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Sample records for fetal fibroblast cell

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

    Science.gov (United States)

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

    2013-01-01

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

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

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

    2013-01-01

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

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

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    Thomas Zuliani

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

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

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    Tokiwa,Takayoshi

    1986-04-01

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

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

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    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.

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

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

    2013-10-01

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

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

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    Zigman Warren B

    2006-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  9. Dogs cloned from fetal fibroblasts by nuclear transfer.

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

    2009-10-01

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

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

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

    2015-01-01

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

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

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

    2014-01-01

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

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

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    Jianhua Gong

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

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

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

    2016-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

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    Fillenwarth Michael J

    2006-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

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

    2015-02-01

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

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

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

    2015-02-15

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

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

    Institute of Scientific and Technical Information of China (English)

    Yu-jie SUN; Xiao HAN

    2004-01-01

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

  1. Plasticity of fetal cartilaginous cells

    OpenAIRE

    Quintin, Aurelie; Schizas, Constantin; Scaletta, Corinne; Jaccoud, Sandra; Applegate, Lee Ann; Pioletti, Dominique P.

    2010-01-01

    Tissue-specific stem cells found in adult tissues can participate to the repair process following injury. However adult tissues, such as articular cartilage and intervertebral disc, have low regeneration capacity, whereas fetal tissues, such as articular cartilage, show high regeneration ability. The presence of fetal stem cells in fetal cartilaginous tissues and their involvement in the regeneration of fetal cartilage is unknown. The aim of the study was to assess the chondrogenic differenti...

  2. Bovine trophectoderm cell lines induced from bovine fibroblasts with reprogramming factors

    Science.gov (United States)

    Bovine trophectoderm (TE) cells were induced [induced bovine trophectoderm-like (iBT)] from bovine fetal liver-derived fibroblasts, and other bovine fetal fibroblasts, after viral-vector transduction with either four or six reprogramming factors (RF), including POU5F1, KLF4, SOX2, C-MYC, SV40 large ...

  3. Establishment and Biological Characteristic Analysis of Fetal Fibroblast Cell Line for Jinhua Pig%金华猪胎儿成纤维细胞系的建立与生物学特性分析

    Institute of Scientific and Technical Information of China (English)

    郝柱; 王颖; 彭静; 沈一飞; 郭晓令; 徐宁迎

    2012-01-01

    建立并保存家畜的成纤维细胞在保护畜禽种质资源研究中发挥着重要作用.本研究以40日龄金华猪胚胎为实验材料,采用组织贴壁法建立了金华猪胎儿成纤维细胞系,并对所建细胞系进行生物学特性研究.结果表明,原代细胞生长迅速,贴壁后2~3d可长满培养瓶,获得的成纤维细胞生长态势良好;细胞生长曲线为典型的S形,细胞群体倍增时间(population doubling time,PDT)约为36h;细胞冻存前、复苏后的活率分别为95.2%和92.8%;细胞中期染色体二倍体(2n=38)占主体约为91%,达到了建立成纤维细胞系的要求;苹果酸脱氢同工酶(malic dehydrogenase,MDH)和乳酸脱氢同工酶(lactic dehydrogenase,LDH)电泳结果表明,本细胞系没有被其他细胞污染,细胞纯度较高;细菌、真菌和支原体三类微生物检测结果均为阴性,细胞没有受到微生物的污染;15代细胞的凋亡率为2.0%,细胞没有大规模的凋亡现象发生;当阳离子脂质体(Lipofectamine 2000)剂量为0.3 μL、荧光蛋白报告质粒(pEGFP-N3)为0.5 μg时转染成纤维细胞的效率最高,可达32.4%.细胞系各项指标均达到美国典型培养中心(American Type Culture Collection,ATCC)标准.金华猪胎儿成纤维细胞系的成功建立使金华猪种质资源在细胞水平得到保存,并可为胚胎克隆以及转基因等研究提供必要的实验材料;也可以为以后其他种质资源在细胞水平上的保存提供理论与技术支持.%Establishment and preservation of livestock fibroblasts play a important role in protecting of animal genetic resources. The fetal fibroblast cell line of Jinhua pig was successfully established from 40 d's embryos by direct explant technique, and the cell morphology, growth dynamic, vitality, chromosome, isoenzymes, microbial inspection, cell apoptosis, as well as the transfection of fluorescent protein report plasmid (pEGFP-N3) had been studied in the line. The

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Swathi Balaji

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

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Human fetal mesenchymal stem cells.

    Science.gov (United States)

    O'Donoghue, Keelin; Chan, Jerry

    2006-09-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2012-05-01

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

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

    Science.gov (United States)

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

    2016-02-03

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

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

    Directory of Open Access Journals (Sweden)

    Dawei Yu

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

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

    Science.gov (United States)

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

    2006-06-01

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

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

    Science.gov (United States)

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

    2001-10-01

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

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

    Science.gov (United States)

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

    2017-05-15

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

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

    DEFF Research Database (Denmark)

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

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Amir Amiri Yekta

    2013-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    陈鸿辉; 唐毅; 李斯明; 沈雁; 刘向荣; 钟灿灿

    2002-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    孙中帅; 雷建园; 广茜茜; 畅继武

    2011-01-01

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

  2. 转双基因(pGH/IGF-Ⅰ)猪胎儿成纤维细胞系的建立及鉴定%Establishment and Identification of a Fetal Fibroblast Cell Line with Double Transgenic (pGH/IGF-Ⅰ) Porcine

    Institute of Scientific and Technical Information of China (English)

    李晓玲; 于光辉; 孙金海

    2016-01-01

    本研究旨在建立转双基因(pG H/IG F‐Ⅰ)猪胎儿成纤维细胞系,保存转双基因猪的成纤维细胞以便于后续细胞水平研究。试验采用胰蛋白酶消化法对猪胎儿躯干组织进行原代培养,通过原代培养、细胞传代、冷冻保存等成功分离出转双基因猪胎儿成纤维细胞,并对细胞进行了形态学观察、冻存前和复苏后细胞活力检测、生长动力学分析、波形蛋白免疫组化及微生物污染检测等生物学特性分析。结果显示,原代细胞经过胰蛋白酶消化和差速离心分离培养出成纤维细胞,冻存前细胞活力为94.3%,冻存3个月后细胞复苏后活力为91.2%;细胞生长总趋势呈“S”型,经历了潜伏期、指数生长期和平台期3个阶段;细胞波形蛋白在成纤维细胞中呈阳性反应;细胞的细菌、真菌、病毒和支原体检测均为阴性。结果表明本试验成功建立了转双基因猪成纤维细胞系。%This study was aimed to establish a fetal fibroblast cell line of double transgenic (pGH/IGF‐Ⅰ) pigs ,and reserve fibroblast cells of double transgenic pigs for the follow‐up study.A method of trypsin digestion was adopted to isolate and culture body tissues from pregnant por‐cine ,and porcine fetal fibroblasts were isolated successfully through primary culture ,subculturing and cryopreservation.T he morphological observation ,determination of viability before cryopreser‐vation and after recovery ,dynamic grow th analysis ,vimentin immunohistochemistry and microbial contamination detection were all done to study the biological characteristics of the cell line.The results showed that the fibroblasts were cultured and isolated successfully by trypsin digestion and differential centrifugation.The cell viability before cryopreservation and after recovery were 94.3% and 91.2% ,respectively.The growth curve was sigmoidal ,and experienced the incubation period ,exponential growth

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Science.gov (United States)

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

    2015-09-28

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

  5. Construction of Fibroblast Growth Factor 5 Gene (FGF5) Hair Follicle Specific Expression Vector and Transfected into Cashmere Goat (Capra hircus) Fetal Fibroblast Cells%绒山羊成纤维细胞生长因子5基因(FGF5)毛囊特异性表达载体的构建及转染胎儿成纤维细胞

    Institute of Scientific and Technical Information of China (English)

    康健; 胡广东; 权富生; 张涌

    2014-01-01

    The quality and yield of cashmere goat wool are closely related to the growth and development of skin hair follicles. This study aimed to construct an eukaryotic expression vector and prove whether it could specifically express fibroblast growth factor 5 gene (FGF5) in the hair follicles of cashmere goat (Capra hircus). The keratin associated protein 6-1 (KAP6-1) promoter and FGF5 gene coding sequence were obtained by PCR. The vector pEGFP-N1-KF, a hair follicles specific expression vector of FGF5, was constructed by inserting the KAP6-1 promoter into the CMV promoter-less pEGFP-N1, and then connecting FGF5 to the downstream of KAP6-1 promoter, using Porcine teschovirus 2A peptide (P2A) to link FGF5 and enhanced green fluorescent protein (EGFP) gene. The constructed vector was transfected into cashmere goat fetal fibroblast cells and two methods were used to verify its functionality including qRT-PCR and Western blot. The enzyme digestion results showed the vector was correctly constructed. The qRT-PCR results indicated that the FGF5 gene could express in fetal fibroblast cells. The Western blotting results confirmed that the fusion protein could be availably spelited into FGF5 and GFP protein by the functionality of P2A. The result showed that the vector pEGFP-N1-KF which could specifically express FGF5 gene was successfully constructed and could normally expressed in cashmere goat fetal fibroblast cells.%绒山羊的绒毛品质、产量与皮肤毛囊的生长发育密切相关。本研究旨在构建绒山羊(Capra hircus)成纤维细胞生长因子5基因(fibroblast growth factor 5, FGF5)的毛囊特异性表达载体,并证明其表达的有效性。分别以绒山羊基因组和cDNA为模板,利用PCR方法克隆角蛋白关联蛋白6-1(keratin associated protein 6-1, KAP6-1)基因的启动子和FGF5基因的编码区序列(coding sequence, CDS),并将这两个元件连接到去除CMV启动子的真核表达载体pEGFP-N1上,FGF5

  6. Identification of circulating fetal cell markers by microarray analysis

    DEFF Research Database (Denmark)

    Brinch, Marie; Hatt, Lotte; Singh, Ripudaman

    2012-01-01

    identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem......OBJECTIVE: Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found...... in the maternal blood circulation at the end of the first trimester. METHOD: Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells...

  7. Hepatocyte growth factor improves direct reprogramming of fibroblasts towards endothelial progenitor cells via ETV2 transduction

    Directory of Open Access Journals (Sweden)

    Phuc Van Pham

    2016-09-01

    Full Text Available Human fibroblasts can be differentiated into endothelial progenitor cells by direct reprogramming via ETV-2 transfection. Previously, we have shown that the efficacy of direct reprogramming can be enhanced by hypoxia treatment. In this study, we aim to investigate whether the efficacy of direct reprogramming of fibroblasts into EPCs via Ets variant gene 2 (ETV2 transfection can be increased with hepatocyte growth factor (HGF treatment. Foreskin-derived fibroblasts were cultured in standard medium (DMEM/F12 supplemented with fetal bovine serum. They were then transduced with a viral vector expressing ETV2 in culture medium supplemented with HGF. The transduced fibroblasts were cultured in endothelial cell medium supplemented with HGF for 28 days. The efficacy of direct reprogramming was evaluated based on expression of CD31 and VEGFR2 markers by transduced cells. Phenotypic and functional characterization of induced EPCs were also confirmed by expression of particular genes and in vitro angiogenesis assays. Our results showed that HGF significantly increased the efficacy of direct reprogramming of fibroblasts towards EPCs via ETV2 transcription factors; efficiency increased from 5.41+/-1.51% for ETV2 transduction alone to 12.31+/-2.15% for ETV2 transduction combined with HGF treatment. These findings suggest the rationale for combined use of ETV2 and HGF in direct in vitro reprogramming of fibroblasts into EPCs. [Biomed Res Ther 2016; 3(9.000: 836-843

  8. 敲除成纤维细胞生长因子5基因对阿尔巴斯绒山羊胎儿皮肤成纤维细胞系的影响%Influences of FGF5 Gene Knockout to Arbas Cashmere Goat Fetal Skin Fibroblast Cell Line

    Institute of Scientific and Technical Information of China (English)

    高蓓; 马玉珍; 尹俊; 周欢敏

    2012-01-01

    In this study, in order to provide a nuclear for somatic cell nuclear transfer and production of transgenic animals, cationic liposome-mediated transfection of homologous recombination FGF5 gene knockout vector to somatic cell. After positive and negative selection obtained 51 cell clones, which remaining 12 clones after propagation, and ultimately get 4 positive cell clones by the molecular indentification of DNA and RNA levels, namely established the FGF5-knockouted Inner Mongolia Arbas cashmere goat fetal skin fibroblast cell line. Compared with normal somatic cell, the four FGF5 -knockouted positive cell clones had occurred very significant changes in cell morphology, traits and growth characteristics: bigger cell volume; fuzzy boundaries, reduced stereoscopic;growth rate dramatic decline;trypsin digestion time required was significantly increased. This study showed that these changes might be due to FGF5 gene knockout, cell transfection, drug selection and other roles.%本研究为体细胞核移植生产转基因动物提供核供体,以阳离子脂质体介导转染同源重组成纤维细胞生长因子5(fibroblast growth factor 5,FGF5)基因打靶载体至体细胞中,经正负双筛选获得了51个细胞克隆,扩繁后剩余12个克隆,最终经DNA及RNA水平的分子生物学鉴定得到4个阳性细胞克隆,即建立了敲除FGF5基因的内蒙古阿尔巴斯绒山羊胎儿皮肤成纤维细胞系.与普通细胞相比,所得的阳性细胞克隆在细胞形态、性状及生长特性等方面均发生了极其显著的变化:细胞体积增大;边界模糊,立体感降低;生长速率明显下降;胰蛋白酶消化所需时间明显增加.研究结果表明上述变化可能是受到了敲除FGF5基因、细胞转染和药物筛选作用等的影响所致.

  9. 胎肝间充质干细胞和皮肤成纤维细胞有限细胞系的建立及肝向分化研究%Establishment and hepatocyte differentiation of fetal liver mesenchymal stem cells and skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    朱争艳; 杜智; 骆莹; 黎娇; 王鹏; 刘彤; 高英堂

    2011-01-01

    Objective To establish a long-term culture system of fetal liver mesenchymal stem cells and skin fibroblasts, and to compare the phenotyping and the hepatocyte differentiation between two types of cells. Methods Fetus liver-derived mesenchymal stem cells were selected by adherence culture, and fibroblasts were isolated and cultured from skin tissue of the same individual. Immunocytochemistry (ICC)and flow cytometry (FCM) were used in detection of the expressions of CD34, CD90 and CD105, and chromosome analysis and soft agar colony formation test in study of biological characteristics in the two types of cells. The induced differentiation of the P3- 30 cell into hepatocyte was performed with hepatic culture medium (HCM) containing hepatocyte growth factor (HGF) , fibroblast growth factor 4 (FGF4) and oncostatin M(OSM). Albumin(ALB), CK18 and CK19 in the induced and non-induced cells were detected by ICC, glycogen stained by PAS, and the expressions of AFP and ALB mRNA by RT-PCR. Results CD90was highly expressed in fetal liver mesenchymal stem cells but barely detectable in skin fibroblasts. Culture in conditioned medium led to hepatocyte-like differentiation in approximately (5.1±0.2)% of fibroblasts versus ( 10.3±0.3)% of fetal liver mesenchymal stem cells (P<0.01). Both of the two types of cells could be cultured in vitro for about 50 passages that appeared karyotypically normal, non-oncogenic but not immortal.Conclusion Mesenchymal stem cells of adult tissues derived from any adult tissue may have a limited lifetime over a long-term culture in vitro, and some from certain adult tissues may show a stronger potential of differentiation to parenchyma cells of the origin.%通过体外长期培养胎肝间充值干细胞和皮肤成纤维细胞,建立两种细胞长期培养体系并比较此两种细胞的表型特征及向肝细胞分化的潜能。方法 从人胎肝中分离贴壁生长的间充质干细胞,同时取同一个体的皮肤组织,分离培

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

    Science.gov (United States)

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

    2011-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Amornrat Tangprasittipap

    2015-11-01

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

  12. Transcriptional and Posttranscriptional Inhibition of Lysyl Oxidase Expression by Cigarette Smoke Condensate in Cultured Rat Fetal Lung Fibroblasts

    Science.gov (United States)

    Gao, Song; Chen, Keyang; Zhao, Yinzhi; Rich, Celeste B.; Chen, Lijun; Li, Sandy J.; Toselli, Paul; Stone, Phillip; Li, Wande

    2005-01-01

    Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0–120 μg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80–120 μg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 μg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 μg CSC/ml. Moreover, 80–120 μg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema. PMID:15933228

  13. 山羊天然抵抗力相关巨噬细胞蛋白基因(Nramp 1)的克隆及其在羊胎儿成纤维细胞中的表达和鉴定%Expression of goat natural resistance associated macrophage protein gene in mice and sheep fetal fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    秦莹; 安星兰; 张学明; 岳占碰; 李子义

    2013-01-01

    The expression of Nramp 1(Slc11a 1) in late phagolysosomes confers resistance to infection with several intracellular pathogens. The antimicrobial actions of Nramp 1 are attributable, in part,to modulation of macrophage immune function and cellular iron metabolism. In order to investigate the function of goat natural resistance associated macrophage protein and to find a effectively way on natural resistance to infection in goat. Nramp 1 gene was cloned from goat spleen by RT-PCR and was inserted into pcDNA3. 1 ( + ),which named pcDNA3. 1(+)-Nramp 1. Getting 60 days small tail han sheep fetal to establish fetal fibroblast cell lines while the linearized vector was transfected into sheep fetal fibroblast cells to gain stably transfected cell lines. However,the identification at the genome and transcription level shows that the exogenous gene successfully integrated into the genome and the gene expression is detected at transcription level. Two transgenic cell lines were carried out and laid a foundation of Nramp 1 protein function researching and antibacterial experiments.%Nramp 1(Slclla 1)表达于晚期吞噬溶酶体中,能够抵抗多种细胞内病原体的感染.Nramp 1主要是通过调节巨噬细胞的免疫功能和细胞铁代谢发挥抗菌作用.为了研究山羊天然抵抗力相关巨噬细胞蛋白的功能,寻找一种在山羊中抵抗胞内菌感染的新途径.通过RT-PCR的方法从山羊脾脏中克隆Nramp 1基因,连接到真核表达载体pcDNA3.1(+)上,命名为pcDNA3.1(+)-Nramp1.取60 d小尾寒羊胎儿,建立胎儿成纤维细胞系,将线性化的载体转染胎儿成纤维细胞,获得稳定转染的细胞系,在基因组和转录水平分别对转基因细胞系进行鉴定,结果显示外源基因成功整合到基因组中并且在转录水平检测到基因表达,最后获得阳性转基因细胞系2株,为Nramp1蛋白的功能研究和抗菌试验奠定了基础.

  14. Investigation of CNTs interaction with fibroblast cells.

    Science.gov (United States)

    Pensabene, V; Vittorio, O; Raffa, V; Menciassi, A; Dario, P

    2007-01-01

    The need for toxicological studies on carbon nanotubes (CNTs) has arisen from the rapidly emerging applications of CNTs well beyond material science and engineering. In order to provide a method to collect data about toxicology, we characterized by Scanning Electron Microscopy (SEM), by Energy Dispersive X-ray Spectrometry (EDS) analysis and by Focused Ion Beam (FIB) microscopy different kinds of treated CNTs. The bio-interaction was investigated seeding Crandell feline kidney fibroblasts with CNT-modified medium; a dedicated sample preparation by FIB has been defined to fix cells. In the present study, the cytotoxic effects of CNTs with 91% and 97% of purity were compared and changes in the growth behaviour of cells after 3 days in culture with modified medium have been recorded, considering also the distribution of CNTs within cells. While lower purified CNTs induced a slight cytotoxic effect, homogeneously suspended CNTs with high purity were less cytotoxic, and the rate of cell growth remained constant. CNTs aggregated in bundles, showed high adhesion on cell membrane. Interestingly, CNTs bundles were observed inside cells, underneath the cell membrane, and despite of that, cells were extended, in good vitality conditions and no cell-degeneration was observed.

  15. Cholesteatoma fibroblasts promote epithelial cell proliferation through overexpression of epiregulin.

    Directory of Open Access Journals (Sweden)

    Mamoru Yoshikawa

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

  16. The number of fetal cells in maternal blood is associated to exercise and fetal gender

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Kirkegaard, Ida; Christensen, Connie Britta;

    were then stained with a cocktail of fetal cell-specific antibodies, identified and counted. Results: Participants carrying male fetuses had higher median number of fcmbs per 30 mL blood than those carrying female fetuses (5 vs. 3, p=0.004). Exercise within 3 hours (1.5 vs. 4, p=0.02) and 24 hours (2......Introduction: We have established a robust method to specifically identify and isolate a placental fetal cell in maternal blood (fcmbs) at a gestational age of 12 weeks. The concentration of these cells, however, varies considerably among pregnant women (median 3 fcmbs/30 mL blood, range 0...... activity was obtained by a questionnaire and a structured interview. The number of fcmbs was assessed in 30 mL blood processed by a proprietary method developed in-house. Fetal cells in the blood, binding to fetal cell specific antibodies, were initially isolated by magnetic cell sorting. The fetal cells...

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Chan-Jung Chang

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

  19. Fetal stem cell transplantation: Past, present, and future.

    Science.gov (United States)

    Ishii, Tetsuya; Eto, Koji

    2014-09-26

    Since 1928, human fetal tissues and stem cells have been used worldwide to treat various conditions. Although the transplantation of the fetal midbrain substantia nigra and dopaminergic neurons in patients suffering from Parkinson's disease is particularly noteworthy, the history of other types of grafts, such as those of the fetal liver, thymus, and pancreas, should be addressed as there are many lessons to be learnt for future stem cell transplantation. This report describes previous practices and complications that led to current clinical trials of isolated fetal stem cells and embryonic stem (ES) cells. Moreover, strategies for transplantation are considered, with a particular focus on donor cells, cell processing, and the therapeutic cell niche, in addition to ethical issues associated with fetal origin. With the advent of autologous induced pluripotent stem cells and ES cells, clinical dependence on fetal transplantation is expected to gradually decline due to lasting ethical controversies, despite landmark achievements.

  20. Fetal stem cell transplantation: Past, present, and future

    Institute of Scientific and Technical Information of China (English)

    Tetsuya; Ishii; Koji; Eto

    2014-01-01

    Since 1928, human fetal tissues and stem cells have been used worldwide to treat various conditions. Although the transplantation of the fetal midbrain substantia nigra and dopaminergic neurons in patients suffering from Parkinson’s disease is particularly noteworthy, the history of other types of grafts, such as those of the fetal liver, thymus, and pancreas, should be addressed as there are many lessons to be learnt for future stem cell transplantation. This report describes previous practices and complications that led to current clinical trials of isolated fetal stem cells and embryonic stem(ES) cells. Moreover, strategies for transplantation are considered, with a particular focus on donor cells, cell processing, and the therapeutic cell niche, in addition to ethical issues associated with fetal origin. With the advent of autologous induced pluripotent stem cells and ES cells, clinical dependence on fetal transplantation is expected to gradually decline due to lasting ethical controversies, despite landmark achievements.

  1. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy.

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

    Full Text Available BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. METHODOLOGY/PRINCIPAL FINDINGS: A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both. The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001 than in SA women with normal karyotype (r = 0.465, P = 0.002 and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037. The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852-0.945 and 0.939 (95% CI, 0.903-0.975, respectively. CONCLUSIONS: Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA

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

    Science.gov (United States)

    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.

  3. Fetal liver cell transplantation : role and nature of the fetal haemopoietic stem cell

    NARCIS (Netherlands)

    B. Löwenberg (Bob)

    1975-01-01

    textabstractFetal liver cell transplantation deserves intensified interest because, according to previous experimental evidence, it may represent a useful approach to reduce or avoid severe Graft-versus-Host (GvH) reactions following treatment with allogeneic haemopoietic cell grafts. The applicatio

  4. In vitro development competence of bovine nuclear transfer embryos derived from Nanog-overexpressing fibroblast cells

    Directory of Open Access Journals (Sweden)

    Xi-bang Zheng, Yan Yun, Yong-ce Hu, Yong Li, Hua-yan Wang, Xiao-ling Ma, Jin-qiang Sui, An-min Lei and Zhong-ying Dou

    2014-04-01

    Full Text Available The purpose of this study was to establish Nanog-expressing cell lines that can be used as donor cells to construct transgenic cloned embryos, and to investigate their in vitro development competence. By reverse transcription-polymerase chain reaction (RT-PCR, the cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge tissues. The gene was inserted into PMD18-T vector using recombination techniques and then subcloned into vector pEGFP-C1. After confirmation by restrictive endonuclease digestion and sequencing, the recombinant plasmid pEGFP-Nanog was transfected into skin fibroblast cells. A stable transfected cell line was successfully established after two months of selection with neomycine (G418. Fluorescence microscopy, RT-PCR, and Western Blotting assays indicated that Nanog mRNA and EGFP-Nanog fusion protein were expressed in these cells. The EGFP-Nanog expressing fibroblast cells and the intact fibroblast cells (BEF422 were respectively used to construct cloned embryos. The results showed that the cleavage rate of recombinant embryos in BEF422 cells was significantly (P<0.05 higher than in EGFP-Nanog expressing cells (82.14 vs 40.38 %, but the blastocyst development rate in the latter was slightly higher than in the former (17.30 vs 14.29% (P<0.05, indicating that Nanog-overexpressed fibroblasts may be a better candidate of donor cells. To our knowledge, this is the first time that Nanog gene has been introduced into fibroblast cells to produce cloned embryos in bovine.

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

    Science.gov (United States)

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

    2013-09-01

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

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

    Science.gov (United States)

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

    2014-02-25

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

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

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Veland, Iben; Schwab, Albrecht;

    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 p...

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  9. Fibroblasts as Efficient Antigen-Presenting Cells in Lymphoid Organs

    Science.gov (United States)

    Kundig, Thomas M.; Bachmann, Martin F.; Dipaolo, Claudio; Simard, John J. L.; Battegay, Manuel; Lother, Heinz; Gessner, Andre; Kuhlcke, Klaus; Ohashi, Pamela S.; Hengartner, Hans; Zinkernagel, Rolf M.

