The Experts below are selected from a list of 34572 Experts worldwide ranked by ideXlab platform
Kohji Nishida - One of the best experts on this subject based on the ideXlab platform.
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Development of Genetically Modified Eliminable Human Dermal Fibroblast Feeder Cells for Ocular Surface Regeneration Medicine
Investigative ophthalmology & visual science, 2013Co-Authors: Tomoyuki Inoue, Fumihiko Takamatsu, Naoyuki Maeda, Yuichi Ohashi, Kohji NishidaAbstract:PURPOSE Cultured human corneal limbal stem/progenitor cells are usually established and maintained on feeder layers. However, animal feeder cells are associated with viral infection, pathogen transmission, and xenogenic contamination. All feeder cells also can be mixed easily into cell-sheet production, causing self-contamination. We developed a line of labeled, immortalized, eliminable human Dermal Fibroblast cells to eliminate these problems. METHODS The enhanced green fluorescent protein gene, human-derived telomerase reverse transcriptase gene, and herpes simplex virus thymidine kinase gene were transfected into human Dermal Fibroblast cells to establish labeled, immortalized, eliminable feeder cells. Established eliminable Dermal Fibroblasts (TERT+TK-D) were treated with mitomycin, cocultured with human limbal stem/progenitor cells to regenerate epithelium sheets, and compared with 3T3 feeder cells. RESULTS Established TERT+TK-D feeder cells maintained immortalization, visualization, and eliminable characteristics during 6 months of continuous passages. The colony-forming efficiency of limbal stem/progenitor cells was similar in the TERT+TK-D group (11.77 ± 0.21%) and the 3T3 group (12.8 ± 1.61%) (P = 0.332). All cell sheets were well stratified into 4 to 5 layers. The TERT+TK-D group colonies and epithelial cell sheets showed weaker staining of corneal epithelium differentiation marker K3 than the 3T3 group and quantitative analysis of mRNA transcripts. Moreover, PCR analysis against the long terminal repeat sequence of the lentiviral vector integrated into the genetically modified feeder cells showed no contamination of ganciclovir-treated regeneration epithelial sheets. CONCLUSIONS Genetically modified, labeled, immortalized, eliminable human Dermal feeder cells are promising substitutes for 3T3 feeder cells for xenogeny-free ocular surface regeneration.
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Development of Genetically Modified Eliminable Human Dermal Fibroblast Feeder Cells for Ocular Surface Regeneration Medicine
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Yingli Li, Tomoyuki Inoue, Fumihiko Takamatsu, Naoyuki Maeda, Yuichi Ohashi, Kohji NishidaAbstract:Cultured human corneal limbal stem/progenitor cells are usually established and maintained on feeder layers. However, animal feeder cells are associated with viral infection, pathogen transmission, and xenogenic contamination. All feeder cells also can be mixed easily into cell-sheet production, causing self-contamination. We developed a line of labeled, immortalized, eliminable human Dermal Fibroblast cells to eliminate these problems. The enhanced green fluorescent protein gene, human-derived telomerase reverse transcriptase gene, and herpes simplex virus thymidine kinase gene were transfected into human Dermal Fibroblast cells to establish labeled, immortalized, eliminable feeder cells. Established eliminable Dermal Fibroblasts (TERT+TK-D) were treated with mitomycin, cocultured with human limbal stem/progenitor cells to regenerate epithelium sheets, and compared with 3T3 feeder cells. Established TERT+TK-D feeder cells maintained immortalization, visualization, and eliminable characteristics during 6 months of continuous passages. The colony-forming efficiency of limbal stem/progenitor cells was similar in the TERT+TK-D group (11.77 ± 0.21%) and the 3T3 group (12.8 ± 1.61%) (P = 0.332). All cell sheets were well stratified into 4 to 5 layers. The TERT+TK-D group colonies and epithelial cell sheets showed weaker staining of corneal epithelium differentiation marker K3 than the 3T3 group and quantitative analysis of mRNA transcripts. Moreover, PCR analysis against the long terminal repeat sequence of the lentiviral vector integrated into the genetically modified feeder cells showed no contamination of ganciclovir-treated regeneration epithelial sheets. Genetically modified, labeled, immortalized, eliminable human Dermal feeder cells are promising substitutes for 3T3 feeder cells for xenogeny-free ocular surface regeneration.
