CEP290 or IFT88 Loss of Function in RPE Suggest a Role for Primary Cilia in Human iPSC-RPE Maturation and Provide Insights into the Mechanism of Ciliopathy-Induced Retinal Degeneration

Abstract

Purpose : Retinal pigment epithelium (RPE), a ciliated monolayer in the back of the eye, is critical for maintaining the health and integrity of adjacent photoreceptors. Our previous studies demonstrate that the primary cilia play important role in the regulation of RPE monolayer maturation. However, a direct genetic link between primary cilium proteins and RPE maturation has not been explored yet. The goal of this study is to determine the role of CEP290 and IFT88, two key primary cilium proteins in RPE development and maturation.

Methods : Healthy or CEP290 mutant human iPSC derived RPE (iPSC-RPE) were grown on semi-permeable transwells to generate a confluent monolayer. Primary cilia in iPSC-RPE were manipulated using doxycycline-inducible IFT88 shRNA lentivirus. The effective knockdown of IFT88 was verified by Western blot. Immunocytochemistry and scanning electron microscopy were used to assess the effect of IFT88 knockdown on primary cilia formation. Gene expression and electrophysiology were used to determine the level of iPSC-RPE monolayer polarization and maturation.

Results : IFT88 knockdown prevented primary cilia formation and yielded sparse apical processes in iPSC-RPE monolayer. In comparison CEP290 mutations led to abnormal primary cilium and apical processes in iPSC-RPE. Gene expression analysis revealed that the RPE-signature genes and adult-specific RPE genes were downregulated in IFT88 knockdown and CEP290 mutant RPE cells. Electrophysiology recordings demonstrated that the suppression of primary cilia with IFT88 shRNA led to significantly decreased RPE transepithelial potential and whole tissue resistance; the electrical responses to physiologically relevant stimuli were also dramatically reduced, indicating deficient RPE maturation and functionality.

Conclusions : Our results demonstrate that IFT88 knockdown and CEP290 mutations severely affected primary cilia formation and profoundly disrupted iPSC-RPE monolayer maturation. This study presents direct evidence for the critical role of primary cilia in RPE maturation, and provides insights into the mechanism of ciliopathy-induced retinal degeneration.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.