral Direct Conversion and In Vivo Reprogramming of Fibroblasts to Functional Neuronal Cells.

    PubMed

    Jin, Yoonhee; Seo, Jungmok; Lee, Jung Seung; Shin, Sera; Park, Hyun-Ji; Min, Sungjin; Cheong, Eunji; Lee, Taeyoon; Cho, Seung-Woo

    2016-09-01

    Triboelectric nanogenerators (TENGs) can be an effective cell reprogramming platform for producing functional neuronal cells for therapeutic applications. Triboelectric stimulation accelerates nonviral direct conversion of functional induced neuronal cells from fibroblasts, increases the conversion efficiency, and induces highly matured neuronal phenotypes with improved electrophysiological functionalities. TENG devices may also be used for biomedical in vivo reprogramming.

  1. Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

    SciTech Connect

    Olive, P.L.; Vanderbyl, S.; MacPhail, S.H. )

    1991-04-01

    Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing.

  2. Phagocytosis and solubilization of fixed cells by metastatic hamster embryo fibroblasts, Nil2C2

    SciTech Connect

    Sakiyama, H.; Nishino, Y.; Nishimura, K.; Noda, Y.; Otsu, H.

    1984-05-01

    When Nil2C2, a metastatic clone derived from hamster embryo fibroblasts (Nil), was inoculated over (/sup 3/H)leucine-labeled fixed cells, Nil2C2 cells solubilized and phagocytosed fixed cells, and the radioactivity was released into the culture medium as trichloroacetic acid-soluble fragments. The solubilization of fixed cells was dependent on both the time of incubation of living cells with fixed cells and the number of living cells inoculated. Nil2C2 cells were shown by autoradiographic and electron microscopic studies to peel off fixed cells and ingest them as large fragments. The solubilization of fixed cells was significantly decreased when plasminogen was depleted from the culture medium. Protease inhibitors such as leupeptin, epsilon-aminocaproic acid, and soybean trypsin inhibitor partially inhibited the proteolysis and phagocytosis of Nil2C2 cells. Mouse peritoneal macrophages activated by Salmonella typhimurium solubilized fixed cells after the addition of 12-O-tetradecanoylphorbol-13-acetate. However, they did not phagocytose fixed cells as large fragments.

  3. Cytotoxic effects of new MTA-based cement formulations on fibroblast-like MDPL-20 cells.

    PubMed

    Garcia, Lucas da Fonseca Roberti; Santos, Alailson Domingos dos; Moraes, João Carlos Silos; Costa, Carlos Alberto de Souza

    2016-01-01

    The present study aimed at evaluating the cytotoxic effects of a novel cement called CER on periodontal fibroblast-like cells of mice (MDPL-20), in comparison with different formulations of Mineral Trioxide Aggregate (MTA), by means of the cell viability test (MTT) and cell morphology analysis. Thirty-two round-shaped samples were fabricated with the following cements: white MTA, white and gray CER and experimental white MTA. The samples were immersed in serum-free culture medium for 24 hours or 7 days (n = 16). The extracts (culture medium + components released from the cements) were applied for 24 hours to previously cultured cells (40.000 cells/cm2) in the wells of 24-well plates. Cells seeded in complete culture medium were used as a negative control. Cell viability was assessed using the MTT assay. Two samples of each cement were used for cell morphology analysis by Scanning Electron Microscopy (SEM). The extracts obtained at the 7-day period presented higher cytotoxicity compared with the 24-hour period (p < 0.05). The gray CER obtained at 24 hours presented the highest cytotoxic effect, whereas the experimental white MTA presented the lowest, similar to the control (p > 0.05). However, at the 7-day period, the experimental white MTA presented no significant difference in comparison with the other cements (p > 0.05). At the 7-day period, CER cement presented cytotoxic effects on fibroblast-like cells, similar to different MTA formulations. However, the immersion period in the culture medium influenced the cytotoxicity of the cements, which was greater for CER cement at 24 hours.

  4. Breast Cancer Cells Induce Cancer-Associated Fibroblasts to Secrete Hepatocyte Growth Factor to Enhance Breast Tumorigenesis

    PubMed Central

    Tyan, Shiaw-Wei; Kuo, Wen-Hung; Huang, Chun-Kai; Pan, Chi-Chun; Shew, Jin-Yuh; Chang, King-Jen; Lee, Eva Y.-H. P.; Lee, Wen-Hwa

    2011-01-01

    It has been well documented that microenvironment consisting of stroma affects breast cancer progression. However, the mechanisms by which cancer cells and fibroblasts, the major cell type in stroma, interact with each other during tumor development remains to be elucidated. Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients. The expression level of hepatocyte growth factor (HGF) in these fibroblasts was positively correlated with their ability to enhance breast tumorigenesis in mice. Deprivation of HGF using a neutralizing antibody reduced CAF-mediated colony formation of human breast cancer cells, indicating that CAFs enhanced cancer cell colony formation mainly through HGF secretion. Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity. The newly gained ability of these “educated” NAFs became irreversible after continuing this process till fourth passage. These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling. PMID:21249190

  5. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    SciTech Connect

    Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  6. Human lung-derived mature mast cells cultured alone or with mouse 3T3 fibroblasts maintain an ultrastructural phenotype different from that of human mast cells that develop from human cord blood cells cultured with 3T3 fibroblasts.

    PubMed Central

    Dvorak, A. M.; Furitsu, T.; Estrella, P.; Ishizaka, T.

    1991-01-01

    Culture systems designed to maintain or develop human mast cells have proved difficult, yet these systems would provide valuable resources for future investigations of human mast cell biology. Cocultures of either isolated mature human lung mast cells (Levi-Schaffer et al., J Immunol 1987, 139:494-500) or human cord blood mononuclear cells (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043) with 3T3 embryonic mouse skin fibroblasts have implicated fibroblasts as an important factor in the successful maintenance and development of human mast cells in vitro. The authors cultured isolated, mature human lung mast cells either with or without 3T3 cells for 1 month and examined their ultrastructural phenotype. Mast cell viability in each circumstance was equivalent, but mast cell yield was improved in the presence of 3T3 cells. The ultrastructural phenotype was identical in both culture systems. Mast cells were shown to maintain the phenotype of their in vivo lung counterparts (ie, scroll granules predominanted, and numerous lipid bodies were present). This ultrastructural phenotype differs from that of mast cells that develop in cocultures of human cord blood cells and 3T3 cells, where developing mast cells with crystalline granules and few lipid bodies prevail, a phenotype much like that of human skin mast cells in vivo (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1750506

  7. Transcription factor–mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells

    PubMed Central

    Najm, Fadi J.; Lager, Angela M.; Zaremba, Anita; Wyatt, Krysta; Caprariello, Andrew V.; Factor, Daniel C.; Karl, Robert T.; Maeda, Tadao; Miller, Robert H.; Tesar, Paul J.

