Abstract
Purpose :
Age-related macular degeneration (AMD) is a leading cause of vision loss after 50 years of age. While genetic studies have identified various polymorphisms associated with AMD predisposition, there still is no clear picture of the molecular and cellular players leading to disease initiation and progression. The main pathological hallmark of AMD is the accumulation of lipid-rich extracellular deposits, namely drusen, below the retinal pigmented epithelium (RPE), leading to retina degeneration. Several risk variants for AMD occur in lipid-related genes, including the apolipoprotein E (APOE) gene, but their role in AMD is unknown. Unfortunately, there are currently no model systems for a rigorous assessment of the interplay between genetic risk factors and drusen pathology. Using induced pluripotent stem cell (iPSC) technology we developed an in vitro human induced outer blood-retina barrier tissue (BRB) to investigate the role of APOE variants on AMD.
Methods :
We generated iPSC-derived RPE, pericytes and endothelial cells and combined them within a transwell system by adding RPE to the apical side and extracellular matrix-encapsulated vascular cells on the basal side. We have also established protocols for reproducing drusen-like deposits in BRBs. Using iPSC lines that were CRISPR-edited to harbor different variants of the APOE gene, we engineered isogenic BRBs with different APOE genotypes within specific cell types.
Results :
Transepithelial electrical resistance of the RPE within BRB evolves and stabilizes at around 4 weeks. Staining of the BRBs showed a monolayer of ZO-1-positive retinal epithelial cells, and an underlying 3D vascular compartment staining positive for endothelial cell marker PECAM1. We found APOE-rich clusters similar to drusen-like deposits between the RPE and vasculature. BRBs engineered with APOE3/3 RPE and APOE2/2 vasculature had larger deposits when compared to BRBs with APOE3/3 RPE and APOE3/3 or APOE4/4 vasculature.
Conclusions :
We developed a human BRB isogenic model to study the effect of genetic risk factors on the development of drusen and other AMD-related phenotypes. We are further expanding this platform by incorporating transcriptomics and lipidomics analysis of the BRBs. This multimodal strategy will establish a versatile platform for modeling AMD and genetic vulnerabilities.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.