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

Abstract

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 knockout (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.

Novel roles for voltage‐gated T‐type Ca2+ and ClC‐2 channels in phagocytosis and angiogenic factor balance identified in human iPSC‐derived RPE

“…We show here that both Kir4.1 and Kir7.1 are localized to the apical microvilli. This is consistent to what has been previously reported in rat 72,73 and bovine 74 RPE cells, and what was recently reported for Kir7.1 in hiPSC‐derived RPE 75 . However, this is the first description of Kir4.1 localization in hiPSC‐derived RPE.…”

“…This is consistent to what has been previously reported in rat 72,73 and bovine 74 RPE cells, and what was recently reported for Kir7.1 in hiPSC-derived RPE. 75 However, this is the first description of Kir4.1 localization in hiPSC-derived RPE. Furthermore, using patient-specific hiPSC-derived RPE it was also suggested that Kir7.1 plays a role in phagocytosis, 75 consistent with its localization in the microvilli and the important role these structures play in POS internalization, thus, we did not investigate this function further.…”

Sensing through Non-Sensing Ocular Ion Channels

“…In vivo KCNJ13 conditional or mosaic KO and knock-down mice exhibited a loss of photoreceptors and abnormal electroretinogram due to reduced or absent Kir7.1 [185][186][187]. A recent study in KCNJ13-KO hiPSC-RPE cells lacking Kir7.1 channel revealed the loss of phagocytic activity as well as the reduced expression of phagocytosis-related genes, which could be a possible cause of retinal degeneration as seen in LCA16 patients [188].…”

Heterogeneity of Potassium Channels in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium

“…K + channels in the RPE are linked to the generation of membrane potential, 3 control of cell volume, 4 transport of ions and water, 2 and buffering of K + concentration following its light-induced changes in the subretinal space. 5 Recently, Kir7.1 was shown to participate in the phagocytosis of photoreceptor outer segments 6-8 and secretion of growth factors. 7 Due to the great importance of K + channels to RPE functionality, understanding their physiology in human pluripotent stem cell (hPSC)-derived RPE is important for the success of cell transplantation therapies.…”

“…Furthermore, hPSC-RPE is being used in healthy and disease-specific cell models to study RPE cell biology and visual disorders. 6 , 8 , 18-30 RPE participates in the pathogenesis of numerous diseases that threaten visual function, such as age-related macular degeneration (AMD), 31 Bestrophinopathy, 32 and Leber congenital amaurosis. 6 , 8 Some of these diseases are directly linked to the misfunctioning of a specific ion channel.…”