    2013-01-01

    Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to “induced” oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies. PMID:23584611

  8. The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.

    PubMed

    Astarita, Jillian L; Cremasco, Viviana; Fu, Jianxin; Darnell, Max C; Peck, James R; Nieves-Bonilla, Janice M; Song, Kai; Kondo, Yuji; Woodruff, Matthew C; Gogineni, Alvin; Onder, Lucas; Ludewig, Burkhard; Weimer, Robby M; Carroll, Michael C; Mooney, David J; Xia, Lijun; Turley, Shannon J

    2015-01-01

    In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

  9. Regulation of matrix metalloproteinase-9 expression between gingival fibroblast cells from old and young rats

    SciTech Connect

    Kim, Su-Jung; Chung, Yong-Koo; Chung, Tae-Wook; Kim, Jeong-Ran; Moon, Sung-Kwon; Kim, Cheorl-Ho Park, Young-Guk

    2009-01-09

    Gingival fibroblast cells (rGF) from aged rats have an age-related decline in proliferative capacity compared with young rats. We investigated G1 phase cell cycle regulation and MMP-9 expression in both young and aged rGF. G1 cell cycle protein levels and activity were significantly reduced in response to interleukin-1{beta} (IL-1{beta}) stimulation with increasing in vitro age. Tumor necrosis factor-{alpha} (TNF-{alpha})-induced matrix metalloproteinase-9 (MMP-9) expression was also decreased in aged rGF in comparison with young rGF. Mutational analysis and gel shift assays demonstrated that the lower MMP-9 expression in aged rGF is associated with lower activities of transcription factors NF-{kappa}B and AP-1. These results suggest that cell cycle dysregulation and down-regulation of MMP-9 expression in rGF may play a role in gingival remodeling during in vitro aging.

  10. Nuclear transfer with apoptotic bovine fibroblasts: can programmed cell death be reprogrammed?

    PubMed

    Miranda, Moyses dos Santos; Bressan, Fabiana Fernandes; De Bem, Tiago Henrique Camara; Merighe, Giovana Krempel Fonseca; Ohashi, Otávio Mitio; King, William Alan; Meirelles, Flavio Viera

    2012-06-01

    Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 μM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of Anx+ cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p>0.05). However, blastocyst formation was affected by the use of Casp-9+ cells (12.3%; p<0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return" for apoptosis may be located around activation of Caspase-9.

  11. Effect of essential oil of Rosa Damascena on human colon cancer cell line SW742

    PubMed Central

    Rezaie-Tavirani, Mostafa; Heydari-Keshel, Saeid; Rezaee, Mohamad Bagher; Zamanian-Azodi, Mona; Rezaei-Tavirani, Majid; Khodarahmi, Reza

    2013-01-01

    Aim In this study, we report the effect of the essential oil of Rosa Damascena on human colon cancer cell line (SW742) and human fibroblast cells. Background Colon cancer is the second most common fatal malignancy. Owing to the existence of many side effects and problems related to common treatments such as surgery, chemotherapy and radiotherapy, alternative treatments are being investigated. Some herbal medicines have shown promising results against different types of cancers. Herbal medicines used have included the use naturally occurring essential oils. Patients and methods The essential oil of Rosa Damascena was obtained by distillation and its effect on SW742 cell-line and fibroblast cells were investigated with cell culture. The cells were cultured and different volumes of essential oil were induced to the cells. After48hincubation, cell survival was measured and using statistical analysis, the findings were evaluated and reported. Results This study showed that soluble part of Rosa Damascena oil increases cell proliferation in high volumes and the non-soluble component decreases cell proliferation. Conclusion The effects of essential oils, such as Rosa Damascena, on cell proliferation require more thorough investigation. PMID:24834241

  12. Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor.

    PubMed

    Schwafertz, Carolin; Schinner, Sven; Kühn, Markus C; Haase, Matthias; Asmus, Amelie; Mülders-Opgenoorth, Birgit; Ansurudeen, Ishrath; Hornsby, Peter J; Morawietz, Henning; Oetjen, Elke; Schott, Matthias; Willenberg, Holger S

    2017-02-05

    Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of β-catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect β-catenin activity in adrenocortical NCIH295R cells. Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of β-catenin and an increase in β-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway. Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced β-catenin-dependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM. Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate β-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.

  13. Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production

    PubMed Central

    Chen, Joseph C.; Erikson, David W.; Piltonen, Terhi T.; Meyer, Michelle R.; Barragan, Fatima; McIntire, Ramsey H.; Tamaresis, John S.; Vo, Kim Chi; Giudice, Linda C.; Irwin, Juan C.