    1995-06-01

    Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

  10. Fetal hemoglobin in sickle cell anemia.

    Science.gov (United States)

    Akinsheye, Idowu; Alsultan, Abdulrahman; Solovieff, Nadia; Ngo, Duyen; Baldwin, Clinton T; Sebastiani, Paola; Chui, David H K; Steinberg, Martin H

    2011-07-07

    Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Some patients with sickle cell disease have exceptionally high levels of HbF that are associated with the Senegal and Saudi-Indian haplotype of the HBB-like gene cluster; some patients with different haplotypes can have similarly high HbF. In these patients, high HbF is associated with generally milder but not asymptomatic disease. Studying these persons might provide additional insights into HbF gene regulation. HbF appears to benefit some complications of disease more than others. This might be related to the premature destruction of erythrocytes that do not contain HbF, even though the total HbF concentration is high. Recent insights into HbF regulation have spurred new efforts to induce high HbF levels in sickle cell disease beyond those achievable with the current limited repertory of HbF inducers.

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

    Institute of Scientific and Technical Information of China (English)

    张玉玲; 刘凤军; 张涌

    2009-01-01

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

  12. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  13. Pre-weaning performance and health of pigs born to cloned (fetal cell derived) swine versus non-cloned swine.

    Science.gov (United States)

    Martin, M; Adams, C; Wiseman, B

    2004-07-01

    The objective of this study was to compare the pre-weaning performance of pigs derived from cloned versus non-cloned parents. Five cloned gilts and one cloned boar were used to produce five litters of pigs. One of five cloned females and the cloned boar were derived from two genetically unmanipulated fetal fibroblast cell lines. The remaining female clones were derived from a fetal fibroblast cell line in which random insertion of a alpha-1,3-galactosyltransferase gene targeting construct had occurred. Fetal cell lines had similar genetic backgrounds and were derived from three different fetuses in three different litters. Five litters of pigs were also generated from matings between two non-cloned boars and five non-cloned gilts. The mean gestation length, mean litter size, mean birth and weaning weights for male and female pigs were similar for litters derived from cloned parents versus non-cloned parents. The proportions of pigs born live and pigs that survived to weaning were also similar for pigs born to cloned as compared to non-cloned parents. In summary, matings between cloned swine derived from fetal fibroblast cell lines yielded litters of pigs that were similar in the number born, piglet birth weight and perinatal and pre-weaning mortality to litters produced by non-cloned swine.

  14. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Reza B Jalili

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  17. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi

    Institute of Scientific and Technical Information of China (English)

    Jai-Hee Moon; June Seok Heo; Jun Sung Kim; Eun Kyoung Jun; Jung Han Lee; Aeree Kim; Jonggun Kim

    2011-01-01

    Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4,Sox2,and Klf4 in combination with c-Myc.Recently,Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells.Here,we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells,and,in combination with Oct4,can replace Sox2,Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells.Furthermore,activation of sonic hedgehog signaling (by Shh,purmorphamine,or oxysterol) compensates for the effects of Bmil,and,in combination with Oct4,reprograms mouse embryonic and adult fibroblasts into iPS cells.One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile,epigenetic status,and in vitro and in vivo differentiation into all three germ layers,as well as teratoma formation and germline transmission in vivo.These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2,KIf4,and N-Myc allows iPS generation via the addition of Oct4.

  18. Fibroblast nemosis induces angiogenic responses of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Enzerink, Anna, E-mail: anna.enzerink@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland); Rantanen, Ville, E-mail: ville.rantanen@helsinki.fi [Computational Systems Biology Laboratory, Institute of Biomedicine and Genome-Scale Biology Research Program, University of Helsinki, P.O. BOX 63, 00014 Helsinki (Finland); Vaheri, Antti, E-mail: antti.vaheri@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland)

    2010-03-10

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Burkard, Michael, E-mail: Michael.burkard@eawag.ch [Griffith University, Environmental Futures Research Institute, Southern Ocean Persistent Organic Pollutants Program, Brisbane, QLD (Australia); Eawag, Swiss Federal Institute of Technology, Dübendorf (Switzerland); Whitworth, Deanne [The University of Queensland, School of Veterinary Science, Gatton, QLD (Australia); Schirmer, Kristin [Eawag, Swiss Federal Institute of Technology, Dübendorf (Switzerland); ETH Zürich, Institute of Biogechemistry and Pollutant Dynamics, Zürich (Switzerland); EPF Lausanne, School of Architecture, Civil and Environmental Engineering, Lausanne (Switzerland); Nash, Susan Bengtson [Griffith University, Environmental Futures Research Institute, Southern Ocean Persistent Organic Pollutants Program, Brisbane, QLD (Australia)

    2015-10-15

    Highlights: • We established and characterised the first humpback whale fibroblast cell lines. • Cell lines have a stable karyotype with 2n = 44. • Exposure to p,p′-DDE resulted in a concentration-dependent loss of cell viability. • p,p′-DDE sensitivity differed considerably from human fibroblasts. • Exposure to a whale blubber extract showed higher sensitivity than to p,p′-DDE alone. - Abstract: 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% CO{sub 2} 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 (EC{sub 50} value) was approximately six times greater than the EC{sub 50} 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

  20. Attachment of epithelial cells and fibroblasts to ceramic materials.

    Science.gov (United States)

    Niederauer, G G; McGee, T D; Keller, J C; Zaharias, R S

    1994-04-01

    This study examined in vitro gingival epithelial and fibroblast cell attachment to ceramic materials made of tricalcium phosphate and/or magnesium aluminate spinel. The composite made of tricalcium phosphate and spinel is called 'osteoceramic'. These ceramics had various compositions and surface structures, which were initially characterized. Cell attachment assays were performed using both cell types to compare cellular response to the ceramic materials. Specimens were also prepared for scanning electron microscopy to investigate cellular morphology. The highest levels of cell attachment for gingival epithelial cells were observed on the rough osteoceramic surface, whereas gingival fibroblasts attached least to the rough osteoceramic surface.

  1. Scientists ID Key Fetal Cells Vulnerable to Zika

    Science.gov (United States)

    ... html Scientists ID Key Fetal Cells Vulnerable to Zika Lab study suggests possible mechanism for birth defects ... 29, 2016 (HealthDay News) -- The devastating mosquito-borne Zika virus can infect cells that play a role ...

  2. Regulation of germ cell meiosis in the fetal ovary.

    Science.gov (United States)

    Spiller, Cassy M; Bowles, Josephine; Koopman, Peter

    2012-01-01

    Fertility depends on correct regulation of meiosis, the special form of cell division that gives rise to haploid gametes. In female mammals, germ cells enter meiosis during fetal ovarian development, while germ cells in males avoid entering meiosis until puberty. Decades of research have shown that meiotic entry, and germ cell sex determination, are not initiated intrinsically within the germ cells. Instead, meiosis is induced by signals produced by the surrounding somatic cells. More recently, retinoic acid (RA), the active derivative of vitamin A, has been implicated in meiotic induction during fetal XX and postnatal XY germ cell development. Evidence for an intricate system of RA synthesis and degradation in the fetal ovary and testis has emerged, explaining past observations of infertility in vitamin A-deficient rodents. Here we review how meiosis is triggered in fetal ovarian germ cells, paying special attention to the role of RA in this process.

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

    OpenAIRE

    2008-01-01

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

  4. Effect of captopril on collagen metabolisms in keloid fibroblast cells.

    Science.gov (United States)

    Chen, Junjie; Zhao, Sha; Liu, Yong; Cen, Ying; Nicolas, Crook

    2016-12-01

    Keloid is a proliferative disease of fibrous tissues. The mechanism and consistently effective treatments of keloid remained unknown. Although there was a report about treating keloid with topical captopril, the further investigation about captopril affecting keloid has not been performed so far. The aim of this study was to analyse the effect of captopril on collagen metabolisms in keloid fibroblast cells, and to provide information for the mechanism and therapy of keloid. To investigate the effects and relative mechanism of captopril on keloid fibroblast cells, we examined the changes of collagen metabolism, expression of angiotensin, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF)-BB and heat shock protein 47 (HSP47), and cellular proliferation in keloid fibroblast cells. We found that all collagen metabolisms, expression of TGF-β1, PDGF-BB and HSP47, and cellular proliferation decreased significantly with effective captopril concentrations in keloid fibroblast cells. With a comprehensive analysis of test results, we proposed that captopril may decrease the expression of angiotensin, PDGF-BB, TGF-β1 and HSP47, and further inhibit proliferation and collagen synthesis of keloid fibroblast cells, which were the key in keloid formation. © 2014 Royal Australasian College of Surgeons.

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

    Science.gov (United States)

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

    2011-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Keiko Miyoshi

    2015-01-01

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

  7. Mechanism of induction of fibroblast to corneal endothelial cell.

    Science.gov (United States)

    Jiang, Yan; Fu, Wei-Cai; Zhang, Lin

    2014-08-01

    To explore mechanism of nduction of fibroblast to corneal endothelial cell. Rabbit conjunctiva fibroblasts were used as feeder cells, rabbit oral mucosa epithelial cells were used as seed cells, and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium. The transformation effect was observed. As concentration of mitomycin C increased, cell survival rate gradually decreased, cell proliferation was obviously inhibited when concentration≥25 μg/mL; 5 days after being treated by 5 μg/mL mitomycin C, cell body was enlarged and extended without cell fusion, however after being treated by 0.5 μg/mL mitomycin C, cell body was significantly proliferated and gradually fused; after 3 weeks of culture, stratified epithelium appeared on rabbit oral mucosa epithelial cells, differentiation layers were 4-5 and were well differentiated, the morphology was similar to corneal endothelial cells; Under electron microscope, surface layer of cells were polygonal, tightly connected to another with microvilli on the border, there was hemidesmosome between basal cells and human denuded amniotic membrane. Fibroblast cells have the potential of multi-directional differentiation, effective induction can promote emergence of intercellular desmosomes between seed cells and emergence of epithelial surface microvilli, and differentiate to the corneal endothelial cell. However, clinical application still needs more research and safety evaluation. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  8. Cell-free fetal DNA and intact fetal cells in maternal blood circulation: implications for first and second trimester non-invasive prenatal diagnosis.

    Science.gov (United States)

    Bischoff, Farideh Z; Sinacori, Mina K; Dang, Dianne D; Marquez-Do, Deborah; Horne, Cassandra; Lewis, Dorothy E; Simpson, Joe Leigh

    2002-01-01

    Both intact fetal cells as well as cell-free fetal DNA are present in the maternal circulation and can be recovered for non-invasive prenatal genetic diagnosis. Although methods for enrichment and isolation of rare intact fetal cells have been challenging, diagnosis of fetal chromosomal aneuploidy including trisomy 21 in first- and second-trimester pregnancies has been achieved with a 50-75% detection rate. Similarly, cell-free fetal DNA can be reliably recovered from maternal plasma and assessed by quantitative PCR to detect fetal trisomy 21 and paternally derived single gene mutations. Real-time PCR assays are robust in detecting low-level fetal DNA concentrations, with sensitivity of approximately 95-100% and specificity near 100%. Comparing intact fetal cell versus cell-free fetal DNA methods for non-invasive prenatal screening for fetal chromosomal aneuploidy reveals that the latter is at least four times more sensitive. These preliminary results do not support a relationship between frequency of intact fetal cells and concentration of cell-free fetal DNA. The above results imply that the concentration of fetal DNA in maternal plasma may not be dependent on circulating intact fetal cells but rather be a product of growth and cellular turnover during embryonic or fetal development.

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  10. Conversion of Fibroblasts to Neural Cells by p53 Depletion

    Directory of Open Access Journals (Sweden)

    Di Zhou

    2014-12-01

    Full Text Available Conversion from fibroblasts to neurons has recently been successfully induced. However, the underlying mechanisms are poorly understood. Here, we find that depletion of p53 alone converts fibroblasts into all three major neural lineages. The induced neuronal cells express multiple neuron-specific proteins and generate action potentials and transmitter-receptor-mediated currents. Surprisingly, depletion does not affect the well-known tumorigenic p53 target, p21. Instead, knockdown of p53 upregulates neurogenic transcription factors, which in turn boosts fibroblast-neuron conversion. p53 binds the promoter of the neurogenic transcription factor Neurod2 and regulates its expression during fibroblast-neuron conversion. Furthermore, our method provides a high efficiency of conversion in late-passage fibroblasts. Genome-wide transcriptional analysis shows that the p53-deficiency-induced neurons exhibit an expression profile different from parental fibroblasts and similar to control-induced neurons. The results may help to understand and improve neural conversion mechanisms to develop robust neuron-replacement therapy strategies.

  11. Cloned goats (Gapra hircus) from foetal fibroblast cell lines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mammalian cloning has been one of the most active research topics in the world.Cloning with in vitro culured foetal fibroblast cells,in comparison with embryonic cells,can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals,but also to utilize the unlimited fibroblast cells to produce large numbers of clonings.The preliminary results are as follows:(i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%,77% and 35%,31%,respectively.There is no significant statistical difference between them.(ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines,which are the first cloned mammals from somatic cells in China.This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development,and a novel technique for the cloning of animals with a high-level expression of transgene(s).

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

    Energy Technology Data Exchange (ETDEWEB)

    Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Nobe, Hiromi [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Department of Physical Therapy, Bunkyo-Gakuin University (Japan); Yoshida, Hiroko [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan); Kolodney, Michael S. [Dermatology Division, Department of Medicine, UCLA, Los Angeles, CA (United States); Paul, Richard J. [Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH (United States); Honda, Kazuo [Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo (Japan)

    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.

  13. Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells

    Institute of Scientific and Technical Information of China (English)

    Claudia Lange; Helge Bruns; Dietrich Kluth; Axel R Zander; Henning C Fiegel

    2006-01-01

    AIM: To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells.METHODS: MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF),hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin,and alpha-fetoprotein (AFP) was performed in different cell populations.RESULTS: Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18,and AFP-RNA over two weeks. At wk 3, MSCs lost hepatooytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential.CONCLUSION: The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also prorides a beneficial environment for expansion and differentiation of FLC.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2009-12-01

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

  16. Circadian-independent cell mitosis in immortalized fibroblasts.

    Science.gov (United States)

    Yeom, Mijung; Pendergast, Julie S; Ohmiya, Yoshihiro; Yamazaki, Shin

    2010-05-25

    Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (approximately 24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-four-hour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells.

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

    Science.gov (United States)

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

    2009-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  19. Application of Allogeneic Fibroblast Cells in Cellular Therapy of Recessive Dystrophic Epidermolysis Bullosa

    Directory of Open Access Journals (Sweden)

    Zare

    2015-09-01

    Full Text Available Context Connective tissue cells include fibroblasts, chondrocytes, adipocyte, and osteocytes. These cells are specialized for the secretion of collagenous extracellular matrix and are responsible for the architectural framework of the human body. Evidence Acquisition Connective tissue cells play a central role in supporting as well as repairing tissues and organs. Fibroblast cell therapy could be used for the treatment of burn wounds, scars, diabetic foot ulcers, acne scars and skin aging. This review focused on biology of fibroblasts and their role in cell therapy of recessive dystrophic epidermolysis bullosa (RDEB. Results Fibroblasts are known to play a pivotal role in skin structure and integrity, and dermal fibroblasts are believed to promote skin regeneration and rejuvenation via collagen production. Conclusions Fibroblasts can be used in transplantations to ameliorate an immune system response, in order to reduce antigen production. Human fibroblasts suppress ongoing mixed lymphocyte reactions (MLRs between lymphocyte cells from two individuals, and supernatant materials from fibroblast cultures suppress MLRs.

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

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

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

  1. Biologicals and Fetal Cell Therapy for Wound and Scar Management

    OpenAIRE

    Hirt-Burri, Nathalie; Ramelet, Albert-Adrien; Raffoul, Wassim; de Buys Roessingh, Anthony; Scaletta, Corinne; Pioletti, Dominique; Applegate, Lee Ann

    2011-01-01

    Few biopharmaceutical preparations developed from biologicals are available for tissue regeneration and scar management. When developing biological treatments with cellular therapy, selection of cell types and establishment of consistent cell banks are crucial steps in whole-cell bioprocessing. Various cell types have been used in treatment of wounds to reduce scar to date including autolog and allogenic skin cells, platelets, placenta, and amniotic extracts. Experience with fetal cells show ...

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

    Directory of Open Access Journals (Sweden)

    Hari H. P. Cohly

    2003-01-01

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

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

    DEFF Research Database (Denmark)

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

    1992-01-01

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

  4. Fetal Gender and Several Cytokines Are Associated with the Number of Fetal Cells in Maternal Blood - An Observational Study

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Kirkegaard, Ida; Petersen, Olav Bjørn

    2014-01-01

    and subsequent identification. RESULTS: Participants carrying male fetuses had a higher median number of fetal cells in maternal blood than those carrying female fetuses (5 vs. 3, p = 0.04). Certain cytokines (RANTES, IL-2 and IL-5) were significantly associated with the number of fetal cells in maternal blood...

  5. Expression of Connexin43 in Rat Epithelial Cells and Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy . And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EAgroup, n=10). EA ( 0.5-1.5 V, 4-16 Hz , 30 min) was applied to"Zusanli"acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected.The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91 % and 29.53 % respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

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

    Directory of Open Access Journals (Sweden)

    Weipeng Wang

    2015-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-11-28

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

  8. Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA).

    Science.gov (United States)

    de Leon, Priscila Marques Moura; Campos, Vinicius Farias; Dellagostin, Odir Antônio; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Collares, Tiago

    2012-02-01

    In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.

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

    Science.gov (United States)

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

    2016-06-01

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

  10. Fetal Cartilage-Derived Cells Have Stem Cell Properties and Are a Highly Potent Cell Source for Cartilage Regeneration.

    Science.gov (United States)

    Choi, Woo Hee; Kim, Hwal Ran; Lee, Su Jeong; Jeong, Nayoung; Park, So Ra; Choi, Byung Hyune; Min, Byoung-Hyun

    2016-01-01

    Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. However, the characteristics and the potential of progenitor cells from fetal tissue remain poorly defined. In this study, we examined cells from human fetal cartilage at 12 weeks after gestation in comparison with bone marrow-derived MSCs or cartilage chondrocytes from young donors (8-25 years old). The fetal cartilage-derived progenitor cells (FCPCs) showed higher yields by approximately 24 times than that of chondrocytes from young cartilage. The morphology of the FCPCs was polygonal at passage 0, being similar to that of the young chondrocytes, but it changed later at passage 5, assuming a fibroblastic shape more akin to that of MSCs. As the passages advanced, the FCPCs showed a much greater proliferation ability than the young chondrocytes and MSCs, with the doubling times ranging from 2∼4 days until passage 15. The surface marker profile of the FCPCs at passage 2 was quite similar to that of the MSCs, showing high expressions of CD29, CD90, CD105, and Stro-1. When compared to the young chondrocytes, the FCPCs showed much less staining of SA-β-gal, a senescence indicator, at passage 10 and no decrease in SOX9 expression until passage 5. They also showed a much greater chondrogenic potential than the young chondrocytes and the MSCs in a three-dimensional pellet culture in vitro and in polyglycolic acid (PGA) scaffolds in vivo. In addition, they could differentiate into adipogenic and osteogenic lineages as efficiently as MSCs in vitro. These results suggest that FCPCs have stem cell properties to some extent and that they are more active in terms of

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    Science.gov (United States)

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...

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

    Science.gov (United States)

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

    2011-08-01

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

  14. Are there fetal stem cells in the maternal brain?

    Institute of Scientific and Technical Information of China (English)

    Osman Demirhan; Necmi (C)ekin; Deniz Ta(s)temir; Erdal Tun(c); Ali irfan Güzel; Demet Meral; Bülent Demirbek

    2013-01-01

    Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother's brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby's stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-05-18

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

  17. Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

    Directory of Open Access Journals (Sweden)

    Kanako Miyabayashi

    Full Text Available Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.

  18. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Ci-Hang; Wang, Xin-Tong [Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan 250012 (China); Ma, Wei [Department of Radiation Oncology, Cancer Hospital, Genaral Hospital of Ningxia Medical University, Yinchuan 750000 (China); Wang, Na-Na; Nesa, Effat un; Wang, Jian-Bo; Wang, Cong; Jia, Yi-Bin; Wang, Kai [Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan 250012 (China); Tian, Hui [Department of Thoracic Surgery, Qilu Hospital of Shandong University, Jinan 250012 (China); Cheng, Yu-Feng, E-mail: qlcyf1965@126.com [Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan 250012 (China)

    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.

  20. Fetal liver stromal cells promote hematopoietic cell expansion

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Kun; Hu, Caihong [Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Zhou, Zhigang [Shanghai 1st People Hospital, Shanghai Jiao Tong University, Shanghai 201620 (China); Huang, Lifang; Liu, Wenli [Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Sun, Hanying, E-mail: shanhum@163.com [Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China)

    2009-09-25

    Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

  1. A FTIR imaging characterization of fibroblasts stimulated by various breast cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Saroj Kumar

    Full Text Available It is well known that the microenvironment plays a major role in breast cancer progression. Yet, the mechanism explaining the transition from normal fibroblasts to cancer-stimulated fibroblasts remains to be elucidated. Here we report a FTIR imaging study of the effects of three different breast cancer cell lines on normal fibroblasts in culture. Fibroblast activation process was monitored by FTIR imaging and spectra compared by multivariate statistical analyses. Principal component analysis evidenced that the fibroblasts stimulated by these cancer cell lines grouped together and remained distinctly separated from normal fibroblasts indicating a modified different chemical composition in the cancer-stimulated fibroblasts. Similar changes in fibroblasts were induced by the various breast cancer cell lines belonging to different sub-types. Most significant changes were observed in the region of 2950 and 1230 cm(-1, possibly related to changes in lipids and in the 1230 cm(-1 area assigned to phosphate vibrations (nucleotides. Interestingly, the cancer-cell induced changes in the fibroblasts also occurred when there was no possible direct contact between the two cell lines in the co-culture. When contact was possible, the spectral changes were similar, suggesting that soluble factors but not direct cell-cell interactions were responsible for fibroblast activation. Overall, the results indicate that IR imaging could be used in the future for analyzing the microenvironment of breast tumors.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

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

  4. Fetal liver hepatic progenitors are supportive stromal cells for hematopoietic stem cells.

    Science.gov (United States)

    Chou, Song; Lodish, Harvey F

    2010-04-27

    Previously we showed that the ~2% of fetal liver cells reactive with an anti-CD3epsilon monoclonal antibody support ex vivo expansion of both fetal liver and bone marrow hematopoietic stem cells (HSCs); these cells express two proteins important for HSC ex vivo expansion, IGF2, and angiopoietin-like 3. Here we show that these cells do not express any CD3 protein and are not T cells; rather, we purified these HSC-supportive stromal cells based on the surface phenotype of SCF(+)DLK(+). Competitive repopulating experiments show that SCF(+)DLK(+) cells support the maintenance of HSCs in ex vivo culture. These are the principal fetal liver cells that express not only angiopoietin-like 3 and IGF2, but also SCF and thrombopoietin, two other growth factors important for HSC expansion. They are also the principal fetal liver cells that express CXCL12, a factor required for HSC homing, and also alpha-fetoprotein (AFP), indicating that they are fetal hepatic stem or progenitor cells. Immunocytochemistry shows that >93% of the SCF(+) cells express DLK and Angptl3, and a portion of SCF(+) cells also expresses CXCL12. Thus SCF(+)DLK(+) cells are a highly homogenous population that express a complete set of factors for HSC expansion and are likely the primary stromal cells that support HSC expansion in the fetal liver.

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

    Science.gov (United States)

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

    2008-02-26

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

  6. Co-cultivation of pancreatic cancer cells with orthotopic tumor-derived fibroblasts: fibroblasts stimulate tumor cell invasion via HGF secretion whereas cancer cells exert a minor regulative effect on fibroblasts HGF production.

    Science.gov (United States)

    Qian, Li-Wu; Mizumoto, Kazuhiro; Maehara, Naoki; Ohuchida, Kenoki; Inadome, Naoki; Saimura, Michiyo; Nagai, Eishi; Matsumoto, Kunio; Nakamura, Toshikazu; Tanaka, Masao

    2003-02-10

    The intensive stromal reaction is one of characteristics of pancreatic exocrine carcinoma. The mutual interaction between pancreatic cancer cells and orthotopic tumor-derived fibroblasts have not been clarified yet. In this study, we sought to elucidate the mechanism underlying the tumor-stromal interaction with an in vitro coculture experimental system. Considerable strong c-Met expression was detected in seven out ten lines of human pancreatic carcinoma cells, as determined by Western blotting. For hepatocyte growth factor (HGF)-production, however, none or only trace amounts of HGF could be detected in those ten cell lines. Of the two lots of tumor-derived fibroblasts obtained from two pancreatic cancer patients, the fibroblasts capable to produce HGF could initiate an apparent invasion-stimulating response in strong c-Met-expressed Suit-2 and Panc-1 cells but not in faint expressed Mia PaCa-2 and BxPC-3 cells. A specialized HGF antagonist, NK4 would effectively inhibit the fibroblast-mediated invasive growth, thus proving the key role of the paracrine-fashioned HGF/c-Met pathway in the tumor-stromal interaction. On the other hand, the regulative action of cancer cells on HGF expression of fibroblasts was also investigated using direct or indirect coculture systems. For the fibroblasts that originally did not produce HGF, cancer cells failed to show any HGF-inductive effect. For the HGF-producing fibroblasts, despite of somewhat upregulation or downregulation in fibroblast HGF expression, the feedback regulation by studied pancreatic cancer cells in both coculture modes were relatively limited. This in vitro study sketched out the interaction between cancerous and stromal compartments with an emphasis on HGF/c-Met signal pathway, thus possibly helping to unveil the more complicated mutual modulation in vivo between pancreatic cancer and host mesenchymal tissues.

