Primary Cilia Regulates Human iPSC-RPE Maturation via Regulation of WNT Signaling

Abstract

Purpose : Induced pluripotent stem cell (iPSC) derivatives often do not fully-mature in vitro, limiting their use in cell therapy and disease modeling. Retinal pigment epithelium (RPE), a ciliated monolayer critical for maintaining the health and integrity of adjacent photoreceptors, is an attractive candidate for stem cell therapies to treat blinding eye diseases including Age-related macular degeneration. The primary cilium serves as a signaling and sensory hub and controls developmental cellular processes in many cell types, but the role of primary cilium in human RPE development remains largely unknown. The goal of this study is to explore the function and underlying mechanism of primary cilium in RPE development and maturation.

Methods : 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 cilium inducers or blockers, or canonical WNT agonist or antagonist. Electrophysiology, immunocytochemistry, electron microscopy, gene expression, and phagocytosis assays were used to determine the polarization and maturity of iPSC-RPE monolayer.

Results : The progressive development of primary cilia in human iPSC-RPE coincides with the formation of RPE tight junctions and epithelial cell morphology. Experimentally enhanced activation of primary cilia led to extensive apical processes, improved pigmentation, increased expression of adult-specific RPE genes, increased phagocytic capability, and significantly enhanced electrical responses that mimic native human RPE, demonstrating improved RPE maturation and functionality. Electrophysiological recordings in combination with immunostaining and phagocytosis using canonical WNT activator or inhibitors revealed that primary cilia-induced iPSC-RPE maturation was regulated through the suppression of canonical WNT signaling.

Conclusions : Our results demonstrate a developmental role for primary cilia in human iPSC-RPE maturation, which is mediated through the suppression of canonical WNT pathway. This study provides a mechanistic tool to mature RPE or other epithelial cell types derived from human iPSCs, and also provides insight into retinal degeneration caused by ciliopathies.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.