Optimizing Biodegradable Scaffolds for Developing iPS Cell Derived RPE Tissue

Abstract

Purpose: Age-related macular degeneration (AMD) is a leading cause of vision loss in the United States. The disease is thought to originate by a malfunctioning retinal pigment epithelium (RPE), which accompanies photoreceptor death. The RPE is a monolayer of cells that performs multiple roles required for survival and proper functionality of the photoreceptors, including fluid level regulation, ion transport, nutrient replenishment, and phagocytosis of photoreceptor outer segments. Potential therapies for AMD consist of replacing the diseased RPE layer. Previous work has shown that transplanting RPE cells on a scaffold rather than cell suspension provides greater viability and integration into the host retina.

Methods: We used a triphasic WNT and TGF treatment protocol to generate fully differentiated and functional RPE from iPS cells. The resulting RPE was cultured as a polarized monolayer on two different types of biodegradable scaffolds: a dual layer poly lacto-co-glycolic acid (PLGA) and a cross-linked electro-spun collagen scaffold. We compared cell viability on collagen and PLGA scaffolds and monitored the degradation of the scaffolds and cell growth over time. RPE tissue structure and functionality was confirmed through electron microscopy, immunostaining, gene expression analysis, fluid transport assays, and electrophysiological analysis.

Results: iPS cell derived RPE was successfully cultured as electrically intact monolayers on biodegradable scaffolds. As compared to collagen, PLGA provides a better substrate for RPE scaffold and helps cells form a confluent monolayer. Electrophysiological experiments show that RPE cells on PLGA develop resistance >100 ohms/cm2, suggesting tight electrical contacts between neighboring cells. RPE cells on PLGA show improved responses to changes in intracellular calcium and for their ability to transport fluid from apical to basal side on the monolayer.

Conclusions: Successful validation of artificial RPE tissue indicates its suitability for implantation into the subretinal space. We are currently testing these scaffolds in animal models. This work will provide a GMP-ready protocol for generating RPE tissues on a scaffold for transplantation as treatment for diseases like AMD.

Keywords: 701 retinal pigment epithelium • 508 electrophysiology: non-clinical