角膜上皮細胞の細胞特性を規定するコア転写因子群の同定

2013 Fiscal Year Annual Research Report

• Project/Area Number: 23390404
• Research Institution: Kyoto Prefectural University of Medicine
• Principal Investigator: 木下 茂 京都府立医科大学, 医学(系)研究科(研究院), 教授 (30116024)
• Co-Investigator(Kenkyū-buntansha): 上野 盛夫 京都府立医科大学, 医学(系)研究科(研究院), 助教 (40426531) ; 川崎 諭 大阪大学, 医学(系)研究科(研究院), 講師 (60347458)
• Project Period (FY): 2011-04-01 – 2014-03-31
• Keywords: 角膜上皮細胞 / 転写因子 / ダイレクトリプログラミング / コア転写因子 / マスターレギュレーター

Research Abstract

前年度にiPS干渉法（Hikichi et al:PNAS, 2013)を用いて、強干渉した20因子をもとに、どの因子の組み合わせが重要かを詳細に検討した。誘導前の細胞はヒト胎児皮膚繊維芽細胞を用いた。20因子をすべて導入するとインテグリンα6陽性、CD71陰性の細胞が数％得られた。インテグリンα6陽性、CD71陰性の細胞はサイズが小さく、またN/C比も大きく、幹細胞の特徴をもった形態を示した。この細胞群の導入効率が最も高い組み合わせで転写因子を導入すると、ケラチン１５陽性細胞を高効率で誘導できることを免疫染色で確認した。そしてこの誘導細胞はKi67陽性であり、Ncadherin陽性細胞であった。これはすなわち、角膜上皮幹細胞様の細胞が誘導できたことを意味する。次に、これらの細胞を羊膜上にまいて角膜上皮分化誘導をおこなった。角膜上皮最終分化マーカーはケラチン12あるが、様々な分化誘導法を行ってもケラチン12の発現を認めることはできなかった。そのため、今度はケラチン12を指標に転写因子の組み合わせを検討した。新生児皮膚繊維芽細胞に様々な転写因子セットを導入し角膜培地で培養すると、10日目に上皮様の形態を示す細胞群を確認した。そして、誘導細胞のほとんどはpan-cytokeratin陽性細胞で上皮細胞であることを確認した。またそのうちの一部は角膜上皮特異的タンパクであるkeratin3陽性、keratin12陽性細胞であった。また、それらの細胞の遺伝子発現レベルをqRT-PCRで確認すると、keratin3, keratin12以外にも、ALDH3A1,　TKT、CDH1, keratin15の発現が上昇していた。一方で、繊維芽細胞マーカーであるVIMの発現は誘導細胞で低下していた。このことから、繊維芽細胞における転写因子セットの強制発現によって、keratin12陽性細胞の誘導、すなわち角膜上皮細胞のダイレクトリプログラミングに成功した。

