KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells
KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells
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DOI：
10.1167/iovs.61.5.38
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发表时间：
2020-05-01
影响因子：
4.4
通讯作者：
Ohuchi, Hideyo
中科院分区：
文献类型：
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作者：
Kanzaki, Yuki;Fujita, Hirofumi;Ohuchi, Hideyo
目的。本研究旨在建立和分析由KCNJ13基因突变引起的Leber先天性黑色素16型(LCA16)的细胞模型。方法设计针对KCNJ13基因靶点的两个引导RNA，利用CRISPR/Cas9系统获得KCNJ13基因敲除(KO)的人诱导多能干细胞(HiPSCs)。将KCNJ13-KO细胞分化为视网膜色素上皮细胞(hiPSC-RPEs)。免疫组织化学染色证实HIPSC-RPEs中存在KCNJ13-KO。通过摄取荧光标记的猪光感受器外段(POSS)来评价HiPSC-RPEs的吞噬活性。结果CRISPR/Cas9系统检测到KCNJ13-KO hiPSCs中KCNJ13基因的大部分翻译区被缺失，证实在由其诱导的RPE细胞中不存在Kir7.1蛋白。RPE标记基因BEST1和CRALBP在野生型(WT)和KCNJ13-KO hiPSC-RPE细胞中均有表达。然而，KCNJ13缺失的HiPSC-RPE细胞的吞噬活性和吞噬相关基因的表达明显低于WT.结论我们成功地用HiPSCs建立了LCA16的RPE模型。我们认为，RPE细胞吞噬POSS需要Kir7.1，而在缺乏Kir7.1的情况下，吞噬功能受损可能与LCA16的视网膜变性有关。
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.