Analysis of developmental stage of photoreceptors derived from human iPSC

2015 Fiscal Year Final Research Report

• Project/Area Number: 26861473
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Ophthalmology
• Research Institution: Nippon Medical School
• Principal Investigator: Homma Kohei 日本医科大学, 医学部, 助教 (80462729)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: ヒトiPS細胞 / 網膜色素変性 / 視細胞 / 細胞移植 / CRISPR/Cas9 / ゲノム編集

Outline of Final Research Achievements

Retinal cell replacement therapy is a promising approach for the treatment of retinal degenerative diseases. To generate donor photoreceptors from human induced-pluripotent stem cells (iPSCs), we successfully inserted fluorescent reporter (E2-crimson) gene at the 3’-end of Crx (photoreceptor specific) gene in human iPSC genome by using CRISPR/Cas9 system. Crx gene and E2-crimson gene were connected with 2A peptide gene. After the translation of Crx-E2-crimson, 2A peptide is cleaved off and E2-crimson is released in the cytosol. Fluorescence of E2-crimson in Crx-expressing cells is detected by fluorescence-activated cell sorter and Crx positive cells are purified by the sorting. By using this gene expression monitoring system, we explored the Notch inhibition signaling during Crx upregulation by the treatment with DAPT, and the diversity of differentiation into rod photoreceptor from Crx-positive cells in vitro.

Free Research Field

神経科学