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

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K11305
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Ophthalmology
• Research Institution: Keio 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
• Keywords: ヒトiPS細胞 / 網膜色素変性症 / ゲノム編集

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.

Free Research Field

神経科学

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。