    2013-01-01

    Objective To determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns. Design In vitro study. Setting University research laboratory. Patient(s) Endometrial biopsies were obtained from premenopausal women. Intervention(s) Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls. Main Outcome Measure(s) Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures. Result(s) Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion. Conclusion(s) This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro. PMID:23849844

  14. miR-31 affects colorectal cancer cells by inhibiting autophagy in cancer-associated fibroblasts

    PubMed Central

    Zhang, Shuyu; Wu, Yong; Wu, Yongyou; Zhao, Kui; Xing, Chungen; Cao, Jianping; Zhu, Hong; Li, Ming; Ye, Zhenyu; Peng, Wei

    2016-01-01

    Autophagy is a double-edged sword in tumor development. Recent studies have found that miRNAs have an inhibitory effect on the regulation of autophagy. It has been reported that miR-31 plays an important role in the development of colorectal cancer. However, what role miR-31 plays in colorectal cancer-associated fibroblasts (CAFs) has not been determined. In this study, we confirmed that the expression of miR-31 in CAFs was higher than in normal colorectal fibroblasts (NFs). We also found that treatment of CAFs with miR-31 mimic inhibited the expression of the autophagy-related genes Beclin-1, ATG, DRAM and LC3. In addition, we found up-regulation of miR-31 significantly affected colorectal cancer cell behaviors, including proliferation, invasion and apoptosis. Also, up-regulation of miR-31 in CAF could increase the radiosensitivity of colorectal cancer cells co-cultured with CAF. In summary, miR-31 can inhibit autophagy in colorectal CAFs, affect colorectal cancer development, and increase the radiosensitivity of colorectal cancer cells co-cultured with CAF. We hypothesize that miR-31 may become a new target of treatments for colorectal cancer. PMID:27793031

  15. Use of lymphoblastoid cell lines to evaluate the hypersensitivity to ultraviolet radiation in Cockayne syndrome

    SciTech Connect

    Otsuka, F.; Tarone, R.E.; Cayeux, S.; Robbins, J.H.

    1984-05-01

    Cockayne syndrome (CS) is a rare autosomal recessive disease characterized by acute sun sensitivity, cachectic dwarfism, and neurologic and skeletal abnormalities. Cultured skin fibroblasts from patients with this disease are known to be hypersensitive to the lethal effects of 254-nm UV radiation. The authors have studied the sensitivity of 254-nm UV radiation of lymphoblastoid lines derived from 3 typical CS patients, 1 atypical CS patient who had a very late age of onset of clinical manifestations, 2 patients who had both xeroderma pigmentosum (XP) and typical CS, and 3 heterozygous parents of these patients. Post-UV survival was determined by the trypan-blue dye-exclusion method. The lymphoblastoid lines from the 3 typical CS patients, the atypical CS patient, and the 2 patients with both CS and XP had decreased post-UV viability in comparison with lines from normal donors. Lines from the heterozygous parents had normal post-UV viability. The post-UV viability of the typical CS lines was similar to that of a XP complementation group C line. The relative post-UV viability of lymphoblastoid lines from the typical CS patients was similar to the relative post-UV survival of their fibroblast lines. The lymphoblastoid line from the atypical CS patient had a post-UV viability similar to that of the typical CS patients. Thus, the relative hypersensitivity of CS patients cells in vitro does not reflect the severity or age of onset of the patients clinical manifestations. The lymphoblastoid lines from the 2 patients who had both CS and XP were significantly more sensitive to the UV radiation than those from patients with only CS. Our studies demonstrate that lymphoblastoid lines from patients with CS are appropriate and useful cell lines for the study of the inherited hypersensitivity to UV radiation.

  16. Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor

    SciTech Connect

    Zhang Xufeng; Wu Guoxiang; Chen, Jian-Quan; Zhang Aimin; Liu Siguo; Jiao Binghua . E-mail: jiaobh@uninet.com.cn; Cheng Guoxiang . E-mail: Chenggx@cngenon.com

    2005-07-22

    Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo{sup r}), replaced the {alpha}-lactalbumin gene in a 210 kb human {alpha}-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock.

  17. Fibroblast growth factor 8 increases breast cancer cell growth by promoting cell cycle progression and by protecting against cell death

    SciTech Connect

    Nilsson, Emeli M.; Brokken, Leon J.S.; Haerkoenen, Pirkko L.

    2010-03-10

    Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.

  18. Anticancer activity of chemically prepared shrimp low molecular weight chitin evaluation with the human monocyte leukaemia cell line, THP-1.

    PubMed

    Salah, R; Michaud, P; Mati, F; Harrat, Z; Lounici, H; Abdi, N; Drouiche, N; Mameri, N

    2013-01-01

    In the present study, anticancer activities of chitin, chitosan and low molecular weight chitin were evaluated using a human tumour cell line, THP-1. A molecular weight-activity relationship and an electrostatic interaction-activity relationship were determined. The cytotoxic effects of chitin and derivatives were also evaluated using a normal human foetal lung fibroblastic cell line, MRC-5 and the specific cytotoxicity of chitin and derivatives to tumour cell lines was demonstrated. The high antitumour effect of low molecular weight of chitin was established.

  19. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  20. c-Ski activates cancer-associated fibroblasts to regulate breast cancer cell invasion.

    PubMed

    Wang, Liyang; Hou, Yixuan; Sun, Yan; Zhao, Liuyang; Tang, Xi; Hu, Ping; Yang, Jiajia; Zeng, Zongyue; Yang, Guanglun; Cui, Xiaojiang; Liu, Manran

    2013-12-01

    Aberrant expression of c-Ski oncoprotein in some tumor cells has been shown to be associated with cancer development. However, the role of c-Ski in cancer-associated fibroblasts (CAFs) of tumor microenvironment has not been characterized. In the current study, we found that c-Ski is highly expressed in CAFs derived from breast carcinoma microenvironment and this CAF-associated c-Ski expression is associated with invasion and metastasis of human breast tumors. We showed that c-Ski overexpression in immortalized breast normal fibroblasts (NFs) induces conversion to breast CAFs by repressing p53 and thereby upregulating SDF-1 in NFs. SDF-1 treatment or p53 knockdown in NFs had similar effects on the activation of NFs as c-Ski overexpression. The c-Ski-activated CAFs show increased proliferation, migration, invasion and contraction compared with NFs. Furthermore, c-Ski-activated CAFs facilitated the migration and invasion of MDA-MB-231 breast cancer cells. Our data suggest that c-Ski is an important regulator in the activation of CAFs and may serve as a potential therapeutic target to block breast cancer progression.

  1. Carcinoma-associated fibroblasts affect sensitivity to oxaliplatin and 5FU in colorectal cancer cells

    PubMed Central

    Gonçalves-Ribeiro, Samuel; Díaz-Maroto, Natalia Guillen; Berdiel-Acer, Mireia; Soriano, Antonio; Guardiola, Jordi; Martínez-Villacampa, Mercedes; Salazar, Ramon; Capellà, Gabriel; Villanueva, Alberto; Martínez-Balibrea, Eva; Molleví, David G.

