Generation and Characterization of Induced Pluripotent Stem Cells and Retinal Organoids From a Leber's Congenital Amaurosis Patient With Novel RPE65 Mutations.

Guilan Li,Ping Xu,Fuhua Peng,Panfeng Wang,Xiaojing Song,Bingbing Xie,Guangjin Pan,Xiufeng Zhong,Guanjie Gao,Jian Ge,Qingjiong Zhang,Tiancheng Zhou

doi:10.3389/fnmol.2019.00212

Abstract

RPE65-associated Leber congenital amaurosis (LCA) is one of highly heterogeneous, early onset, severe retinal dystrophies with at least 130 gene mutation sites identified. Their pathogenicity has not been directly clarified due to lack of diseased cells. Here, we generated human-induced pluripotent stem cells (hiPSCs) from one putative LCA patient carrying two novel RPE65 mutations with c.200T>G (p.L67R) and c.430T>C (p.Y144H), named RPE65-hiPSCs, which were confirmed to contain the same mutations. The RPE65-hiPSCs presented typical morphological features with normal karyotype, expressed pluripotency markers, and developed teratoma in NOD-SCID mice. Moreover, the patient hiPSCs were able to differentiate toward retinal lineage fate and self-form retinal organoids with layered neural retina. All major retinal cell types including photoreceptor and retinal pigment epithelium (RPE) cells were also acquired overtime. Compared to healthy control, RPE cells from patient iPSCs had lower expression of RPE65, but similar phagocytic activity and VEGF secretion level. This study provided the valuable patient specific, disease targeted retinal organoids containing photoreceptor and RPE cells, which would facilitate the study of personalized pathogenic mechanisms of disease, drug screening, and cell replacement therapy.

Highlights

Leber’s congenital amaurosis (LCA) is a group of recessively inherited retinal dystrophies (IRDs) with severe visual impairment, accounts for >5% of all retinal dystrophy and 20% of legal blindness in school-age children
A Leber congenital amaurosis (LCA) patient was diagnosed by clinical standards with compound heterozygotes RPE65 gene mutations
This study was approved by the ethics committee of the Zhongshan Ophthalmic Center of Sun Yat-sen University and was conducted in accordance with the Declaration of Helsinki

Summary

Introduction

Leber’s congenital amaurosis (LCA) is a group of recessively inherited retinal dystrophies (IRDs) with severe visual impairment, accounts for >5% of all retinal dystrophy and 20% of legal blindness in school-age children. At least 20 mutation genes such as CEP290, GUCY2D, CRB1, and RPE65 have been identified in patients with LCA. These genes were proved to express in photoreceptors or retinal pigment epithelium (RPE) cells of retina, and involved in disparate functional pathways including photoreceptor morphogenesis, visual phototransduction, and visual cycle (Wang et al, 2015; Jacobson et al, 2016; Kumaran et al, 2017). RPE65 is a 65 kDa isomer hydrolase synthesized in RPE cells It catalyzes isomerization of all-trans retinyl esters into the chromophore 11-cis retinol which is transported into photoreceptors to participate in visual phototransduction (Jin et al, 2005). Many variants in this gene have been documented, their pathogenicity have not been directly clarified partly due to lack of human in vitro disease models (Astuti et al, 2016; Bernardis et al, 2016)

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