Analysis of Primary Cilia Phenotypes in Patient-Derived iPSC-RPE

Abstract

Purpose : Purpose
Primary cilia serve as extracellular protrusions, promoting communication between cells. The primary cilium has three main structures: the axoneme, the transition zone, and the basal body. Defective ciliary proteins and structure impede the proper function of genes, leading to ciliopathies. Patients with ciliopathies are likely to suffer from retinal degeneration. Retinal degeneration is a common phenotype among ciliopathies, affecting various tissues in the eye, including the photoreceptors and the retinal pigment epithelium. Previous research has shown the crucial role of cilia in the development and maturation of the RPE. However, research is limited regarding quantifiable data on cilia and the basal body in the RPE monolayer. Here, we develop a method to quantify the ciliary phenotype by measuring the ciliary structure and localization of ciliary proteins.

Methods : Methods
We differentiated iPSC from nine ciliopathy patients into RPE monolayer (iRPE) and performed immunohistochemistry for the ciliary axoneme (ARL13B), the basal body (γ-tubulin, Pericentrin) and intraflagellar transport (IFT88). We developed a MATLAB script to analyze ciliary proteins along the axoneme. ImageJ was used to quantify the cilia present in each cell and their respective lengths.

Results : Results
Patient iRPE had a lower abundance of cilia and impaired ciliary length, thus signifying a defective process of ciliogenesis. Cilia with a curved and truncated morphology were found among some of the patient lines by staining for Arl13B. The analysis for GT335 (a key component of the transition zone) showed decreased specificity across ciliopathy lines. Subsequent quantification of IFT88 (important for ciliary protein transport) exhibited accumulation in the ciliary tip suggesting a malfunction in ciliary trafficking, implying defective signaling in cilia. γ-tubulin showed normal composition of the basal body. Meanwhile, Pericentrin showed a disorganized structure of the pericentriolar matrix, indicating a malfunction in protein regulation at the base of the cilium.

Conclusions : Conclusion
Quantification methods using MATLAB and ImageJ were established to visualize ciliary defects in iRPE from ciliopathy patients. Given that the primary cilium is important for the RPE, studying its condition and contribution to the phenotype is crucial.

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