Development of the method for ex vivo expansion of corneal epithelial cells

• Project/Area Number: 24592675
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Ophthalmology
• Research Institution: Osaka University (2013-2015); Kyoto Prefectural University of Medicine (2012)
• Principal Investigator: KAWASAKI Satoshi 大阪大学, 医学(系)研究科(研究院), 特任准教授(常勤) (60347458)
• Co-Investigator(Kenkyū-buntansha): UENO Morio 京都府立医科大学, 医学(系)研究科(研究院), 助教 (40426531)
• Project Period (FY): 2012-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 再生医療 / 膠様滴状角膜ジストロフィ / レチノブラストーマ / 細胞老化 / 創薬 / 不死化 / 幹細胞 / TACSTD2遺伝子 / p53 / Rb / 膠状滴状角膜ジストロフィ / 角膜上皮細胞 / 癌遺伝子

Outline of Final Research Achievements

In regenerative medicine, efficient cell proliferation is a very important factor. In the present study, we focused on constructing cells for the screening of small-molecule drugs that enhance cell proliferation. Cells were constructed to express the well-known tumor-suppressor retinoblastoma (pRb) protein by introducing the RB1 gene that is regulated under the control of tetracycline to an osteosarcoma cell line. As expected, when pRb protein was expressed in the constructed cell by adding tetracycline, cell proliferation arrest and cell senescence was induced in the cells. If cell proliferation can be observed when simultaneously adding tetracycline and certain small-molecule drugs to the cells, the small-molecule drug may have an inhibitory effect on the pRb protein in terms of the arrest of cell proliferation. Among the 48 clones that we obtained, the best clone achieved 0.8 in Z’ value. The cells may be useful for the screening of small-molecule drugs to inhibit pRb protein.

