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Investigation of disease mechanisms by fluorescent labeling of photoreceptor derived from retinitis pigmentosa patient-derived induced pluripotent stem cells

Research Project

Project/Area Number 16K11305
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Ophthalmology
Research InstitutionKeio University

Principal Investigator

Homma Kohei  慶應義塾大学, 医学部(信濃町), 特任助教 (80462729)

Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
KeywordsヒトiPS細胞 / 網膜色素変性症 / ゲノム編集 / 網膜視細胞 / 疾患iPS細胞 / ノックイン / 移植・再生医療 / 遺伝学 / 再生医学 / 脳神経疾患 / 発生・分化
Outline of Final Research Achievements

In this research, several human induced pluripotent stem cells (hiPSCs) were used to investigate the disease mechanism of retinitis pigmentosa (RP). Genomic mutation of RP patient derived hiPSCs were confirmed by genomic PCR and conventional sequencing. Genome of these cell lines (and wild-type control hiPSCs) were edited with CRISPR-Cas9(clustered regularly interspaced short palindromic repeats / CRISPR associated proteins). In these cell lines, photoreceptor specific promoter GFP expression cassette were inserted in both alleles of AAVS1 sites, safe harbor for gene insertion, thus derived photoreceptors from these hiPSC lines were fluorescently labeled with GFP. By using these cell lines, the gene expressions and the cellular function could be investigated specifically in photoreceptors. These fluorescently labelled cells could be used for the investigation of disease mechanisms and the drug screening.

Academic Significance and Societal Importance of the Research Achievements

網膜色素変性症や加齢黄斑変性症などの視細胞変性疾患は,緑内障,糖尿病網膜症に次いで日本国内の主要な失明原因となっている(厚生労働省資料)。これらの視細胞変性疾患では、様々な遺伝子の変異により、網膜で光を感受する視細胞が細胞死を引き起こすが、この細胞死のメカニズムについては、ほとんど明らかではない。本研究で作製された遺伝子改変のヒトiPS細胞のラインから分化したGFP陽性の視細胞を用いることによって、より詳細に細胞死のメカニズムを調べることができ、またGFPによるドラッグスクリーニングを検討することができるようになった。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (20 results)

All 2018 2017 2016 Other

All Int'l Joint Research (1 results) Journal Article (6 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 4 results,  Open Access: 2 results,  Acknowledgement Compliant: 2 results) Presentation (12 results) (of which Int'l Joint Research: 3 results) Patent(Industrial Property Rights) (1 results)

  • [Int'l Joint Research] National Eye Institute(米国)

    • Related Report
      2016 Research-status Report
  • [Journal Article] Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.2017

    • Author(s)
      Ishii T, Homma K, Mano A, Akagi T, Shigematsu Y, Shimoda Y, Inoue H, Kakinuma Y, Kaneda M.
    • Journal Title

      J Neurophysiol.

      Volume: 118 Issue: 4 Pages: 1952-1961

    • DOI

      10.1152/jn.00506.2016

    • Related Report
      2017 Research-status Report
    • Peer Reviewed
  • [Journal Article] Bicistronic 2A-peptide-based co-expression reporter knock-in strategy by CRISPR/Cas9 system: application to the labeling of specific cell lineages and gene expression monitoring.2017

    • Author(s)
      Homma K
    • Journal Title

      Stem Cell Transl Investig

      Volume: 4:e1551.

    • DOI

      10.14800/scti.1551

    • Related Report
      2017 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] CRISPR/Cas9ゲノム編集による網膜視細胞の蛍光標識技術とその応用2017

    • Author(s)
      本間耕平
    • Journal Title

      BIO Clinica

      Volume: Vol.32 No.6 Pages: 75-77

    • Related Report
      2017 Research-status Report
  • [Journal Article] Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.2017

    • Author(s)
      Homma K(corresponding author), Usui S, Kaneda M.
    • Journal Title

      Genes to Cells.

      Volume: 22 Issue: 3 Pages: 250-264

    • DOI

      10.1111/gtc.12472

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.2016

    • Author(s)
      Kaewkhaw R, Swaroop M, Homma K, Nakamura J, Brooks M, Kaya KD, Chaitankar V, Michael S, Tawa G, Zou J, Rao M, Zheng W, Cogliati T, Swaroop A.
    • Journal Title

      Invest Ophthalmol Vis Sci.

