Elucidation of molecular mechanism of corneal epithelial stem cells using functional miRNA analysis

• Project/Area Number: 17K11487
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Ophthalmology
• Research Institution: Kyoto Prefectural University of Medicine
• Principal Investigator: NAKAMURA TAKAHIRO 京都府立医科大学, 医学(系)研究科(研究院), 客員講師 (30411078)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 角膜上皮 / 幹細胞 / miRNA / 再生医療

Outline of Final Research Achievements

We created a comprehensive functional miRNA profile at the single-cell level and confirmed that reproducibility of miRNA 205 was significantly upregulated in human corneal epithelial stem cells in multiple human corneal epithelial cell groups. Next, a genetically modified mouse of miRNA-205 was prepared. The miRNA-205 knockout mouse was fatal during the embryonic period due to skin hypoplasia. Morphological and cytobiological analyzes of corneal epithelium during the embryonic period and up to 5 days after birth were performed. Compared with WT, slight changes such as the degree of stratification were observed, but there were no obvious morphological or cell biological differences. These results suggest that miRNA205 has different functions in embryonic and adult corneal epithelial stem cells.

Academic Significance and Societal Importance of the Research Achievements

近年の精力的な基礎研究から、タンパク質をコードしていないnon-coding RNAの一種であるmiRNAは生体内の様々な部位に存在し、発生・分化・癌化などの生命現象や幹細胞分化制御に深く関与していることが報告されている。生体内で角膜は視覚を司る重要な組織であり、その角膜上皮幹細胞の単一細胞レベルでの網羅的な機能性miRNAプロファイルの作成の試みは、世界的に見ても初めての試みであり、極めて独創的・先駆的である。また、miRNAレベルでの遺伝子改変マウスの作成の試みは、いまだ報告が少なく、遺伝改変技術を用いた生体内での恒常性維持機構の解明、分子レベルでの機能解析に大きく貢献したと考える。

