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      Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis


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          X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.


          • hiPSC-derived retinal organoid model recapitulates key features of XLRS

          • CRISPR/Cas9 correction normalizes RS1 secretion and retinal development

          • Transcriptome analysis links XLRS to other hereditary retinopathies


          Chiou, Schlaeger, and colleagues use hiPSC-derived retinal organoids to model X-linked juvenile retinoschisis. They show that patient hiPSC-derived retinal organoids replicate key pathologies observed in patients, including retinal splitting and photoreceptor deficit. The observed abnormalities were normalized in organoids derived from isogenic CRISPR/Cas9 gene-corrected hiPSCs. This validated XLRS in vitro model could be used to test and optimize therapeutic approaches.

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          Most cited references 38

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          A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions.

          Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.
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            iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration.

            Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.
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              Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells

              The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.

                Author and article information

                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                24 October 2019
                12 November 2019
                24 October 2019
                : 13
                : 5
                : 906-923
                [1 ]Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan
                [2 ]Institute of Pharmacology, National Yang-Ming University, Taipei 11221, Taiwan
                [3 ]Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA
                [4 ]School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan
                [5 ]Institute of Food Safety and Health Risk Assessment, National Yang-Ming University, Taipei 11221, Taiwan
                [6 ]Department of Ophthalmology, Taipei Veterans General Hospital, Taipei 11217, Taiwan
                [7 ]Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan
                [8 ]Cancer Progression Research Center, National Yang-Ming University, Taipei 11221, Taiwan
                [9 ]Shiley Eye Institute, University of California San Diego, La Jolla, CA 92093, USA
                [10 ]School of Medicine, Fu-Jen Catholic University, New Taipei City 24205, Taiwan
                [11 ]Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA
                [12 ]Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan
                [13 ]Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan
                [14 ]Department of Ophthalmology, Shin Kong Wu Ho-Su Memorial Hospital & Fu-Jen Catholic University, Taipei 11101, Taiwan
                [15 ]Genomic Research Center, Academia Sinica, Taipei 11529, Taiwan
                Author notes
                [∗∗ ]Corresponding author shchiou@ 123456vghtpe.gov.tw
                © 2019 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).



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