Research Products

• [Journal Article] Co-ordinated ocular development from hiPSCs and recovery of anterior eye function. (2016)
  • Author(s): Hayashi R, Ishikawa Y, Sasamoto Y, Katori R, Nomura N, Ichikawa T, Araki S, Soma T, Kawasaki S, Sekiguchi K, Quantock A, Tsujikawa M, & Nishida K.
  • Journal Title: Nature Volume: 17;531(7594) Issue: 7594 Pages: 376-80
  • DOI: 10.1038/nature17000
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes. (2016)
  • Author(s): Sasamoto Y, Hayashi R, Park S, Saito-Adachi M, Suzuki Y, Kawasaki S, Quantock A, Nakai K, Tsujikawa M, and Nishida K
  • Journal Title: Scientific Reports Volume: 22;6:20807 Issue: 1 Pages: 20807-20807
  • DOI: 10.1038/srep20807
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Novel TACSTD2 mutation in gelatinous drop-like corneal dystrophy. (2015)
  • Author(s): Jongkhajornpong P, Lekhanont K, Ueta M, Kitazawa K, Kawasaki S, Kinoshita S.
  • Journal Title: Hum Genome Var. Volume: 2 Issue: 1 Pages: 5047-5047
  • DOI: 10.1038/hgv.2015.47
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in the Fuchs endothelial corneal dystrophy (2015)
  • Author(s): Okumura N, Minamiyama R, Ho L, Kay EP, Kawasaki S, Tourtas T, Schlötzer-Schrehardt U, KruseF, Young RD, Quantock AJ, Kinoshita S, Koizumi N
  • Journal Title: Laboratory Investigation Volume: 95 Issue: 11 Pages: 1291-1304
  • DOI: 10.1038/labinvest.2015.111
  • Peer Reviewed / Open Access / Int'l Joint Research
• [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 Issue: 1 Pages: 385-97
  • DOI: 10.1172/jci71488
  • Peer Reviewed / Open Access
• [Journal Article] Ocular forward light scattering and corneal backward light scattering in patients with dry eye. (2014)
  • Author(s): Koh S, Maeda N, Ikeda C, Asonuma S, Mitamura H, Oie Y, Soma T, Tsujikawa M, Kawasaki S, Nishida K
  • Journal Title: Invest Ophthalmol Vis Sci Volume: 55 Pages: 6601-6
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [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 Issue: 8 Pages: 5701-11
  • DOI: 10.1167/iovs.12-11043
  • 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 Issue: 7 Pages: 1396-1407
  • DOI: 10.1002/stem.1390
  • Peer Reviewed
• [Journal Article] Clinical outcomes of phototherapeutic keratectomy in eyes with Thiel-Behnke corneal dystrophy. (2013)
  • Author(s): Hieda O
  • Journal Title: American Journal of Ophthalmology Volume: 155 Issue: 1 Pages: 66-72
  • DOI: 10.1016/j.ajo.2012.06.022
  • Peer Reviewed
• [Journal Article] A novel mutation (p.Glu1389AspfsX16) of the phosphoinositide kinase, FYVE finger containing gene found in a Japanese patient with fleck corneal dystrophy. (2012)
  • Author(s): Kawasaki S, Yamasaki K, Nakagawa H, et al.
  • Journal Title: Mol Vis Volume: 18 Pages: 2954-2960
  • Peer Reviewed
• [Journal Article] Establishment Of A Human Conjunctival Epithelial Cell Line Lacking The Functional TACSTD2 Gene. (2012)
  • Author(s): Kinoshita S, Kawasaki S, Kitazawa K, et al.
  • Journal Title: Trans Am Ophthalmol Soc Volume: 110 Pages: 166-177
  • Peer Reviewed
• [Presentation] PCRから遺伝子クローニング、不死化細胞の樹立まで～日々の実験に おけるちょっとしたコツ～ (2014)
  • Author(s): 川崎 諭
  • Organizer: 日本眼科学会
  • Place of Presentation: 帝国ホテル
  • Year and Date: 2014-04-02 – 2014-04-06
• [Presentation] GDLD患者由来の不死化角膜上皮細胞におけるタイトジャンクション関連タンパクの発現の検討 (2013)
  • Author(s): 北澤耕司、川崎諭、篠宮克彦、青井啓太、松田彰、舟木俊成、中司美奈、山崎健太 、海老原伸行、村上晶、羽室淳爾、木下茂
  • Organizer: 第117回日本眼科学会
  • Place of Presentation: 東京国際フォーラム（日本）
• [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 ShinomiyaA Matsuda, T Funaki, M Nakatsukasa, J HamuroA Murakami, S Kinoshita
  • Organizer: Association for research in vision and ophthalmology
  • Place of Presentation: Washington state Convention Center （米国）
• [Presentation] 膠様滴状角膜ジストロフィに対する遺伝子治療の可能性についての検討 (2013)
  • Author(s): 川崎諭、北澤耕司、篠宮克彦 、山崎健太 、中司美奈 、辻川元一 、西田幸二 、木下茂
  • Organizer: 第６７回臨床眼科学会
  • Place of Presentation: パシフィコ横浜 （日本）
• [Presentation] 2. Establishment Of Immortalized Corneal And Conjunctival Epithelial Cells Lacking The Functional Tacstd2 Gene (2012)
  • Author(s): Koji Kitazawa, Satoshi Kawasaki, Katsuhiko Shinomiya, Akira Matsuda, Toshinari Funaki, Mina Nakatsukasa, Kenta Yamazaki, Nobuyuki Ebihara, Akira Murakami, Shigeru Kinoshita
  • Organizer: ARVO
  • Place of Presentation: Florida, USA
• [Presentation] The Molecular Mechanism Of The Tacstd2 Protein In Regulating The Subcellular Localization And Protein Stability Of Claudin Proteins (2012)
  • Author(s): Satoshi Kawasaki, Mina Nakatsukasa, Kenta Yamasaki, Koji Kitazawa, Katsuhiko Shinomiya, Shigeru Kinoshita
  • Organizer: ARVO
  • Place of Presentation: Florida, USA
• [Presentation] 8. Establishment of Immortalized Corneal Epithelial Cells Lacking the Functional TACSTD2 Gene as an in vitro Model for Gelatinous Drop-Like Dystrophy. (2012)
  • Author(s): Satoshi Kawasaki, Koji Kitazawa, Katsuhiko Shinomiya, Akira Matsuda, Toshinari Funaki, Kenta et al.
  • Organizer: ISER
  • Place of Presentation: Berlin, Germany
• [Book] 角膜疾患 外来でこう診てこう治せ (2015)
  • Author(s): 木下茂
  • Total Pages: 338
  • Publisher: メジカルレビュー社