  7. Interactions between fibroblastic reticular cells and B cells promote mesenteric lymph node lymphangiogenesis.

    Science.gov (United States)

    Dubey, Lalit Kumar; Karempudi, Praneeth; Luther, Sanjiv A; Ludewig, Burkhard; Harris, Nicola L

    2017-08-28

    Lymphatic growth (lymphangiogenesis) within lymph nodes functions to promote dendritic cell entry and effector lymphocyte egress in response to infection or inflammation. Here we demonstrate a crucial role for lymphotoxin-beta receptor (LTβR) signaling to fibroblastic reticular cells (FRCs) by lymphotoxin-expressing B cells in driving mesenteric lymph node lymphangiogenesis following helminth infection. LTβR ligation on fibroblastic reticular cells leads to the production of B-cell-activating factor (BAFF), which synergized with interleukin-4 (IL-4) to promote the production of the lymphangiogenic factors, vascular endothelial growth factors (VEGF)-A and VEGF-C, by B cells. In addition, the BAFF-IL-4 synergy augments expression of lymphotoxin by antigen-activated B cells, promoting further B cell-fibroblastic reticular cell interactions. These results underlie the importance of lymphotoxin-dependent B cell-FRC cross talk in driving the expansion of lymphatic networks that function to promote and maintain immune responsiveness.The growth of lymph nodes in response to infection requires lymphangiogenesis. Dubey et al. show that the mesenteric lymph node lymphangiogenesis upon helminth infection depends on the signaling loop between the B and fibroblastic reticular cells (FRCs), whereby the FRCs respond to lymphotoxin secreted by B cells by releasing B cell activating factor.

  8. Fetal Research

    Science.gov (United States)

    Hansen, John T.; Sladek, John R.

    1989-11-01

    This article reviews some of the significant contributions of fetal research and fetal tissue research over the past 20 years. The benefits of fetal research include the development of vaccines, advances in prenatal diagnosis, detection of malformations, assessment of safe and effective medications, and the development of in utero surgical therapies. Fetal tissue research benefits vaccine development, assessment of risk factors and toxicity levels in drug production, development of cell lines, and provides a source of fetal cells for ongoing transplantation trials. Together, fetal research and fetal tissue research offer tremendous potential for the treatment of the fetus, neonate, and adult.

  9. Immature hematopoietic stem cells undergo maturation in the fetal liver.

    Science.gov (United States)

    Kieusseian, Aurelie; Brunet de la Grange, Philippe; Burlen-Defranoux, Odile; Godin, Isabelle; Cumano, Ana

    2012-10-01

    Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.

  10. Fetal microchimeric cells in blood of women with an autoimmune thyroid disease.

    Directory of Open Access Journals (Sweden)

    Trees Lepez

    Full Text Available CONTEXT: Hashimoto's thyroiditis (HT and Graves' disease (GD, two autoimmune thyroid diseases (AITD, occur more frequently in women than in men and show an increased incidence in the years following parturition. Persisting fetal cells could play a role in the development of these diseases. OBJECTIVE: Aim of this study was to detect and characterize fetal cells in blood of postpartum women with and without an AITD. PARTICIPANTS: Eleven patients with an AITD and ten healthy volunteers, all given birth to a son maximum 5 years before analysis, and three women who never had been pregnant, were included. None of them had any other disease of the thyroid which could interfere with the results obtained. METHODS: Fluorescence in situ hybridization (FISH and repeated FISH were used to count the number of male fetal cells. Furthermore, the fetal cells were further characterized. RESULTS: In patients with HT, 7 to 11 fetal cells per 1.000.000 maternal cells were detected, compared to 14 to 29 fetal cells in patients with GD (p=0.0061. In patients with HT, mainly fetal CD8(+ T cells were found, while in patients with GD, fetal B and CD4(+ T cells were detected. In healthy volunteers with son, 0 to 5 fetal cells were observed, which was significantly less than the number observed in patients (p<0,05. In women who never had been pregnant, no male cells were detected. CONCLUSION: This study shows a clear association between fetal microchimeric cells and autoimmune thyroid diseases.

  11. Schwann cell cultures from human fetal dorsal root ganglia

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  12. Gene expression during development of fetal and adult Leydig cells.

    Science.gov (United States)

    Dong, Lei; Jelinsky, Scott A; Finger, Joshua N; Johnston, Daniel S; Kopf, Gregory S; Sottas, Chantal M; Hardy, Matthew P; Ge, Ren-Shan

    2007-12-01

    In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

  13. Pluripotent male germline stem cells from goat fetal testis and their survival in mouse testis.

    Science.gov (United States)

    Hua, Jinlian; Zhu, Haijing; Pan, Shaohui; Liu, Chao; Sun, Junwei; Ma, Xiaoling; Dong, Wuzi; Liu, Weishuai; Li, Wei

    2011-04-01

    Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis, and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4, Sox2, C-myc, and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3, STRA8, and germ cell marker-VASA, and haploid marker-FE-J1, PRM1, indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However, most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice, and showed that mGSCs can colonize, self-renew, and differentiate into germ cells. Thus, we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro, providing a promising platform for generation of transgenic goat for production of specific humanized proteins.

  14. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

    Directory of Open Access Journals (Sweden)

    Aroem Naruni

    2016-01-01

    .Extracellular matrix provides tissue integrity, acts as a native scaffold for cell attachment and interaction and alsoserves as reservoir for growth factors. The aim of this experiment w asto achieve the deposition and decellularizationof ECM.Methods: Cells crowder have been developed to increase ECM deposit in the primary breast fibroblast cells layerobtained from isolation of single cell from breast mammoplasty specimen. Five hundred kDa dextran was addedinto DMEM medium containing 0.5% fetal bovine serum (FBS and 100 μm of L-ascorbic acid 2-phosphate. Afterseven days, cells were lysed by sodium deoxycholate (DOC. Results were observed in a fluorescence microscope.Results: Extracellular matrix deposition and decellularization of primary breast fibroblast cells were detected byusing extracellular matrix protein, fibronectin via rabbit anti human fibronectin and anti-rabbit IgG conjugated withAlexa Fluor 488.Conclusion: Dextran sulphate increased extracellular matrix deposit in primary breast fibroblast cell layer and thetreatment with sodium deoxycolate lysed cells resulted in extracellular matrix decellularization. (Health ScienceJournal of Indonesia 2015;6:43-7Keywords: extracellular matrix, breast cancer stem cell, breast fibroblast cell

  17. Fetal stem cells and skeletal muscle regeneration: a therapeutic approach

    Directory of Open Access Journals (Sweden)

    Michela ePozzobon

    2014-08-01

    Full Text Available More than 40% of the body mass is represented by muscle tissue, which possesses the innate ability to regenerate after damage through the activation of muscle specific stem cell, namely satellite cells. Muscle diseases, in particular chronic degenerative state of skeletal muscle such as dystrophies, lead to a perturbation of the regenerative process, which causes the premature exhaustion of satellite cell reservoir due to continue cycles of degeneration/regeneration. Nowadays, the research is focused on different therapeutic approaches, ranging from gene and cell to pharmacological therapy, but still there is not a definitive cure in particular for genetic muscle disease. Taking this in mind, in this article we will give special consideration to muscle diseases and the use of fetal derived stem cells as new approach for therapy. Cells of fetal origin, from cord blood to placenta and amniotic fluid, can be easily obtained without ethical concern, expanded and differentiated in culture, and possess immunemodulatory properties. The in vivo approach in animal models can be helpful to study the mechanism underneath the operating principle of the stem cell reservoir, namely the niche, which holds great potential to understand the onset of muscle pathologies.

  18. Uses of cell free fetal DNA in maternal circulation.

    Science.gov (United States)

    Hill, Melissa; Barrett, Angela N; White, Helen; Chitty, Lyn S

    2012-10-01

    For over a decade, researchers have focused their attention on the development of non-invasive prenatal diagnosis tests based on cell-free fetal DNA circulating in maternal blood. With the possibility of earlier and safer testing, non-invasive prenatal diagnosis has the potential to bring many positive benefits to prenatal diagnosis. Non-invasive prenatal diagnosis for fetal sex determination for women who are carriers of sex-linked conditions is now firmly established in clinical practice. Other non-invasive prenatal diagnosis-based tests are set to follow, as future applications, such as the detection of single-gene disorders and chromosomal abnormalities, are now well within reach. Here, we review recent developments in non-invasive prenatal diagnosis for genetic conditions and chromosomal abnormalities, and provide an overview of research into ethical concerns, social issues and stakeholder view points.

  19. The role of STAT1 for crosstalk between fibroblasts and colon cancer cells

    Directory of Open Access Journals (Sweden)

    Pawan eKaler

    2014-04-01

    Full Text Available Signaling between tumor cells and the associated stroma has an important impact on cancer initiation and progression. The tumor microenvironment has a paradoxical role in tumor progression and fibroblasts, a major component of the tumor stroma, have been shown to either inhibit or promote cancer development. In this study we established that normal intestinal fibroblasts activate STAT1 signaling in colon cancer cells and, in contrast to cancer- associated fibroblasts, inhibit growth of tumor cells. Treatment of 18Co fibroblasts with the proinflammatory cytokine TNF interfered with their ability to trigger STAT1 signaling in cancer cells. Accordingly, intestinal myofibroblasts isolated from patients with Ulcerative colitis (UC or Crohn’s disease (CD, which are activated and produce high levels of TNF, failed to stimulate STAT1 signaling in tumor cells, demonstrating that activated myofibroblasts lose the ability to trigger growth-inhibitory STAT1 signaling in tumor cells. Finally, we confirmed that silencing of STAT1 in tumor cells alters the crosstalk between tumor cells and fibroblasts, suggesting STAT1 as a novel link between intestinal inflammation and colon cancer. We demonstrated that normal fibroblasts restrain the growth of carcinoma cells, at least in part, through the induction of STAT1 signaling in cancer cells. We showed that changes in the microenvironment, as they occur in inflammatory bowel disease, alter the crosstalk between carcinoma cells and fibroblasts, perturb the homeostasis of intestinal tissue and thereby contribute to tumor progression.

  20. Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells.

    Science.gov (United States)

    Kilcoyne, Karen R; Smith, Lee B; Atanassova, Nina; Macpherson, Sheila; McKinnell, Chris; van den Driesche, Sander; Jobling, Matthew S; Chambers, Thomas J G; De Gendt, Karel; Verhoeven, Guido; O'Hara, Laura; Platts, Sophie; Renato de Franca, Luiz; Lara, Nathália L M; Anderson, Richard A; Sharpe, Richard M

    2014-05-06

    Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

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

    Directory of Open Access Journals (Sweden)

    Rong Lu

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

  2. Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues.

    Science.gov (United States)

    Hackett, Catherine H; Greve, Line; Novakofski, Kira D; Fortier, Lisa A

    2012-07-01

    Cellular pluripotency is associated with expression of the homeobox transcription factor genes NANOG, SOX2, and POU5F1 (OCT3/4 protein). Some reports suggest that mesenchymal progenitor cells (MPCs) may express increased quantities of these genes, creating the possibility that MPCs are more "pluripotent" than other adult cell types. The objective of this study was to determine whether equine bone marrow-derived MPCs had gene expression or DNA methylation patterns that differed from either early fetal-derived or terminally differentiated adult cells. Specifically, this study compared DNA methylation of the NANOG and SOX2 promoter regions and concurrent gene expression of NANOG, SOX2, and POU5F1 in equine induced pluripotent stem (iPS) cells, fetal fibroblasts, fetal brain cells, adult chondrocytes, and MPCs. Results indicate that NANOG and POU5F1 were not detectable in appreciable quantities in tissues other than the equine iPS cell lines. Equine iPS cells expressed large quantities of all three genes examined. Significantly increased quantities of SOX2 were noted in iPS cells and both fetal-derived cell types compared with adult cells. MPCs and adult chondrocytes expressed equivalent, low quantities of SOX2. Further, NANOG and SOX2 expression inversely correlated with the DNA methylation pattern in the promoter region, such that as gene expression increased, DNA methylation decreased. The equine iPS cell lines examined demonstrated DNA methylation and gene expression patterns that were consistent with pluripotency features described in other species. Results do not support previous reports that NANOG, SOX2, and POU5F1 are poised for increased activity in MPCs compared with other adult cells.

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

    Science.gov (United States)

    Guillot, Pascale V

    2016-02-01

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

  4. Cell surface carbohydrate changes during embryonic and fetal skin development

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Holbrook, K; Clausen, H

    1986-01-01

    Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N...... expressed at the early stages of development, but may later be modified either by sialylation or fucosylation into blood group H or Lex, or by Ley substances, respectively. The orderly and well-defined changes observed during skin differentiation are in agreement with other studies, which have demonstrated...... and granular cells in the epithelium. Lex stained both basal cells and intermediate cells positively, until keratinization around week 20 EGA. Ley is never expressed on basal cells. It is weakly expressed by intermediate cells from week 14 EGA. Our study demonstrates that N-acetyllactosamine is maximally...

  5. Fetal hemoglobin in sickle cell anemia: a glass half full?

    Science.gov (United States)

    Steinberg, Martin H; Chui, David H K; Dover, George J; Sebastiani, Paola; Alsultan, Abdulrahman

    2014-01-23

    Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia by inhibiting deoxy sickle hemoglobin (HbS) polymerization. The blood concentration of HbF, or the number of cells with detectable HbF (F-cells), does not measure the amount of HbF/F-cell. Even patients with high HbF can have severe disease because HbF is unevenly distributed among F-cells, and some cells might have insufficient concentrations to inhibit HbS polymerization. With mean HbF levels of 5%, 10%, 20%, and 30%, the distribution of HbF/F-cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells can have polymer-inhibiting, or protective, levels of HbF of ∼10 pg; with lower HbF, few or no protected cells can be present. Only when the total HbF concentration is near 30% is it possible for the number of protected cells to approach 70%. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have enough HbF to thwart HbS polymerization is the most critical predictor of the likelihood of severe sickle cell disease.

  6. Two-colour immunocytochemical staining of gamma (gamma) and epsilon (epsilon) type haemoglobin in fetal red cells

    NARCIS (Netherlands)

    Mesker, WE; Velzen, MCMOV; Oosterwijk, JC; Bernini, LF; Golbus, MS; Kanhai, HHH; Van Ommen, GJB; Tanke, HJ

    1998-01-01

    We have developed a two-colour immunocytochemical staining method for the detection of fetal and embryonic haemoglobin in erythroid cells. The method was applied to study these haemoglobin types in fetal red cells. Specimens from fetal blood (10 weeks), cord blood and fetal liver (14 weeks) as well

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

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

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

  8. Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

    Science.gov (United States)

    Fishman, V S; Shnayder, T A; Orishchenko, K E; Bader, M; Alenina, N; Serov, O L

    2015-01-01

    Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

    Itoi, M; Amagai, T

    1998-01-10

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

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

    Science.gov (United States)

    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.

  12. Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner.

    Science.gov (United States)

    Kruglova, Anna A; Matveeva, Natalia M; Gridina, Maria M; Battulin, Nariman R; Karpov, Anton; Kiseleva, Elena V; Morozova, Ksenia N; Serov, Oleg L

    2010-06-01

    Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

  13. Fetal hemoglobin accumulation in vitro. Effect of adherent mononuclear cells.

    OpenAIRE

    Javid, J; Pettis, P K

    1983-01-01

    In clonal cultures of erythroid burst-forming units (BFU-E) obtained from blood, the accumulation of fetal and adult hemoglobins (Hb F and Hb A) was measured by radioligand immunoassay. Inclusion of adherent mononuclear cells in the culture promoted a striking increase in the relative amount of Hb F in each of 44 experiments with 14 donors. In two-thirds of the instances, this was accounted for by a selective increase in the absolute amount of Hb F. The differential effect on Hb F and Hb A ac...

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

    Science.gov (United States)

    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.

  15. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow

    DEFF Research Database (Denmark)

    Potocnik, A J; Brakebusch, C; Fässler, R

    2000-01-01

    Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that beta1 integrin-deficient HSCs from the para-aortic splanchnopleura and the fetal blood had...... hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult beta1 integrin null HSCs isolated from mice carrying loxP-tagged beta1 integrin alleles and ablated for beta1 integrin expression by retroviral cre transduction...... failed to engraft irradiated recipient mice. Moreover, absence of beta1 integrin resulted in sequestration of HSCs in the circulation and their reduced adhesion to endothelioma cells. These findings define beta1 integrin as an essential adhesion receptor for the homing of HSCs....

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Vandekerckhove, J; Bauw, G; Vancompernolle, K

    1990-01-01

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

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

    Science.gov (United States)

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

    2011-09-15

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

  19. Cell-free fetal DNA in maternal plasma and noninvasive prenatal diagnosis

    OpenAIRE

    Ester Silveira Ramos

    2006-01-01

    The noninvasive nature of the detection of fetal DNA in the maternal circulation represents the greatest advantage over the conventional methods of prenatal diagnosis. The applications of this methodology involve the detection of the fetal sex, and diagnosis, intra-uterine treatment, and evaluation of the prognosis of many diseases. Fetal cells detected in the maternal circulation have also been shown to be implicated in autoimmune diseases and to represent a potential source of stem cells. O...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-10-07

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

  1. Mitochondrial Oxidative Stress due to Complex I Dysfunction Promotes Fibroblast Activation and Melanoma Cell Invasiveness

    Directory of Open Access Journals (Sweden)

    Maria Letizia Taddei

    2012-01-01

    Full Text Available Increased ROS (cellular reactive oxygen species are characteristic of both fibrosis and tumour development. ROS induce the trans-differentiation to myofibroblasts, the activated form of fibroblasts able to promote cancer progression. Here, we report the role of ROS produced in response to dysfunctions of mitochondrial complex I, in fibroblast activation and in tumour progression. We studied human fibroblasts with mitochondrial dysfunctions of complex I, leading to hyperproduction of ROS. We demonstrated that ROS level produced by the mutated fibroblasts correlates with their activation. The increase of ROS in these cells provides a greater ability to remodel the extracellular matrix leading to an increased motility and invasiveness. Furthermore, we evidentiated that in hypoxic conditions these fibroblasts cause HIF-1α stabilization and promote a proinvasive phenotype of human melanoma cells through secretion of cytokines. These data suggest a possible role of deregulated mitochondrial ROS production in fibrosis evolution as well as in cancer progression and invasion.

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

    Institute of Scientific and Technical Information of China (English)

    Yao-wen WANG; Ji-hao REN; Kun XIA; Shu-hui WANG; Tuan-fang YIN; Ding-hua XIE; Li-hua LI

    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.

  3. Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells.

    Science.gov (United States)

    Ishii, Takamichi; Yasuchika, Kentaro; Ikai, Iwao

    2013-01-01

    Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45(-)CD49f(+/-)Thy1(+)gp38(+) mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell-cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity.

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

    Science.gov (United States)

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

    2014-10-01

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

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

    Science.gov (United States)

    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).

  6. Renal fibroblast-like cells in Goodpasture syndrome rats.

    Science.gov (United States)

    Okada, H; Inoue, T; Kanno, Y; Kobayashi, T; Ban, S; Kalluri, R; Suzuki, H

    2001-08-01

    The extent of renal fibrosis is the best predictor for functional outcomes in a variety of progressive renal diseases. Interstitial fibroblast-like cells (FbLCs) are presumably involved in the fibrotic process. However, such FbLCs have never been well characterized in the kidney. We characterized renal FbLCs in the nephritic kidney (in which the number of FbLCs and extracellular matrix accumulation were significantly increased) with regards to their expression of phenotypic and functional markers using day 49 Goodpasture syndrome (GPS) rats. Within the renal cortical interstitium, there were a number of alpha-smooth muscle actin(+) (alpha-SMA(+)) FbLCs, negative for vimentin (VIM) and transforming growth factor-beta 1, and not equipped with well-developed rough endoplasmic reticulum and actin-stress fibers. All of these findings were incompatible with the typical features of granulation tissue alpha-SMA(+) myofibroblasts. On the other hand, FbLCs negative for alpha-SMA and VIM produced alpha1(I) procollagen in the nephritic kidney. A number of FbLC populations reside within the cortical interstitium of the kidney in GPS rats, each of which is likely to have developed independently in response to the local conditions of the nephritic kidney, contributing to renal fibrogenesis. Further studies are needed to clarify the key type of FbLC that orchestrates other members to produce renal fibrosis.

  7. 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

    Energy Technology Data Exchange (ETDEWEB)

    Fuchigami, Takao [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kibe, Toshiro [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Nishizawa, Yoshiaki [Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Hijioka, Hiroshi; Fujii, Tomomi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ueda, Masahiro [Natural Science Centre for Research and Education, Kagoshima University, 1-21-24 Koorimoto, Kagoshima 890-8580 (Japan); Nakamura, Norifumi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kiyono, Tohru [Department of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuouku, Tokyo 104-0045 (Japan); Kishida, Michiko, E-mail: kmichiko@m2.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    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

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

    Science.gov (United States)

    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

  9. Continual cell deformation induced via attachment to oriented fibers enhances fibroblast cell migration.

    Directory of Open Access Journals (Sweden)

    Sisi Qin

    Full Text Available 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.

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

    Science.gov (United States)

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

    2011-01-01

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

  11. Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver

    Science.gov (United States)

    Orelio, Claudia; Peeters, Marian; Haak, Esther; van der Horn, Karin; Dzierzak, Elaine

    2009-01-01

    Background Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. Design and Methods We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1−/− embryos. Results We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1−/− embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. Conclusions The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells. PMID

  12. Cadherin-9 is a novel cell surface marker for the heterogeneous pool of renal fibroblasts.

    Directory of Open Access Journals (Sweden)

    Cornelia Thedieck

    Full Text Available BACKGROUND: Interstitial fibroblasts are a minor, but nevertheless very important, component of the kidney. They secrete and remodel extracellular matrix and they produce active compounds such as erythropoietin. However, studying human renal fibroblasts has been hampered by the lack of appropriate surface markers. METHODS AND FINDINGS: The expression of cadherin-9 in various human renal cell lines and tissues was studied on the mRNA level by RT-PCR and on the protein level with the help of newly generated cadherin-9 antibodies. The classical type II cadherin-9, so far only described in the neural system, was identified as a reliable surface marker for renal fibroblasts. Compared to FSP1, a widely-used cytosolic renal fibroblast marker, cadherin-9 showed a more restricted expression pattern in human kidney. Under pathological conditions, cadherin-9 was expressed in the stroma of renal cell carcinoma, but not in the tumor cells themselves, and in renal fibrosis the percentage of cadherin-9-positive cells was clearly elevated 3 to 5 times compared to healthy kidney tissue. Induction of epithelial mesenchymal transition in renal epithelial cells with cyclosporin-A, which causes renal fibrosis as a side effect, induced cadherin-9 expression. Functional studies following siRNA-mediated knockdown of cadherin-9 revealed that it acts in the kidney like a typical classical cadherin. It was found to be associated with catenins and to mediate homophilic but not heterophilic cell interactions. CONCLUSIONS: Cadherin-9 represents a novel and reliable cell surface marker for fibroblasts in healthy and diseased kidneys. Together with the established marker molecules FSP1, CD45 and alpha smooth muscle actin, cadherin-9 can now be used to differentiate the heterogenic pool of renal fibroblasts into resident and activated fibroblasts, immigrated bone marrow derived fibroblast precursors and cells in different stages of epithelial mesenchymal transition.

  13. Fetal microchimeric cells in autoimmune thyroid diseases: harmful, beneficial or innocent for the thyroid gland?

    Science.gov (United States)

    Lepez, Trees; Vandewoestyne, Mado; Deforce, Dieter

    2013-01-01

    Autoimmune thyroid diseases (AITD) show a female predominance, with an increased incidence in the years following parturition. Fetal microchimerism has been suggested to play a role in the pathogenesis of AITD. However, only the presence of fetal microchimeric cells in blood and in the thyroid gland of these patients has been proven, but not an actual active role in AITD. Is fetal microchimerism harmful for the thyroid gland by initiating a Graft versus Host reaction (GvHR) or being the target of a Host versus Graft reaction (HvGR)? Is fetal microchimerism beneficial for the thyroid gland by being a part of tissue repair or are fetal cells just innocent bystanders in the process of autoimmunity? This review explores every hypothesis concerning the role of fetal microchimerism in AITD.

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

    Directory of Open Access Journals (Sweden)

    L. V. Didenko

    2015-01-01

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

  15. Development and trafficking function of haematopoietic stem cells and myeloid cells during fetal ontogeny

    NARCIS (Netherlands)

    Heinig, Kristina; Sage, Fanny; Robin, Catherine; Sperandio, Markus

    2015-01-01

    Fetal haematopoiesis is a highly regulated process in terms of time and location. It is characterized by the emergence of specific cell populations at different extra-and intraembryonic anatomical sites. Trafficking of haematopoietic stem cells (HSCs) between these supportive niches is regulated by

  16. Development and trafficking function of haematopoietic stem cells and myeloid cells during fetal ontogeny

    NARCIS (Netherlands)

    Heinig, Kristina; Sage, Fanny; Robin, Catherine; Sperandio, Markus

    2015-01-01

    Fetal haematopoiesis is a highly regulated process in terms of time and location. It is characterized by the emergence of specific cell populations at different extra- and intraembryonic anatomical sites. Trafficking of haematopoietic stem cells (HSCs) between these supportive niches is regulated by

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

    Science.gov (United States)

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

    2017-04-20

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

  18. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

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

    Energy Technology Data Exchange (ETDEWEB)

    Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

    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

  20. Purification and characterization of fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA)

    DEFF Research Database (Denmark)

    Hokland, P; Rosenthal, P; Griffin, J D

    1983-01-01

    Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual...... antigen. Furthermore, using methanol-fixed cells, it could be shown that approximately 20% contained intracytoplasmic mu chains (cyto-mu) and that approximately 15% were positive for the terminal transferase enzyme (TdT) marker. The CALLA+ fetal cells thus closely resemble the childhood acute...... that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells. Udgivelsesdato: 1983-Jan-1...

  1. Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide.

    Directory of Open Access Journals (Sweden)

    Stefanie Siegert

    Full Text Available Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/- mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation.