    2016-01-01

    The importance of tumor microenvironment (TME) as a relevant contributor to cancer progression and its role in the development of de novo resistance to targeted therapies has become increasingly apparent. However, the mechanisms of microenvironment-mediated drug resistance for nonspecific conventional chemotherapeutic agents, such as platinum compounds or antimetabolites, are still unclear. Here we describe a mechanism induced by soluble factors released by carcinoma-associated fibroblasts (CAFs) that induce the translocation of AKT, Survivin and P38 to the nucleus of tumor cells. These changes are guided to ensure DNA repair and the correct entrance and exit from mitosis in the presence of chemotherapy. We used conditioned media (CM) from normal-colonic fibroblasts and paired CAFs to assess dose response curves of oxaliplatin and 5-fluorouracil, separately or combined, compared with standard culture medium. We also evaluated a colony-forming assay and cell death to demonstrate the protective role of CAF-CM. Immunofluorescence confirmed the translocation of AKT, P38 and Survivin to the nucleus induced by CAF-soluble factors. We also have shown that STAT3 or P38 inhibition provides a promising strategy for overcoming microenvironment-mediated resistance. Conversely, pharmacologic AKT inhibition induces an antagonistic effect that relieves a cMET and STAT3-mediated compensatory feedback that might explain the failure of AKT inhibitors in the clinic so far. PMID:27517495

  2. A cell-cycle independent role for p21 in regulating synovial fibroblast migration in rheumatoid arthritis.

    PubMed

    Woods, James M; Klosowska, Karolina; Spoden, Darrin J; Stumbo, Nataliya G; Paige, Douglas J; Scatizzi, John C; Volin, Michael V; Rao, Malathi S; Perlman, Harris

    2006-01-01

    Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21(-/-)) and were examined with respect to growth and migration. p21(-/-) and wild-type (WT) FLS were compared with respect to migration towards chemoattractants found in RA synovial fluid in the presence and absence of cell cycle inhibitors. Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21(-/-) FLS display a marked increase (3.1-fold; p < 0.05) in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.

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

    PubMed

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

    2016-08-01

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

  4. Comparison between fibroblast wound healing and cell random migration assays in vitro.

    PubMed

    Ascione, Flora; Vasaturo, Angela; Caserta, Sergio; D'Esposito, Vittoria; Formisano, Pietro; Guido, Stefano

    2016-09-10

    Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques.

  5. Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

    PubMed Central

    Bun, Philippe; Liu, JunJun; Turlier, Hervé; Liu, ZengZhen; Uriot, Karen; Joanny, Jean-François; Coppey-Moisan, Maïté

    2014-01-01

    Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development. PMID:25028874

  6. Cancer-associated-fibroblasts and tumour cells: a diabolic liaison driving cancer progression.

    PubMed

    Cirri, Paolo; Chiarugi, Paola

    2012-06-01

    Several recent papers have now provided compelling experimental evidence that the progression of tumours towards a malignant phenotype does not depend exclusively on the cell-autonomous properties of cancer cells themselves but is also deeply influenced by tumour stroma reactivity, thereby undergoing a strict environmental control. Tumour microenvironmental elements include structural components such as the extracellular matrix or hypoxia as well as stromal cells, either resident cells or recruited from circulating precursors, as macrophages and other inflammatory cells, endothelial cells and cancer-associated fibroblasts (CAFs). All these elements synergistically play a specific role in cancer progression. This review summarizes our current knowledge on the role of CAFs in tumour progression, with a particular focus on the biunivocal interplay between CAFs and cancer cells leading to the activation of the epithelial-mesenchymal transition programme and the achievement of stem cell traits, as well as to the metabolic reprogramming of both stromal and cancer cells. Recent advances on the role of CAFs in the preparation of metastatic niche, as well as the controversial origin of CAFs, are discussed in light of the new emerging therapeutic implications of targeting CAFs.

  7. Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA

    PubMed Central

    Lao, Zheng; Kelly, Catherine J.; Yang, Xiang-Yang; Jenkins, W. Timothy; Toorens, Erik; Ganguly, Tapan; Evans, Sydney M.; Koch, Cameron J.

    2015-01-01

    Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2–3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids. PMID:26208323

  8. Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

    NASA Astrophysics Data System (ADS)

    Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

    2010-02-01

    Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

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

    PubMed Central

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

    1995-01-01

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

  10. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  11. Human Subperitoneal Fibroblast and Cancer Cell Interaction Creates Microenvironment That Enhances Tumor Progression and Metastasis

    PubMed Central

    Yokota, Mitsuru; Ishii, Genichiro; Saito, Norio; Aoyagi, Kazuhiko; Sasaki, Hiroki; Ochiai, Atsushi

    2014-01-01

    Backgrounds Peritoneal invasion in colon cancer is an important prognostic factor. Peritoneal invasion can be objectively identified as periotoneal elastic laminal invasion (ELI) by using elastica stain, and the cancer microenvironment formed by the peritoneal invasion (CMPI) can also be observed. Cases with ELI more frequently show distant metastasis and recurrence. Therefore, CMPI may represent a particular milieu that facilitates tumor progression. Pathological and biological investigations into CMPI may shed light on this possibly distinctive cancer microenvironment. Methods We analyzed area-specific tissue microarrays to determine the pathological features of CMPI, and propagated subperitoneal fibroblasts (SPFs) and submucosal fibroblasts (SMFs) from human colonic tissue. Biological characteristics and results of gene expression profile analyses were compared to better understand the peritoneal invasion of colon cancer and how this may form a special microenvironment through the interaction with SPFs. Mouse xenograft tumors, derived by co-injection of cancer cells with either SPFs or SMFs, were established to evaluate their active role on tumor progression and metastasis. Results We found that fibrosis with alpha smooth muscle actin (α-SMA) expression was a significant pathological feature of CMPI. The differences in proliferation and gene expression profile analyses suggested SPFs and SMFs were distinct populations, and that SPFs were characterized by a higher expressions of extracellular matrix (ECM)-associated genes. Furthermore, compared with SMFs, SPFs showed more variable alteration in gene expressions after cancer-cell-conditioned medium stimulation. Gene ontology analysis revealed that SPFs-specific upregulated genes were enriched by actin-binding or contractile-associated genes including α-SMA encoding ACTA2. Mouse xenograft tumors derived by co-injection of cancer cells with SPFs showed enhancement of tumor growth, metastasis, and capacity for

