Abstract
Purpose :
Artificial outer blood-retinal barrier (oBRB) models present numerous applications in age-related macular degeneration (AMD) disease modeling. A thorough understanding of behaviors by different cell populations in the model has significant implications on the understanding of disease progression. Here, iPSC-derived epithelial cells (RPE) and endothelial cells (EC) within a 3D-bioprinted model of the oBRB were analyzed using single-cell RNA-seq to begin determining how printed choroid and RPE carry out specialized functions compared to their in-vivo counterparts.
Methods :
EC, Pericytes, and fibroblasts were suspended in gelatin-based bio-ink, bioprinted onto biodegradable poly(lactic, co-glycolic) acid scaffolds and matured for one week. Afterward, RPE was seeded on the opposite sides of the membrane. The completed tissues were cultured for 6 weeks before enzymatic tissue dissociation and cell isolation using magnetic assisted cell sorting. Single-cell suspensions were used to generate barcoded single-cell gel bead-in-emulsions. Following cDNA amplification and processing, libraries were read using an Illumina HiSeq 3000. The Cellranger software package, Seurat R package, and the Biowulf computing cluster were used to process single-cell sequencing data. Gene set enrichment analysis was performed using GSEA v4.0.3, curated Gene Ontology sets and EnrichR.
Results :
Differential expression (DE) analysis of RPE gene expression show a general increase in RPE signature genes in the bioprinted oBRB compared to transwell monolayer culture. tSNE analysis revealed broadly homogenous populations of RPE in oBRB and monolayer cultures. DE analysis of EC populations suggested increases in blood vessel maturation, choroidal maturation, and angiogenesis genes in the oBRB model. tSNE analysis suggested that EC in the oBRB model separated into populations at different stages of blood vessel maturation and an inflammatory population.
Conclusions :
RNA-seq data suggests that the RPE monolayer acts as a homogeneous cell population within the 3D bioprinted tissue, while ECs show a greater degree of separation into differential stages of vascular maturation within the tissues. The variable stages of maturation are likely a consequence of a dynamic signaling environment within the printed choroid which would promote angiogenesis, maturation and inflammation.
This is a 2021 ARVO Annual Meeting abstract.