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Kohei Homma, Nami Ozato, Kazuo Tsubota, Hideyuki Okano, Yoko Ozawa; Analysis of photoreceptor degeneration by using promoter Nrl reporter knock-in human induced pluripotent stem cell lines. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2865.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis Pigmentosa (RP) is a group of inherited diseases causing retinal photoreceptor degeneration. Although the several attempts to rescue the photoreceptors have been made, the effective treatments were limited. To analyze the RP disease mechanisms, we applied rod photoreceptor specific reporter (Nrl promoter) knock-in human induced pluripotent stem cell (hiPSC) lines derived from several RP patients. This study investigates cellular functions and gene expression profiles during its degeneration.
Control hiPSCs (454E2, 201B7; RIKEN) and hiPSCs derived from patients with either Rhodopsin mutation, RP9 or RP1 (RP-iPSCs), and mitochondria DNA (mtDNA)-mutation, A3243G (MELAS-iPSCs) were cultured for the reporter knock-in experiment. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) was used to induce DNA double strand break at AAVS1 site. Promoter Nrl GFP expression cassette and antibiotic resistant gene were inserted at AAVS1 site by the homologous recombination with donor vectors. Established knock-in iPSCs were screened by antibiotic drug treatment. These cell lines were differentiated into retinal organoids by the three dimensional retinal differentiation culture for fluorescence activated cell sorting (FACS) to analyze the gene expression patterns in the reporter positive cells. The study adhered to the tenets of the Declaration of Helsinki, was approved by the Ethics Committee of Keio University School of Medicine (Tokyo, Japan; 20080016).
We confirmed the correct gene insertion at AAVS1 site by genome PCR and sequencing, and established several reporter knock-in Control iPSCs, RP-iPSCs and MELAS-iPSCs. The immunohistochemistry of retinal organoids at Day 150 derived from control hiPSCs showed fluorescent reporters, and the gene expressions in the fluorescent reporter cells collected by FACS indicated rod photoreceptor phenotype. Retinal organoids derived from RP-iPSCs and MELAS-iPSCs were also differentiated to compare with those of control hiPSC.
The reporter knock-in approach which could be applied to any of the patient derived hiPSCs was established to analyze photoreceptor degeneration in vitro.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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