Retinal Pigment Epithelium Modulates Endothelial Cell, Fibroblast, and Pericyte Gene Expression Patterns in 3D Bioprinted Tissues

Abstract

Purpose : Age-related macular degeneration (AMD) disease processes are thought to initiate at the Retinal Pigment Epithelial Cell (RPE)/choroid interface. However, how each tissue contributes to disease pathogenesis is not known. We hypothesized that the presence of RPE would reduce gene expression patterns indicative of AMD within 3D bioprinted Outer Blood Retinal Barrier (oBRB) models. Here, we utilized 3D bioprinted oBRB models and single-cell RNA-seq to assess how the presence or absence of RPE affects endothelial cell (EC), fibroblast, and pericyte gene expression patterns within the context of AMD pathogenesis.

Methods : iPSC-derived EC, iPSC-derived fibroblasts, and ocular pericytes were encapsulated in a fibrinogen and gelatin-based hydrogel solution and 3D-printed onto degradable PLGA scaffolds or PET membranes to generate choroid-like tissues. iPSC-RPE were seeded on the apical side of scaffolds 7 days after bioprinting. After 6 weeks of maturation, tissues were digested using dispase, collagenase, and RNAse. Single-cell suspensions were retrieved and fixed to generate barcoded single-cell emulsions and cDNA libraries. The Illumina HiSeq 3000, Cellranger package, the Seurat R package, and the Biowulf computing cluster analyzed single-cell sequencing data. Gene set enrichment analysis was performed using EnrichR and curated using Gene Ontology 2021 ontology sets. EC, fibroblast and pericyte populations were identified within 3D oBRB models using UMAP clustering, EnrichR, and the expression of key signature genes by each cell type.

Results : In EC populations, the presence of RPE in tissues enhanced expression of key choriocapillaris signature genes while the absence of RPE enhanced expression of vascular morphogenesis genes and AMD signature genes. In fibroblast populations, the presence of RPE downregulated senescence and aging genes while enhancing the expression of genes associated with melanin synthesis, phenol synthesis, and retinoid metabolic processes; the absence of RPE enriched genes related to AMD and ECM synthesis. Pericytes demonstrated similar effects on ECM synthesis genes as fibroblasts when RPE were absent.

Conclusions : Combined, these data suggest that the presence of RPE changes EC specification toward choriocapillaris, downregulates senescence and aging in fibroblasts, and downregulates AMD-associated gene expression patterns within oBRB models.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.