Current Status of Research Progress

Reason

25年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

25年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis (2014)
  • Author(s): Nakamura T, Hamuro J, Takaishi M, Simmons S, Maruyama K, Zaffalon A, Bentley AJ, Kawasaki S, Nagata-Takaoka M, Fullwood NJ, Itami S, Sano S, Ishii M, Barrandon Y, Kinoshita S.
  • Journal Title: J Clin Invest Volume: １ Pages: 385-97
  • DOI: 10.1172/JCI71488
  • Peer Reviewed
• [Journal Article] Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing. (2014)
  • Author(s): Okumura N, Nakano S, Kay EP, Numata R, Ota A, Sowa Y, Sakai T, Ueno M, Kinoshita S, Koizumi N.
  • Journal Title: Invest Ophthalmol Vis Sci. Volume: １ Pages: 318-29
  • DOI: 10.1167/iovs.13-12225
  • Peer Reviewed
• [Journal Article] Clinical outcomes in descemet stripping automated endothelial keratoplasty with internationally shipped precut donor corneas. (2014)
  • Author(s): Nakagawa H, Inatomi T, Hieda O, Sotozono C, Yokoi N, Iliakis B, Miller TD, Ulrickson C, Kinoshita S.
  • Journal Title: Am J Ophthalmol Volume: １ Pages: 50-55.e1
  • DOI: 10.1016/j.ajo.2013.09.016
  • Peer Reviewed
• [Journal Article] Cultivation of Corneal Endothelial Cells on a Pericellular Matrix Prepared from Human Decidua-Derived Mesenchymal Cells. (2014)
  • Author(s): Numata R, Okumura N, Nakahara M, Ueno M, Kinoshita S, Kanematsu D, Kanemura Y, Sasai Y, Koizumi N
  • Journal Title: Plos One Volume: 2 Pages: e88169
  • DOI: 10.1371/journal.pone.0088169
  • Peer Reviewed
• [Journal Article] Visual improvement following cultivated oral mucosal epithelial transplantation (2013)
  • Author(s): Sotozono C, Inatomi T, Nakamura T, Koizumi N, Yokoi N, Ueta M, Matsuyama K, Miyakoda K, Kaneda H, Fukushima M, Kinoshita S
  • Journal Title: Ophthalmology Volume: 120(1) Pages: 193-200
  • Peer Reviewed
• [Journal Article] Establishment of a human corneal epithelial cell line lacking the functional TACSTD2 gene as an in vitro model for gelatinous drop-like dystrophy. (2013)
  • Author(s): Kitazawa K, Kawasaki S, Shinomiya K, Aoi K, Matsuda A, Funaki T, Yamasaki K, Nakatsukasa M, Ebihara N, Murakami A, Hamuro J, Kinoshita S.
  • Journal Title: Invest Ophthalmol Vis Sci. Volume: ８ Pages: 5701-11
  • DOI: 10.1167/iovs.12-11043
  • Peer Reviewed
• [Journal Article] Classification of secondary corneal amyloidosis and involvement of lactoferrin. (2013)
  • Author(s): Araki-Sasaki K, Hirano K, Osakabe Y, Kuroda M, Kitagawa K, Mishima H, Obata H, Yamada M, Maeda N, Nishida K, Kinoshita S.
  • Journal Title: Ophthalmology. Volume: ６ Pages: 1166-72
  • DOI: 10.1016/j.ophtha.2012.11.047
  • Peer Reviewed
• [Journal Article] Corneal Endothelial Cell Fate is Maintained by LGR5 via the Regulation of Hedgehog and Wnt Pathway. (2013)
  • Author(s): Hirata-Tominaga K, Nakamura T, Okumura N, Kay EP, Barrandon Y, Koizumi N, Kinoshita S
  • Journal Title: Stem Cells Volume: 7 Pages: 1396-1407
  • DOI: 10.1002/stem.1390.
  • Peer Reviewed
• [Journal Article] Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium. (2013)
  • Author(s): Nakahara M, Okumura N, Kay EP, Hagiya M, Imagawa K, Hosoda Y, Kinoshita S, Koizumi N.
  • Journal Title: PLOS ONE Volume: 7 Pages: e69009.
  • DOI: 10.1371/journal.pone.0069009
  • Peer Reviewed
• [Journal Article] Human periodontal ligament cell sheets cultured on amniotic membrane substrate (2013)
  • Author(s): Adachi K, Amemiya T, Nakamura T, Honjyo KI, Kumamoto S, Yamamoto T, Bentley AJ, Fullwood NJ, Kinoshita S, Kanamura N
  • Journal Title: Oral Dis Volume: 8 Pages: Epub
  • DOI: 10.1111/odi.12176
  • Peer Reviewed
• [Presentation] ヒト角膜上皮細胞シートの安定的な製造を可能にするシート作製技術の開発 (2014)
  • Author(s): 135. 前田謙一, 横山修一, 細田勇喜, 渡部俊介, 今井究, 中村隆宏, 木下茂
  • Organizer: 第13回日本再生医療学会総会
  • Place of Presentation: 京都
  • Year and Date: 20140305-20140305
• [Presentation] LRIG1による角膜の恒常性維持機構の解析 (2014)
  • Author(s): 中村隆宏, 羽室淳爾, 高石樹朗, 丸山和一, 永田真帆, 川崎論, 板見智, 佐野栄紀, 石井優, 木下茂
  • Organizer: 角膜カンファランス2014
  • Place of Presentation: 沖縄
  • Year and Date: 20140131-20140131
• [Presentation] 角膜上皮細胞におけるR-spondin1の機能解析 (2014)
  • Author(s): 大倉翔貴, 中村隆宏, 畑友衣子, 小林正和, 永田真帆, 小泉範子, 木下茂
  • Organizer: 角膜カンファランス2014
  • Place of Presentation: 沖縄
  • Year and Date: 20140131-20140131
