Abstract
Purpose :
Mutations in the CRB1 can induce severe retinal dystrophies, but the underlying pathogenesis remains poorly defined. In this study, we generated a CRB1 mutation disease model of retinal pigment epithelial (RPE) cells and investigated the pathological mechanism of the disease.
Methods :
Induced pluripotent stem cells (iPSCs) of a retinitis pigmentosa (RP) patient carrying the CRB1 mutation were induced into RPE cells. The expressions of RPE-specific markers were examined by immunofluorescence staining. Phagocytic ability was assessed by measuring the internalized fluorescein isothiocyanate (FITC)-labeled photoreceptor outer segments (POS). For the detection of cellular barrier function, the iRPE cells were seeded on 24-transwell inserts at a density of 1 x 104 cells/insert. Subsequently, transepithelial electrical resistance (TER) measurement was performed with a volt-ohm meter and a permeability assay was conducted by measuring the apical-to-basal movements of FITC dextran.
Results :
Mature RPE cells derived from a CRB1-RP patient and normal iPSC cell lines displayed a classic cobblestone-like appearance, with pigmented and polygonal morphology. Immunofluorescence staining results also revealed that RPE biomarkers were expressed in the RP-iRPE and NC-iRPE groups, including tight junction marker ZO-1 and transcription factor marker MITF (Fig 1A). These results suggested that an adequate disease model was successfully established in this study. In addition, we observed the typical hexagonal structure of the NC-iRPE group, while irregular, abnormal morphology in the RP-iRPE group via immunofluorescence staining of the ZO-1 marker. The internalized localizations of FITC-labeled POS phagocytosis in the two groups were observed and the results showed that the number of internalized POS was markedly reduced in the RP-iRPE group (Fig 1B). After 2 weeks in transwell inserts, the TEER values of the RPE monolayer in the RP-iRPE group were lower than those in the NC-iRPE group (Fig 1C). In addition, a permeability assay was performed and the result showed that the FITC molecule penetrated from the apical-to-basal side in the RP-iRPE group more than in the NC-iRPE group (Fig 1D).
Conclusions :
In this study, we demonstrated that RPE cells with the CRB1 mutation displayed irregular morphology and aberrant phagocytosis and barrier function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.