  12. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  13. Selective staining of actin in live human dermal fibroblast cells using quantum dots

    NASA Astrophysics Data System (ADS)

    Adurkar, Udita S.; Agrawal, Amit; Nie, Shuming

    2005-03-01

    Semiconductor quantum dots (QD) are nanometer size fluorophores with improved brightness, resistance against photobleaching and narrow emission bands. These properties make QDs ideal for ultrasensitive imaging of biomolecules in living cells, in multiplexed format. By conjugating QDs with a delivery agent such as TAT peptide and a target-recognition element such as an antibody, we have delivered and imaged target-specific fluorescent probes in living cells. In this work, we demonstrate staining of actin filaments in living Human Dermal Fibroblast (HDF) cells using QD probes functionalized with monoclonal actin antibody. Actin probes were developed by coupling streptavidin coated QDs (λem = 605 nm, QDC Corp.) to biotinylated monoclonal β-actin antibody. Antibody molecules on QDs were conjugated with the TAT peptide. Finally, HDF cells were incubated with the QD-actin antibody-TAT construct. As expected, the characteristic fine streaks of actin filaments were observed in the cells and on the periphery of the cells, similar to phalloidin staining of actin filaments in fixed cells. Using a similar approach, one may image cellular components, proteins or nucleic acids, in a living cell, in real time.

  14. Biofunctionalized 3-D Carbon Nano-Network Platform for Enhanced Fibroblast Cell Adhesion

    PubMed Central

    Chowdhury, A. K. M. Rezaul Haque; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

    2017-01-01

    Carbon nanomaterials have been investigated for various biomedical applications. In most cases, however, these nanomaterials must be functionalized biologically or chemically due to their biological inertness or possible cytotoxicity. Here, we report the development of a new carbon nanomaterial with a bioactive phase that significantly promotes cell adhesion. We synthesize the bioactive phase by introducing self-assembled nanotopography and altered nano-chemistry to graphite substrates using ultrafast laser. To the best of our knowledge, this is the first time that such a cytophilic bio-carbon is developed in a single step without requiring subsequent biological/chemical treatments. By controlling the nano-network concentration and chemistry, we develop platforms with different degrees of cell cytophilicity. We study quantitatively and qualitatively the cell response to nano-network platforms with NIH-3T3 fibroblasts. The findings from the in vitro study indicate that the platforms possess excellent biocompatibility and promote cell adhesion considerably. The study of the cell morphology shows a healthy attachment of cells with a well-spread shape, overextended actin filaments, and morphological symmetry, which is indicative of a high cellular interaction with the nano-network. The developed nanomaterial possesses great biocompatibility and considerably stimulates cell adhesion and subsequent cell proliferation, thus offering a promising path toward engineering various biomedical devices. PMID:28287138

  15. Key players in pancreatic cancer-stroma interaction: Cancer-associated fibroblasts, endothelial and inflammatory cells

    PubMed Central

    Nielsen, Michael Friberg Bruun; Mortensen, Michael Bau; Detlefsen, Sönke

    2016-01-01

    Pancreatic cancer (PC) is the most aggressive type of common cancers, and in 2014, nearly 40000 patients died from the disease in the United States. Pancreatic ductal adenocarcinoma, which accounts for the majority of PC cases, is characterized by an intense stromal desmoplastic reaction surrounding the cancer cells. Cancer-associated fibroblasts (CAFs) are the main effector cells in the desmoplastic reaction, and pancreatic stellate cells are the most important source of CAFs. However, other important components of the PC stroma are inflammatory cells and endothelial cells. The aim of this review is to describe the complex interplay between PC cells and the cellular and non-cellular components of the tumour stroma. Published data have indicated that the desmoplastic stroma protects PC cells against chemotherapy and radiation therapy and that it might promote the proliferation and migration of PC cells. However, in animal studies, experimental depletion of the desmoplastic stroma and CAFs has led to more aggressive cancers. Hence, the precise role of the tumour stroma in PC remains to be elucidated. However, it is likely that a context-dependent therapeutic modification, rather than pure depletion, of the PC stroma holds potential for the development of new treatment strategies for PC patients. PMID:26973408

  16. Biofunctionalized 3-D Carbon Nano-Network Platform for Enhanced Fibroblast Cell Adhesion

    NASA Astrophysics Data System (ADS)

    Chowdhury, A. K. M. Rezaul Haque; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

    2017-03-01

    Carbon nanomaterials have been investigated for various biomedical applications. In most cases, however, these nanomaterials must be functionalized biologically or chemically due to their biological inertness or possible cytotoxicity. Here, we report the development of a new carbon nanomaterial with a bioactive phase that significantly promotes cell adhesion. We synthesize the bioactive phase by introducing self-assembled nanotopography and altered nano-chemistry to graphite substrates using ultrafast laser. To the best of our knowledge, this is the first time that such a cytophilic bio-carbon is developed in a single step without requiring subsequent biological/chemical treatments. By controlling the nano-network concentration and chemistry, we develop platforms with different degrees of cell cytophilicity. We study quantitatively and qualitatively the cell response to nano-network platforms with NIH-3T3 fibroblasts. The findings from the in vitro study indicate that the platforms possess excellent biocompatibility and promote cell adhesion considerably. The study of the cell morphology shows a healthy attachment of cells with a well-spread shape, overextended actin filaments, and morphological symmetry, which is indicative of a high cellular interaction with the nano-network. The developed nanomaterial possesses great biocompatibility and considerably stimulates cell adhesion and subsequent cell proliferation, thus offering a promising path toward engineering various biomedical devices.

  17. Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells.

