Abstract
Purpose :
The primary cilium is a microtubule-based antenna-like structure extending from the cell surface, important for the development and function of almost all cells in the human body. Mutations in various ciliary proteins are associated with a multitude of syndromic and non-syndromic diseases, collectively called ciliopathies. Retinal degeneration is the most common phenotype across all ciliopathies. It has already been shown that primary cilia are important in the development and maturation of the retinal pigment epithelium (RPE), a highly polarized cell monolayer in the back of the eye. However, to what extend the RPE is affected by different ciliopathy mutations is not well known. Here, we seek to understand the role of different cilia-associated genes in RPE maturation and polarization.
Methods :
We generated iPSC-derived RPE (iRPE) from nine patients with different ciliopathy-associated retinal degeneration including Bardet-Biedl syndrome, Joubert syndrome, Usher syndrome, autosomal dominant retinitis pigmentosa and Leber’s congenital amaurosis. iRPE cells were characterized using flow cytometry, immunostaining, transepithelial resistance (TER), RPE cell shape metrics, scanning and transmission electron microscopy.
Results :
Flow cytometry showed that more than 95% of differentiated cells expressed TYRP1 in all samples, an early RPE marker. RPE maturity and polarity was assessed by staining for RPE markers RPE65, Ezrin and Collagen IV. The gradual increase in TER over 5 weeks of maturation of the monolayer confirmed the junctional integrity of the iRPE. Machine learning based analysis of ciliopathy iRPE shape showed that all patient lines have a bigger cell size, less hexagonal and more elongated cells compared to controls with no ciliopathy mutations. Structural analysis using scanning and transmission electron microscopy revealed sparse and shorter apical processes in patient cells. Analysis of the primary cilia in the ciliopathy iRPE showed changes in the ciliary length indicating a malfunctioning cilium.
Conclusions :
We provide the first human in vitro RPE disease models for a broad variety of ciliopathies. We will perform further characterization of the ciliopathy iRPE cells to investigate the underlying molecular defects across all diseases. This work will shed light on how the primary cilium impacts RPE differentiation, maturation, and function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.