  2. Transplantation of mouse fetal liver cells for analyzing the function of hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Gudmundsson, Kristbjorn Orri; Stull, Steven W; Keller, Jonathan R

    2012-01-01

    Hematopoietic stem cells are defined by their ability to self-renew and differentiate through progenitor cell stages into all types of mature blood cells. Gene-targeting studies in mice have demonstrated that many genes are essential for the generation and function of hematopoietic stem and progenitor cells. For definitively analyzing the function of these cells, transplantation studies have to be performed. In this chapter, we describe methods to isolate and transplant fetal liver cells as well as how to analyze donor cell reconstitution. This protocol is tailored toward mouse models where embryonic lethality precludes analysis of adult hematopoiesis or where it is suspected that the function of fetal liver hematopoietic stem and progenitor cells is compromised.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2013-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Hidetatsu Outani

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

  6. Fetal cell microchimerism develops through the migration of fetus-derived cells to the maternal organs early after implantation.

    Science.gov (United States)

    Sunami, Rei; Komuro, Mayuko; Yuminamochi, Tsutomu; Hoshi, Kazuhiko; Hirata, Shuji

    2010-03-01

    Fetus-derived cells are present in the blood and tissues of the maternal body over a long period of time, even after delivery, resulting in fetal cell microchimerism. The exact process by which fetal cells cross the placental barrier to enter the maternal circulation is unclear. The objective of this paper was to determine the time during pregnancy that fetal cells with multilineage potential migrate to the maternal organs. Wild type female mice were crossbred with male transgenic mice, expressing enhanced green fluorescent protein (EGFP). Total hysterectomies were performed at different time points of pregnancy. On day 60 after surgery, mice were injected with either streptozotocin (STZ) to induce insulin-dependent diabetes mellitus, or vehicle. Detection and quantification of fetal cells were then undertaken in a variety of maternal organs via fluorescent microscopy and quantitative PCR amplification of the gfp transgene. In vehicle control mice, fetal cells were detected only in the maternal bone marrow. However on day 30 after STZ injection, fetal cells were detected not only in bone marrow but also in the maternal pancreas, liver and kidney. Histological analysis showed differentiated fetal cells within the pancreatic acinar cells, hepatocytes and tubular epithelial cells. Their morphological appearance was indistinguishable from their maternal counterparts, and their frequency in these organs was constant, regardless of the timing of hysterectomy. These results indicate that most fetal cells with multilineage potential in maternal tissues migrate to the maternal body early after implantation, and thereafter sustain their population over the long term after delivery.

  7. Sex-specific differences in fetal germ cell apoptosis induced by ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Guerquin, M.J.; Duquenne, C.; Coffigny, H.; Rouiller-Fabre, V.; Lambrot, R.; Habert, R.; Livera, G. [CEA, DSV/DRR/SEGG/LDRG, Laboratory of Differentiation and Radiobiology of the Gonads, Unit of Gametogenesis and Genotoxicity, F-92265 Fontenay aux Roses (France); Guerquin, M.J.; Duquenne, C.; Coffigny, H.; Rouiller-Fabre, V.; Lambrot, R.; Habert, R.; Livera, G. [Univ. Paris 7-Denis Diderot, UFR of Biology, UMR-S 566, F-92265 Fontenay aux Roses (France); Guerquin, M.J.; Duquenne, C.; Coffigny, H.; Rouiller-Fabre, V.; Lambrot, R.; Habert, R.; Livera, G. [INSERM, U566, F-92265 Fontenay aux Roses (France); Bakalska, M. [Institute of Experimental Morphology and Anthropology, Bulgarian Academy of Sciences, Sofia (Bulgaria); Frydman, R. [Univ Paris-Sud, Clamart F-92140 (France); Frydman, R. [AP-HP, Service de Gynecologie-Obstetrique et Medecine de la Reproduction, Hopital Antoine Beclere, Clamart F-92141 (France); Frydman, R. [INSERM, U782, Clamart F-92140 (France)

    2009-07-01

    Background: We have previously shown that male human fetal germ cells are highly radiosensitive and that their death depends on p53 activation. Male germ cell apoptosis was initiated with doses as low as 0.1 Gy and was prevented by pifithrin {alpha}, a p53 inhibitor. In this study, we investigated the radiosensitivity of early female and male fetal proliferating germ cells. Methods and results: Both male and female fetal germ cells displayed a similar number of {gamma}H2AX foci in response to ionizing radiation (IR). In organ culture of human fetal ovaries, the germ cells underwent apoptosis only when exposed to high doses of IR (1.5 Gy and above). Accumulation of p53 was detected in irradiated male human fetal germ cells but not in female ones. Inhibition of p53 with pifithrin {alpha} did not affect oogonia apoptosis following irradiation. IR induced apoptosis similarly in mouse fetal ovaries in organ culture and in vivo during oogonial proliferation. Germ cell survival in testes from p53 knockout or p63 knockout mice exposed to IR was better than wild-type, whereas female germ cell survival was unaffected by p53 or p63 knockout. Conclusions: These findings show that pre-meiotic male and female fetal germ cells behave differently in response to a genotoxic stress-irradiation with oogonia being less sensitive and undergoing p53-independent apoptosis. (authors)

  8. Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    DEFF Research Database (Denmark)

    Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

    2017-01-01

    Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the o......Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming...... and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig....

  9. Purification and characterization of fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA)

    DEFF Research Database (Denmark)

    Hokland, P; Rosenthal, P; Griffin, J D

    1983-01-01

    Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual...... lymphoblastic leukemia cell with respect to surface marker phenotype. A population of CALLA- cells devoid of mature erythroid and myeloid surface markers was found to contain higher numbers of TdT+ cells but lower numbers of cyto-mu, B1, and Ia+ cells than the CALLA+ subset. In vitro analysis of normal...... antigen. Furthermore, using methanol-fixed cells, it could be shown that approximately 20% contained intracytoplasmic mu chains (cyto-mu) and that approximately 15% were positive for the terminal transferase enzyme (TdT) marker. The CALLA+ fetal cells thus closely resemble the childhood acute...

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

    Directory of Open Access Journals (Sweden)

    Sascha Schäuble

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

  11. Deletion of Calponin 2 in Mouse Fibroblasts Increases Myosin II-Dependent Cell Traction Force.

    Science.gov (United States)

    Hossain, M Moazzem; Zhao, Guangyi; Woo, Moon-Sook; Wang, James H-C; Jin, Jian-Ping

    2016-11-01

    Cell traction force (CTF) plays a critical role in controlling cell shape, permitting cell motility, and maintaining cellular homeostasis in many biological processes such as angiogenesis, development, wound healing, and cancer metastasis. Calponin is an actin filament-associated cytoskeletal protein in smooth muscles and multiple types of non-muscle cells. An established biochemical function of calponin is the inhibition of myosin ATPase in smooth muscle cells. Vertebrates have three calponin isoforms. Among them, calponin 2 is expressed in epithelial cells, endothelial cells, macrophages, myoblasts, and fibroblasts and plays a role in regulating cytoskeleton activities such as cell adhesion, migration, and cytokinesis. Knockout (KO) of the gene encoding calponin 2 (Cnn2) in mice increased cell motility, suggesting a function of calponin 2 in modulating CTF. In this study, we examined fibroblasts isolated from Cnn2 KO and wild-type (WT) mice using CTF microscopy. Primary mouse fibroblasts were cultured on polyacrylamide gel substrates embedded with fluorescent beads to measure root-mean-square traction, total strain energy, and net contractile movement. The results showed that calponin 2-null fibroblasts exhibit traction force greater than that of WT cells. Adherent calponin 2-null fibroblasts de-adhered faster than the WT control during mild trypsin treatment, consistent with an increased CTF. Blebbistatin, an inhibitor of myosin II ATPase, is more effective upon an alteration in cell morphology when calponin 2 is present in WT fibroblasts than that on Cnn2 KO cells, indicating their additive effects in inhibiting myosin motor activity. The novel finding that calponin 2 regulates myosin-dependent CTF in non-muscle cells demonstrates a mechanism for controlling cell motility-based functions.

  12. Lin28b Regulates Fetal Regulatory T Cell Differentiation through Modulation of TGF-β Signaling.

    Science.gov (United States)

    Bronevetsky, Yelena; Burt, Trevor D; McCune, Joseph M

    2016-12-01

    Immune tolerance between the fetus and mother represents an active process by which the developing fetus must not mount immune responses to noninherited Ags on chimeric maternal cells that reside in fetal tissue. This is, in part, mediated by the suppressive influence of CD4(+)FOXP3(+)CD25(+) regulatory T cells (Tregs). Fetal secondary lymphoid organs have an increased frequency of Tregs and, as compared with adult T cells, fetal naive CD4(+) T cells exhibit a strong predisposition to differentiate into Tregs when stimulated. This effect is mediated by the TCR and TGF-β pathways, and fetal T cells show significantly increased Treg differentiation in response to anti-CD3 and TGF-β stimulation. Naive fetal T cells also exhibit increased signaling through the TGF-β pathway, with these cells demonstrating increased expression of the signaling mediators TGF-βRI, TGF-βRIII, and SMAD2, and higher levels of SMAD2/SMAD3 phosphorylation. Increased fetal Treg differentiation is mediated by the RNA-binding protein Lin28b, which is overexpressed in fetal T cells as compared with adult cells. When Lin28b expression is decreased in naive fetal T cells, they exhibit decreased Treg differentiation that is associated with decreased TGF-β signaling and lowered expression of TGF-βRI, TGF-βRIII, and SMAD2. Lin28b regulates the maturation of let-7 microRNAs, and these TGF-β signaling mediators are let-7 targets. We hypothesize that loss of Lin28b expression in fetal T cells leads to increased mature let-7, which causes decreased expression of TGF-βRI, TGF-βRIII, and SMAD2 proteins. A reduction in TGF-β signaling leads to reduced Treg numbers. Copyright © 2016 by The American Association of Immunologists, Inc.

  13. Interaction of cambial dermal cells (fibroblasts) and epidermis in morphofunctional area of mouse skin.

    Science.gov (United States)

    Yavisheva, T M; Shcherbakov, S D; Golubeva, I S; Sharafutdinov, G Z

    2007-11-01

    Epidermis and dermis form an integral morphofunctional area, where cambial cells proliferate and their first-division daughter cells differentiate. An important feature of this area is different rates of the development of daughter cells (fibroblasts) in the dermis and epidermis, which is greater in the epidermis. This asymmetry results in the prevalence of first epidermal daughter cells and, hence, their effect on cambial cells, and then of stromal daughter cells and their effects on cambial cells. The regulator factors of epidermal daughter cells promote unblocking of the major polarity axis of cambial (mother) cell, while stromal cells (fibroblasts) induce their polarization along the major axis and the onset of mitosis. In the dermis and epidermis, division of cambial cells is asymmetric; a prominent role in the formation of mother and daughter cells is given to the basal membrane as an elastic support. Mother and daughter cells form ring-like structures generating electric field that can promote differentiation of the daughter cells.

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

    Science.gov (United States)

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

    2016-01-01

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

  15. Cell-Free RNA Is a Reliable Fetoplacental Marker in Noninvasive Fetal Sex Determination

    NARCIS (Netherlands)

    Mersy, E.; Faas, B.H.W.; Spierts, S.; Houben, L.M.; Macville, M.V.; Frints, S.G.; Paulussen, A.D.; Veltman, J.A.

    2015-01-01

    BACKGROUND: Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally

  16. Cell-Free RNA Is a Reliable Fetoplacental Marker in Noninvasive Fetal Sex Determination

    NARCIS (Netherlands)

    Mersy, E.; Faas, B.H.W.; Spierts, S.; Houben, L.M.; Macville, M.V.; Frints, S.G.; Paulussen, A.D.; Veltman, J.A.

    2015-01-01

    BACKGROUND: Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally

  17. Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma

    NARCIS (Netherlands)

    M.G.H.M. Grootkerk-Tax; A.A. Soussan; M. de Haas; P.A. Maaskant-van Wijk; C.E. van der Schoot

    2006-01-01

    BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHD psi mothers and that RHD psi fetuses are correctly typed. Owing to

  18. Fibroblast gene expression profile reflects the stage of tumour progression in oral squamous cell carcinoma.

    Science.gov (United States)

    Lim, Kue Peng; Cirillo, Nicola; Hassona, Yazan; Wei, Wenbin; Thurlow, Johanna K; Cheong, Sok Ching; Pitiyage, Gayani; Parkinson, E Ken; Prime, Stephen S

    2011-03-01

    Oral cancer is a highly aggressive malignancy with poor prognosis. This study examined the behaviour of fibroblast strains from normal oral mucosa, dysplastic epithelial tissue, and genetically stable (minimal copy number alterations-CNA; minimal loss of heterozygosity-LOH; wild-type p53; wild-type p16INK4A) and unstable (extensive CNA and LOH; inactivation of p53 and p16INK4A) oral squamous cell carcinoma (OSCC). Fibroblasts from genetically unstable OSCC relative to the other fibroblast subtypes grew more slowly and stimulated the invasion of a non-tumourigenic keratinocyte cell line into fibroblast-rich collagen gels. To understand these findings, genome-wide transcriptional profiles were generated using the GeneChip(®) cDNA whole transcript microarray platform. Principal component analysis showed that the fibroblasts could be distinguished according to the stage of tumour development. Tumour progression was associated with down-regulation of cell cycle- and cytokinesis-related genes and up-regulation of genes encoding transmembrane proteins including cell adhesion molecules. Gene expression was validated in independent fibroblast strains using qRT-PCR. Gene connectivity and interactome-transcriptome associations were determined using a systems biology approach to interrogate the gene expression data. Clusters of gene signatures were identified that characterized genetically unstable and stable OSCCs relative to each other and to fibroblasts from normal oral mucosa. The expression of highly connected genes associated with unstable OSCCs, including those that encode α-SMA and the integrin α6, correlated with poor patient prognosis in an independent dataset of head and neck cancer. The results of this study demonstrate that fibroblasts from unstable OSCCs represent a phenotypically distinguishable subset that plays a major role in oral cancer biology. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

    Science.gov (United States)

    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  20. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    Science.gov (United States)

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

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

    Directory of Open Access Journals (Sweden)

    Won-Gyu Bae

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Takayuki Ueno

    2015-01-01

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

  3. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1

    NARCIS (Netherlands)

    de Haan, G; Weersing, E; Dontje, B; van Os, R; Bystrykh, LV; Vellenga, E; Miller, G

    2003-01-01

    The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed pr

  4. Physical exercise decreases the number of fetal cells in maternal blood

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Kirkegaard, Ida; Christensen, Connie Britta;

    liability company). Fetal cells in the blood, bound to fetal cell specific markers, were initially isolated by magnetic cell sorting, then stained with a cocktail of intracellular antibodies, identified and counted. Information about 6 variables reflecting the physical activity of the participants......Physical exercise decreases the number of fetal cells in maternal blood J. M. Schlütter1, I. Kirkegaard1, B. Christensen2, S. Kølvraa3, N. Uldbjerg1 1. Department of Gynecology and Obstetrics, Aarhus University Hospital, Skejby, Aarhus N, Denmark. 2. FCMB ApS, Vejle, Denmark. 3. Department...... of Clinical Genetics, Vejle Hospital, Vejle, Denmark Objectives We have established a robust method to specifically identify and isolate a subgroup of fetal cells in maternal blood (fcmb) at a gestational age of 12 weeks. The concentration of these cells, however, varies considerably among pregnant women...

  5. Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways.

    Science.gov (United States)

    Götherström, Cecilia; Lundqvist, Andreas; Duprez, Ida Rasmusson; Childs, Richard; Berg, Louise; le Blanc, Katarina

    2011-03-01

    Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties. We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γ. Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC. Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.

  6. Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta.

    Science.gov (United States)

    Sardesai, Varda S; Shafiee, Abbas; Fisk, Nicholas M; Pelekanos, Rebecca A

    2017-04-01

    Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender-discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi-derived MSC (CV-MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV-MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070-1084.

  7. FIP200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells.

    Science.gov (United States)

    Liu, Fei; Lee, Jae Y; Wei, Huijun; Tanabe, Osamu; Engel, James D; Morrison, Sean J; Guan, Jun-Lin

    2010-12-02

    Little is known about whether autophagic mechanisms are active in hematopoietic stem cells (HSCs) or how they are regulated. FIP200 (200-kDa FAK-family interacting protein) plays important roles in mammalian autophagy and other cellular functions, but its role in hematopoietic cells has not been examined. Here we show that conditional deletion of FIP200 in hematopoietic cells leads to perinatal lethality and severe anemia. FIP200 was cell-autonomously required for the maintenance and function of fetal HSCs. FIP200-deficient HSC were unable to reconstitute lethally irradiated recipients. FIP200 ablation did not result in increased HSC apoptosis, but it did increase the rate of HSC proliferation. Consistent with an essential role for FIP200 in autophagy, FIP200-null fetal HSCs exhibited both increased mitochondrial mass and reactive oxygen species. These data identify FIP200 as a key intrinsic regulator of fetal HSCs and implicate a potential role for autophagy in the maintenance of fetal hematopoiesis and HSCs.

  8. Isolation, in vitro culture and identification of a new type of mesenchymal stem cell derived from fetal bovine lung tissues.

    Science.gov (United States)

    Hu, Pengfei; Pu, Yabin; Li, Xiayun; Zhu, Zhiqiang; Zhao, Yuhua; Guan, Weijun; Ma, Yuehui

    2015-09-01

    Lung‑derived mesenchymal stem cells (LMSCs) are considered to be important in lung tissue repair and regenerative processes. However, the biological characteristics and differentiation potential of LMSCs remain to be elucidated. In the present study, fetal lung‑derived mesenchymal stem cells (FLMSCs) were isolated from fetal bovine lung tissues by collagenase digestion. The in vitro culture conditions were optimized and stabilized and the self‑renewal ability and differentiation potential were evaluated. The results demonstrated that the FLMSCs were morphologically consistent with fibroblasts, were able to be cultured and passaged for at least 33 passages and the cell morphology and proliferative ability were stable during the first 10 passages. In addition, FLMSCs were found to express CD29, CD44, CD73 and CD166, however, they did not express hematopoietic cell specific markers, including CD34, CD45 and BOLA‑DRα. The growth kinetics of FLMSCs consisted of a lag phase, a logarithmic phase and a plateau phase, and as the passages increased, the proliferative ability of cells gradually decreased. The majority of FLMSCs were in G0/G1 phase. Following osteogenic induction, FLMSCs were positive for the expression of osteopontin and collagen type I α2. Following neurogenic differentiation, the cells were morphologically consistent with neuronal cells and positive for microtubule‑associated protein 2 and nestin expression. It was concluded that the isolated FLMSCs exhibited typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their proliferation and the maintenance of stemness. The present study illustrated the potential application of lung tissue as an adult stem cell source for regenerative therapies.

  9. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  10. Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver

    OpenAIRE

    Orelio, Claudia; Peeters, Marian; Haak, Esther; van der Horn, Karin; Dzierzak, Elaine

    2009-01-01

    Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is know...

  11. Sexual dimorphism in epigenomicresponses of stem cells to extreme fetal growth

    Science.gov (United States)

    Delahaye, Fabien; Wijetunga, N. Ari; Heo, Hye J.; Tozour, Jessica N.; Zhao, Yong Mei; Greally, John M.; Einstein, Francine H.

    2014-01-01

    Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory. We tested whether heritable epigenetic processes in long-lived CD34+ hematopoietic stem/progenitor cells (HSPCs) showed evidence for re-programming associated with the extremes of fetal growth. Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function. We find a sexually dimorphic response; intrauterine growth restriction (IUGR) is associated with substantially greater epigenetic dysregulation in males, whereas large for gestational age (LGA) growth predominantly affects females. The findings are consistent with extreme fetal growth interacting with variable fetal susceptibility to influence cellular aging and metabolic characteristics through epigenetic mechanisms, potentially generating biomarkers that could identify infants at higher risk for chronic disease later in life. PMID:25300954

  12. Sexual dimorphism in epigenomic responses of stem cells to extreme fetal growth.

    Science.gov (United States)

    Delahaye, Fabien; Wijetunga, N Ari; Heo, Hye J; Tozour, Jessica N; Zhao, Yong Mei; Greally, John M; Einstein, Francine H

    2014-10-10

    Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory. We tested whether heritable epigenetic processes in long-lived CD34(+) haematopoietic stem/progenitor cells showed evidence for re-programming associated with the extremes of fetal growth. Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function. We find a sexually dimorphic response; intrauterine growth restriction is associated with substantially greater epigenetic dysregulation in males, whereas large for gestational age growth predominantly affects females. The findings are consistent with extreme fetal growth interacting with variable fetal susceptibility to influence cellular ageing and metabolic characteristics through epigenetic mechanisms, potentially generating biomarkers that could identify infants at higher risk for chronic disease later in life.

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

    Directory of Open Access Journals (Sweden)

    Sally Metsuyanim

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

  14. Candidate sequence variants and fetal hemoglobin in children with sickle cell disease treated with hydroxyurea.

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    Nancy S Green

    Full Text Available BACKGROUND: Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin. METHODOLOGY/PRINCIPAL FINDINGS: In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively, with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels. CONCLUSIONS/SIGNIFICANCE: These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease.

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

    Directory of Open Access Journals (Sweden)

    Maucksch C

    2012-01-01

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

  16. 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.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Yannick Gache

    Full Text Available 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

  18. Fetal cell carcinogenesis of the thyroid: a modified theory based on recent evidence.

    Science.gov (United States)

    Takano, Toru

    2014-01-01

    Thyroid cancer cells were believed to be generated by multi-step carcinogenesis, in which cancer cells are derived from thyrocytes, via multiple incidences of damage to their genome, especially in oncogenes or anti-oncogenes that accelerate proliferation or foster malignant phenotypes, such as the ability to invade the surrounding tissue or metastasize to distant organs, until a new hypothesis, fetal cell carcinogenesis, was presented. In fetal cell carcinogenesis, thyroid tumor cells are assumed to be derived from three types of fetal thyroid cell which only exist in fetuses or young children, namely, thyroid stem cells (TSCs), thyroblasts and prothyrocytes, by proliferation without differentiation. Genomic alternations, such as RET/PTC and PAX8-PPARγ1 rearrangements and a mutation in the BRAF gene, play an oncogenic role by preventing thyroid fetal cells from differentiating. Fetal cell carcinogenesis effectively explains recent molecular and clinical evidence regarding thyroid cancer, including thyroid cancer initiating cells (TCICs), and it underscores the importance of identifying a stem cells and clarifying the molecular mechanism of organ development in cancer research. It introduces three important concepts, the reverse approach, stem cell crisis and mature and immature cancers. Further, it implies that analysis of a small population of cells in a cancer tissue will be a key technique in establishing future laboratory tests. In the contrary, mass analysis such as gene expression profiling, whole genomic scan, and proteomics analysis may have definite limitations since they can only provide information based on many cells.

  19. Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation.

    Science.gov (United States)

    Liao, Gary J W; Gronowski, Ann M; Zhao, Zhen

    2014-01-20

    The identification of cell-free fetal DNA (cffDNA) in maternal circulation has made non-invasive prenatal testing (NIPT) possible. Maternal plasma cell free DNA is a mixture of maternal and fetal DNA, of which, fetal DNA represents a minor population in maternal plasma. Therefore, methods with high sensitivity and precision are required to detect and differentiate fetal DNA from the large background of maternal DNA. In recent years, technical advances in the molecular analysis of fetal DNA (e.g., digital PCR and massively parallel sequencing (MPS)) has enabled the successful implementation of noninvasive testing into clinical practice, such as fetal sex assessment, RhD genotyping, and fetal chromosomal aneuploidy detection.With the ability to decipher the entire fetal genome from maternal plasma DNA, we foresee that an increased number of non-invasive prenatal tests will be available for detecting many single-gene disorders in the near future. This review briefly summarizes the technical aspects of the NIPT and application of NIPT in clinical practice.

  20. Fetal Sex Determination Using Cell-Free Fetal Dna (cffDNA in Maternal Blood

    Directory of Open Access Journals (Sweden)

    I Nyoman Hariyasa Sanjaya

    2016-06-01

    Full Text Available Background: Prenatal test has routinely performed in antenatal care and has become a part of the obstetric care feature in many countries. Prenatal test is divided into screening and diagnostic test. Recently, the early noninvasive method in order to found and lessen the risk factors of pregnancy loss, has been studied. One of the methods is molecular test using cffDNA which has many screening purpose such as sex determination, aneuploidy, paternal inherited genetic disorder, fetus rhesus, and performed early at 7 weeks of pregnancy. Objective: The purpose of this study is to measure diagnostic value of cffDNA in determining fetal sex prenatally. Methods: In a diagnostic test study, 18 randomized samples were selected and divided based on fetal gender confirmed at birth. The group consisted of 9 pregnant women with male babies and 9 pregnant women with female babies. CffDNA then isolated from maternal blood sample and specific region in Y chromosome termed SRY is detected by PCR and electrophoresis. The data obtained analyzed both descriptively for baseline characteristic and analytically to determine its diagnostic value. Results: This study found significant correlation between SRY detection in cffDNA with male fetal phenotype (p<0.05. The sensitivity of the method is 100% with 89% specificity. In addition, we found 9.09 values for positive likelihood ratio (LR+ and 0 for negative likelihood ratio (LR-. Moreover, the result yielded 100% positive predictive value (PPV+ and 88.8% of negative predictive value (PPV-. Conclusion: This study proofed that cffDNA have a great diagnostic value to determine fetal sex prenatally. However, further study with several group of gestational age mother and better matching is required to further confirm the diagnostic potential of cffDNA 

  1. Reactivation of fetal hemoglobin in thalassemia and sickle cell disease

    Directory of Open Access Journals (Sweden)

    Sandro Eridani

    2014-09-01

    Full Text Available Considerable attention has been recently devoted to mechanisms involved in the perinatal hemoglobin switch, as it was long ago established that the survival of fetal hemoglobin (HbF production in significant amount can reduce the severity of the clinical course in severe disorders like β-thalassemia and sickle cell disease (SCD. For instance, when β-thalassemia is associated with hereditary persistence of fetal hemoglobin (HPFH the disease takes a mild course, labeled as thalassemia intermedia. The same clinical amelioration occurs for the association between HPFH and SCD. As for the mechanism of this effect, some information has been obtained from the study of natural mutations at the human β-globin locus in patients with increased HbF, like the Corfu thalassemia mutations. Important evidence came from the discovery that drugs capable of improving the clinical picture of SCD, like decitabine ad hydroxycarbamide, are acting through the reactivation, to some extent, of HbF synthesis. The study of the mechanism of action of these compounds was followed by the identification of some genetic determinants, which promote this event. In particular, among a few genetic factors involved in this process, the most relevant appears the BCL11A gene, which is now credited to be able to silence γ-globin genes in the perinatal period by interaction with several erythroid-specific transcription factors and is actually considered as a barrier to HbF reactivation by known HbF inducing agents. Epigenetics is also a player in the process, mainly through DNA demethylation. This is certified by the recent demonstration that hypomethylating agents such as 5-azacytidine and decitabine, the first compounds used for HbF induction by pharmacology, act as irreversible inhibitors of demethyltransferase enzymes. Great interest has also been raised by the finding that several micro-RNAs, which act as negative regulators of gene expression, have been implicated in the

  2. The cytokine regulation of SPARC production by rabbit corneal epithelial cells and fibroblasts in vitro.

    Science.gov (United States)

    Abe, Kosuke; Hibino, Tsuyoshi; Mishima, Hiroshi; Shimomura, Yoshikazu

    2004-03-01

    SPARC (osteonectin/BM40) is detected in the corneal stroma during the wound-healing process. To understand the metabolism of SPARC in the cornea, we investigated the effects of cytokines and growth factors on SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. Rabbit corneal epithelial cells or fibroblasts were cultured for 3 days with serum-containing minimal essential medium (MEM), then subcultured for 3 days on serum-free MEM with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), or interleukin-1beta (IL-1beta). SPARC concentration in the medium was measured by the ELISA method using anti-SPARC monoclonal antibody. The concentration of SPARC in the conditioned medium of the epithelial cells depended on either cell numbers or cultivation periods. When EGF was added to the medium, the amount of SPARC in the medium decreased. The addition of IL-1beta, PDGF, or TGF-beta did not affect SPARC synthesis by the epithelial cells. The production of SPARC by rabbit corneal fibroblasts was low compared with that by epithelial cells. However, the synthesis of SPARC by corneal fibroblasts was significantly enhanced by the addition of TGF-beta. The addition of IL-1beta, PDGF, or EGF slightly increased SPARC synthesis by corneal fibroblasts. Cytokines and growth factors modulate SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. These results suggest that cytokines and growth factors modulate cell-matrix interaction in corneal wound healing, possibly by regulating SPARC synthesis.

