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KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells

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KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells Biochemistry and Molecular Biology KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells Yuki Kanzaki,1,2 Hirofumi Fujita,2 Keita Sato,2 Mio Hosokawa,1 Hiroshi Matsumae,1 Fumio Shiraga,1 Yuki Morizane,1 and Hideyo Ohuchi2 1Department of Ophthalmology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan 2Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Correspondence: Yuki Morizane, PURPOSE. The purpose of this study was to establish and analyze a cell model of Leber Department of Ophthalmology, congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene Okayama University Graduate encoding Kir7.1, an inward-rectifying potassium ion channel. School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, METHODS. The two guide RNAs specific to the target sites in the KCNJ13 gene were 700-8558, Japan; designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) [email protected]. were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differ- Hideyo Ohuchi, Department of entiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC- Cytology and Histology, Okayama RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed University Graduate School of using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Medicine, Dentistry, and Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain Pharmaceutical Sciences, Okayama, Japan; reaction. [email protected]. RESULTS. Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO Received: November 24, 2019 hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not Accepted: April 24, 2020 present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as Published: May 21, 2020 BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE Citation: Kanzaki Y, Fujita H, Sato K, cells. However, phagocytic activity and expression of phagocytosis-related genes in the et al. KCNJ13 gene deletion impairs KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. cell alignment and phagocytosis in CONCLUSIONS. We succeeded in generating an RPE model of LCA16 using hiPSCs. We retinal pigment epithelium derived suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired from human-induced pluripotent phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration stem cells. Invest Ophthalmol Vis Sci. 2020;61(5):38. found in LCA16. https://doi.org/10.1167/iovs.61.5.38 Keywords: Kir7.1, KCNJ13, human-induced pluripotent cells, retinal pigment epithelium, phagocytosis etinal pigment epithelium (RPE) has multiple functions, There are many questions regarding the pathophysio- R such as ion exchange in the subretinal space, main- logic mechanism of LCA16. Previous studies using KCNJ13 tenance of the visual cycle, secretion of trophic factors conditional knock-out (KO),13 genetic mosaic KO,9 and (e.g., vascular endothelial growth factor), and formation knock-down8 mice revealed that deletion or decrease of the blood–retinal barrier. The RPE also has an impor- of the Kir7.1 protein induces photoreceptor loss and tant role in the continuous renewal of photoreceptor outer abnormal changes in the electroretinogram. These find- segments (POSs) through phagocytosis.1,2 Leber congeni- ings suggest that Kir7.1 dysfunction in RPE cells in tal amaurosis (LCA) is a genetic disease with progressive the subretinal space may cause structural and functional severe visual impairment, retinal structural abnormalities, abnormalities in the retina; however, how the dysfunc- and abnormal electroretinograms.3–5 In LCA, there have tion of Kir7.1 in the RPE causes LCA symptoms remains been numerous known genetic alterations, one of which, unclear. LCA16, was found to be associated with KCNJ13 gene muta- Here, we aimed to establish an RPE cell model of LCA16 tions.3–5 The KCNJ13 gene encodes the Kir7.1 protein, an by deleting the KCNJ13 gene using the CRISPR/Cas9 system inward-rectifying potassium ion channel, which is expressed in human-induced pluripotent stem cells (hiPSCs). KCNJ13- by the apical microvilli of RPE cells in ocular tissues.6,7 deficient hiPSCs were generated and induced to differentiate Kir7.1 regulates potassium ions in the subretinal space8 and into hiPSC-RPE cells, where the functions of KCNJ13 were is known to be involved in photoreceptor maintenance.9–12 analyzed. Copyright 2020 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/28/2021 Kir7.1 Regulates RPE Alignment and Phagocytosis IOVS | May 2020 | Vol. 61 | No. 5 | Article 38 | 2 FIGURE 1. Establishment of KCNJ13-KO hiPSC. (A) KCNJ13 gene locus and the target sites of designed gRNAs. Schematic diagram of gRNA