Induced Pluripotent Stem Cell Modeling of Best Disease and Autosomal Recessive Bestrophinopathy

Abstract

Purpose: To understand the pathophysiology of Best disease (BD) and autosomal recessive bestrophinopathy (ARB) by establishing an in vitro model using human induced pluripotent stem cell (iPSC). Materials and Methods: Human iPSC lines were generated from mononuclear cells in peripheral blood of one ARB patient, one autosomal dominant BD patient, and two normal controls. Immunocytochemistry and reverse transcriptase polymerase chain reaction in iPSC lines were conducted to demonstrate the pluripotent markers. After the differentiation of iPSC into functional retinal pigment epithelium (RPE), morphological characteristics of the RPE were evaluated using confocal microscopy and immunocytochemistry. The rates of fluid flow across iPSC-RPE monolayer were measured to compare apical to basal fluid transports by RPE. RNA sequencing was performed on iPSC-RPE to identify the differences in gene expression profiles, and specific gene sets were tested using Gene Set Enrichment Analysis. Results: Morphological characteristics, gene expression, and epithelial integrity of ARB iPSC were comparable to those of BD patient or normal control. Fluid transport from apical to basal was significantly decreased in ARB iPSC-RPE compared with BD iPSC-RPE or control iPSC-RPE. Gene Set Enrichment Analysis confirmed that ARB iPSC-RPE exhibited significant enrichments of epithelial-mesenchymal transition gene set and TNF-α signaling via NF-κB gene set compared to control iPSC-RPE or BD iPSC-RPE. Conclusion: A human iPSC model of ARB showed a functional deficiency rather than anatomical defects. ARB may be caused by RPE dysfunction following BEST1 mutation.

Testing Mitochondrial-Targeted Drugs in iPSC-RPE from Patients with Age-Related Macular Degeneration

“…iPSC-RPE can also provide the opportunity for modeling disease on a patient-by-patient basis. This was shown in previous studies where patient-specific iPSC-RPE lines were used to investigate both monogenetic retinal diseases [34][35][36][37][38] as well as disease arising from both genetic and environmental factors, including AMD [15,24,25,39,40].…”

Human pluripotent stem cells for the modelling of retinal pigment epithelium homeostasis and disease: A review

“…49 BD-iPSC-derived RPE cells with various BEST1 mutations possess various functional defects, including a delayed degradation of POS, increased oxidative stress following chronic POS feeding, 50,51 defective chloride conductance, 52 or decreased fluid transport. 53 Work with BD-iPSC-derived RPE has also permitted exploring the potential of gene augmentation to treat BD. Dominantly inherited disorders are commonly excluded as candidates for gene replacement therapies, as it is challenging to silence the dominant allele without altering wild-type gene expression.…”

“…BEST1 encodes a calcium‐activated chloride channel found in the RPE, with disease‐associated mutations clustering within regions encoding calcium or chloride‐ion binding sites 49 . BD‐iPSC‐derived RPE cells with various BEST1 mutations possess various functional defects, including a delayed degradation of POS, increased oxidative stress following chronic POS feeding, 50,51 defective chloride conductance, 52 or decreased fluid transport 53 . Work with BD‐iPSC‐derived RPE has also permitted exploring the potential of gene augmentation to treat BD.…”

Structure and Function of the Bestrophin family of calcium-activated chloride channels

“…Over 250 disease-causing mutations have been identified in the VMD2 gene, and iPSC-RPE-based models have been used to identify a wide array of cellular dysfunctions in patient-derived tissues with increased throughput. The wide range of dysfunctions induced by Best1 disease-causing mutations include a reduced ability of the RPE to efficiently phagocytose or process photoreceptor outer segment, changes in intracellular Ca 2+ handling, altered fluid flux, reduced Best1 protein expression, ER quality control-dependent and -independent mechanisms, increased or decreased anion permeability, as well as direct effects to the channel’s CaCC activity [ 31 , 108 , 109–114 ]. These models have enabled diverse therapeutic strategies, which include the development of pharmacological chaperones to restore Best1 expression, utilizing CRISPR/Cas9 to repair mutant BEST1 genes, and a curcumin-based nanomedicine approach to increase Best1 mRNA and protein levels [ 109 , 113 , 115 , 116 ].…”