Abstract
Purpose :
To establish a well-defined 3D corneal organoid model that will be useful in studying ocular surface diseases, their genetic modeling and screening of pharmaceutical drugs. We performed single-cell RNA sequencing (scRNA-seq) of a human donor cornea and a cornea organoid developed in culture from induced pluripotent stem cells (iPSC) to elucidate corneal cell populations represented in the organoid.
Methods :
One cornea organoid (7 month old), and a healthy donor (41 year old) cornea (Lions Eye Institute for Transplant and Research, FL) were digested for 5 h with Collagenase type I (2 mg/ml) in complete DMEM-F12 containing 5% FBS followed by Accutase treatment for 20 min, washed and re-suspended in PBS with 0.04% BSA. Live cells were counted by trypan-blue exclusion in a Countess® II automated cell counter. For scRNA-seq, the cells were captured and libraries generated using Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10x Genomics). Libraries were run on an Illumina HiSeq 4000 as 150-bp paired-end reads. The Cell Ranger Single-Cell Software Suite v3.01 was used to perform sample demultiplexing, barcode processing and single-cell 3’ gene counting. Quality control filtering was applied to remove any cells with fewer than 1000 reads or greater that 15% mitochondrial reads. The 10x Chromium Single Cell 3’ Library & Gel Bead Kit v2 was used to capture cells from the donor cornea (10,000 cells) and the organoid (2792 cells). The Loupe Browser 4.2.0 was used to analyze and visualize the data.
Results :
We detected 11 and 18 cell clusters in the cornea organoid and donor cornea, respectively. Among these, the cornea organoid revealed clusters of epithelial cells expressing MUC1 and MUC16, a small set of TP63+ cells and a stromal cell cluster expressing COL1A1, COL5A, and a small subset of these expressing LUM and KERA. Unlike the cornea, the organoid also contained cells expressing KRT13 and MUC4, indicative of cornea atypical epithelial differentiation. We also found the expression of SARS-CoV2 receptors (ACE2 and TMPRSS2) in the organoid and the donor cornea.
Conclusions :
The cornea organoid displayed considerable overlap with the cornea with respect to stromal cell and some epithelial cell markers. However, the organoid also harbored a cluster indicative of dermal and conjunctival differentiation.
This is a 2021 ARVO Annual Meeting abstract.