start-ver=1.4 cd-journal=joma no-vol=61 cd-vols= no-issue=5 article-no= start-page=38 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=202005 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells en-subtitle= kn-subtitle= en-abstract= kn-abstract=Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel.
Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction.
Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT.
Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16. en-copyright= kn-copyright= en-aut-name=KanzakiYuki en-aut-sei=Kanzaki en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=FujitaHirofumi en-aut-sei=Fujita en-aut-mei=Hirofumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=SatoKeita en-aut-sei=Sato en-aut-mei=Keita kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HosokawaMio en-aut-sei=Hosokawa en-aut-mei=Mio kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=MatsumaeHiroshi en-aut-sei=Matsumae en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShiragaFumio en-aut-sei=Shiraga en-aut-mei=Fumio kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=MorizaneYuki en-aut-sei=Morizane en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=OhuchiHideyo en-aut-sei=Ohuchi en-aut-mei=Hideyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=2 en-affil=Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= affil-num=3 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=4 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=5 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=6 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=7 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= affil-num=8 en-affil=Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences kn-affil= en-keyword=Kir7.1 kn-keyword=Kir7.1 en-keyword=KCNJ13 kn-keyword=KCNJ13 en-keyword=human-induced pluripotent cells kn-keyword=human-induced pluripotent cells en-keyword=retinal pigment epithelium kn-keyword=retinal pigment epithelium en-keyword=phagocytosis kn-keyword=phagocytosis END