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

    Institute of Scientific and Technical Information of China (English)

    Jiang Lu; Dongsheng Li; Kehuan Lu

    2012-01-01

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

  4. Nanoscale topography-induced modulation of fundamental cell behaviors of rabbit corneal keratocytes, fibroblasts, and myofibroblasts.

    Science.gov (United States)

    Pot, Simon A; Liliensiek, Sara J; Myrna, Kathern E; Bentley, Ellison; Jester, James V; Nealey, Paul F; Murphy, Christopher J

    2010-03-01

    Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

  5. Influence of hyperthermia on the phosphorylation of ribosomal protein S6 from human skin fibroblasts and meningioma cells

    DEFF Research Database (Denmark)

    Richter, W W; Zang, K D; Issinger, O G

    1983-01-01

    Skin fibroblasts and meningioma cells, derived from primary cultures of the same patients have been used to study the influence of hyperthermia on (i) cell morphology and (ii) phosphorylation pattern of ribosomal and ribosome-associated proteins. Incubation of tumour cells and fibroblasts up to 7 h...

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

    Science.gov (United States)

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

    2013-01-01

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

  7. [Detection of fetal nucleated red blood cells in the maternal circulation by Kleihauer test].

    Science.gov (United States)

    Liu, Wei-Yu; Jin, Chun-Lian; Liu, Li-Ying; Lin, Chang-Kun; Wang, Yan; Sun, Kai-Lai

    2007-03-01

    Maternal blood was obtained from 18 pregnant women at 7 to 25 weeks of gestation. After Percoll discontinuous density gradient centifugation, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. Positive fetal cells appeared with an intense red cytoplasmic staining while maternal cells with adult haemoglobin were colourless. Individual positive NRBC was collected by micromanipulator and whole genome amplification was then performed to determine sex and STR status. This allowed the simultaneous verification of the fetal origin of NRBC and prenatal diagnosis of genetic diseases. The non-invasive prenatal genetic diagnosis of 9 fetuses at high risk of Duchenne muscular dystrophy (DMD) was completed successfully. The Kleihauer test is a rapid, simple and direct chemical staining method to select fetal cells and can be applied in prenatal diagnosis.

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

    Science.gov (United States)

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

    2010-04-01

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

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

    Science.gov (United States)

    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

  10. In vitro characteristics of pulmonary neuroendocrine cells isolated from rabbit fetal lung. I. Effects of culture media and nerve growth factor.

    Science.gov (United States)

    Cutz, E; Yeger, H; Wong, V; Bienkowski, E; Chan, W

    1985-12-01

    not detectable after separation into single cells, culture, or exposure to NGF. This study demonstrates that NE cells can be isolated from rabbit fetal lung and maintained in short-term culture. Low concentrations of fetal bovine serum enhanced the in vitro maintenance of NE cells, whereas NGF had no such effect. A feeder layer may be also important, since NE cells were closely associated with lung epithelial or fibroblast-like cells. The formation of neurite-like processes appears to be an expression of paracrine/paraneuron-type cell differentiation not mediated by NGF.(ABSTRACT TRUNCATED AT 400 WORDS)

  11. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day of ge...... on GFA-content was seen any longer, although some few weakly GFA positive cells could be observed in all permanent cell lines. Fetal rat brain cells therefore seem to become less responsive to this differentiation inducer during neoplastic transformation in cell culture....

  12. Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

    OpenAIRE

    1999-01-01

    We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5–14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10–30 nM thiamine (in the range of norma...

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

    Directory of Open Access Journals (Sweden)

    Paola Brun

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

  14. Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

    Science.gov (United States)

    Demirci, Selami; Doğan, Ayşegül; Apdik, Hüseyin; Tuysuz, Emre Can; Gulluoglu, Sukru; Bayrak, Omer Faruk; Şahin, Fikrettin

    2017-06-15

    Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

  15. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

    Science.gov (United States)

    Mold, Jeff E; Venkatasubrahmanyam, Shivkumar; Burt, Trevor D; Michaëlsson, Jakob; Rivera, Jose M; Galkina, Sofiya A; Weinberg, Kenneth; Stoddart, Cheryl A; McCune, Joseph M

    2010-12-17

    Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

  16. Fetal cells in maternal blood: state of the art for non-invasive prenatal diagnosis.

    Science.gov (United States)

    Ho, S S; O'Donoghue, K; Choolani, M

    2003-09-01

    In Singapore, 1 in 5 pregnancies occur in mothers > 35 years old and genetic diseases, such as thalassaemia, are common. Current methods for the diagnosis of aneuploidy and monogenic disorders require invasive testing by amniocentesis, chorion villus biopsy or fetal blood sampling. These tests carry a procedure-related risk of miscarriage that is unacceptable to many couples. Development of non-invasive methods for obtaining intact fetal cells would allow accurate prenatal diagnosis for aneuploidy and single gene disorders, without the attendant risks associated with invasive testing, and would increase the uptake of prenatal diagnosis by women at risk. Isolation of fetal erythroblasts from maternal blood should allow accurate non-invasive prenatal diagnosis of both aneuploidies and monogenic disorders. Expression of gamma-globin in maternal erythroblasts and the inability to locate fetal erythroblasts reliably in all pregnancies have prevented its clinical application. In the absence of a highly specific fetal cell marker, enrichment, identification and diagnosis--the 3 components of non-invasive prenatal diagnosis--have clearly defined objectives. Since fetal cells are rare in maternal blood, the sole purpose of enrichment is yield--to recover as many fetal cells as possible--even if purity is compromised at this stage. In contrast, the primary goal of identification is specificity; absolute certainty of fetal origin is required at this stage if the ultimate objective of diagnosis, accuracy, is to be achieved. This review summarises the current state of the art of non-invasive prenatal diagnosis using fetal erythroblasts enriched from maternal blood.

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

    Science.gov (United States)

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

    2015-01-01

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

  18. Fetal hepatic progenitors support long-term expansion of hematopoietic stem cells.

    Science.gov (United States)

    Chou, Song; Flygare, Johan; Lodish, Harvey F

    2013-05-01

    We have developed a coculture system that establishes DLK(+) fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In 1-week cultures supplemented with serum and supportive cytokines, both cocultured DLK(+) fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase in HSC numbers. In 2- and 3-week cultures DLK(+) cells, but not their conditioned medium, continuously and significantly (>20-fold) expanded both hematopoietic stem and progenitor cells. Physical contact between HSCs and DLK(+) cells was crucial to maintaining this long-term expansion. Similar HSC expansion (approximately sevenfold) was achieved in cocultures using a serum-free, low cytokine- containing medium. In contrast, DLK(-) cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver.

  19. Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells

    DEFF Research Database (Denmark)

    Byskov, A G; Fenger, M; Westergaard, L

    1993-01-01

    We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes. We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days. In this study MIS media are spent culture...... are fixed, squashed, and DNA-stained. In these preparations germ cells and somatic cells can be distinguished, and the number of germ cells in the different stages of meiosis is counted as is the number of somatic cells in mitosis. MIS activity is defined to be present in a medium when meiosis is induced...... in male germ cells during culture. We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner. In addition, MIS media and forskolin acted synergistically by inducing meiosis. Female germ cells seem to be unaffected by the various culture media...

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Antonio Miguel

    2014-02-01

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

  2. Characterization and radiosensitivity of fibroblasts derived from squamous cell carcinomas of the head and neck, and the surrounding oral mucosa

    Energy Technology Data Exchange (ETDEWEB)

    Stausboel-Groen, B.; Moeller Bentzen, S. [Danish Cancer Society, Aarhus (Denmark). Dept. of Experimental Clinical Oncology; Overgaard, J. [Danish Cancer Society, Aarhus (Denmark). Dept. of Experimental Clinical Oncology]|[Danish Cancer Society, Aarhus (Denmark). Dept. of Oncology

    1998-12-31

    Recently, extensive stromal fibroblast contamination has been reported in the modified Courtenay-Mills soft agar clonogenic assay for cellular in vitro radiosensitivity in tumour biopsies. The aim of the present study was to evaluate the hypothesis that an immunocytochemical analysis added to the modified Courtenay-Mills soft agar clonogenic assay provides a measure of both fibroblast and tumour cell radiosensitivity. Therefore, fibroblast derived from squamos cell carcinomas of the head and neck, and from the surrounding oral mucosa were compared for immunocytochemistry, DNA ploidy, plating efficiency and surviving fraction of cells after a radiation dose of 2 Gy. The results of our study suggest that the stromal fibroblast derived from tumour biopsies are representative of normal fibroblasts with respect to the characteristics examined using mucosal fibroblasts as normal controls. (orig.)

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

    Institute of Scientific and Technical Information of China (English)

    马玉珍; 任宇; 周雪原; 刘东军; 旭日干

    2011-01-01

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

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

    Science.gov (United States)

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

    2009-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

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

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

  7. Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Su, Xiaohu; Ling, Yu; Liu, Chunxia; Meng, Fanhua; Cao, Junwei; Zhang, Li; Zhou, Huanmin; Liu, Zongzheng; Zhang, Yanru

    2015-08-01

    We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.

  8. Endogenous type C viral gene expression in cultures of fetal diploid baboon cells treated with 5'-bromodeoxyuridine

    Energy Technology Data Exchange (ETDEWEB)

    Lavelle, G.; Kennel, S.J.; Foote, L.J.

    1981-04-30

    Cultures of fetal diploid baboon fibroblasts treated with 5-bromodeoxyuridine synthesized protein antigenically related to baboon endogenous type C viral gag gene product, p28. Radioimmunoassays detected p28 antigenic specificities indistinguishable from those of purified virus. However, viral RNA-dependent DNA polymerase was not detected in culture fluids, and infectious virus was rarely recovered by cocultivation with susceptible heterologous cells. Extracellular particles containing p28 were not readily detected, further indicating that viral gag gene-coded proteins were synthesized independently of whole virus. Normal cultures of the same baboon cells exhibited endogenous expression of a glycoprotein antigenically related to BEV gp70, suggesting differential regulation of the endogenous gag and env gene-coded products. Baboon cell cultures exogenously infected with BEV produced extracellular particles having viral p28 and gp70 as measured by radioimmunoassays of culture fluids. Since induced cultures have about 10% positive cells versus close to 100% for infected culture, the amount of p28 per producing cell was about the same in both cell populations.

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2012-02-01

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

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

    Science.gov (United States)

    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.

  12. Tissue Engineering of Tendons: A Comparison of Muscle-Derived Cells, Tenocytes, and Dermal Fibroblasts as Cell Sources.

    Science.gov (United States)

    Chen, Bo; Ding, Jinping; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Wang, Bin

    2016-03-01

    The rapid development of tendon tissue-engineering technology may offer an alternative graft for reconstruction of severe tendon losses. One critical factor for tendon tissue engineering is the optimization of seed cells. Little is known about the optimal cell source for engineered tendons. The aim of this study was to compare mouse muscle-derived cells, dermal fibroblasts, and tenocytes and determine the optimal cell source for tendon tissue engineering. Mouse muscle-derived cells, dermal fibroblasts, and tenocytes were isolated and cultured in vitro. At passage 1, cellular morphology, cell proliferation, and tenogenic marker expression were evaluated. After seeding on the polyglycolic acid scaffolds for 2 weeks in vitro and 12 weeks in vivo, histologic qualities, ultrastructure, and biomechanical characteristics were evaluated. Proliferation and cellular morphology were similar for dermal fibroblasts and tenocytes, whereas muscle-derived cells proliferated faster than the other two groups. With regard to the phenotype difference between them, muscle-derived cells and tenocytes shared the gene expression of SCX, TNMD, GDF-8, and Col-I, but with MyoD gene expression only in muscle-derived cells. In contrast to dermal fibroblast and tenocyte constructed tendons, neotendon with muscle-derived cells exhibited better aligned collagen fibers, more mature collagen fibril structure, and stronger mechanical properties, whereas no significant difference in the dermal fibroblast and tenocyte groups was observed. Although dermal fibroblasts are candidates for tendon tissue engineering because they are similar to tenocytes in proliferation and neotendon formation, muscle-derived cells appear to be the most suitable cells for further study and development of engineered tendon.

  13. Does Fetal antigen 1 (FA1) identify cells with regenerative, endocrine and neuroendocrine potentials?

    DEFF Research Database (Denmark)

    Jensen, Charlotte Floridon; Jensen, Charlotte Harken; Thorsen, Poul

    2000-01-01

    in the subcellular localisation indicating differential post-translational/post-transcriptional modifications during fetal development. FA1 may be a new marker of cellular subtypes with a regenerative potential and of specific cells with endocrine or neuroendocrine functions. Udgivelsesdato: 2000-Aug......, the localisation of FA1/dlk was analysed in embryonic and fetal tissues between week 5 to 25 of gestation and related to germinal origin and development. FA1 was observed in endodermally derived hepatocytes, glandular cells of the pancreas anlage, and in respiratory epithelial cells. FA1 was also present...... in mesodermally derived cells of the renal proximal tubules, adrenal cortex, Leydig and Hilus cells of the testes and ovaries, fetal chondroblasts, and skeletal myotubes. Ectodermally derived neuro- and adenohypophysial cells, cells in the floor of the 3rd ventricle and plexus choroideus were also FA1 positive...

  14. The prognostic significance of cancer-associated fibroblasts in esophageal squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Sang Yun Ha

    Full Text Available BACKGROUND: Cancer-associated fibroblasts (CAF are activated fibroblasts in the cancer stroma and play an important role in cancer progression. Some reports have indicated the correlation between the expression of CAF markers and adverse prognosis in several cancers. However, no reports have studied CAF phenotype and its clinical relevance in esophageal squamous cell carcinoma (ESCC. METHODS: We investigated CAF phenotype of ESCC based on histology and immunohistochemical expressions of five CAF markers such as fibroblast activation protein (FAP, smooth muscle actin (SMA, fibroblast-specific protein-1 (FSP1, platelet-derived growth factor receptor (PDGFRα, and PDGFRβ in 116 ESCC tissue samples. Besides, we also examined the correlation of the CAF phenotype with clinical relevance as well as other cancer-microenvironment related factors. RESULTS: Histologically immature CAF phenotype was correlated with poor prognosis (p<0.001 and associated with increased microvessel density, increased tumor associated macrophages, and epithelial to mesenchymal transition. CAF markers were characteristically expressed in stromal fibroblast close to tumor cells and the expression pattern of 5 CAF markers was highly heterogeneous in every individual cases. Of five CAF markers, SMA, FSP1, and PDGFRα were unfavorable prognostic indicators of ESCC. The number of positive CAF markers was greater in ESCC with immature CAFs than in those with mature ones. CONCLUSIONS: Our results demonstrate that histologic classification of CAF phenotype is a reliable and significant prognostic predictor in ESCC. CAF markers have the potential to be diagnostic and therapeutic targets in ESCC.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-01-15

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  18. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  19. CD34+ cells in human intestine are fibroblasts adjacent to, but distinct from, interstitial cells of Cajal

    DEFF Research Database (Denmark)

    Vanderwinden, J M; Rumessen, J J; De Laet, M H;

    1999-01-01

    and confocal microscopy. CD34 immunoreactivity identified previously unrecognized cells closely adjacent to, but distinct from, the Kit immunoreactive ICC. These CD34 immunoreactive cells expressed the fibroblast marker prolyl 4-hydroxylase-whereas ICC did not-and were also distinct from smooth muscle cells...

  20. Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells.

    Science.gov (United States)

    Nayak, Pritha S; Wang, Yulian; Najrana, Tanbir; Priolo, Lauren M; Rios, Mayra; Shaw, Sunil K; Sanchez-Esteban, Juan

    2015-05-27

    Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury. Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively. TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4. These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.

  1. POSTEMBRYONIC SUBTOTIPOTENT STEM CELLS DERIVED FROM A VARIETY OF FETAL TISSUES HAVE MULTIPLE DIFFERENTIATION POTENTIAL AND GREATLY CONTRIBUTE TO STEM CELL PLASTICITY

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To isolate and culture PSSCs from different tissues and determine their characteristics anddifferentiation potential in vitro. Methods: PSSCs were isolated and cultured from human aborted fetal bone mar-row, liver, skin, skeletal muscle, lung and pancreas. Morphology and biological activities were assessed. Pheno-types were analyzed by FACS and immunohistochemieal staining. We tested the potential of PSSCs to differentiateinto multiple cell lineages, such as bone, cartilage, fat, muscle, nerve , endothelial cell and hematopoietic progeni-tor cells. Results: PSSCs could be isolated from human aborted fetal above. PSSCs were a population of adherent cells characterized by a typical fibroblast - like morphology. PSSCs had few endoplasmic reticulum and mitochon- drias. It could be expanded by successive cycles of trypsinization, seeding, and culture ex vitro. PSSCs had a capa-bility of passaging up to 30 times without displaying significant changes in morphology, with a 2 -fold increase in cell number after each passage. Cell cycle analysis revealed that more than 90 % of cells were in the G0/G1 phases, while a small population of cells were actively engaged in proliferation. These cells were positively stained by FITC labeled CD44, CD29, CD13, but negative for CD34, HLA- DR. The culture- expanded PSSCs have multilineage differentiation potential giving rise to cells of osteogenic, chondrogenic, adipogenic, myogenic, neurogenic, hematopoietic and endotielial lineages. Conclusion:PSSCs may still remain in a number of tissues after embryonic development,could be identified by their phenotypic and functional characteristics, and contribute significantly to multipotent differentiation outside the tissue of origin.

  2. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  3. Characterization of fetal keratinocytes, showing enhanced stem cell-like properties: a potential source of cells for skin reconstruction.

    Science.gov (United States)

    Tan, Kenneth K B; Salgado, Giorgiana; Connolly, John E; Chan, Jerry K Y; Lane, E Birgitte

    2014-08-12

    Epidermal stem cells have been in clinical application as a source of culture-generated grafts. Although applications for such cells are increasing due to aging populations and the greater incidence of diabetes, current keratinocyte grafting technology is limited by immunological barriers and the time needed for culture amplification. We studied the feasibility of using human fetal skin cells for allogeneic transplantation and showed that fetal keratinocytes have faster expansion times, longer telomeres, lower immunogenicity indicators, and greater clonogenicity with more stem cell indicators than adult keratinocytes. The fetal cells did not induce proliferation of T cells in coculture and were able to suppress the proliferation of stimulated T cells. Nevertheless, fetal keratinocytes could stratify normally in vitro. Experimental transplantation of fetal keratinocytes in vivo seeded on an engineered plasma scaffold yielded a well-stratified epidermal architecture and showed stable skin regeneration. These results support the possibility of using fetal skin cells for cell-based therapeutic grafting.

  4. Characterization of Fetal Keratinocytes, Showing Enhanced Stem Cell-Like Properties: A Potential Source of Cells for Skin Reconstruction

    Science.gov (United States)

    Tan, Kenneth K.B.; Salgado, Giorgiana; Connolly, John E.; Chan, Jerry K.Y.; Lane, E. Birgitte

    2014-01-01

    Summary Epidermal stem cells have been in clinical application as a source of culture-generated grafts. Although applications for such cells are increasing due to aging populations and the greater incidence of diabetes, current keratinocyte grafting technology is limited by immunological barriers and the time needed for culture amplification. We studied the feasibility of using human fetal skin cells for allogeneic transplantation and showed that fetal keratinocytes have faster expansion times, longer telomeres, lower immunogenicity indicators, and greater clonogenicity with more stem cell indicators than adult keratinocytes. The fetal cells did not induce proliferation of T cells in coculture and were able to suppress the proliferation of stimulated T cells. Nevertheless, fetal keratinocytes could stratify normally in vitro. Experimental transplantation of fetal keratinocytes in vivo seeded on an engineered plasma scaffold yielded a well-stratified epidermal architecture and showed stable skin regeneration. These results support the possibility of using fetal skin cells for cell-based therapeutic grafting. PMID:25254345

  5. Characterization of Fetal Keratinocytes, Showing Enhanced Stem Cell-Like Properties: A Potential Source of Cells for Skin Reconstruction

    Directory of Open Access Journals (Sweden)

    Kenneth K.B. Tan

    2014-08-01

    Full Text Available Epidermal stem cells have been in clinical application as a source of culture-generated grafts. Although applications for such cells are increasing due to aging populations and the greater incidence of diabetes, current keratinocyte grafting technology is limited by immunological barriers and the time needed for culture amplification. We studied the feasibility of using human fetal skin cells for allogeneic transplantation and showed that fetal keratinocytes have faster expansion times, longer telomeres, lower immunogenicity indicators, and greater clonogenicity with more stem cell indicators than adult keratinocytes. The fetal cells did not induce proliferation of T cells in coculture and were able to suppress the proliferation of stimulated T cells. Nevertheless, fetal keratinocytes could stratify normally in vitro. Experimental transplantation of fetal keratinocytes in vivo seeded on an engineered plasma scaffold yielded a well-stratified epidermal architecture and showed stable skin regeneration. These results support the possibility of using fetal skin cells for cell-based therapeutic grafting.

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

    Directory of Open Access Journals (Sweden)

    Thon-Hon Vincent G

    2012-09-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Insulin-like growth factors act synergistically with basic fibroblast growth factor and nerve growth factor to promote chromaffin cell proliferation

    DEFF Research Database (Denmark)

    Frödin, M; Gammeltoft, S

    1994-01-01

    We have investigated the effects of insulin-like growth factors (IGFs), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) on DNA synthesis in cultured chromaffin cells from fetal, neonatal, and adult rats by using 5-bromo-2'-deoxyuridine (BrdUrd) pulse labeling for 24 or 48 h......% of fetal, 20% of neonatal, and 2% of adult chromaffin cells. The ED50 value of IGF-I- and IGF-II-stimulated BrdUrd labeling in neonatal chromaffin cells was 0.3 nM and 0.8 nM, respectively. In neonatal and adult chromaffin cells, addition of 1 nM bFGF or 2 nM NGF stimulated nuclear BrdUrd incorporation...... to approximately the same level as 10 nM IGF-I or IGF-II. However, the response to bFGF or NGF in combination with either IGF-I or IGF-II was more than additive, indicating that the combined effect of the IGFs and bFGF or NGF is synergistic. The degree of synergism was 2- to 4-fold in neonatal chromaffin cells...

  12. Effects of carbon nanotubes in a chitosan/collagen-based composite on mouse fibroblast cell proliferation.

    Science.gov (United States)

    Zhao, Wen; Yu, Wenwen; Zheng, Jiawei; Wang, Ying; Zhang, Zhiyuan; Zhang, Dongsheng

    2014-01-01

    This study investigated the in vitro cytocompatibility of carbon nanotubes (CNTs) in a chitosan/collagen-based composite. Mouse fibroblasts were cultured on the surface of a novel material consisting of CNTs in a chitosan/collagen-based composite (chitosan/collagen+CNTs group). Chitosan/collagen composites without CNTs served as the control material (chitosan/collagen group) and cells cultured normally in tissue culture plates served as blank controls (blank control group). Cell adhesion and proliferation were observed, and cell apoptosis was measured. The doubling time (DT1) of cells was significantly shorter in the chitosan/collagen+CNTs group than in the chitosan/collagen group, and that in the chitosan/collagen group was shorter than in the blank control group. The CNTs in the chitosan/collagen-based composites promoted mouse fibroblast adhesion, producing a distinct cytoskeletal structure. At 24 h after culture, the cytoskeleton of the cells in the chitosan/collagen+CNTs group displayed typical fibroblastic morphology, with clear microfilaments. Cells in the chitosan/collagen group were typically round, with an unclear cytoskeleton. The blank control group even had a few unattached cells. At 4 days after incubation, no early apoptosis of cells was detected in the blank control group, whereas early apoptosis of cells was observed in the chitosan/collagen+CNTs and chitosan/collagen groups. No significant difference in the proportion of living cells was detected among the three groups. After entering the plateau stage, the average cell number in the chitosan/collagen+CNTs group was similar to that in the chitosan/collagen group and significantly smaller than that in the blank control group. Early apoptosis of cells in the blank control group was not detectable. There were significant differences in early apoptosis among the three groups. These results suggest that CNTs in a chitosan/collagen-based composite did not cause significant cytotoxic effects on mouse

  13. Effect of hydroxyurea on G gamma chain fetal hemoglobin synthesis by sickle-cell disease patients

    Directory of Open Access Journals (Sweden)

    Teixeira S.M.