    PubMed

    Pan, Zhi; Lee, Wilson; Slutsky, Lenny; Clark, Richard A F; Pernodet, Nadine; Rafailovich, Miriam H

    2009-04-01

    The effects of exposure of human dermal fibroblasts to rutile and anatase TiO(2) nanoparticles are reported. These particles can impair cell function, with the latter being more potent at producing damage. The exposure to nanoparticles decreases cell area, cell proliferation, mobility, and ability to contract collagen. Individual particles are shown to penetrate easily through the cell membrane in the absence of endocytosis, while some endocytosis is observed for larger particle clusters. Once inside, the particles are sequestered in vesicles, which continue to fill up with increasing incubation time till they rupture. Particles coated with a dense grafted polymer brush are also tested, and, using flow cytometry, are shown to prevent adherence to the cell membrane and hence penetration of the cell, which effectively decreases reactive oxygen species (ROS) formation and protects cells, even in the absence of light exposure. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating can potentially play an important role in protecting cells and tissue from damage.

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

    SciTech Connect

    Wakatsuki, Masaru

    2012-09-01

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

  19. Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

    PubMed Central

    Pagano, RE; Takeichi, M

    1977-01-01

    The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions. PMID:407233

  20. The influence of sintering temperature on the proliferation of fibroblastic cells in contact with HA-bioceramics.

    PubMed

    Frayssinet, P; Rouquet, N; Fages, J; Durand, M; Vidalain, P O; Bonel, G

    1997-06-05

    HA-ceramics used in human surgery as osteoconductive surfaces show a great variety of characteristics. Certain characteristics such as grain size, porosity, and surface area, are controlled by the sintering temperature of the slurry. We grew L-929 fibroblast cells on HA-ceramic disks that had been sintered at different temperatures ranging from 850 degrees-1350 degrees C. The cell line growth rate was lower on ceramic disks than on the culture-grade polystyrene used as a negative control. Cell growth correlated with the ceramic sintering temperature although no significant difference in the cell adhesion to the different ceramics was shown. Growth rate on ceramics sintered at low temperatures (850 degrees and 950 degrees C) was negative whereas it was positive on disks sintered at higher temperatures. When the cells were separated from the disks by a polycarbonate membrane, the growth rate was negative on those membranes in contact with low-temperature sintered disks and positive on the high-temperature sintered disks. The calcium and phosphorus concentration in the culture medium in contact with ceramics sintered below 1050 degrees C decreased during the culture period. Ceramics sintered between 1100 degrees and 1250 degrees C brought about an increase in Ca and P concentrations while ceramics sintered at higher temperatures did not induce any changes. SEM examination of the 850 degrees and 1200 degrees C sintered ceramics showed that the 850 degrees C sintered ceramics consisted of small grains with pores between them and the 1200 degrees C sintered ceramics were made of larger grains without any visible pores, thereby decreasing the surface of material in contact with the culture medium. This difference in surface area was confirmed by the fact that the amount of albumin absorbed onto the ceramic was dependent on the sintering temperature. In conclusion, the modification of the culture medium brought about by high-surfaced ceramics could influence the growth of

  1. Photosensitization of skin fibroblasts and HeLa cells by three chlorin derivatives: Role of chemical structure and delivery vehicle.

    PubMed

    Postigo, Fernando; Sagristá, M Luisa; De Madariaga, M Africa; Nonell, Santi; Mora, Margarita

    2006-05-01

    The chemical nature of the sensitizer and its selective uptake by malignant cells are decisive to choose an appropriate biocompatible carrier, able to preserve the photosensitizing characteristics of the dye. In this paper we demonstrate the photodynamic properties of three chlorins, derived from chlorophyll a, and the usefulness of liposomal carriers to design pharmaceutical formulations. The chlorins have been quantitatively incorporated into stable liposomes obtained from a mixture of L-alpha-palmitoyloleoylphosphatidylcholine and L-alpha-dioleoylphosphatidylserine in a 13.5:1.5 molar ratio (POPC/OOPS-liposomes). The chlorin uptake by skin fibroblasts increases steadily, reaching in all cases a plateau level dependent on both the chlorin structure and the vehicle employed. The photophysical properties of the three chlorins in THF are nearly identical and fulfill the requirements for a PDT photosensitizer. Incorporation of chlorins into liposomes induces important changes in their photophysics, but does not impair their cellular uptake or their cell photosensitization ability. In fact we observe in the cells the same photophysical behavior as in THF solution. Specifically, we demonstrate, by recording the near-IR phosphorescence of 1O2, that the chlorins are able to photosensitize the production of 1O2 in the cell membrane. The cell-photosensitization efficiency depended on the chlorin and cell line nature, the carrier, and the length of pre-incubation and post-irradiation periods. The high photodynamic activity of chlorin-loaded liposomes and the possibility to design liposomal carriers to achieve a specific target site favors this approach to obtain an eventual pharmaceutical formulation.

  2. Canine distemper virus utilizes different receptors to infect chicken embryo fibroblasts and vero cells.

    PubMed

    Chen, Jun; Liang, Xiu; Chen, Pei-fu

    2011-04-01

    Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts (CEF) is a common method to develop attenuated live vaccines with full security. Canine distemper virus (CDV) also does this, but the mechanisms and particular receptors remain unclear. Virus overlay protein blot assays were carried out on CEF membrane proteins, which were extracted respectively with a Mem-PER™ kit, a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method, and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells, indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

  3. Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling

    PubMed Central

    Ramachandra, Rashmi; Namburi, Ramesh B; Ortega-Martinez, Olga; Shi, Xiaofeng; Zaia, Joseph; Dupont, Sam T; Thorndyke, Michael C; Lindahl, Ulf; Spillmann, Dorothe

    2014-01-01

    Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates. PMID:24253764

  4. Parkinson's disease and Alzheimer's disease: hypersensitivity to X rays in cultured cell lines.

    PubMed Central

    Robbins, J H; Otsuka, F; Tarone, R E; Polinsky, R J; Brumback, R A; Nee, L E

    1985-01-01

    Fibroblast and/or lymphoblastoid lines from patients with several inherited primary neuronal degenerations are hypersensitive to DNA-damaging agents. Therefore, lymphoblastoid lines were irradiated from patients with sporadic Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis. The mean survival values of the eight Parkinson's disease and of the six Alzheimer's disease lines, but not of the five amyotrophic lateral sclerosis lines, were less than that of the 28 normal lines. Our results with Parkinson's disease and Alzheimer's disease cells can be explained by a genetic defect arising as a somatic mutation during embryogenesis, causing defective repair of the X-ray type of DNA damage. Such a DNA repair defect could cause an abnormal accumulation of spontaneously occurring DNA damage in Parkinson's disease and Alzheimer's disease neurons in vivo, resulting in their premature death. PMID:3876409

  5. Neural cell surface differentiation antigen gp130(RB13-6) induces fibroblasts and glioma cells to express astroglial proteins and invasive properties.

