Abstract
Purpose :
The cornea is a highly specialized tissue, that is crucial for vision. Currently, there is a need for in vitro models of the cornea to study infections, wound healing, drug screening, and as sources for transplant cells. Here, we report on the identification of distinct corneal epithelium as well as other important corneal cells through single cell RNA sequencing (scRNAseq) analysis of a 3D eye organoid.
Methods :
3D eye organoids were grown from H9 ESCs, seeding 3000 cells/well in a 96 ultra-low bind u-bottom plate in mTeSR media supplemented with 2.5% Matrigel and 40nM Thiazovivin (THIA) on day 0. Deafferentation was initiated at day 2 by changing to differentiation media (DM): GMEM with 10% knock out serum replacement, 1mM Sodium pyruvate, 1mM non-essential amino acids, 2mM L-glutamate, 1% penicillin-streptomycin solution and 55μM β-mercapto ethanol. Corneal specification was enhanced at day 32-46 by changing to DM:CnT-PR (1:1) supplemented with 10ng/mL keratinocyte growth factor (KGF) and 40nM THIA, before changing to maintenance media; DMEM:F12, 2% B-27 supplement, 10ng/ml KGF and 2μM THIA at day 48. At day 153, one organoid was digested with 2.5mg/mL collagenase II to form a single cell suspension. The single cell suspension was processed for 10x scRNAseq. The data was clustered by guided clustering using the Seurat package in R, and the resulting clusters were annotated using the Enricher online database and expression of specific gene markers.
Results :
scRNAseq data revealed clusters that could be annotated as 11 different cell populations, among which were a corneal epithelium population, corneal stroma, and corneal endothelial like cells. The corneal epithelium had a high expression of corneal keratins (KRT12, KRT5, KRT15) as well as expression of mucus (MUC16), E-cadherin (CDH1) and eye filed marker PAX6. The corneal stroma- and corneal endothelium-like cells had high expression of collagen (e.g. COL8A1), hyaluronan acid synthase (HAS2), KERA, mesenchymal, and hematopoietic markers CD44, CD34 and CD133 (PROM1).
Conclusions :
We report on the generation of 3D eye organoids from hESCs that mature to form cells from the cornea. These organoids can be used as tools to study infections, wound healing, and pharmacological screening in a cost-effective way with reduced need for animal experiments.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.