    2003-01-01

    Full Text Available Hydroxyurea is used for sickle-cell disease patients in order to increase fetal hemoglobin synthesis and consequently decrease the severity of pain episodes. Fetal hemoglobin, which is formed by gamma-globin chains A and G, is present in a constant composition throughout fetal development: about 75% of Ggamma and 25% of Agamma. In contrast, adult red cells contain about 40% of Ggamma and 60% of Agamma. In the present study, we analyzed the effect of hydroxyurea induction on the gamma chain composition of fetal hemoglobin in 31 sickle-cell disease patients treated with hydroxyurea. The control group was composed of 30 sickle-cell disease patients not treated with hydroxyurea in clinical steady state. The patients were older than 13 years and were not matched for age. All patients were seen at Hemocentro/UNICAMP and Boldrini Infantile Center, Campinas, SP, Brazil. The levels of total hemoglobin were significantly higher in patients treated with hydroxyurea (mean ± SD, 9.6 ± 2.16 g/dl than in untreated patients (8.07 ± 0.91 g/dl. Fetal hemoglobin levels were also higher in treated patients (14.16 ± 8.31% than in untreated patients (8.8 ± 4.09%, as was the Ggamma/Agamma ratio (1.45 ± 0.78 vs 0.98 ± 0.4, P < 0.005. The increase in the Ggamma/Agamma ratio in patients treated with hydroxyurea suggests the prevalence of a pattern of fetal hemoglobin synthesis, whereas patients not treated with hydroxyurea maintain the adult pattern of fetal hemoglobin synthesis. Because no correlation was observed between the Ggamma/Agamma ratio and total hemoglobin or fetal hemoglobin levels, the increase in Ggamma chain synthesis may not imply a higher production of hemoglobin.

  14. Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

    Science.gov (United States)

    Fernandez Vallone, Valeria; Leprovots, Morgane; Strollo, Sandra; Vasile, Gabriela; Lefort, Anne; Libert, Frederick; Vassart, Gilbert; Garcia, Marie-Isabelle

    2016-05-01

    Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2(+) cells generating gastric spheroids differed from adult glandular Lgr5(+) stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5(+) stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2(+) cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2(+) cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

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

    OpenAIRE

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

    1996-01-01

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

  16. The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

    DEFF Research Database (Denmark)

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir;

    2006-01-01

    The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface...

  17. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    Science.gov (United States)

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-04-30

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis.

  18. Corneal Fibroblasts as Sentinel Cells and Local Immune Modulators in Infectious Keratitis

    Directory of Open Access Journals (Sweden)

    Ken Fukuda

    2017-08-01

    Full Text Available The cornea serves as a barrier to protect the eye against external insults including microbial pathogens and antigens. Bacterial infection of the cornea often results in corneal melting and scarring that can lead to severe visual impairment. Not only live bacteria but also their components such as lipopolysaccharide (LPS of Gram-negative bacteria contribute to the development of inflammation and subsequent corneal damage in infectious keratitis. We describe the important role played by corneal stromal fibroblasts (activated keratocytes as sentinel cells, immune modulators, and effector cells in infectious keratitis. Corneal fibroblasts sense bacterial infection through Toll-like receptor (TLR–mediated detection of a complex of LPS with soluble cluster of differentiation 14 (CD14 and LPS binding protein present in tear fluid. The cells then initiate innate immune responses including the expression of chemokines and adhesion molecules that promote the recruitment of inflammatory cells necessary for elimination of the infecting bacteria. Infiltrated neutrophils are activated by corneal stromal collagen and release mediators that stimulate the production of pro–matrix metalloproteinases by corneal fibroblasts. Elastase produced by Pseudomonas aeruginosa (P. aeruginosa activates these released metalloproteinases, resulting in the degradation of stromal collagen. The modulation of corneal fibroblast activation and of the interaction of these cells with inflammatory cells and bacteria is thus important to minimize corneal scarring during treatment of infectious keratitis. Pharmacological agents that are able to restrain such activities of corneal fibroblasts without allowing bacterial growth represent a potential novel treatment option for prevention of excessive scarring and tissue destruction in the cornea.

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

    Science.gov (United States)

    Singh, Mahipal; Sharma, Anil K

    2011-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Hyun Jeong [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Park, Sahng Wook [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Hojeong [Department of Anatomy, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Park, Sang-Kyu, E-mail: 49park@kd.ac.kr [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Yoon, Dojun, E-mail: mozart@kd.ac.kr [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)

    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.

  1. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

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    Tahereh Talaei-Khozani

    2014-03-01

    Full Text Available Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA and 5-Aza-2-Deoxycytidine (5-aza-dC. The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function.

  2. Povidone-iodine Solutions Inhibit Cell Migration and Survival of Osteoblasts, Fibroblasts, and Myoblasts.

    Science.gov (United States)

    Liu, James X; Werner, Jordan A; Buza, John A; Kirsch, Thorsten; Zuckerman, Joseph D; Virk, Mandeep S

    2017-04-12

    In vitro laboratory study. The purpose of this study was to identify the effect of dilute povidone-iodine (PVI) solutions on human osteoblast, fibroblast and myoblast cells in vitro. Dilute PVI wound lavage has been used successfully in spine and joint arthroplasty procedures to prevent post-operative surgical site infection, but their biologic effect on host cells is largely unknown. Human primary osteoblasts, fibroblasts, and myoblasts were expanded in cell culture and subjected to various concentrations of PVI (0%, 0.001%, 0.01%, 0.1%, 0.35%, 1%) for 3 minutes. To assess the effect of PVI on cell migration, a scratch assay was performed, in which a "scratch" was made by a standard pipette tip in a cell monolayer following PVI exposure, and time to closure of the scratch was evaluated. Cell survival and proliferation was measured 48 hours post-PVI exposure using a cell viability and cytotoxicity assay. Closure of the scratch defect in all cell monolayers was achieved in PVI concentrations PVI concentrations of ≥ 0.1%. PVI concentrations PVI ≥ 0.1% had cell survival rates of less than 6% (p PVI (0.35%) exerts a pronounced cytotoxic effect on osteoblasts, fibroblast, and myoblasts in vitro. Further investigation is required to systematically study the effect of PVI on tissue healing in vivo and also determine a safe and clinically potent concentration for PVI lavage. N/A.

  3. Cardiac Fibroblasts Aggravate Viral Myocarditis: Cell Specific Coxsackievirus B3 Replication

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    Diana Lindner

    2014-01-01

    Full Text Available Myocarditis is an inflammatory disease caused by viral infection. Different subpopulations of leukocytes enter the cardiac tissue and lead to severe cardiac inflammation associated with myocyte loss and remodeling. Here, we study possible cell sources for viral replication using three compartments of the heart: fibroblasts, cardiomyocytes, and macrophages. We infected C57BL/6j mice with Coxsackievirus B3 (CVB3 and detected increased gene expression of anti-inflammatory and antiviral cytokines in the heart. Subsequently, we infected cardiac fibroblasts, cardiomyocytes, and macrophages with CVB3. Due to viral infection, the expression of TNF-α, IL-6, MCP-1, and IFN-β was significantly increased in cardiac fibroblasts compared to cardiomyocytes or macrophages. We found that in addition to cardiomyocytes cardiac fibroblasts were infected by CVB3 and displayed a higher virus replication (132-fold increase compared to cardiomyocytes (14-fold increase between 6 and 24 hours after infection. At higher virus concentrations, macrophages are able to reduce the viral copy number. At low virus concentration a persistent virus infection was determined. Therefore, we suggest that cardiac fibroblasts play an important role in the pathology of CVB3-induced myocarditis and are another important contributor of virus replication aggravating myocarditis.

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

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    Abdolmaleki A

    2013-04-01

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

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

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    Fernanda Gonçalves BASSO

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

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

    Science.gov (United States)

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

    2016-11-28

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

  7. Allogeneic fetal stem cell transplantation to child with psychomotor retardation: A case report

    OpenAIRE

    2016-01-01

    Introduction. The consequences of autologous and allogeneic stem cell transplantation (stem cells of hematopoiesis), applied in adults and children suffering from leukemia or some other malignant disease, are well-known and sufficiently recognizable in pediatric clinical practice regardless of the indication for the treatment. However, the efficacy of fetal stem cell transplantation is unrecognizable when the indications are psychomotor retardation and epil...

  8. Cyclical cell stretching of skin-derived fibroblasts downregulates connective tissue growth factor (CTGF) production.

    Science.gov (United States)

    Kanazawa, Yuichiro; Nomura, Jun; Yoshimoto, Shinya; Suzuki, Toshikazu; Kita, Kazuko; Suzuki, Nobuo; Ichinose, Masaharu

    2009-01-01

    Delayed healing of skin wounds can be caused by wound instability, whereas appropriate massage or exercise prevents sclerosis and scar contracture. However, the mechanism by which wound healing is related to mechanical stress has not been fully elucidated. The present study aimed to identify whether mechanical stretching of fibroblasts reduces their production of extracellular matrix. We transferred skin fibroblasts into collagen-coated elastic silicone chambers, cultured them on a stretching apparatus, and used RT-PCR to examine the effects of mechanical stretching on the expression levels of 17 genes related to extracellular matrix production and growth factor secretion. We found that connective tissue growth factor (CTGF) was downregulated after 24 hr of cell stretching. Specifically, the CTGF mRNA and protein levels were 50% and 48% of the control levels, respectively. These findings suggest that cyclic stretching of fibroblasts contributes to anti-fibrotic processes by reducing CTGF production.

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

    Science.gov (United States)

    Belgiovine, Cristina; Chiodi, Ilaria; Mondello, Chiara

    2011-11-01

    Studying neoplastic transformation of telomerase immortalized human fibroblasts (cen3tel), we found that the transition from normal to tumorigenic cells was associated with the loss of growth contact inhibition, the acquisition of an epithelial-like morphology and a change in actin organization, from stress fibers to cortical bundles. We show here that these variations were paralleled by an increase in N-cadherin expression and relocalization of different adhesion molecules, such as N-cadherin, α-catenin, p-120 and β-catenin. These proteins presented a clear membrane localization in tumorigenic cells compared to a more diffuse, cytoplasmic distribution in primary fibroblasts and non-tumorigenic immortalized cells, suggesting that tumorigenic cells could form strong cell-cell contacts and cell contacts did not induce growth inhibition. The epithelial-like appearance of tumorigenic cells did not reflect a mesenchymal-epithelial transition; in fact, cen3tel cells expressed vimentin and did not express cytokeratins at all transformation stages. Moreover, they did not express epithelial proteins such as occluding and claudin-1. In contrast, ZO-1 showed higher levels and a more defined membrane localization in tumorigenic cells compared to non-tumorigenic cells; this confirms its role in adherens junction formation in mesenchymal cells and is in agreement with the strong cell-cell contact formation by neoplastically transformed cells. Finally, we found α-catenin and ZO-1 nuclear localization in non-transformed cells, suggestive of possible additional roles of these proteins besides cell junction formation.

  10. Enhanced fibroblast cell adhesion on Al/Al{sub 2}O{sub 3} nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Aktas, O.C. [INM - Leibniz-Institute for New Materials, CVD/Biosurfaces Division, Campus D2 2, 66123 Saarbruecken (Germany); Sander, M. [Saarland University, Biological Experimental Physics Department, P.O. Box 151150, 66041 Saarbruecken (Germany); Miro, M.M.; Lee, J.; Akkan, C.K.; Smail, H. [INM - Leibniz-Institute for New Materials, CVD/Biosurfaces Division, Campus D2 2, 66123 Saarbruecken (Germany); Ott, A. [Saarland University, Biological Experimental Physics Department, P.O. Box 151150, 66041 Saarbruecken (Germany); Veith, M., E-mail: Michael.veith@inm-gmbh.de [INM - Leibniz-Institute for New Materials, CVD/Biosurfaces Division, Campus D2 2, 66123 Saarbruecken (Germany); Saarland University, Institute for Inorganic Chemistry, P.O. Box 151150, 66041 Saarbruecken (Germany)

    2011-02-01

    Biological cells stick together via transmembrane proteins, which are linked to receptor molecules of the extracellular matrix (ECM). This specific biochemical adhesion plays a leading role in many cellular processes, among them cell differentiation, morphogenesis, and wound healing. Various medical applications require endogen cells to bind to an exogene substrate as in the case of an implant. Coatings with proteins that naturally belong to the ECM are known to enhance the cell adhesion. However, the choice of inorganic materials, which promote cell adhesion, is limited. Here, we report on a new engineered surface composed of Al/Al{sub 2}O{sub 3} bi-phasic nanowires (NWs), which promotes the adhesion of fibroblast cells. Fibroblasts grow well on this inorganic layer and keep proliferating. Using the cell monolayer rheology (CMR) technique, we show that the adhesion of fibroblasts on Al/Al{sub 2}O{sub 3} NWs is comparable to fibronectin coated surfaces. To our knowledge, this is one of the strongest cell adhesions on an inorganic surface, which has been reported on so far, since it compares to bio-organic layers such as fibronectin.

  11. Enhanced fibroblast cell adhesion on Al/Al2O3 nanowires

    Science.gov (United States)

    Aktas, O. C.; Sander, M.; Miró, M. M.; Lee, J.; Akkan, C. K.; Smail, H.; Ott, A.; Veith, M.

    2011-02-01

    Biological cells stick together via transmembrane proteins, which are linked to receptor molecules of the extracellular matrix (ECM). This specific biochemical adhesion plays a leading role in many cellular processes, among them cell differentiation, morphogenesis, and wound healing. Various medical applications require endogen cells to bind to an exogene substrate as in the case of an implant. Coatings with proteins that naturally belong to the ECM are known to enhance the cell adhesion. However, the choice of inorganic materials, which promote cell adhesion, is limited. Here, we report on a new engineered surface composed of Al/Al2O3 bi-phasic nanowires (NWs), which promotes the adhesion of fibroblast cells. Fibroblasts grow well on this inorganic layer and keep proliferating. Using the cell monolayer rheology (CMR) technique, we show that the adhesion of fibroblasts on Al/Al2O3 NWs is comparable to fibronectin coated surfaces. To our knowledge, this is one of the strongest cell adhesions on an inorganic surface, which has been reported on so far, since it compares to bio-organic layers such as fibronectin.

  12. Middermal Elastolysis: Dermal Fibroblasts Cooperate with Inflammatory Cells to the Elastolytic Disorder

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    Giovanna De Cunto

    2017-01-01

    Full Text Available Little is known about the cause and pathophysiology of middermal elastolysis (MDE. In this condition, variable inflammatory infiltrate may be present or not together with loss of elastic fibres in the middermis that spares both papillary and lower reticular dermis. MDE may be a consequence of abnormal extracellular matrix degradation related to an imbalance between elastolytic enzymes released from inflammatory and resident cells and their naturally occurring inhibitors. However, the cause of this imbalance is still an object of investigation. In order to shed light on the role of fibroblasts in MDE, we used fibroblast cultures from MDE and control subjects to evaluate matrix metalloproteinases (MMPs and their major inhibitor TIMP-1, which in combination with neutrophil or macrophage proteases released in inflamed areas may influence the elastolytic burden. We demonstrate that fibroblasts derived from MDE produce in vitro low levels of TIMP-1, the major inhibitor of MMPs. Elevated levels of MMP-2, MMP-14, and TIMP-2 capable to activate in a cooperative manner pro-MMP-2 are present in MDE tissue samples. Additionally, significant reaction for MMP-1 is present in the same MDE areas. These data all together suggest that ECM changes in MDE are due to cooperation of different cell populations (i.e., inflammatory cells and fibroblasts.

  13. Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts.

    Science.gov (United States)

    Akbari, Amir; Li, Yunyuan; Kilani, Ruhangiz T; Ghahary, Aziz

    2012-11-01

    During the early stage of wound healing process, blood clots can be served as a temporary extracellular matrix (ECM) to let skin cell migration and proliferation. The red blood cells are generally thought as inert bystanders in the early and inflammatory phase of wound healing. Here, we provide evidence that red blood cells (RBC) also play an important role in modulation of key ECM components such as type-I collagen, α-smooth muscle actin, fibronectin, and matrix metalloproteinases (MMPs). In this study, we used western blot analysis and showed a significant increase in the level of MMP-1, 2, 3. Furthermore, we found that RBC lysate significantly down-regulates type-I collagen and α-smooth muscle actin while up-regulates fibronectin expression in dermal fibroblasts. To further explore the mechanism by which RBC lysate modulates MMP-1 expression, the effect of inhibitors for three MAPK signaling pathways on RBC inducing MMP-1 expression by dermal fibroblasts were tested. The result showed that the inhibitor of ERK1/2 could abrogate the stimulatory effect of RBC lysate on MMP-1 expression in dermal fibroblasts. Consistently, RBC treatment results in an increase of ERK1/2 phosphorylation in dermal fibroblast. In conclusion, these findings suggest that RBC lysate can modulate the expression of MMPs and key ECM components which are important in healing process.

  14. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta is a specific regulator of fibroblast motility and contractility.

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    Latha Satish

    Full Text Available Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF and platelet derived growth factor (PDGF stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (alpha-SMA expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less alpha-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular

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

    Science.gov (United States)

    Ohshima, Susumu; Seyama, Atsushi

    2012-09-01

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

  16. Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

    Science.gov (United States)

    Bressan, F F; Miranda, M S; Bajgelman, M C; Perecin, F; Mesquita, L G; Fantinato-Neto, P; Merighe, G F K; Strauss, B E; Meirelles, F V

    2013-04-01

    Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed

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

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    Jordan R Plews

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

  18. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

    Science.gov (United States)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Cancer-associated fibroblasts as another polarized cell type of the tumor microenvironment

    Directory of Open Access Journals (Sweden)

    Martin eAugsten

    2014-03-01

    Full Text Available Tumor- or cancer-associated fibroblasts (CAFs are one of the most abundant stromal cell types in different carcinomas and comprise a heterogeneous cell population. Classically, CAFs are assigned with pro-tumorigenic effects stimulating tumor growth and progression. More recent studies demonstrated also tumor-inhibitory effects of CAFs suggesting that tumor-residing fibroblasts exhibit a similar degree of plasticity as other stromal cell types. Reciprocal interactions with the tumor milieu and different sources of origin are emerging as two important factors underlying CAF heterogeneity. This review highlights recent advances in our understanding of CAF biology and proposes to expand the term of cellular ´polarization´, previously introduced to describe different activation states of various immune cells, onto CAFs to reflect their phenotypic diversity.

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

    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.

  2. Increased fetal insulin concentrations for one week fail to improve insulin secretion or β-cell mass in fetal sheep with chronically reduced glucose supply.

    Science.gov (United States)

    Lavezzi, Jinny R; Thorn, Stephanie R; O'Meara, Meghan C; LoTurco, Dan; Brown, Laura D; Hay, William W; Rozance, Paul J

    2013-01-01

    Maternal undernutrition during pregnancy and placental insufficiency are characterized by impaired development of fetal pancreatic β-cells. Prolonged reduced glucose supply to the fetus is a feature of both. It is unknown if reduced glucose supply, independent of other complications of maternal undernutrition and placental insufficiency, would cause similar β-cell defects. Therefore, we measured fetal insulin secretion and β-cell mass following prolonged reduced fetal glucose supply in sheep. We also tested whether restoring physiological insulin concentrations would correct any β-cell defects. Pregnant sheep received either a direct saline infusion (CON = control, n = 5) or an insulin infusion (HG = hypoglycemic, n = 5) for 8 wk in late gestation (75 to 134 days) to decrease maternal glucose concentrations and reduce fetal glucose supply. A separate group of HG fetuses also received a direct fetal insulin infusion for the final week of the study with a dextrose infusion to prevent a further fall in glucose concentration [hypoglycemic + insulin (HG+I), n = 4]. Maximum glucose-stimulated insulin concentrations were 45% lower in HG fetuses compared with CON fetuses. β-Cell, pancreatic, and fetal mass were 50%, 37%, and 40% lower in HG compared with CON fetuses, respectively (P < 0.05). Insulin secretion and β-cell mass did not improve in the HG+I fetuses. These results indicate that chronically reduced fetal glucose supply is sufficient to reduce pancreatic insulin secretion in response to glucose, primarily due to reduced pancreatic and β-cell mass, and is not correctable with insulin.

  3. Design and Fabrication a Microfluidic Device for Fetal Cells Dielectrophoretic Properties Characterization

    Energy Technology Data Exchange (ETDEWEB)

    Xu Guolin [Institute of Bioengineering and Nanotechnologies, 31 Biopolis, Way, The Nanos, hashmark 04-01, Singapore 138669 (Singapore); Chan, M B [Nanyang Technological University, Singapore. 16 Nanyang Drive, Singapore 637722 (Singapore); Yang, Charles [Nanyang Technological University, Singapore. 16 Nanyang Drive, Singapore 637722 (Singapore); Sukumar, P [National University Hospital, Singapore. 10 Medical Drive, Singapore 117597 (Singapore); Choolani, M [National University Hospital, Singapore. 10 Medical Drive, Singapore 117597 (Singapore); Ying, Jackie Y [Institute of Bioengineering and Nanotechnologies, 31 Biopolis, Way, The Nanos, hashmark 04-01, Singapore 138669 (Singapore)

    2006-04-01

    The present work presents a microfluidic device with interdigitated microelectrode and microchannel for fetal nucleated red blood cell dielectrophoresis properties characterization using crossover frequency method. To obtain the electric field and its gradient along the microchannel, simulation study was done by using MAXWELL{sup TM} software. Results show maximum electric field and gradient are obtained near the electrode edge and they are affected by electrode width and the electrode gap. The crossover frequency should be obtained by keeping the cell moving near the electrode edge. The device has been successfully used in fetal cell characterization with better than 1KHz frequency repeatability, which is about 2% of the measured crossover frequency.

  4. Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage.

    Science.gov (United States)

    Zhao, Q; Qiu, Y G; Tian, J T; Wang, C S; An, T Z

    2016-11-17

    The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P M-embryos were octoploid before the first cleavage. Furthermore, 81.25% of the blastomeres of blastocysts developed from the M-embryos showed abnormal ploidy compared with those developed from the G1-embryos (22.55%). However, some of the blastomeres remained diploid in all the M-embryos tested. A portion of the blastomeres restored normal diploidy in some of the M-embryos at the blastocyst stage. This finding provides an explanation for M-embryos developing to term.

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

    Science.gov (United States)

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

    2016-08-01

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

  6. Biology of the Sertoli Cell in the Fetal, Pubertal, and Adult Mammalian Testis.

    Science.gov (United States)

    Chojnacka, Katarzyna; Zarzycka, Marta; Mruk, Dolores D

    A healthy man typically produces between 50 × 10(6) and 200 × 10(6) spermatozoa per day by spermatogenesis; in the absence of Sertoli cells in the male gonad, this individual would be infertile. In the adult testis, Sertoli cells are sustentacular cells that support germ cell development by secreting proteins and other important biomolecules that are essential for germ cell survival and maturation, establishing the blood-testis barrier, and facilitating spermatozoa detachment at spermiation. In the fetal testis, on the other hand, pre-Sertoli cells form the testis cords, the future seminiferous tubules. However, the role of pre-Sertoli cells in this process is much less clear than the function of Sertoli cells in the adult testis. Within this framework, we provide an overview of the biology of the fetal, pubertal, and adult Sertoli cell, highlighting relevant cell biology studies that have expanded our understanding of mammalian spermatogenesis.

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

    Directory of Open Access Journals (Sweden)

    Xingjia Wu

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

  8. Circadian-independent cell mitosis in immortalized fibroblasts

    OpenAIRE

    Yeom, Mijung; Pendergast, Julie S.; Ohmiya, Yoshihiro; Yamazaki, Shin

    2010-01-01

    Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (~24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is ...

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

    Science.gov (United States)

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

    2010-03-29

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

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

    Directory of Open Access Journals (Sweden)

    Humidah Alanazi

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ji-Yong Jung

    2015-08-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Advanced Research of Fibroblast Growth Factor Receptor 
in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Dan PU

    2013-11-01

    Full Text Available Lung cancer is severely threatening human health. In recent years, the treatment for lung adenocarcinoma has made a great progress, targeted therapy has been widely applied in clinic, and benefits amount of patients. However, in squamous cell lung cancer, the incidence of epidermal growth factor receptor (EGFR gene mutant and ALK fusion gene are low,and targeted therapy like Tarceva and crizotinib, can hardly work. Since the fibroblast growth factors (fibroblast growth factor, FGF pathway is considered to be related to tumor cell proliferation, metastasis and angiogenesis, more and more researches proved the amplification of fibroblast growth factor receptor (FGFR in squamous cell lung cancer. Experiments in vivo and in vitro found that blocking FGF pathway could reduce the proliferation of tumor cells and inhibit metastasis. The FGF pathway might be a new target for treatment of squamous cell lung cancer. This article reviews the effect of FGFR in tumorigenesis,as well as the prospect as a therapeutic target in non-small cell lung cancer.

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

    Science.gov (United States)

    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.

  15. Isolation and analysis of SSEA-4 positive cells derived from fetal marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LIU Daqing; PEI Xuetao; YANG Yinxiang; GAO Yanhong; YUAN Hongfeng; QIN Lipeng; WANG Yunfang; NAN Xue; SHI Shuangshuang; YUE Wen

    2006-01-01

    A big issue in stem cell research is to derive prospective totipotential stem cells. In this study, fMSC-SSEA-4 cells expressing SSEA-4 antigen were isolated from fetal marrow masenchymal stem cells (fMSCs) using immunomagnetic bead sorting technique. The totipotent cells were identified and their biological characteristics were further studied. The expression of Oct-4 and SSEA-4, carcino- genicity, and the ability to differentiation of fMSC- SSEA-4 cells were evaluated to verify the totipotent potential. fMSC-SSEA-4 cells were isolated successfully from fMSCs (2.5% among fMSCs), while no obvious differences were seen in morphology, growth curve, cell cycle and immunophenotype, Oct-4 and SSEA-4 expression between fMSC-SSEA-4 cells and fMSCs. fMSC-SSEA-4 cells showed normal diploid chromosome karyotype and no carcinoma was induced after inoculation into nude mice. fMSC- SSEA-4 cells could be induced to fat cells, osteogenic cells and neuron-like cells in vitro with different induced factors. The results indicated that there may be a few totipotent cells among the fMSCs and it may offer the experimental basis for the further study and application of fMSCs.

