title: Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.
abstract: Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs.
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Japan.
authors.forename: G
authors.initials: G
authors.lastname: Yu
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Japan Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Okawa
authors.affiliation: Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Okita
authors.affiliation: Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kamano
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Japan.
authors.forename: F
authors.initials: F
authors.lastname: Wang
authors.affiliation: Division of Dental Pharmacology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Saeki
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yatani
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Japan Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Japan egu@dent.tohoku.ac.jp.
authors.forename: H
authors.initials: H
authors.lastname: Egusa
mesh_terms: D000328:Adult; D000818:Animals; D064592:Autografts / Q000166:cytology; D015815:Cell Adhesion Molecules / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D018899:CpG Islands / Q000235:genetics; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003470:Culture Media; D016895:Culture Media, Serum-Free; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology*; D005881:Gingiva / Q000166:cytology*; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D007335:Insulin-Like Growth Factor II / Q000032:analysis; D059785:Karyotype; D007797:Laminin / Q000032:analysis; D051379:Mice; D008875:Middle Aged; D016685:Mitomycin / Q000494:pharmacology; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000032:analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012867:Skin / Q000166:cytology; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D003470:Culture Media; D016895:Culture Media, Serum-Free; D018398:Homeodomain Proteins; D007335:Insulin-Like Growth Factor II; D007797:Laminin; D016685:Mitomycin; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D000097794:Kalinin; C486780:NANOG protein, human; C494173:POU5F1 protein, human; C097043:laminin alpha5
keywords: culture media; feeder cells; gingiva; insulin-like growth factor II; regenerative medicine; serum-free