• [Presentation] シリコンオイル注入眼に併発した帯状角膜変性に対しPTKを施行した2例 (2014)
  • Author(s): 宇山紘史, 中村隆宏, 石田和寛, 外園千恵, 稲富勉, 木下茂, 栗本康夫
  • Organizer: 角膜カンファランス2014
  • Place of Presentation: 沖縄
  • Year and Date: 20140130-20140130
• [Presentation] 先進医療としての羊膜移植の現状と有効性 (2014)
  • Author(s): 森川恵輔, 外園千恵, 稲富勉, 中村隆宏, 横井則彦, 松尾俊康, 木下茂
  • Organizer: 角膜カンファランス2014
  • Place of Presentation: 沖縄
  • Year and Date: 20140130-20140130
• [Presentation] 眼表面扁平上皮腫瘍におけるLRIG1発現解析 (2014)
  • Author(s): 永田真帆, 中村隆宏, 外園千恵, 稲富勉, 横井則彦, 木下茂
  • Organizer: 角膜カンファランス2014
  • Place of Presentation: 沖縄
  • Year and Date: 2014-01-31
• [Presentation] ヒト角膜上皮細胞シートの安定的な製造を可能にするシート作製技術の開発 (2014)
  • Author(s): 前田謙一, 横山修一, 細田勇喜, 渡部俊介, 今川究, 中村隆宏, 木下茂
  • Organizer: 角膜カンファランス2014
  • Place of Presentation: 沖縄
  • Year and Date: 2014-01-30
• [Presentation] The Biological Characteristics and Clinical Application of Human (2013)
  • Author(s): Shigeru Kinoshita
  • Organizer: Kyoto International Workshop for Visual Science 2013
  • Place of Presentation: Kyoto
  • Year and Date: 20131104-20131104
• [Presentation] Investigation of Gene Therapy Using Immortalized Cells Derived from a Gelatinous Drop-Like Corneal Dystrophy Patient (2013)
  • Author(s): K Kitazawa, S Kawasaki, K Aoi, K Shinomiya, A Matsuda, T Funaki, M Nakatsukasa, J Hamuro, S Kinoshita
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130509-20130509
• [Presentation] Integral Analysis of Gene Signatures and MicroRNA Expression of Cultured Human Corneal Endothelial Cells in Relation to Their Functions, Cell Senescence, Epithelial-Mesenchymal Transition, and Fibrosis. (2013)
  • Author(s): Kazuko Asada, Munetoyo Toda, Michio Hagiya, Kana Nakata, Morio Ueno, Naoki Okumura, Noriko Koizumi, Junji Hamuro, Shigeru Kinoshita
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130507-20130507
• [Presentation] Cell Line of Fuchs' Corneal Dystrophy Produces an Abnormal Extracellular Matrix. (2013)
  • Author(s): Leona Ho, Naoki Okumura, EunDuck P. Kay, Kenta Yamasaki, Satoshi Kawasaki, Theofilos Tourtas, Ursula Schlotzer-Schrehardt, Friedrich E. Kruse, Shigeru Kinoshita, Noriko Koizumi
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506
• [Presentation] Rho-kinase inhibitor enhances corneal endothelial cell proliferation via p27 degradation. (2013)
  • Author(s): Ryohei Numata, Naoki Okumura, EunDuck P. Kay, Makiko Nakahara, Shinichiro Nakano, Morio Ueno, Shigeru Kinoshita, Noriko Koizumi
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506
• [Presentation] Proliferation Propensity of Cultured Human Corneal Endothelial Cells and Their Plasticity Dictated by Culture Microenvironments. (2013)
  • Author(s): Munetoyo Toda, Kana Nakata, kazuko asada, Michio Hagiya, Morio Ueno, Naoki Okumura, Noriko Koizumi, Junji Hamuro, Shigeru Kinoshita:
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506
• [Presentation] Cell-injection Therapy Using Rho Kinase Inhibitor in a Corneal Endothelial Dysfunction Rabbit Model (2013)
  • Author(s): Junji Kitano, Naoki Okumura, EunDuck P. Kay, Morio Ueno, Junji Hamuro, Shigeru Kinoshita, Noriko Koizumi
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506
• [Presentation] Rho kinase inhibitor promotes cell adhesion of corneal endothelial cells through inhibiting phosphorylation of MLC. (2013)
  • Author(s): Yuki Tsujimoto, Naoki Okumura, EunDuck P. Kay, Ryohei Numata, Shigeru Kinoshita, Noriko Koizumi
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506
• [Presentation] Efficacy and Safety Evaluation of Cell-Injection Therapy using Cultivated Human Corneal Endothelial Cells. (2013)
  • Author(s): Noriko Koizumi, Naoki Okumura, Takashi Shiina, Shingo Suzuki, Shinichiro Nakamura, Yuji Sakamoto, Kenta Yamasaki, Morio Ueno, Junji Hamuro, Shigeru Kinoshita
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506
• [Presentation] Development of a Human Cultivated Nasal Mucosal Epithelial Cell Sheet for the Treatment of Severe Ocular Surface Diseases. (2013)
  • Author(s): Masakazu Kobayashi, Takahiro Nakamura, Makoto Yasuda, Yuiko Hata, Shoki Okura, Naoki Okumura, Noriko Koizumi, Hisao Yasu, Shigeru Kinoshita
  • Organizer: The Association for Research in Vision and Ophthalmology 2013
  • Place of Presentation: Seattle
  • Year and Date: 20130506-20130506