  16. A new marker set that identifies fetal cells in maternal circulation with high specificity

    DEFF Research Database (Denmark)

    Hatt, Lotte; Brinch, Marie; Singh, Ripudaman;

    2014-01-01

    OBJECTIVE: Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. METHOD: Blood samples from 78 normal pregnancies...

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

    Science.gov (United States)

    Treutlein, Barbara

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

  18. Prolonged expression of the c-kit receptor in germ cells of intersex fetal testes

    DEFF Research Database (Denmark)

    Rajpert-De Meyts, Ewa; Jørgensen, N; Müller, Jørn

    1996-01-01

    Stem cell factor (SCF) and its receptor Kit encoded by the c-kit proto-oncogene are crucial for the development and migration of primordial germ cells in rodents. The expression of Kit has been examined immunohistochemically in gonads obtained from five specimens of fetal tissues with intersex...

  19. Expression of immunohistochemical markers for testicular carcinoma in situ by normal human fetal germ cells

    DEFF Research Database (Denmark)

    Jørgensen, N; Rajpert-De Meyts, E; Graem, N;

    1995-01-01

    -like alkaline phosphatase, the protooncogene c-kit protein product, and the antigens for the monoclonal antibodies TRA-1-60 and M2A. The relative numbers of fetal germ cells that demonstrated positive reaction with the markers were calculated. RESULTS: The vast majority of the germ cells (75-100%) in the first...

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

    Science.gov (United States)

    Christensen, Claus; Berezin, Vladimir; Bock, Elisabeth

    2011-10-26

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

  1. Noninvasive Prenatal Diagnosis of Fetal Sex by Single-cell PEP-PCR Method

    Institute of Scientific and Technical Information of China (English)

    王陶然; 陈汉平; 马庭元

    2004-01-01

    Summary: A new method for noninvasive prenatal diagnosis of fetal sex was developed by using single-cell PEP-PCR techniques. Micromamipulation techniques were used to obtain single fetal cells from 273 maternal blood samples. The genome of single cells was preamplified by PEP and SRY genes were analyzed by PCR method. The SRY genes of 149 samples were detected by the new method among 153 samples carrying male fetus, while 119 out of 120 samples carrying female fetus were proved negative for SRY genes. The sensitivity and specificity of the new method were 97.39% and 99.17 % respectively and the correct rate was 98.17 %. The new method has the advantage of high sensitivity and specificity in noninvasive prenatal diagnosis of fetal sex and provides the basis of other researches such as sex-linked inherited diseases.

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

    Science.gov (United States)

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

    2013-01-01

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

  3. Prevention of Simian Virus 40 Tumors by Hamster Fetal Tissue: Influence of Parity Status of Donor Females on Immunogenicity of Fetal Tissue and on Immune Cell Cytotoxicity

    Science.gov (United States)

    Girardi, Anthony J.; Reppucci, Phyllis; Dierlam, Peggy; Rutala, William; Coggin, Joseph H.

    1973-01-01

    Fetal tissue from primiparous hamsters prevented simian virus 40 (SV40) tumorigenesis in male hamsters, whereas fetal tissue from multiparous hamsters did not. The parity status of normal (uninoculated) hamsters also influenced the cytotoxicity of their lymphoid cells against tumor cells. Lymph node cells from nonpregnant primiparous and multiparous animals were cytotoxic in microcytotoxicity tests against SV40, polyoma, and adenovirus 7 tumor cells, but were not active against control BHK cells. Lymph node cells from virgin female donors were inactive. Peritoneal exudate cells from these donors reacted in similar fashion against SV40 tumor cells in vitro and in adoptive transfer tests in vivo. However, the cytotoxicity of peritoneal exudate cells from multiparous hamsters was greatly reduced during pregnancy, a time when noncytotoxic humoral antibody reactive with surface antigen of SV40 tumor cells is present. This humoral antibody is not detected during first pregnancy, and peritoneal exudate cells obtained from pregnant primiparous hamsters demonstrated a high degree of cytotoxicity. PMID:4346032

  4. Cell free fetal DNA testing in maternal blood of Romanian pregnant women

    Directory of Open Access Journals (Sweden)

    Viorica E Radoi

    2015-10-01

    Full Text Available Background: The discovery of circulating fetal DNA in maternal blood led to the discovery of new strategies to perform noninvasive testing for prenatal diagnosis. Objective: The purpose of the study was to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y by analysis of fetal cell-free DNA from maternal blood, without endangering pregnancy. Materials and Methods: This retrospective study has been performed in Bucharest at Medlife Maternal and Fetal Medicine Department between 2013-2014. In total 201 women were offered noninvasive prenatal test. Maternal plasma samples were collected from women at greater than 9 weeks of gestation after informed consent and genetics counseling. Results: From 201 patients; 28 (13.93% had screening test with high risk for trisomy 21, 116 (57.71% had advanced maternal age, 1 (0.49% had second trimester ultrasound markers and the remaining 56 patients (27.86% performed the test on request. Of those patients, 189 (94.02% had a “low risk” result (99% risk all for trisomy 21 (T21. T21 was confirmed by amniocentesis in 1 patient and the other 4 patients declined confirmation. The 7 remaining patients (3.48% had a low fetal fraction of DNA. Conclusion: It is probably that prenatal diagnosis using fetal DNA in maternal blood would play an increasingly role in the future practice of prenatal testing because of high accuracy.

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

    Directory of Open Access Journals (Sweden)

    Zhongqiu Li

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    GuoXB; DengFR

    2002-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

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

  9. On-chip constructive cell-Network study (I: Contribution of cardiac fibroblasts to cardiomyocyte beating synchronization and community effect

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    Yasuda Kenji

    2011-05-01

    Full Text Available Abstract Backgrounds To clarify the role of cardiac fibroblasts in beating synchronization, we have made simple lined-up cardiomyocyte-fibroblast network model in an on-chip single-cell-based cultivation system. Results The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts showed (1 propagation velocity of electrophysiological signals decreased a magnitude depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2 fluctuation of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and (3 the synchronized cardiomyocytes connected by fibroblasts sometimes loses their synchronized condition and recovered to synchronized condition, in which the length of asynchronized period was shorter less than 30 beats and was independent to their cultivation time, whereas the length of synchronized period increased according to cultivation time. Conclusions The results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly enhance and contribute to beating interval stability and synchronization. This might also mean that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte 'community effect', which enhances synchronization and stability of their beating rhythms.

  10. Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss.

    Science.gov (United States)

    Ramadoss, Jayanth; Lunde, Emilie R; Ouyang, Nengtai; Chen, Wei-Jung A; Cudd, Timothy A

    2008-08-01

    Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

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

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    Adrián Ruiz-Villalba

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

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

    OpenAIRE

    2013-01-01

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

  13. Fetal haemoglobin in sickle-cell disease: from genetic epidemiology to new therapeutic strategies.

    Science.gov (United States)

    Lettre, Guillaume; Bauer, Daniel E

    2016-06-18

    Sickle-cell disease affects millions of individuals worldwide, but the global incidence is concentrated in Africa. The burden of sickle-cell disease is expected to continue to rise over the coming decades, adding to stress on the health infrastructures of many countries. Although the molecular cause of sickle-cell disease has been known for more than half a century, treatment options remain greatly limited. Allogeneic haemopoietic stem-cell transplantation is the only existing cure but is limited to specialised clinical centres and remains inaccessible for most patients. Induction of fetal haemoglobin production is a promising strategy for the treatment of sickle-cell disease. In this Series paper, we review scientific breakthroughs in epidemiology, genetics, and molecular biology that have brought reactivation of fetal haemoglobin to the forefront of sickle-cell disease research. Improved knowledge of the regulation of fetal haemoglobin production in human beings and the development of genome editing technology now support the design of innovative therapies for sickle-cell disease that are based on fetal haemoglobin.

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

    Science.gov (United States)

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

    2013-10-01

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

  15. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

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    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  16. Self-renewal and pluripotency is maintained in human embryonic stem cells by co-culture with human fetal liver stromal cells expressing hypoxia inducible factor 1alpha.

    Science.gov (United States)

    Ji, Lei; Liu, Yu-xiao; Yang, Chao; Yue, Wen; Shi, Shuang-shuang; Bai, Ci-xian; Xi, Jia-fei; Nan, Xue; Pei, Xue-Tao

    2009-10-01

    Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Recent evidence suggests that stem cells are localized in the microenvironment of low oxygen. We hypothesized that hypoxia could maintain the undifferentiated phenotype of embryonic stem cells. We have co-cultured a human embryonic cell line with human fetal liver stromal cells (hFLSCs) feeder cells stably expressing hypoxia-inducible factor-1 alpha (HIF-1alpha), which is known as the key transcription factor in hypoxia. The results suggested HIF-1alpha was critical for preventing differentiation of hES cells in culture. Consistent with this observation, hypoxia upregulated the expression of Nanog and Oct-4, the key factors expressed in undifferentiated stem cells. We further demonstrated that HIF-1alpha could upregulate the expression of some soluble factors including bFGF and SDF-1alpha, which are released into the microenvironment to maintain the undifferentiated status of hES cells. This suggests that the targets of HIF-1alpha are secreted soluble factors rather than a cell-cell contact mechanism, and defines an important mechanism for the inhibition of hESCs differentiation by hypoxia. Our findings developed a transgene feeder co-culture system and will provide a more reliable alternative for future therapeutic applications of hES cells.

  17. RNA-Seq Reveals the Angiogenesis Diversity between the Fetal and Adults Bone Mesenchyme Stem Cell.

    Science.gov (United States)

    Zhao, Xin; Han, Yingmin; Liang, Yu; Nie, Chao; Wang, Jian

    2016-01-01

    In this research, we used RNA sequencing (RNA-seq) to analyze 23 single cell samples and 2 bulk cells sample from human adult bone mesenchyme stem cell line and human fetal bone mesenchyme stem cell line. The results from the research demonstrated that there were big differences between two cell lines. Adult bone mesenchyme stem cell lines showed a strong trend on the blood vessel differentiation and cell motion, 48/49 vascular related differential expressed genes showed higher expression in adult bone mesenchyme stem cell lines (Abmsc) than fetal bone mesenchyme stem cell lines (Fbmsc). 96/106 cell motion related genes showed the same tendency. Further analysis showed that genes like ANGPT1, VEGFA, FGF2, PDGFB and PDGFRA showed higher expression in Abmsc. This work showed cell heterogeneity between human adult bone mesenchyme stem cell line and human fetal bone mesenchyme stem cell line. Also the work may give an indication that Abmsc had a better potency than Fbmsc in the future vascular related application.

  18. Fibroblast and lymphoblast gene expression profiles in schizophrenia: are non-neural cells informative?

    Directory of Open Access Journals (Sweden)

    Nicholas A Matigian

    Full Text Available Lymphoblastoid cell lines (LCLs and fibroblasts provide conveniently derived non-neuronal samples in which to investigate the aetiology of schizophrenia (SZ using gene expression profiling. This assumes that heritable mechanisms associated with risk of SZ have systemic effects and result in changes to gene expression in all tissues. The broad aim of this and other similar studies is that comparison of the transcriptomes of non-neuronal tissues from SZ patients and healthy controls may identify gene/pathway dysregulation underpinning the neurobiological defects associated with SZ. Using microarrays consisting of 18,664 probes we compared gene expression profiles of LCLs from SZ cases and healthy controls. To identify robust associations with SZ that were not patient or tissue specific, we also examined fibroblasts from an independent series of SZ cases and controls using the same microarrays. In both tissue types ANOVA analysis returned approximately the number of differentially expressed genes expected by chance. No genes were significantly differentially expressed in either tissue when corrected for multiple testing. Even using relaxed parameters (p or = 2-fold change between the groups of SZ cases and controls common to both LCLs and fibroblasts. We conclude that despite encouraging data from previous microarray studies assessing non-neural tissues, the lack of a convergent set of differentially expressed genes associated with SZ using fibroblasts and LCLs indicates the utility of non-neuronal tissues for detection of gene expression differences and/or pathways associated with SZ remains to be demonstrated.

  19. Bone Morphogenetic Proteins stimulate mammary fibroblasts to promote mammary carcinoma cell invasion.

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    Philip Owens

    Full Text Available Bone Morphogenetic Proteins (BMPs are secreted cytokines that are part of the Transforming Growth Factor β (TGFβ superfamily. BMPs have been shown to be highly expressed in human breast cancers, and loss of BMP signaling in mammary carcinomas has been shown to accelerate metastases. Interestingly, other work has indicated that stimulation of dermal fibroblasts with BMP can enhance secretion of pro-tumorigenic factors. Furthermore, treatment of carcinoma-associated fibroblasts (CAFs derived from a mouse prostate carcinoma with BMP4 was shown to stimulate angiogenesis. We sought to determine the effect of BMP treatment on mammary fibroblasts. A large number of secreted pro-inflammatory cytokines and matrix-metallo proteases (MMPs were found to be upregulated in response to BMP4 treatment. Fibroblasts that were stimulated with BMP4 were found to enhance mammary carcinoma cell invasion, and these effects were inhibited by a BMP receptor kinase antagonist. Treatment with BMP in turn elevated pro-tumorigenic secreted factors such as IL-6 and MMP-3. These experiments demonstrate that BMP may stimulate tumor progression within the tumor microenvironment.

  20. Late appearance of a type I alveolar epithelial cell marker during fetal rat lung development.

    Science.gov (United States)

    Danto, S I; Zabski, S M; Crandall, E D

    1994-10-01

    Recent studies in fetal lung using immunological and molecular probes have revealed type I and type II cell phenotypic markers in primordial lung epithelial cells prior to the morphogenesis of these cell types. We have recently developed monoclonal antibodies specific for adult type I cells. To evaluate further the temporal appearance of the type I cell phenotype during alveolar epithelial cell ontogeny, we analyzed fetal lung development using one of our monoclonal antibodies (mAb VIII B2). The epitope recognized by mAb VIII B2 first appears in the canalicular stage of fetal lung development, at approx. embryonic day 19 (E19), in occasional, faintly stained tubules. Staining with this type I cell probe becomes more intense and more widespread with increasing gestational age, during which time the pattern of staining changes. Initially, all cells of the distal epithelial tubules are uniformly labelled along their apical and basolateral surfaces. As morphological differentiation of the alveolar epithelium proceeds, type I cell immunoreactivity appears to become restricted to the apical surface of the primitive type I cells in a pattern approaching that seen in the mature lung. We concurrently analyzed developing fetal lung with an antiserum to surfactant apoprotein-A (alpha-SP-A). Consistent with the findings of others, labeling of SP-A was first detectable in scattered cuboidal cells at E18. Careful examination of the double-labeled specimens suggested that some cells were reactive with both the VIII B2 and SP-A antibodies, particularly at E20. Confocal microscopic analysis of such sections from E20 lung confirmed this impression. Three populations of cells were detected: cells labeled only with alpha-SP-A, cells labeled only with mAb VIII B2, and a smaller subset of cells labeled by both.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Ricardo Chaves Vilela

    2012-09-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2014-08-06

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

  4. [Prenatal diagnosis with fetal cells in maternal blood: report of experiences in Basal].

    Science.gov (United States)

    Holzgreve, W; Troeger, C; Schatt, S; Vial, Y; Louwen, F; Gloning, K; Hahn, S

    1998-10-24

    Currently prenatal diagnosis relies on invasive procedures such as chorion villus sampling (CVS) or amniocentesis (AC). Many parents are reluctant to expose themselves and their child to the small, but significant risk posed by these procedures to mother and child. There is, hence, a great need for a risk-free non-invasive alternative. To achieve this goal most research has been focussed on enriching fetal cells from the blood of pregnant women. The erythroblast has emerged as the target cell of choice, since it is abundant in the early fetus, rare in normal adult blood, and since it has a very short half life, there is no risk of obtaining cells from previous pregnancies. Most enrichment protocols rely either on magnetic- or fluorescent activated cell sorting (MACS and FACS) using fetal specific antibodies. These enriched cells can be examined by FISH (fluorescence in-situ hybridisation) for the presence of the most common fetal chromosomal aneuploidies (13, 18, 21, X and Y) or by polymerase chain reaction (PCR) on singly manipulated cells for genetic disorders. The efficacy in detecting fetal aneuploidies is currently being evaluated in a phase II clinical trial under the auspices of the NIH-NICHD, the so-called NIFTY Trial, in which our group is a participant. By modifying our enrichment protocols we have recently been able to obtain detection sensitivities of almost 80%, thereby renewing our optimism that this methodology provides a solid basis for an effective non-invasive prenatal diagnostic test.

  5. Allogeneic fetal stem cell transplantation to child with psychomotor retardation: A case report

    Directory of Open Access Journals (Sweden)

    Dajić Katerina

    2016-01-01

    Full Text Available Introduction. The consequences of autologous and allogeneic stem cell transplantation (stem cells of hematopoiesis, applied in adults and children suffering from leukemia or some other malignant disease, are well-known and sufficiently recognizable in pediatric clinical practice regardless of the indication for the treatment. However, the efficacy of fetal stem cell transplantation is unrecognizable when the indications are psychomotor retardation and epilepsy. Case Outline. With the exception of neurological psychiatric problems, a boy aged 9.5 years was in good general health before transplantation with allogeneic fetal stem cells. The main aim of allogeneic fetal stem cell transplantation was treatment of psychomotor retardation and epilepsy. After 13 months of treatment, he was admitted to hospital in a very serious, life-threatening condition due to sepsis and severe pleuropneumonia. The humoral immunity in the boy was adequate, unlike cellular immunity. The immune imbalance in terms of predominance of T-suppressor lymphocytes contributes to delayed and late development of sepsis and severe pleuropneumonia. The boy still shows the same severity of psychomotor retardation, dyslalia, epilepsy, strabismus and amblyopia. Conclusion. Implementation of fetal stem cell therapy for unconfirmed indications abuses the therapeutic approach, harms patients, misleads parents, and brings financial harm to the healthcare system of any country, including Serbia.

  6. Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

    Science.gov (United States)

    Baerenwaldt, Anne; von Burg, Nicole; Kreuzaler, Matthias; Sitte, Selina; Horvath, Edit; Peter, Annick; Voehringer, David; Rolink, Antonius G; Finke, Daniela

    2016-03-15

    Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4β7(-) and α4β7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life.

  7. Indirect coculture of stem cells with fetal chondrons using PCL electrospun nanofiber scaffolds.

    Science.gov (United States)

    Nikpou, Parisa; Soleimani Rad, Jafar; Mohammad Nejad, Daryoush; Samadi, Nasser; Roshangar, Leila; Navali, Amir Mohammad; Shafaei, Hajar; Nozad Charoudeh, Hojjatollah; Danandeh Oskoei, Neda; Soleimani Rad, Sara

    2017-03-01

    In vitro coculture system provides a powerful tool for tissue engineering. In this study, we evaluated the gene expressions of human adipose-derived stem cells (ASCs) on polycaprolactone (PCL) scaffold in coculture model with fetal chondrons. Electrospun PCL scaffolds (900 nm fiber diameter) were created and human infrapatellar fat pad-adipose-derived stem cells (IPFP-ASCs) were seeded on these scaffolds. Scanning electron microscopy (SEM) showed attachment of human IPFP-ASCs to scaffold. IPFP-ASCs on scaffolds were cocultured with fetal chondrons in transwell. Gene expressions were investigated using real-time polymerase chain reaction (real-time PCR). In comparison with control group, the expression level of collagen type 2 and aggrecan were significantly decreased but Indian Hedgehog(IHH) significantly increased (P fetal chondrons are tending toward osteogenesis rather than chondrogenesis.

  8. Relationships of CD163 and CD169 positive cell numbers in the endometrium and fetal placenta with type 2 PRRSV RNA concentration in fetal thymus.

    Science.gov (United States)

    Novakovic, Predrag; Harding, John C S; Ladinig, Andrea; Al-Dissi, Ahmad N; MacPhee, Daniel J; Detmer, Susan E

    2016-08-05

    Several routes of porcine reproductive and respiratory virus PRRSV transmission across the porcine diffuse epitheliochorial placentation have been proposed, but none have been proven. The objectives of this study were to investigate associations between numbers of CD163 and CD169 positive macrophages, cathepsin positive areolae, and type 2 PRRSV load at the maternal-fetal interface in order to examine important factors related to transplacental infection. On gestation day 85 ± 1, naïve pregnant gilts were inoculated with PRRSV (n = 114) or were sham inoculated (n = 19). At 21 days post-inoculation (dpi), dams and their litters were humanely euthanized and necropsied. Samples of the maternal-fetal interface (uterus with fully attached placenta) and fetal thymus were collected for analysis by RT-qPCR to quantify PRRSV RNA concentration. The corresponding paraffin-embedded uterine tissue sections were subjected to immunohistochemistry for PRRSV nucleocapsid N protein, CD163, CD169, and cathepsin. Our findings confirm significant increases in the numbers of PRRSV, CD163 and CD169 positive cells at the maternal-fetal interface during type 2 PRRSV infection in pregnant gilts. PRRSV load in fetal thymus was positively related to CD163(+) cell count in endometrium and negatively related to CD163(+) cell count in placenta, but unrelated to CD169 counts or cathepsin positive areolae. The endometrium:placenta ratio of CD163 cells, and to a lesser extent CD169 cells, was significantly associated with an increase fetal viral load in thymus. These findings suggest a more important role for CD163(+) cells following trans-placental PRRSV infection, but dichotomous responses in endometrium and placenta for both CD163 and CD169 cells.

  9. Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle.

    Science.gov (United States)

    Lei, Hulong; Yu, Bing; Huang, Zhiqing; Yang, Xuerong; Liu, Zehui; Mao, Xiangbing; Tian, Gang; He, Jun; Han, Guoquan; Chen, Hong; Mao, Qian; Chen, Daiwen

    2013-02-01

    Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P cells (P fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.

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

    OpenAIRE

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

    2011-01-01

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

  11. Effects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocytes

    OpenAIRE

    Hu, Li; ZHAO, JIAJIA; Liu, Jiarong; Gong, Niya; Chen, Lili

    2013-01-01

    Adipose stem cell-conditioned medium (ASC-CM) has been successfully used to treat multiple types of tissue and organ defects, including skin wounds both in vitro and in vivo. However, the mechanisms through which ASC-CM promotes wound healing remain unclear. We hypothesized that the wound healing effect of ASC-CM is mediated in part by the promotion of the migration of vascular endothelial cells, fibroblasts and keratinocytes, the three cell types essential for wound healing. We reported that...

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

    OpenAIRE

    Patwardhan, Juilee; Bhatt, Purvi

    2015-01-01

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

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

    OpenAIRE

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

    1995-01-01

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

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

    OpenAIRE

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2013-05-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  17. SCANNING ELECTRON MICROSCOPIC STUDY OF FETAL CHICKEN CALVARIAL OSTEOBLAST-LIKE CELLS CULTURED IN VITRO

    Institute of Scientific and Technical Information of China (English)

    柴本甫; 汤雪明; 徐荣辉; 朱雅萍

    1993-01-01

    Three types of osteoblast-like cells with different cnfigurations could be ob-tained through culturing fetal chicken calvaria in vitro. They were spindle-shaped cells,globular cells, and polygonal or squamous cells. With passage of culture time, there werechanges in configuration so that the spindle-shaped cells and the globular cells turnedgradually into squamous cells, in quantity which increased greatly to produce confluenceand multi-layer formation of cells, and in function as evidenced by emergence ofintracytoplasmic granules, reflecting collagen synthesis.

  18. CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo

    Directory of Open Access Journals (Sweden)

    Benjamin T. Spike

    2014-04-01

    Full Text Available Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers.

  19. CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo

    Science.gov (United States)

    Spike, Benjamin T.; Kelber, Jonathan A.; Booker, Evan; Kalathur, Madhuri; Rodewald, Rose; Lipianskaya, Julia; La, Justin; He, Marielle; Wright, Tracy; Klemke, Richard; Wahl, Geoffrey M.; Gray, Peter C.

    2014-01-01

    Summary Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers. PMID:24749068

  20. The many roads to Rome: induction of neural precursor cells from fibroblasts.

    Science.gov (United States)

    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.

  1. Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells.

    Science.gov (United States)

    Castoria, G; Giovannelli, P; Di Donato, M; Ciociola, A; Hayashi, R; Bernal, F; Appella, E; Auricchio, F; Migliaccio, A

    2014-12-04

    The functions of androgen receptor (AR) in stromal cells are still debated in spite of the demonstrated importance of these cells in organ development and diseases. Here, we show that physiological androgen concentration (10 nM R1881 or DHT) fails to induce DNA synthesis, while it consistently stimulates cell migration in mesenchymal and transformed mesenchymal cells. Ten nanomolar R1881 triggers p27 Ser10 phosphorylation and its stabilization in NIH3T3 fibroblasts. Activation of Rac and its downstream effector DYRK 1B is responsible for p27 Ser10 phosphorylation and cell quiescence. Ten nanomolar androgen also inhibits transformation induced by oncogenic Ras in NIH3T3 fibroblasts. Overexpression of an AR mutant unable to interact with filamin A, use of a small peptide displacing AR/filamin A interaction, and filamin A knockdown indicate that the androgen-triggered AR/filamin A complex regulates the pathway leading to p27 Ser10 phosphorylation and cell cycle arrest. As the AR/filamin A complex is also responsible for migration stimulated by 10 nM androgen, our report shows that the androgen-triggered AR/filamin A complex controls, through Rac 1, the decision of cells to halt cell cycle and migration. This study reveals a new and unexpected role of androgen/AR signalling in coordinating stromal cell functions.

