CRB1 mutations cause structural and molecular defects in patient-derived retinal pigment epithelium cells

Abstract

Mutations in the CRB1 gene can cause retinitis pigmentosa (RP), Leber congenital amaurosis, and other retinopathies, with retinal pigment epithelium (RPE) being a primary affected cell type. However, the effects of CRB1 variants on RPE cells remain poorly defined. Here, for the first time, we report an in vitro model of patient-specific RPE cells carrying the CRB1 mutations (c.2249G > A and c.2809G > A) to study CRB1-associated RP disease. The patient-derived RPE cells exhibited irregular cell morphology, sparse apical microvilli, abnormal tight junctions, and reduced expression of RPE markers. We also observed that impaired barrier function and phagocytosis lead to increased apical-to-basal movement of fluorescent molecules in disease RPE cells. Notably, transcriptomic analysis revealed decreased expression of cell junction-related genes. In addition, aggregated RPE cells on polydimethylsiloxane (PDMS) microwells significantly enhanced RPE phenotype and cell survival, which was associated with anti-epithelial-mesenchymal transition, anti-aging, and anti-apoptosis. In this study, our results reveal that CRB1-mutated RPE cells generated using RP patient-derived iPSCs could recapitulate the genotype-phenotype features of the disease and provide insights into the pathogenesis of CRB1-associated RPE cells. In addition, our study developed a cell aggregation culture method based on PDMS microwell platforms for the production of highly active and mature iPSC-derived RPE cells.