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YUKI KANZAKI, Hirofumi Fujita, Keita Sato, Mio Hosokawa, Hiroshi Matsumae, Fumio Shiraga, Yuki Morizane, Hideyo Ohuchi; KCNJ13 gene deletion impairs phagocytosis in retinal pigment epithelium derived from human-induced pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1510.
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© ARVO (1962-2015); The Authors (2016-present)
Leber's congenital amaurosis type 16 (LCA 16) is a retinal degenerative disease caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward rectifying potassium ion channel. In LCA 16, it is speculated that phagocytosis, one of the functions of retinal pigment epithelial cells (RPE), is impaired, but the details are unknown. The purpose of this study is to establish a cell model of LCA16 and analyze the phagocytic ability of the cell.
The two gRNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knockout (KO) human-induced pluripotent stem cells (hiPSC) were generated using the CRISPR/Cas9 system. The KCNJ13 KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPE). The KCNJ13 KO in hiPSC-RPE was confirmed by immunostaining. Phagocytic activity of hiPSC-RPE was assessed using uptake of fluorescently labeled porcine photoreceptor outer segments (POS). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction (PCR).
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 hiPSC-RPE cells. Expression of RPE marker genes such as BEST1and CRALBP was retained in the wild type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity was significantly reduced to 23% compared to WT (p < 0.001) and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells such as ITGBV, CD81, and MERTK were significantly reduced compared to those of WT (p < 0.05 for all).
We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POS by RPE cells and that impaired phagocytosis in the absence of Kir7.1 likely leads to the retinal degeneration found in LCA16.
This is a 2020 ARVO Annual Meeting abstract.
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