  2. Transmission of HIV-1 by primary human uterine epithelial cells and stromal fibroblasts.

    Science.gov (United States)

    Asin, Susana N; Fanger, Michael W; Wildt-Perinic, Dunja; Ware, Patricia L; Wira, Charles R; Howell, Alexandra L

    2004-07-15

    Women can become infected with human immunodeficiency virus type 1 (HIV-1) after the heterosexual transmission of virus from an infected male partner. To understand the events that result in transmission of HIV-1 across the female reproductive tract, we characterized the life-cycle events of HIV-1 in primary cultures of human uterine epithelial cells and stromal fibroblasts. Epithelial cells and stromal fibroblasts released virus particles after exposure to either X4- or R5-tropic strains of HIV-1. Virus released by these cells was able to infect CD4(+) T cells. When exposed to an X4-tropic strain of HIV-1, these cells supported HIV-1 reverse transcription, integration, and viral DNA transcription. When exposed to an R5-tropic strain, however, these cells released unmodified virus. These data suggest that uterine cells are targets for productive infection with X4-tropic strains and release unmodified R5-tropic viruses that would then be able to infect submucosal target cells, including T cells and macrophages.

  3. Cytotoxic effects of new MTA-based cement formulations on fibroblast-like MDPL-20 cells.

    Science.gov (United States)

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

    Directory of Open Access Journals (Sweden)

    Hany Mohamed Aly Ahmed

    2011-01-01

    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.

  5. Fetal Nerve Cell Transplantation in Early Post-Resuscitation Period in Rats

    Directory of Open Access Journals (Sweden)

    Damira Tazhibayeva

    2015-02-01

    Full Text Available Introduction. Fetal cell transplantation is a promising biomedical approach for disease treatment; however, the use of fetal cell therapy is still experimental. This research was deemed a necessity to provide evidence-based research for the application of cell transplantation as a treatment method. The aim of this study was to evaluate the effect of fetal nerve cell transplantation in rat survivors (and non-survivors after clinical death by mechanical asphyxia.Methods. 68 white laboratory rats were divided into two groups of identical age and sex: a control group of 12-month adult male rats (n = 26 and an experimental group (n = 42. Rats were fixed under ether anesthesia. We then blocked the oral and nasal regions with cotton wool soaked in saline solution. A four-minute clinical death though acute mechanical asphyxia was simulated by applying the method of N. Shim. After the 4-minute clinical death, we resuscitated the rats using external cardiac massage and artifical respiration. Suspension of the fetal nerve cells was injected intraperitoneally at 1mm3 per 25g at the time of cardiac activity restoration. Lactate dehydrogenase (LDH and creatine phosphokinase (CPK levels were examined in the homogenate cerebral cortex of reanimated animals. We recorded the survival rate during the post-resuscitation period and analyzed the integrative brain functions using anxiety-phobic status and latent inhibition.Results. After fetal nerve cell transplantation, the enzymatic reactions in the experimental group became normal with a significant decrease in LDH and an increase in CPK levels compared to the control group. In the control group, 10 rats died and 16 lived (62% survival rate, while 7 rats died and 35 lived (83% survival rate in the experimental group during the first 7 days. Rats that did not receive the treatment tended to die sooner than those in the experimental group. As a result of transplantation, the anxiety level in the experimental group

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

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    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}.

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

    Science.gov (United States)

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

    2011-06-14

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

  8. Fetal vs adult mesenchymal stem cells achieve greater geneexpression, but less osteoinduction

    Institute of Scientific and Technical Information of China (English)

    Juan E Santiago-Torres; Rebecca Lovasz; Alicia L Bertone

    2015-01-01

    AIM To investigate adenoviral transduction inmesenchymal stem cells (MSCs) and effects onstemness in vitro and function as a cell therapy in vivo .METHODS: Bone marrow-derived adult and fetal MSCwere isolated from an equine source and expandedin monolayer tissue culture. Polyethylenimine (PEI)-mediated transfection of pcDNA3-eGFP or adenoviraltransduction of green fluorescent protein (GFP)was evaluated in fetal MSCs. Adenoviral-mediatedtransduction was chosen for subsequent experiments. Allexperiments were carried out at least in triplicate unlessotherwise noted. Outcome assessment was obtained byflow cytometry or immunohystochemistry and includedtransduction efficiency, cell viability, stemness (i.e. ,cell proliferation, osteogenic and chondrogenic celldifferentiation), and quantification of GFP expression.Fetal and adult MSCs were then transduced with anadenoviral vector containing the gene for the bonemorphogenic protein 2 (BMP2). In vitro BMP2 expressionwas assessed by enzyme linked immunosorbent assay.In addition, MSC-mediated gene delivery of BMP2 wasevaluated in vivo in an osteoinduction nude mousequadriceps model. New bone formation was evaluated bymicroradiography and histology.RESULTS: PEI provided greater transfection andviability in fetal MSCs than other commercial chemicalreagents. Adenoviral transduction efficiency wassuperior to PEI-mediated transfection of GFP in fetalMSCs (81.3% ± 1.3% vs 35.0% ± 1.6%, P 〈 0.05) andwas similar in adult MSCs (78.1% ± 1.9%). Adenoviraltransduction provided significantly greater expressionof GFP in fetal than adult MSCs (7.4 ± 0.1 vs 4.4 ± 0.3millions of mean fluorescence intensity units, P 〈 0.01)as well as significantly greater in vitro BMP2 expression(0.16 pg/cell-day vs 0.10 pg/cell-day, P 〈 0.01). Fractionof fetal MSC GFP positive cells decreased significantlyfaster than adult MSCs (1.15% ± 0.05% vs 11.4% ±2.1% GFP positive at 2 wk post-transduction, P 〈 0

  9. Gingival fibroblasts as a promising source of induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hiroshi Egusa

    Full Text Available BACKGROUND: Induced pluripotent stem (iPS cells efficiently generated from accessible tissues have the potential for clinical applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort. METHODOLOGY/PRINCIPAL FINDINGS: We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts (GFs via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells or three factors (the same as GF-iPS-4F cells, but without the c-Myc oncogene; GF-iPS-3F cells without drug selection. iPS cells were also generated from primary human gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic stem (ES cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity. CONCLUSIONS/SIGNIFICANCE: These results suggest that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications

  10. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    Science.gov (United States)

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  11. Establishment and Characterization of Fibroblast Cell Line Derived from Siberian Tiger (Panthera tigris altaica).

    Science.gov (United States)

    Liu, Changqing; Guo, Yu; Liu, Dan; Guan, Weijun; Ma, Yuehui

    2010-06-01

    The Siberian tiger ear marginal tissue fibroblast cell line (STF34) from 34 samples was successfully established using primary explants technique and cell cryoconservation technology. STF34 cells were adherent, with a population doubling time of 24 h. Chromosome analysis showed that 90.2%-91.6% of cells were diploid (2n = 38). Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that STF34 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses, and mycoplasmas were all negative. Every index of the STF34 cell line meets all the standard quality controls of American Type Culture Collection. Not only has the germline of this important Siberian tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome, and somacloning research.

  12. Aging Impairs the Proliferative Capacity of Cardiospheres, Cardiac Progenitor Cells and Cardiac Fibroblasts: Implications for Cell Therapy

    Directory of Open Access Journals (Sweden)

    Jianqin Ye

    2013-09-01

    Full Text Available Introduction: Cardiospheres (CS are self-assembling clusters of cells that can be grown from cardiac tissue. They contain a heterogeneous cell population that includes cardiac progenitor cells (CPCs and cardiac fibroblasts. CS and CPCs have been shown to improve cardiac function after myocardial infarction (MI in experimental models and are now being studied in clinical trials. The effects of aging on the proliferative capacity of CS and CPCs, and the paracrine signaling between cell types, remain incompletely understood. Methods and Results: We compared the growth of CS from young and aging murine hearts at baseline and following MI. The number of CS from young and aging hearts was similar at baseline. However, after MI, young hearts had a dramatic increase in the number of CS that grew, but this proliferative response to MI was virtually abolished in the aging heart. Further, the proportion of cells within the CS that were CPCs (defined as Sca-1(stem cell antigen-1+/CD45− was significantly lower in aging hearts than young hearts. Thus the number of available CPCs after culture from aging hearts was substantially lower than from young hearts. Cardiac fibroblasts from aging hearts proliferated more slowly in culture than those from young hearts. We then investigated the interaction between aging cardiac fibroblasts and CPCs. We found no significant paracrine effects on proliferation between these cell types, suggesting the impaired proliferation is a cell-autonomous problem. Conclusions: Aging hearts generate fewer CPCs, and aging CPCs have significantly reduced proliferative potential following MI. Aging cardiac fibroblasts also have reduced proliferative capacity, but these appear to be cell-autonomous problems, not caused by paracrine signaling between cell types.

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

    Science.gov (United States)

    Yang, Eun-Jung; Bang, Sa-Ik

    2017-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Liu Wei

    2010-06-01

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

  15. Induction of growth and proliferation of fibroblast cells in magnetic field

    Directory of Open Access Journals (Sweden)

    Naghmeh Ezatti

    2015-02-01

    Full Text Available Background: Tissue engineering is generally defined as developing and changing the laboratory growth of molecules and cells in tissues or organs to replace and repair the damaged part of body. This study was carried out to stimulate the growth of cultured fibroblast cells by a physical electromagnetic method. Methods: First, an air-core coil was prepared and the cell culture plate was placed comfortably into the mold, then the plate containing the culture medium and human fibroblast cell along with air-core coil were placed in an incubator and then connected to the power supply. Thus, the sample underwent electromagnetic field at different times, and cell proliferation was studied by MTTassay. Results: Microscopic images indicated that the cells undergoing electromagnetic field (0.35 amps had a significant growth compared to the cells in control group in a definit range of stimulation. Conclusion: In conclusion, electromagnetic stimulation in a definite range led to cell proliferation and could be used as a positive factor in tissue engineering.

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

    Science.gov (United States)

    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

  17. Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

    Science.gov (United States)

    Ho, Sherry Sze Yee; Barrett, Angela; Thadani, Henna; Asibal, Cecille Laureano; Koay, Evelyn Siew-Chuan; Choolani, Mahesh

    2015-07-01

    Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  19. Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment.

    Science.gov (United States)

    Ghosn, Eliver Eid Bou; Waters, Jeffrey; Phillips, Megan; Yamamoto, Ryo; Long, Brian R; Yang, Yang; Gerstein, Rachel; Stoddart, Cheryl A; Nakauchi, Hiromitsu; Herzenberg, Leonore A

    2016-01-12

    B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  1. Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

    NARCIS (Netherlands)

    Oosterwijk, JC; Mesker, WE; Ouwerkerk-Van Velzen, MCM; Knepfle, CFHM; Wiesmeijer, KC; Beverstock, GC; Van Ommen, GJB; Tanke, HJ; Kanhai, HHH

    1998-01-01

    In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and

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

    Energy Technology Data Exchange (ETDEWEB)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

    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}.

  3. Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions.

    Science.gov (United States)

    Klimchenko, Olena; Di Stefano, Antonio; Geoerger, Birgit; Hamidi, Sofiane; Opolon, Paule; Robert, Thomas; Routhier, Mélanie; El-Benna, Jamel; Delezoide, Anne-Lise; Boukour, Siham; Lescure, Bernadette; Solary, Eric; Vainchenker, William; Norol, Françoise

    2011-03-17

    The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

  4. Human fetal chromaffin cells: a potential tool for cell pain therapy.

    Science.gov (United States)

    Jozan, Suzanne; Aziza, Jacqueline; Châtelin, Sophie; Evra, Corinne; Courtade-Saïdi, Monique; Parant, Olivier; Sol, Jean Christophe; Zhou, Huafang; Lazorthes, Yves

    2007-06-01

    Transplantation of adrenal medulla cells has been proposed in the treatment of various conditions. Indeed, these cells possess a bipotentiality: neural and neuroendocrine, which could be exploited for brain repair or pain therapy. In a previous study, we characterized these human cells in vitro over 7-10 gestational weeks (GW) [Zhou, H., Aziza, J., Sol, J.C., Courtade-Saidi, M., Chatelin, S., Evra, C., Parant, O., Lazorthes, Y., and Jozan, S., 2006. Cell therapy of pain: Characterization of human fetal chromaffin cells at early adrenal medulla development. Exp. Neurol. 198, 370-381]. We report here our results on the extension to 23 GW. This developmental period can be split into three stages. During the first stage (7-10 GW), we observed in situ that extra-adrenal surrounding cells display the same morphology and phenotype as the intra-adrenal chromaffin cells. We also found that the intra-adrenal chromaffin cells could be committed in vitro towards an adrenergic phenotype using differentiating agents. During the second stage (11 to 15-16 GW), two types of cells (Type 1 and Type 2 cells) were identified morphologically both inside and outside the gland. Interestingly, we noted that the Type 2 cells stem from the Type 1 cells. However, during this developmental period only the intra-adrenal Type 2 cells will evolve towards an adrenergic phenotype. In the third stage (17-23 GW), we observed the ultimate location of the medulla gland. Both the in situ results and the in vitro experiments indicate that particular procedures need to be implemented prior transplantation of chromaffin cells. First, in order to obtain a large number of immature chromaffin cells, they must be isolated from the intra and extra-adrenal gland and should then be committed towards an adrenergic phenotype in vitro for subsequent use in pain therapy. This strategy is under investigation in our laboratory.

  5. Biphasic electrical currents stimulation promotes both proliferation and differentiation of fetal neural stem cells.

    Directory of Open Access Journals (Sweden)

    Keun-A Chang

    Full Text Available The use of non-chemical methods to differentiate stem cells has attracted researchers from multiple disciplines, including the engineering and the biomedical fields. No doubt, growth factor based methods are still the most dominant of achieving some level of proliferation and differentiation control--however, chemical based methods are still limited by the quality, source, and amount of the utilized reagents. Well-defined non-chemical methods to differentiate stem cells allow stem cell scientists to control stem cell biology by precisely administering the pre-defined parameters, whether they are structural cues, substrate stiffness, or in the form of current flow. We have developed a culture system that allows normal stem cell growth and the option of applying continuous and defined levels of electric current to alter the cell biology of growing cells. This biphasic current stimulator chip employing ITO electrodes generates both positive and negative currents in the same culture chamber without affecting surface chemistry. We found that biphasic electrical currents (BECs significantly increased the proliferation of fetal neural stem cells (NSCs. Furthermore, BECs also promoted the differentiation of fetal NSCs into neuronal cells, as assessed using immunocytochemistry. Our results clearly show that BECs promote both the proliferation and neuronal differentiation of fetal NSCs. It may apply to the development of strategies that employ NSCs in the treatment of various neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.

  6. Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Yukihiro Nishikawa

    Full Text Available Withaferin A (WA, a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD. WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L. Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

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

    Science.gov (United States)

    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.

  8. The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

    Science.gov (United States)

    Wang, Mei; Wu, Chunping; Guo, Yu; Cao, Xiaojuan; Zheng, Wenwei; Fan, Guo-Kang

    2017-05-01

    Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no

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

    Directory of Open Access Journals (Sweden)

    Zhisen Shen

    2013-01-01

    Full Text Available 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.

  10. Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum

    OpenAIRE

    Ziegler, A; Everett, H.; Hamza, E; Garbani, M; Gerber, V.; Marti, E; Steinbach, F

    2016-01-01

    Background: Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. ...

  11. Fibroblast growth factor 9 activates akt and MAPK pathways to stimulate steroidogenesis in mouse leydig cells.

    Science.gov (United States)

    Lai, Meng-Shao; Cheng, Yu-Sheng; Chen, Pei-Rong; Tsai, Shaw-Jenq; Huang, Bu-Miin

    2014-01-01

    Fibroblast growth factor 9 (FGF9) is a multifunctional polypeptide belonging to the FGF family and has functions related to bone formation, lens-fiber differentiation, nerve development, gap-junction formation and sex determination. In a previous study, we demonstrated that FGF9 stimulates the production of testosterone in mouse Leydig cells. In the present study, we used both primary mouse Leydig cells and MA-10 mouse Leydig tumor cells to further investigate the molecular mechanism of FGF9-stimulated steroidogenesis. Results showed that FGF9 significantly activated steroidogenesis in both mouse primary and tumor Leydig cells (psteroidogenesis in mouse Leydig cells. In conclusion, FGF9 specifically activated the Akt and ERK1/2 in normal mouse Leydig cells and the Akt, JNK and ERK1/2 in MA-10 mouse Leydig tumor cells to stimulate steroidogenesis.

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

    Directory of Open Access Journals (Sweden)

    Tan Jenny

    2005-03-01

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

  13. Piezoelectric Drop-on-Demand Inkjet Printing of Rat Fibroblast Cells: Survivability Study and Pattern Printing

    CERN Document Server

    Li, Er Qiang; Thoroddsen, Sigurdur Tryggvi

    2013-01-01

    A novel piezoelectric, drop-on-demand (DOD) inkjet system has been developed and used to print L929 rat fibroblast cells. We investigate the survivability of the cells subjected to the large stresses during the printing process. These stresses are varied by changing the diameter of the orifice (36 to 119 microns) through which the cells are dispensed, as well as changing the electrical pulse used to drive the piezoelectric element. It is shown that for the smallest 36 microns diameter orifice, cell survival rates fall from 95% to approximately 76% when the ejection velocity is increased from 2 to 16 m/s. This decrease in survival rates is less significant when the larger orifice diameters of 81 microns and 119 microns are used. Analysis shows that there is a clear inverse relationship between cell survival rates and the mean shear rates during drop formation. By using the same printing set-up, fibroblast cells are printed onto alginate and collagen into patterns. Printed cells are cultured over a period of da...

  14. Deficient recovery from potentially lethal damage in some gamma-irradiated human fibroblast cell strains

    Energy Technology Data Exchange (ETDEWEB)

    Arlett, C.F.; Priestley, A. (Medical Research Council, Brighton (UK). Cell Mutation Unit)

    1984-01-01

    The repair of potentially lethal damage following treatment with gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0.5% foetal calf serum. Normal cells were found to be competent in the repair of PLD. Ataxia-telangiectasia cells were deficient as was a heterozygote suggesting that a failure to repair PLD may make it possible to detect such heterozygotes. Fibroblasts from Huntington's disease patients were either slightly or no more sensitive than cells from normal individuals. Cultures from two individuals in the former class showed limited capacity to repair PLD but cells from the latter class were as competent as normals. Thus assays of radiosensitivity where conditions allow for the repair of PLD may maximise small differences in sensitivity. Cells taken from three patients suffering from Basal Cell Naevus Syndrome were also shown to be defective in the repair of PLD. The existence of such a defect may be related to the increased frequency of basal cell cancer observed in exposed fields following irradiation of such individuals.

  15. Fast deposition of hydroxyapatite coating on titanium to modify cell affinity of corneal fibroblast in vitro

    Institute of Scientific and Technical Information of China (English)

    WANG Xiaoping; MA Xiao; WANG Leyun; DU Xuan; HUANG Yifei; CUI Fuzhai

    2007-01-01

    By two step acid-alkali pretreatment and lmmersing into supersaturated calcification solution,hydroxyapatite (HA)coating was deposited on titanium(Ti)discs.The composition,surface morphology and cross-section of the coating were analyzed by X-ray diffraction(XRD)and scanning electron microscopy (SEM).Fibroblasts of rabbit cornea were seeded on HA coated Ti disc,pure Ti disc and glass.Cell adhesion,proliferation and morphology were detected at 24,48 and 72h,respectively.It is shown for the first time that HA coating can significantly enhance the adhesion and proliferation of rabbit corneal fibroblast in comparison with that of pure Ti.

  16. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    Science.gov (United States)

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process. Images Fig. 2. Fig. 3. PMID:6315397

  17. Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

    Science.gov (United States)

    Korpela, J; Kulomaa, M; Tuohimaa, P; Vaheri, A

    1983-01-01

    Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.

  18. Stem cells decreased neuronal cell death after hypoxic stress in primary fetal rat neurons in vitro.

    Science.gov (United States)

    Sakai, Tetsuro; Xu, Yan

    2012-01-01

    To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h nad then were returned to a normoxic condition. The study group then received rat umbilical cord matrix-derived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rα (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK α1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

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

    Science.gov (United States)

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

    2008-02-01

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

  20. Ontological differences in first compared to third trimester human fetal placental chorionic stem cells.

    Directory of Open Access Journals (Sweden)

    Gemma N Jones

    Full Text Available Human mesenchymal stromal/stem cells (MSC isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC. We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.

  1. Fetal Sex Determination using Non-Invasive Method of Cell-free Fetal DNA in Maternal Plasma of Pregnant Women During 6th– 10th Weeks of Gestation

    Science.gov (United States)

    Zargari, Maryam; Sadeghi, Mohammad Reza; Shahhosseiny, Mohammad Hassan; Kamali, Koroush; Saliminejad, Kyomars; Esmaeilzadeh, Ali; Khorshid, Hamid Reza Khorram

    2011-01-01

    In previous years, identification of fetal cells in maternal blood circulation has caused a new revolution in non-invasive method of prenatal diagnosis. Low number of fetal cells in maternal blood and long-term survival after pregnancy limited the use of fetal cells in diagnostic and clinical applications. With the discovery of cell-free fetal DNA (cffDNA) in plasma of pregnant women, access to genetic material of the fetus had become possible to determine early gender of a fetus in pregnancies at the risk of X-linked genetic conditions instead of applying invasive methods. Therefore in this study, the probability of detecting sequences on the Y chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women at 6th to 10th weeks of gestation and then the fetal DNA was extracted from the plasma. Nested PCR was applied to detect the sequences of single copy SRY gene and multi copy DYS14 & DAZ genes on the Y chromosome of the male fetuses. At the end, all the obtained results were compared with the actual gender of the newborns. In 40 out of 42 born baby boys, the relevant gene sequences were identified and 95.2% sensitivity was obtained. Non-invasive early determination of fetal gender using cffDNA could be employed as a pre-test in the shortest possible time and with a high reliability to avoid applying invasive methods in cases where a fetus is at the risk of genetic diseases. PMID:23407464

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    Objective:To estimate electroporation (EP) influence on malignant and normal cells.Methods:Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following:250,1000,1750,2500 V/cm;50 μs by5 impulses for every case. The viability of cells after EP was estimated byMTT assay. The ultrastructural analysis was observed by transmission electron microscope (ZeissEM900). Results:In the current study we observed the intracellular effect followingEP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated byEP. Conversely, we showed thatEP in some conditions can stimulate cells to proliferation. Some changes induced byEP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters ofEP (250 and1000 V/cm). After applying higher electric field intensities (2500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications afterEP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters ofEP.Conclusions:We can claim thatEP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude thatEP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.

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

    Science.gov (United States)

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

    2011-04-01

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

  4. Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape.

    Directory of Open Access Journals (Sweden)

    Mirjam Ochsner

    Full Text Available BACKGROUND: Various physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D cell cultures. Cells anchored in a three-dimensional (3-D microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D. METHODOLOGY/PRINCIPAL FINDINGS: Arrays of 5 or 10 microm deep microwells were fabricated in polydimethylsiloxane (PDMS. The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D or trapped in microwells (3-D of controlled size, shape, and wall rigidity. On rigid substrates (Young's Modulus = 1 MPa, cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area and total surface areas of adhesion (microwell bottom plus wall surface area that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa, regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant. CONCLUSION/SIGNIFICANCE: These data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity and topographical (shape and dimensionality information differently when cell

  5. Fetal and adult liver stem cells for liver regeneration and tissue engineering.

    Science.gov (United States)

    Fiegel, H C; Lange, Claudia; Kneser, U; Lambrecht, W; Zander, A R; Rogiers, X; Kluth, D

    2006-01-01

    For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.

  6. Adsorption of fibronectin and vitronectin onto Primaria and tissue culture polystyrene and relationship to the mechanism of initial attachment of human vein endothelial cells and BHK-21 fibroblasts.

    Science.gov (United States)

    Steele, J G; Dalton, B A; Johnson, G; Underwood, P A

    1995-09-01

    The two cell culture substrata, tissue culture polystyrene (TCPS) and Primaria, were compared in order to determine whether a nitrogen-containing surface such as Primaria attracts cells by a different mechanism to an oxygen-containing surface (TCPS). The amounts of vitronectin (Vn) and fibronectin (Fn) which adsorb from the fetal bovine serum (FBS) component of the culture medium onto Primaria and TCPS were determined. Primaria adsorbed two- to threefold more Fn than TCPS, but adsorbed similar amounts of Vn from medium containing FBS. The Fn and Vn binding sites on Primaria were distinct, as adsorption was non-competitive between these two proteins. The amounts of Fn and Vn that adsorbed onto the two surfaces were compared to the concentration dependence of the cell attachment activities of Fn and Vn. Whereas the amounts of Fn which adsorbed onto TCPS were suboptimal for cell attachment, Primaria adsorbed an Fn surface density that was supraoptimal for attachment of human vein endothelial cells and BHK-21 fibroblasts. We conclude that Primaria differs from TCPS in that both Fn and Vn mediate initial cell attachment to Primaria when the culture medium contains FBS, whereas cell attachment to TCPS is dependent upon Vn.

  7. Characterization of a novel fibroblast-like cell line from rainbow trout and responses to sublethal anoxia

    DEFF Research Database (Denmark)

    Ossum, Carlo Gunnar; Hoffmann, Else Kay; Vijayan, M.M.;

    2004-01-01

    A novel fibroblast-like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation...... suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress....

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

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

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

  9. Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation.

    Science.gov (United States)

    Weber, Benedikt; Emmert, Maximilian Y; Behr, Luc; Schoenauer, Roman; Brokopp, Chad; Drögemüller, Cord; Modregger, Peter; Stampanoni, Marco; Vats, Divya; Rudin, Markus; Bürzle, Wilfried; Farine, Marc; Mazza, Edoardo; Frauenfelder, Thomas; Zannettino, Andrew C; Zünd, Gregor; Kretschmar, Oliver; Falk, Volkmar; Hoerstrup, Simon P

    2012-06-01

    Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2007-11-01

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

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

    Science.gov (United States)

    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