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

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

doi:10.3389/fnmol.2019.00212

Cited by 31 publications

(16 citation statements)

References 39 publications

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“…Here, we have analyzed the disease phenotype and expression profile within a staging system, which is largely consistent with their findings. Based on the staging system, the RO disease models established to date have all been early-or mid-stage (Parfitt David et al, 2016;Shimada et al, 2017;Teotia et al, 2017;Buskin et al, 2018;Deng et al, 2018;Guo et al, 2019;Huang et al, 2019;Li et al, 2019;Quinn et al, 2019). For example, in a previous RPGR-RP model established in our lab (Deng et al, 2018), impaired gene expression was found as early as D90, and impaired photoreceptor morphology was observed at approximately D150.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, retinogenesis can be recapitulated in ROs derived from pluripotent stem cells and analyzed by transcriptomic analysis (Kaewkhaw et al, 2015;Voelkner et al, 2016), and single-cell RNA-seq (Collin et al, 2019;Kim et al, 2019;Mao et al, 2019). Thus, patient-specific iPSCs combined with a three-dimensional (3D) culture system can generate unlimited cell sources for personalized drug testing and organ replacement (Shimada et al, 2017;Buskin et al, 2018;Deng et al, 2018;Guo et al, 2019;Li et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa

Gao

Lei

Han

et al. 2020

Front. Cell Dev. Biol.

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Although an increasing number of disease genes have been identified, the exact cellular mechanisms of retinitis pigmentosa (RP) remain largely unclear. Retinal organoids (ROs) derived from the induced pluripotent stem cells (iPSCs) of patients provide a potential but unvalidated platform for deciphering disease mechanisms and an advantageous tool for preclinical testing of new treatments. Notably, early-onset RP has been extensively recapitulated by patient-iPSC-derived ROs. However, it remains a challenge to model late-onset disease in a dish due to its chronicity, complexity, and instability. Here, we generated ROs from late-onset RP proband-derived iPSCs harboring a PDE6B mutation. Transcriptome analysis revealed a remarkably distinct gene expression profile in the patient ROs at differentiation day (D) 230. Changes in the expression genes regulating cGMP hydrolysis prompted the elevation of cGMP levels, which was verified by a cGMP enzyme-linked immunosorbent assay (ELISA) in patient ROs. Furthermore, significantly higher cGMP levels in patient ROs than in control ROs at D193 and D230 might lead to impaired formation of synaptic connections and the connecting cilium in photoreceptor cells. In this study, we established the first late-onset RP model with a consistent phenotype using an in vitro cell culture system and provided new insights into the PDE6B-related mechanism of RP.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa

Gao

Lei

Han

et al. 2020

Front. Cell Dev. Biol.

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“…Similarly, retinal organoids were also derived from a patient-specific iPSC line with compound heterozygous CRB1 mutations (c.1892A > G and c.2548G > A) [ 61 ]. To replicate the RPE65 -associated LCA and AlPL1 -LCA phenotype in a 3D in vitro system, patient-specific iPSCs were generated that were later used to generate organoids [ 62 , 63 ]. A similar study explored pathogenic splicing variants of the ABCA4 gene, a transporter protein responsible for Stargardt’s disease [ 64 ].…”

Section: The Rise Of Retinal Organoids–technical Development and Amentioning

confidence: 99%

The Rise of Retinal Organoids for Vision Research

Sharma

Krohne

Busskamp

2020

IJMS

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Retinal degenerative diseases lead to irreversible blindness. Decades of research into the cellular and molecular mechanisms of retinal diseases, using either animal models or human cell-derived 2D systems, facilitated the development of several therapeutic interventions. Recently, human stem cell-derived 3D retinal organoids have been developed. These self-organizing 3D organ systems have shown to recapitulate the in vivo human retinogenesis resulting in morphological and functionally similar retinal cell types in vitro. In less than a decade, retinal organoids have assisted in modeling several retinal diseases that were rather difficult to mimic in rodent models. Retinal organoids are also considered as a photoreceptor source for cell transplantation therapies to counteract blindness. Here, we highlight the development and field’s improvements of retinal organoids and discuss their application aspects as human disease models, pharmaceutical testbeds, and cell sources for transplantations.

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“…To overcome these obstacles, in vitro studies evaluating the correct production and expression of the RPE65 protein (e.g., immunoblot, RT-qPCR, and RNA-sequencing), as well as its enzymatic activity, are advantageous. The evaluation of isomerhydrolase activity of RPE65 can be measured using iPS-derived retinal pigment epithelium (RPE) cells from patients, hES-derived RPE cells modified by site-directed mutagenesis, or transfected cells with a mutated RPE65 expression vector [14,25,68,69].…”

Section: Evaluation Of the Rpe65 Variants Pphe83leu And Pgly187glu mentioning

confidence: 99%

“…Recently, Li and colleagues (2019) presented new perspectives for personalized functional studies utilizing retinal organoids containing RPE and photoreceptor cells derived from human-induced pluripotent stem cells [69]. Despite the need for further studies to evaluate the ability of these organoids to mimic the phenotype and molecular aspects of the retina, the generation of these organoids can still enhance the study of cell replacement therapies.…”

Section: Evaluation Of the Rpe65 Variants Pphe83leu And Pgly187glu mentioning

confidence: 99%

Pathogenicity Reclassification of RPE65 Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy

Motta

Martin

Porto

et al. 2019

Genes

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A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T>C (p.Phe83Leu) and c.560G>A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

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

Cited by 31 publications

References 39 publications

Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa

Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa

The Rise of Retinal Organoids for Vision Research

Pathogenicity Reclassification of RPE65 Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy

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