Developing functional and authenticated RPE tissue from iPS cells

National Eye Institute

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Abstract

Human iPSC-derived retinal pigment epithelium (RPE) cells do not fully mature in vitro and often continue to express certain markers. This limits their use as accurate disease models for adult-onset conditions and reduces their effectiveness as cell therapy candidates for degenerative eye diseases. To overcome this, we developed methods to fully polarize and mature iPSC-derived RPE cells. Using mouse models with defective ciliogenesis, we demonstrated that the primary cilium is essential for inducing RPE polarization. In these mice, loss of ciliogenesis resulted in sustained activation of canonical WNT signaling, at least through postnatal day one, preventing proper polarization. Building on this, we tested three drugs to modulate ciliogenesis in iPSC-RPE: Aphidicolin (a cell cycle blocker that induces two primary cilia per cell); PGE2 (enhances ciliogenesis by promoting ciliary protein trafficking), and HPI-4 (a dynein ATPase inhibitor that blocks ciliary trafficking and inhibits cilia function). Treatment with aphidicolin or PGE2 significantly improved apical process formation, epithelial morphology, and expression of RPE-specific markers compared to untreated or HPI-4-treated cells. Gene expression analysis showed that aphidicolin shifted cells toward a more adult-like phenotype. Despite overall improved ciliogenesis across the monolayer, fewer than 5% of cells developed double cilia, consistent with Ki67 labeling that confirmed <5% of cells were actively dividing. Electrophysiological analysis of intact RPE monolayers demonstrated significantly greater polarization in treated versus untreated cultures. Mechanistically, we found that non-canonical WNT signaling mediates RPE polarization induced by ciliogenesis. Using antibodies to PKC-δ (a key mediator of non-canonical WNT) and phospho-MLC (a downstream effector), we confirmed pathway activation upon cilium induction. Pharmacological inhibition of PKC-δ further validated that primary cilia promote RPE polarization through non-canonical WNT signaling. These findings provide new insight into developmental pathways required for complete RPE polarization and offer a strategy to generate fully mature, functional RPE cells for disease modeling and regenerative therapies. In addition, we discovered that iPSC-derived RPE cultures typically consist of a mixture of macular- and peripheral-type RPE cells. Through high-throughput screening, we developed methods to generate pure populations of either cell type. Using a machine learning–based algorithm, we quantified RPE cell shape metrics and compared these to native RPE, enabling us to classify and guide iPSC-RPE differentiation toward specific lineages. Ongoing work with iPSC-RPE derived from ciliopathy patients is further revealing the pathways that govern complete RPE maturation and polarization.