    PubMed

    Deissler, H; Blass-Kampmann, S; Bruyneel, E; Mareel, M; Rajewsky, M F

    1999-04-01

    Transient expression of the differentiation and tumor cell surface antigen gp130(RB13-6) characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea-induced neurooncogenesis. gp130(RB13-6) is as a member of an emerging protein family of ecto-phosphodiesterases/nucleotide pyrophosphatases that includes PC-1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130(RB13-6) in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130(RB13-6) (NIH-3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130(RB13-6). The effect of gp130(RB13-6) expression was analyzed in terms of overall cell morphology, the expression of glial cell-specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130(RB13-6)-positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh-like patterns. Contrary to the parental NIH-3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130(RB13-6)-positive and -negative cells, expression of gp130(RB13-6) induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy-1, Ran-2, and S-100. In accordance with its expression in the immature rat nervous system, gp130(RB13-6) may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.

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

    PubMed

    Bonifacio, Laura N; Jarstfer, Michael B

    2010-09-01

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

  7. Computational Modeling Predicts Simultaneous Targeting of Fibroblasts and Epithelial Cells Is Necessary for Treatment of Pulmonary Fibrosis

    PubMed Central

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; Linderman, Jennifer J.; Moore, Bethany B.; Kirschner, Denise E.

    2016-01-01

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGF-β1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. Thus, a two-hit therapeutic intervention strategy

  8. Computational modeling predicts simultaneous targeting of fibroblasts and epithelial cells is necessary for treatment of pulmonary fibrosis

    SciTech Connect

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; Linderman, Jennifer J.; Moore, Bethany B.; Kirschner, Denise E.

    2016-06-23

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGFβ1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. In conclusion, a two

  9. Computational modeling predicts simultaneous targeting of fibroblasts and epithelial cells is necessary for treatment of pulmonary fibrosis

    DOE PAGES

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; ...

    2016-06-23

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited withmore » enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGFβ1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. In conclusion, a two-hit therapeutic

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  11. Tumor-associated fibroblast-conditioned medium induces CDDP resistance in HNSCC cells

    PubMed Central

    Steinbichler, Teresa Bernadette; Metzler, Veronika; Pritz, Christian; Riechelmann, Herbert; Dudas, Jozsef

    2016-01-01

    Objective EMT (epithelial to mesenchymal transition) contributes to tumor progression and metastasis. We aimed to investigate the effects of EMT on CDDP resistance in HNSCC (head and neck squamous cell carcinoma)-cells. Methods EMT was induced using conditioned medium from a tumor cell/fibroblast co-culture. HNSCC cells were alternatively treated with TGF-β1. The response to CDDP was evaluated with viability and clonogenic assays. Results Treatment of SCC-25/Detroit 562 cells with conditioned medium increased viability of the tumor cells. Moreover, it doubled the IC50 of CDDP of SCC-25 cells from 6.2 μM to 13.1 μM (p < 0.001). The IC50 of CDDP of Detroit 562 cells was increased following treatment with conditioned medium from 13.1 μM to 26.8 μM (p < 0.01). Colony forming ability after treatment with 5 or 10 μM CDDP was significantly higher in HNSCC cells treated with co-culture conditioned medium than in controls (p < 0.05). Treatment with TGF-β1 had no effect on the IC50 of CDDP (p > 0.1). Conclusions Cell free medium from a co-culture was able to induce EMT in HNSCC cells. Co-culture treated HNSCC cells revealed increased viability and were less sensitive to CDDP treatment. TGF-β1 also induced a mesenchymal phenotype, but did not alter resistance to CDDP in HNSCC cells. PMID:26497215

  12. Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression

    PubMed Central

    Xu, Wen Wen; Li, Bin; Guan, Xin Yuan; Chung, Sookja K.; Wang, Yang; Yip, Yim Ling; Law, Simon Y. K.; Chan, Kin Tak; Lee, Nikki P. Y.; Chan, Kwok Wah; Xu, Li Yan; Li, En Min; Tsao, Sai Wah; He, Qing-Yu; Cheung, Annie L. M.

    2017-01-01

    Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression. PMID:28186102

  13. Induction of cancer-associated fibroblast-like cells by carbon nanotubes dictates its tumorigenicity

    PubMed Central

    Luanpitpong, Sudjit; Wang, Liying; Castranova, Vincent; Dinu, Cerasela Zoica; Issaragrisil, Surapol; Chen, Yi Charlie; Rojanasakul, Yon

    2016-01-01

    Tumor microenvironment has been recognized as a key determinant of tumor formation and metastasis, but how tumor microenvironment is affected by nanomaterials is essentially unknown. Here, we investigated whether carbon nanotubes (CNTs), a widely used nanomaterial with known carcinogenic potential, can affect cancer-associated fibroblasts (CAFs), which are a key component of tumor microenvironment that provides necessary support for tumor growth. We show for the first time that single-walled CNT and to a lesser extent multi-walled and its COOH-functionalized form induced CAF-like cells, which are non-tumorigenic in animals, but promote tumor growth of human lung carcinoma and CNT-transformed lung epithelial cells. The mechanism by which CNT-induced CAF-like cells promote tumor growth involved the acquisition of cancer stem cells (CSCs) in cancer population. Gene knockdown experiments showed that an expression of podoplanin on CAF-like cells is essential for their effects, indicating the functional role of CAF-like cells and podoplanin in CNT tumorigenic process. Our findings unveil a novel mechanism of CNT-induced carcinogenesis through the induction of CAF-like cells that support CSCs and drive tumor formation. Our results also suggest the potential utility of podoplanin as a mechanism-based biomarker for rapid screening of carcinogenicity of CNTs and related nanomaterials for their safer design. PMID:27996035

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

    PubMed Central

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

    2016-01-01

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

  15. Sister chromatid exchange induction and cell cycle inhibition by aniline and its metabolites in human fibroblasts

    SciTech Connect

    Wilmer, J.L.; Kligerman, A.D.; Erexson, G.L.