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generation of corneal epithelial cells from induced pluripotent stem cells derived from human Dermal Fibroblast and corneal limbal epithelium
PLOS ONE, 2012Co-Authors: Ryuhei Hayashi, Yuki Ishikawa, Tomofumi Kageyama, Kuniko Takashiba, Tsuyoshi Fujioka, Motokazu Tsujikawa, Hiroyuki Miyoshi, Masayuki Yamato, Yukio Nakamura, Kohji NishidaAbstract:Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult Dermal Fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6+/K12+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.
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a novel method of culturing human oral mucosal epithelial cell sheet using post mitotic human Dermal Fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction
British Journal of Ophthalmology, 2010Co-Authors: Ryuhei Hayashi, Masayuki Yamato, Ryo Takagi, Hiroshi Takayanagi, Yasuo Tano, Kohji NishidaAbstract:Background/aims To cultivate human oral mucosal epithelial cell sheets with post-mitotic human Dermal Fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction. Methods Human oral mucosal epithelial cells obtained from three healthy volunteers were cultured with x-ray-treated Dermal Fibroblasts (Fibroblast group) and NIH/3T3 feeder layers (3T3 group) on temperature-responsive culture dishes. Media were supplemented using clinically approved products. Colony-forming efficiency was determined in both groups. Histological and immunohistochemical analyses were performed for cell sheets. Cell viability and purity of cell sheets were evaluated by flow cytometry. Results Colony-forming efficiency in the Fibroblast group was similar to that in the 3T3 group. All cell sheets were well stratified and harvested successfully. The expression patterns of keratin 1, 3/76, 4, 10, 12, 13, 15, ZO-1 and MUC16 were equivalent in both groups. The percentage of p63-positive cells in the Fibroblast group (46.1±4.2%) was significantly higher than that in the 3T3 group (30.7±7.6%) (p=0.038, t test). The cell viability and purity were similar between the two groups. Conclusion This novel culture method using Dermal Fibroblasts and pharmaceutical agents provides a safe cell processing system without xenogenic feeder cells for ocular surface reconstruction.
Chloé C. Féral - One of the best experts on this subject based on the ideXlab platform.
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Dermal Fibroblast SLC3A2 Deficiency Leads to Premature Aging and Loss of Epithelial Homeostasis.
The Journal of investigative dermatology, 2018Co-Authors: Floriane S. Tissot, Soline Estrach, Etienne Boulter, Laurence Cailleteau, Lionel Tosello, Laetitia Seguin, Sabrina Pisano, Stéphane Audebert, Olivier Croce, Chloé C. FéralAbstract:Skin homeostasis relies on fine-tuning of epidermis–dermis interactions and is affected by aging. While extracellular matrix (ECM) proteins, such as integrins, are involved in aging, the molecular basis of the skin changes needs to be investigated further. Here, we showed that integrin co-receptor, SLC3A2, required for cell proliferation, is expressed at the surface of resting Dermal Fibroblasts in young patients and is reduced drastically with aging. In vivo SLC3A2 Dermal Fibroblast deletion induced major skin phenotypes resembling premature aging. Knockout mice (3 months old) presented strong defects in skin elasticity due to altered ECM assembly, which impairs epiDermal homeostasis. SLC3A2 Dermal Fibroblast loss led to an age-associated secretome profile, with 77% of identified proteins belonging to ECM and ECM-associated proteins. ECM not only contributes to skin mechanical properties, but it is also a reservoir of growth factors and bioactive molecules. We demonstrate that Dermal Fibroblast SLC3A2 is required for ECM to fully exert its structural and reservoir role allowing proper and efficient TGF-β localization and activation. We identified SLC3A2 as a protective controller of Dermal ECM stiffness and quality required to maintain the epidermis to dermis interface as functional and dynamic.
