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

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          Abstract

          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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          Most cited references28

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          Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase.

          Mice homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa. In affected animals the retinal rod photoreceptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left. Degeneration is preceded by accumulation of cyclic GMP in the retina and is correlated with deficient activity of the rod photoreceptor cGMP-phosphodiesterase. We have recently isolated a candidate complementary DNA for the rd gene from a mouse retinal library and completed the characterization of cDNAs encoding all subunits of bovine photoreceptor phosphodiesterase. The candidate cDNA shows strong homology with a cDNA encoding the bovine phosphodiesterase beta subunit. Here we present evidence that the candidate cDNA is the murine homologue of bovine phosphodiesterase beta cDNA. We conclude that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterase beta subunit.
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            Organoids — Preclinical Models of Human Disease

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              Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines

              Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation. Summary: Live morphological staging reveals common strengths and limitations of differentiating 3D retinal organoid cultures across multiple human pluripotent stem cell lines.
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                Author and article information

                Contributors
                Journal
                Front Cell Dev Biol
                Front Cell Dev Biol
                Front. Cell Dev. Biol.
                Frontiers in Cell and Developmental Biology
                Frontiers Media S.A.
                2296-634X
                06 March 2020
                2020
                : 8
                : 128
                Affiliations
                [1] 1Laboratory of Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University , Wenzhou, China
                [2] 2National Center for International Research in Regenerative Medicine and Neurogenetics, National Clinical Research Center for Ophthalmology, State Key Laboratory of Ophthalmology, Optometry and Visual Science , Wenzhou, China
                Author notes

                Edited by: Daniele Bottai, University of Milan, Italy

                Reviewed by: Olivier Goureau, INSERM U968 Institut de la Vision, France; Jan Wijnholds, Leiden University Medical Center, Netherlands

                *Correspondence: Zi-Bing Jin, jinzb@ 123456mail.eye.ac.cn

                These authors have contributed equally to this work

                This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology

                Article
                10.3389/fcell.2020.00128
                7068133
                32211407
                9d3f5dd0-c691-421c-ae99-626e23a43e40
                Copyright © 2020 Gao, Lei, Han, He, Jin, Zhang and Jin.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 22 November 2019
                : 13 February 2020
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 38, Pages: 14, Words: 0
                Categories
                Cell and Developmental Biology
                Original Research

                late onset,retinitis pigmentosa,ipscs,pde6b,retinal organoids

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