Research Products

• [Journal Article] Direct Reprogramming Into Corneal Epithelial Cells Using a Transcriptional Network Comprising PAX6, OVOL2, and KLF4 (2019)
  • Author(s): Kitazawa Koji、Hikichi Takafusa、Nakamura Takahiro、Nakamura Masahiro、Sotozono Chie、Masui Shinji、Kinoshita Shigeru
  • Journal Title: Cornea Volume: 38 Issue: 1 Pages: S34-S41
  • DOI: 10.1097/ico.0000000000002074
  • Peer Reviewed / Open Access
• [Journal Article] Injection of Cultured Cells with a ROCK Inhibitor for Bullous Keratopathy． (2018)
  • Author(s): Kinoshita S，Koizumi N，Ueno M，Okumura N，Imai K，Tanaka H，Yamamoto Y，Nakamura T，Inatomi T，Bush J，Toda M，Hagiya M，Yokota I，Teramukai S，Sotozono C，Hamuro J．
  • Journal Title: N Engl J Med Volume: 378 Issue: 1 Pages: 995-1003
  • DOI: 10.1056/nejm192802231980115
  • Peer Reviewed
• [Journal Article] Injection of Cultured Cells with a ROCK Inhibitor for Bullous Keratopathy (2018)
  • Author(s): Kinoshita Shigeru、Koizumi Noriko、Ueno Morio、Okumura Naoki、Imai Kojiro、Tanaka Hiroshi、Yamamoto Yuji、Nakamura Takahiro、Inatomi Tsutomu、Bush John、Toda Munetoyo、Hagiya Michio、Yokota Isao、Teramukai Satoshi、Sotozono Chie、Hamuro Junji
  • Journal Title: New England Journal of Medicine Volume: 378 Issue: 11 Pages: 995-1003
  • DOI: 10.1056/nejmoa1712770
  • Peer Reviewed / Open Access
• [Journal Article] PAX6 regulates human corneal epithelium cell identity (2017)
  • Author(s): Kitazawa Koji、Hikichi Takafusa、Nakamura Takahiro、Sotozono Chie、Kinoshita Shigeru、Masui Shinji
  • Journal Title: Experimental Eye Research Volume: 154 Pages: 30-38
  • DOI: 10.1016/j.exer.2016.11.005
  • Peer Reviewed / Open Access
• [Journal Article] Unique circumferential peripheral keratitis in relapsing polychondritis (2017)
  • Author(s): Motozawa Naohiro、Nakamura Takahiro、Takagi Seiji、Fujihara Masashi、Hirami Yasuhiko、Ishida Kazuhiro、Sotozono Chie、Kurimoto Yasuo
  • Journal Title: Medicine Volume: 96 Issue: 41 Pages: e7951-e7951
  • DOI: 10.1097/md.0000000000007951
  • Peer Reviewed / Open Access
• [Journal Article] Expression of IL-33 in ocular surface epithelium induces atopic keratoconjunctivitis with activation of group 2 innate lymphoid cells in mice (2017)
  • Author(s): Imai Yasutomo、Hosotani Yuka、Ishikawa Hiroto、Yasuda Koubun、Nagai Makoto、Jitsukawa Orie、Gomi Fumi、Nakanishi Kenji、Yoshimoto Tomohiro、Nakamura Takahiro、Yamanishi Kiyofumi
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 10053-10053
  • DOI: 10.1038/s41598-017-10227-y
  • Peer Reviewed / Open Access
• [Journal Article] The effect of topical application of 0.15% ganciclovir gel on cytomegalovirus corneal endotheliitis. (2016)
  • Author(s): Koizumi N, Miyazaki D, Inoue T, Ohtani F, Kandori-Inoue M, Inatomi T, Sotozono C, Nakagawa H, Horikiri T, Ueta M, Nakamura T, Inoue Y, Ohashi Y, Kinoshita S.
  • Journal Title: Br J Ophthalmology Volume: May 3 Issue: 2 Pages: 114-119
  • DOI: 10.1136/bjophthalmol-2015-308238
  • Peer Reviewed / Open Access
• [Presentation] VCP ATPase阻害剤による角膜組織保護効果． (2018)
  • Author(s): 永田真帆，中村隆宏，池田華子，垣塚彰，中小路真美，石田学，外園千恵，木下茂．
  • Organizer: 角膜カンファランス2018
• [Presentation] CRISPR/Cas9 を用いてKLF4 をノックアウトしたヒト角膜上皮細胞の検討． (2018)
  • Author(s): 北澤耕司，増田皓介，村上怜，中村隆宏，木下茂，外園千恵．
  • Organizer: 第122回日本眼科学会総会
• [Presentation] Ocular surface reconstructoin using defined culture system for severe ocular surface diseases. (2017)
  • Author(s): Nakamura T
  • Organizer: 2nd international Stevens-Johnson syndrome symposium
  • Int'l Joint Research / Invited
• [Presentation] OVOL2 maintains atranscriptional program in human corneal epithelial cells. (2017)
  • Author(s): Kitazawa K，Hikichi T，Nakamura T，Sotozono C，Kinoshita S，Masui S
  • Organizer: 2017 Annual Meeting of the Association for Research in Vision and Ophthalmology
  • Int'l Joint Research
• [Presentation] PAX6, OVOL2, KLF4 coordinately contribute the reprogramming of the corneal epithelial cellsTranscription factors PAX6, OVOL2, and KLF4 coordinately contribute to the reprogramming of corneal epithelial cells. (2017)
  • Author(s): Murakami R，Kitazawa K，Hikichi T，Nakamura T，Sotozono C，Kinoshita S，Masui S
  • Organizer: 2017 Annual Meeting of the Association for Research in Vision and Ophthalmology
  • Int'l Joint Research
• [Presentation] Ocular surface reconstruction using defined culture system for severe ocular surface diseases-Next generation of COMET for global expansion. (2017)
  • Author(s): Nakamura T
  • Organizer: 3rd Chulalongkorn Eye Center (CEC) and Kyoto Prefectural University of Medicine (KPUM) Joint Meeting
  • Int'l Joint Research / Invited