    1981-01-01

    Sister chromatid exchange (SCE) and cell cycle analyses in human fibroblasts were used to ascertain the relative genotoxicity and cytotoxicity of aniline and its metabolites. Significant increases (P0.05) in SCE frequencies were found with aniline HCl, o-aminophenol, N-phenylhydroxylamine, and trimethymelamine (TEM). On an SCE/mmole basis at the highest concentrations examined, o-aminophenol was 270 times more potent than aniline in inducing SCE, whereas TEM was about 390 times more potent than o-aminophenol. Furthermore, fibroblasts treated with o-aminophenol responded in a dose-dependent fashion and exhibited a 2-fold increase in SCE frequency. N-phenylhydroxylamine induced a less clear-cut, dose related increase in SCE frequency with a 1.4-fold elevation. Only marginal increases in SCE were observed with aniline at the highest doses. Using these data, we propose that aniline may exert its turmorigenic potential in rats through the production of both genotoxic and cytotoxic metabolities. (JMT)

  16. Recurrent trisomy and Robertsonian translocation of chromosome 14 in murine iPS cell lines.

    PubMed

    Chen, Qian; Shi, Xiaoyun; Rudolph, Cornelia; Yu, Yong; Zhang, Ding; Zhao, Xiaoyu; Mai, Sabine; Wang, Gang; Schlegelberger, Brigitte; Shi, Qinghua

    2011-10-01

    Induced pluripotent stem (iPS) cells have greatly provoked people's interest due to their enormous potential of clinical applications. Increasing care is taken with the genetic safety of iPS cells. However, up to now, the chromosomal integrity of murine iPS (miPS) cells has been largely unknown. We have observed recurrent trisomy and/or Robertsonian translocation (Rb) of chromosome 14 in six out of nine independent miPS cell lines from three laboratories by G-banding, fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) analyses, while all the miPS cell lines were derived from mouse embryonic fibroblasts (MEFs) or neural precursor cells (NPCs) with a normal karyotype. The miPS cells with trisomy and/or Rb of chromosome 14 showed growth advantage over the miPS cells with a normal karyotype. We found a significantly higher frequency of Rbs in the miPS cell lines induced with c-Myc than those without c-Myc. Our findings demonstrate that miPS cell lines have the propensity for chromosomal aberrations and there is an obvious correlation between the extent of chromosomal aberrations in miPS cells and the transcriptional factors used for their reprogramming. Therefore, our study raises awareness of the need for improvements of the induction conditions of miPS cells in order to avoid the chromosomal aberrations and ensure future safe applications.

  17. Core Transcription Factors, MicroRNAs, and Small Molecules Drive Transdifferentiation of Human Fibroblasts Towards The Cardiac Cell Lineage

    PubMed Central

    Christoforou, Nicolas; Chakraborty, Syandan; Kirkton, Robert D.; Adler, Andrew F.; Addis, Russell C.; Leong, Kam W.

    2017-01-01

    Transdifferentiation has been described as a novel method for converting human fibroblasts into induced cardiomyocyte-like cells. Such an approach can produce differentiated cells to study physiology or pathophysiology, examine drug interactions or toxicities, and engineer cardiac tissues. Here we describe the transdifferentiation of human dermal fibroblasts towards the cardiac cell lineage via the induced expression of transcription factors GATA4, TBX5, MEF2C, MYOCD, NKX2–5, and delivery of microRNAs miR-1 and miR-133a. Cells undergoing transdifferentiation expressed ACTN2 and TNNT2 and partially organized their cytoskeleton in a cross-striated manner. The conversion process was associated with significant upregulation of a cohort of cardiac-specific genes, activation of pathways associated with muscle contraction and physiology, and downregulation of fibroblastic markers. We used a genetically encoded calcium indicator and readily detected active calcium transients although no spontaneous contractions were observed in transdifferentiated cells. Finally, we determined that inhibition of Janus kinase 1, inhibition of Glycogen synthase kinase 3, or addition of NRG1 significantly enhanced the efficiency of transdifferentiation. Overall, we describe a method for achieving transdifferentiation of human dermal fibroblasts into induced cardiomyocyte-like cells via transcription factor overexpression, microRNA delivery, and molecular pathway manipulation. PMID:28071742

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. The Immunomodulatory Small Molecule Imiquimod Induces Apoptosis in Devil Facial Tumour Cell Lines

    PubMed Central

    Darby, Jocelyn M.; Tovar, Cesar; Lyons, A. Bruce; Woods, Gregory M.

    2016-01-01

    The survival of the Tasmanian devil (Sarcophilus harrisii) is threatened by devil facial tumour disease (DFTD). This transmissible cancer is usually fatal, and no successful treatments have been developed. In human studies, the small immunomodulatory molecule imiquimod is a successful immunotherapy, activating anti-tumour immunity via stimulation of toll-like receptor-7 (TLR7) signaling pathways. In addition, imiquimod is a potent inducer of apoptosis in human tumour cell lines via TLR7 independent mechanisms. Here we investigate the potential of imiquimod as a DFTD therapy through analysis of treated DFTD cell lines and Tasmanian devil fibroblasts. WST-8 proliferation assays and annexin V apoptosis assays were performed to monitor apoptosis, and changes to the expression of pro- and anti-apoptotic genes were analysed using qRT-PCR. Our results show that DFTD cell lines, but not Tasmanian devil fibroblasts, are sensitive to imiquimod-induced apoptosis in a time and concentration dependent manner. Induction of apoptosis was accompanied by down-regulation of the anti-apoptotic BCL2 and BCLXL genes, and up-regulation of the pro-apoptotic BIM gene. Continuous imiquimod treatment was required for these effects to occur. These results demonstrate that imiquimod can deregulate DFTD cell growth and survival in direct and targeted manner. In vivo, this may increase DFTD vulnerability to imiquimod-induced TLR7-mediated immune responses. Our findings have impr