このエントリーをはてなブックマークに追加
ID 60103
FullText URL
Thumnail fulltext.pdf 5.45 MB
Author
Kanzaki, Yuki Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
Fujita, Hirofumi Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaken ID researchmap
Sato, Keita Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
Hosokawa, Mio Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kaken ID
Matsumae, Hiroshi Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
Shiraga, Fumio Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kaken ID researchmap
Morizane, Yuki Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kaken ID
Ohuchi, Hideyo Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kaken ID researchmap
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.
Keywords
Kir7.1
KCNJ13
human-induced pluripotent cells
retinal pigment epithelium
phagocytosis
Published Date
2020-05
Publication Title
Investigative Ophthalmology & Visual Science
Volume
volume61
Issue
issue5
Publisher
Association for Research in Vision and Ophthalmology
Start Page
38
ISSN
0146-0404
NCID
AA00683736
Content Type
Journal Article
language
英語
OAI-PMH Set
岡山大学
Copyright Holders
Copyright 2020 The Authors
File Version
publisher
PubMed ID
DOI
Web of Science KeyUT
Related Url
isVersionOf https://doi.org/10.1167/iovs.61.5.38
License
https://creativecommons.org/licenses/by-nc-nd/4.0/
Funder Name
Ministry of Education, Culture, Sports, Science and Technology
助成番号
15K10475
18K09410
19K18878