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Dermal Fibroblast SLC3A2 Deficiency Leads to Premature Aging and Loss of Epithelial Homeostasis
JOURNAL OF INVESTIGATIVE DERMATOLOGY, 2018Co-Authors: Floriane S. Tissot, Soline Estrach, Etienne Boulter, Laurence Cailleteau, Lionel Tosello, Laetitia Seguin, Sabrina Pisano, Stéphane Audebert, Olivier Croce, Chloé C. FéralAbstract:Skin homeostasis relies on fine-tuning of epidermis-dermis interactions and is affected by aging. While extracellular matrix (ECM) proteins, such as integrins, are involved in aging, the molecular basis of the skin changes needs to be investigated further. Here, we showed that integrin co-receptor, SLC3A2, required for cell proliferation, is expressed at the surface of resting Dermal Fibroblasts in young patients and is reduced drastically with aging. In vivo SLC3A2 Dermal Fibroblast deletion induced major skin phenotypes resembling premature aging. Knockout mice (3 months old) presented strong defects in skin elasticity due to altered ECM assembly, which impairs epiDermal homeostasis. SLC3A2 Dermal Fibroblast loss led to an age-associated secretome profile, with 77% of identified proteins belonging to ECM and ECM-associated proteins. ECM not only contributes to skin mechanical properties, but it is also a reservoir of growth factors and bioactive molecules. We demonstrate that Dermal Fibroblast SLC3A2 is required for ECM to fully exert its structural and reservoir role allowing proper and efficient TGF-beta localization and activation. We identified SLC3A2 as a protective controller of Dermal ECM stiffness and quality required to maintain the epidermis to dermis interface as functional and dynamic.
Deirdre R Coombe - One of the best experts on this subject based on the ideXlab platform.
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in vitro expansion of keratinocytes on human Dermal Fibroblast derived matrix retains their stem like characteristics
Scientific Reports, 2019Co-Authors: Chee Wai Wong, Beverley F Kinnear, Radoslaw M Sobota, Rajkumar Ramalingam, Catherine F Legrand, Danielle E Dye, Michael Raghunath, Birgitte E Lane, Deirdre R CoombeAbstract:The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human Dermal Fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that Dermal Fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epiDermal sheets with a potential clinical use.
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in vitro expansion of keratinocytes on human Dermal Fibroblast derived matrix retains their stem like characteristics
bioRxiv, 2018Co-Authors: Chee Wai Wong, Beverley F Kinnear, Radoslaw M Sobota, Rajkumar Ramalingam, Catherine F Legrand, Danielle E Dye, Michael Raghunath, Birgitte E Lane, Deirdre R CoombeAbstract:The long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human Dermal Fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the Dermal Fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.
Liu Wenhua - One of the best experts on this subject based on the ideXlab platform.
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establishment and characterization of pygmy killer whale feresa attenuata Dermal Fibroblast cell line
PLOS ONE, 2018Co-Authors: Sun Yajing, Wang Jingzhen, I. R. Rajput, Edmond Sanganyado, Huang Ying, Li Ping, Liu WenhuaAbstract:The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The Dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as Fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the Fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW Dermal Fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.
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establishment and characterization of pygmy killer whale feresa attenuata Dermal Fibroblast cell line
PLOS ONE, 2018Co-Authors: Sun Yajing, Wang Jingzhen, I. R. Rajput, Edmond Sanganyado, Huang Ying, Li Ping, Liu WenhuaAbstract:The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The Dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as Fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the Fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW Dermal Fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.
I. R. Rajput - One of the best experts on this subject based on the ideXlab platform.
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establishment and characterization of pygmy killer whale feresa attenuata Dermal Fibroblast cell line
PLOS ONE, 2018Co-Authors: Sun Yajing, Wang Jingzhen, I. R. Rajput, Edmond Sanganyado, Huang Ying, Li Ping, Liu WenhuaAbstract:The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The Dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as Fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the Fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW Dermal Fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.
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establishment and characterization of pygmy killer whale feresa attenuata Dermal Fibroblast cell line
PLOS ONE, 2018Co-Authors: Sun Yajing, Wang Jingzhen, I. R. Rajput, Edmond Sanganyado, Huang Ying, Li Ping, Liu WenhuaAbstract:The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The Dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as Fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the Fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW Dermal Fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.