Abstract
Purpose :
We performed scRNA-sequencing of human donor corneas and cornea organoids derived from induced pluripotent stem cells (iPSC) and matured in culture for 4 months to elucidate the transcriptomic cell fate of human cornea organoids.
Methods :
Three cornea organoids and 3 healthy donor corneas (Lions Eye Institute for Transplant and Research, FL) were digested with collagenase to prepare single cell suspension. Libraries were generated using Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10x Genomics). The 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 quality control filtering and integrating. The integrated dataset has 55,241 cells in the gene-cell-barcode matrix that includes a total 25,885 and 29,356 cells from the 3 organoids and 3 human corneas, respectively.
Results :
The transcriptomic data showed high reproducibility within each sample category. Differential gene expression analysis between the two sample categories showed 559 genes to be uniquely expressed in the organoid, 389 in the cornea and 971 genes expressed in common. An integrated analysis of the corneas and the organoids yielded 30 distinct cell clusters (CL). Of these, 14 CL were detected in the cornea and 10 in the organoid samples (Fig.1). The organoids harbor cell clusters representing corneal epithelium (KRT5), stroma (LUM) and endothelium (TJP1) with minor population of myofibroblast-like cells (5%) and immune cells (CCL3). Unlike the cornea, where the largest group consists of stromal cells, the organoid shows almost equal proportion of epithelial (28%), stromal (33%) and endothelial (31%) cell contribution. Interestingly, we also found the organoid cell fates to be consistent with that of a developing cornea that shows high expression of SIX3 and PAX6. The organoids also have low expression of KERA, KRT12, ALDH3A1 and KLF4, known to be expressed in the adult cornea.
Conclusions :
Our work, provides for the first time, a transcriptomic map of human cornea organoids at a single cell resolution. The organoids recapitulate epithelial, endothelial and stromal cell fates of the developing cornea. These well characterized organoids offer a 3D platform to investigate corneal development, diseases and cell-based therapies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.