      Volume: 57(5) Issue: 5 Pages: ORSFl1-ORSFl1

    • DOI

      10.1167/iovs.15-17639

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] ゲノム編集技術を用いた患者iPS細胞由来視細胞変性メカニズム解析2016

    • Author(s)
      本間耕平
    • Journal Title

      BIO Clinica

      Volume: Vol.31 No.11 Pages: 51-53

    • Related Report
      2016 Research-status Report
  • [Presentation] Monitoring the developing photoreceptors in the hiPSC-derived three dimension retinal organoid culture using bicistronic 2A-peptide-based co-expression reporter knock-in system2018

    • Author(s)
      Homma K., Ozato N., Tsubota K., Okano H., Ozawa Y.
    • Organizer
      The Association for Research in Vision and Ophthalmology
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 留学のすゝめ2018 日本人研究者が世界で活躍するためのプラットフォーム構築に向けて2018

    • Author(s)
      赤木紀之,本間耕平
    • Organizer
      第91回日本生化学会大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] ゲノム編集ノックインヒトiPS細胞による錐体視細胞分化過程解析2018

    • Author(s)
      本間耕平,小澤洋子
    • Organizer
      第22回視覚科学フォーラム
    • Related Report
      2018 Annual Research Report
  • [Presentation] Gene expression analysis of cone photoreceptors derived from human induced pluripotent stem cells in the three dimensional retinal differentiation culture2018

    • Author(s)
      本間耕平,小澤洋子
    • Organizer
      第41回日本神経科学学会
    • Related Report
      2018 Annual Research Report
  • [Presentation] ノックインヒトiPS細胞による3次元網膜オルガノイド形成の可視化2018

    • Author(s)
      本間耕平,尾里納美,小澤洋子
    • Organizer
      第17回日本再生医療学会
    • Related Report
      2018 Annual Research Report 2017 Research-status Report
  • [Presentation] ノックインヒトiPS細胞による視細胞分化過程の遺伝子発現解析2018

    • Author(s)
      本間耕平
    • Organizer
      第22回眼科分子生物学研究会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Bicistronic 2A-peptide-based co-expression reporter knock-in hiPSC lines revealed gene expression profiles during human photoreceptor differentiation.2017

    • Author(s)
      Homma K., Ozawa Y.
    • Organizer
      Society for Neuroscience 2017
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] Bicistronic 2A-peptide-based co-expression reporter knock-in strategy for hiPSC: Application to the labeling of specific cell lineages and gene expression monitoring2017

    • Author(s)
      Homma K.
    • Organizer
      IRCMS mini-symposium
    • Related Report
      2017 Research-status Report
    • Int'l Joint Research
  • [Presentation] Bicistronic 2A-peptide-based co-expression reporter knock-in strategy for the labeling of human induced pluripotent stem cell-derived photoreceptors.2017

    • Author(s)
      本間耕平,小澤洋子
    • Organizer
      第40回日本分子生物学会年会
    • Related Report
      2017 Research-status Report
  • [Presentation] Bicistronic 2A-peptide-based co-expression reporter knock-in hiPSC lines revealed gene expression profiles during human photoreceptor differentiation.2017

    • Author(s)
      本間耕平
    • Organizer
      日本生物物理学会第55回年会
    • Related Report
      2017 Research-status Report
  • [Presentation] Analysis of gene expression patterns in photoreceptors derived from knock-in reporter human iPSC lines2016

    • Author(s)
      Kohei Homma, Makoto Kaneda
    • Organizer
      第39回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2016-11-30
    • Related Report
      2016 Research-status Report
  • [Presentation] 視細胞関連遺伝子レポーターノックインヒトiPS細胞を用いた視細胞分化の解析2016

    • Author(s)
      本間耕平
    • Organizer
      視覚科学フォーラム 第20回研究会
    • Place of Presentation
      大阪大学
    • Year and Date
      2016-08-25
    • Related Report
      2016 Research-status Report
  • [Patent(Industrial Property Rights)] 標的遺伝子のモニター方法、ベクターシステム、細胞、及び標的遺伝子編集キット2016

    • Inventor(s)
      本間 耕平
    • Industrial Property Rights Holder
      本間 耕平
    • Industrial Property Rights Type
      特許
    • Industrial Property Number
      2016-179955
    • Filing Date
      2016-09-14
    • Related Report
      2016 Research-status Report

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Published: 2016-04-21   Modified: 2022-02-16  

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