targeting the human KCNJ13 locus. The black rectangle represents the protein coding region of the KCNJ13 gene. The white rectangle represents the untranslated region of the KCNJ13 gene. The two gRNA sequences are designed to delete exons 2 and 3 from the KCNJ13 gene. (B) PCR products of induced pluripotent stem cell line (iPSC) clones whose KCNJ13 genes were edited by the CRISPR/Cas9 system. PCR was performed using primers to confirm that the intended gene editing occurred inthe KCNJ13 locus. The 5129-bp PCR product was observed in the WT iPSC, and the approximately 840-bp PCR fragment was produced in the mutant iPSC. The bi-allelic gene KO was present in clone no. 1. (C) KCNJ13 DNA sequence obtained from WT and KCNJ13-KO iPSCs. KCNJ13-KO was confirmed in clone no. 1, and different mutations occurred in each allele. (D) The amino acid sequences deduced from the nucleotide sequences of the WT and KO alleles are shown. The WT sequence encodes for 360 amino acids. The sequence of allele 1 encodes for 57 amino acids, and that of allele 2 encodes for 62 amino acids. METHODS the 3UTR (Fig. 1A). Two CRISPR RNA (crRNA) complemen- tary to the target sequences and the trans-activating CRISPR Culture of Human iPS Cells RNA (tracrRNA) were obtained from Integrated DNA Tech- The hiPSC line 454E2,14 generated from healthy human nologies, Inc. (Coralville, IA, USA). The crRNA and tracr- dental pulp cells, was obtained from the RIKEN BioRe- RNA were each annealed to construct gRNA according to source Center (Ibaraki, Japan). The hiPSCs were maintained the manufacturer’s instructions. We performed gene editing 17,18 and differentiated as previously described15 (Supplementary of hiPSCs according to previous studies (see Supplemen- Methods). tary Methods). Immunofluorescence Gene Editing of hiPSCs Via CRISPR/Cas9 System Immunostaining was performed according to previous By utilizing CRISPRdirect (https://crispr.dbcls.jp),16 more reports19,20 with minor modifications (see Supplementary than two candidate target sequences for guide RNA (gRNA) Methods for details). We confirmed expression of undif- were found in the human KCNJ13 gene (NM_001172416) ferentiated marker genes in proliferating hiPSCs and local- obtained from the Ensemble database (http://asia.ensembl. ization of the Kir7.1 protein in the RPE differentiated org/index.html). To ensure specificity, the hit sequence from intact hiPSCs (hiPSC-RPE). To visualize the cellular showing one (“1”) in the column “20 mer + PAM,” which polarity and nuclei, hiPSC-RPE cells were co-stained with indicates a perfect match with the intended target site, was rhodamine (tetramethylrhodamine)–phalloidin, anti-RPE65, selected. Only RFX1 was listed as a candidate off-target gene. and 4,6-diamidino-2-phenylindole (DAPI). In the phagocy- To delete most of the KCNJ13 gene, two target sequences tosis assay, hiPSC-RPE cells were stained by anti-ZO-1 or were determined; one was localized to approximately 100 anti-ezrin to identify the location of incorporated POSs. bases downstream of the start codon, and the other was in Immunocytochemical staining was analyzed and images Downloaded from iovs.arvojournals.org on 09/28/2021 Kir7.1 Regulates RPE Alignment and Phagocytosis IOVS | May 2020 | Vol. 61 | No. 5 | Article 38 | 3 were taken on an LSM 780 confocal microscope equipped Phagocytosis Assay with ZEN 2009 software (Carl Zeiss Microscopy GmbH, Jena, 24 Germany). Phagocytosis assay was performed according to Davis et al. The hiPSC-RPE cells were seeded in 12-well Transwells at 5 × 105 cells/well. After 2 and 4 weeks, the hiPSC-RPE cells were supplemented with 2.5 μg/ml human MFG-E8 (R&D Western Blot Analysis Systems, Minneapolis, MN, USA) and 2 μg/ml human Protein Western blot analysis was performed according to previ- S (Aniara Diagnostica, West Chester, OH, USA) and chal- ous studies.19,21 Primary antibodies used were as follows: lenged for 5 hours with 10 POSs per cell. Subsequently, the anti-BEST1 (1:1000; Abcam, Cambridge, UK), anti-CRALBP Transwell membrane on which the hiPSC-RPE cells were (1:1000; Abcam), and anti-cyclophilin B (1:1000; Cell Signal- cultured was removed, and the cells were processed for ing Technology, Danvers, MA, USA). immunostaining with anti-ZO-1 or anti-ezrin and nuclear staining with DAPI. The number of POSs below ZO-1 or ezrin was counted as internalized POSs. The number of inter- nalized POSs per cell and the number of POSs per area Counting of Protruded Cells Per Unit Area (μm2) were measured. The mean number of internalized The hiPSC-RPE cells were seeded onto Transwells (Corning, POSs in the KCNJ13-null hiPSC-RPE was normalized to wild- Inc., Corning, NY, USA) and observed under a microscope at type (WT).
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