Deciphering Shared Phenotypic Traits in iPSC-Derived RPE from Ciliopathy Patients with Mutation-Specific Variations

Abstract

Purpose : The primary cilium, a microtubule-based antenna-like structure in all human cell types, is associated with a spectrum of syndromically heterogeneous developmental disorders - ciliopathies. Retinal degeneration is a typical manifestation of these disorders, leading to gradual vision loss. Primary cilia are important for the development and function of the retinal pigment epithelium (RPE), a highly polarized monolayer important for the integrity of the retina. However, the extent to which different ciliary mutations affect the RPE remains unclear. This study aims to identify shared phenotypes and mutation-specific variations in RPE affected by ciliopathy-associated mutations.

Methods : We generated induced pluripotent stem cell (iPSC)-derived RPE (iRPE) from nine patients harboring mutations in various ciliary proteins (BBS1, BBS10, BBS16, CEP290, LCA5, MYO7A, and PRPF31), each associated with various degrees of retinal degeneration. Evaluating ciliopathy patient iRPE involved a comprehensive analysis for morphological, structural, and functional integrity aspects, allowing us to discern both differential and shared RPE phenotypes in ciliopathy patients.

Results : Patient iRPE revealed heterogeneous alterations in primary cilia abundance, length, shape, and functionality. We detected abnormal cell morphology in patient iRPE lines using machine learning-based single-cell analysis. Evaluation of cell polarization, assessed through staining for mature RPE markers (RPE65, EZRIN, and COL IV), in addition to scanning and transmission electron microscopy, suggests differential abnormalities in apical microvilli among patient lines. Furthermore, the observed loss of epithelial phenotype was characterized by compromised junctional integrity, upregulated expression of vimentin and gene expression changes indicative of epithelial-to-mesenchymal transition (EMT).

Conclusions : This study presents the first human in vitro RPE disease models spanning a wide spectrum of ciliopathies. We found that in patient-derived iRPE, EMT and RPE immaturity are initiated independent of the mutation. Identifying shared molecular targets within iRPE derived from ciliopathy patients with various mutations holds promise for developing mutation-agnostic gene and drug-based therapeutic strategies aiming to address the current absence of treatment options for ciliopathy-associated retinal degeneration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.