Abstract
Purpose :
Cornea cell subtypes, especially limbal stem cells (LSCs) are important for the development, growth, and repair of the cornea; however, so far, LSCs specific markers have not been identified. B-cell or T-cell receptors are important for transplantation rejection, however, the characterization of corneal immune repertoires(IR) has rarely been investigated. We applied single- cell RNA and V(D)J sequencing to explore LSCs and IR simultaneously in the same precious donated corneas, analyzed the potential LSCs and dominant V(D)J type, and verified them by RNA in situ sequencing.
Methods :
Three freshly donated whole corneas were separately processed. RNA and VDJ library preparation were performed according to the manufacturer’s protocol, afterwards, followed by single-cell 5’-mRNA and VDJ sequencing. Reads produced from the 5′ gene expression profiling were aligned to the GRCh38 genome, and cell clusters were visualized using T-distributed stochastic neighbor embedding in Seurat(t-SNE). VDJ contig assembly, annotation, and clonotype analysis were performed using “cellranger vdj”. RNA in situ sequencing was amplified to verify the cell marker location.
Results :
Single-cell transcriptomics of 17218 whole corneal cells revealed 26 cell subtypes, and a subcluster (0.3% of total cells) was identified as LSCs based on the other known markers stating putative epithelial stem cells. Gpha2 was discovered and validated as a potential LSC markers based on its enriched expression pattern and spatial localization in the limbal zone by RNA in situ sequencing. Simultaneously, less than 100 cells for each sample were profiled for VDJ sequencing, which is not sufficient for further bioinformatics analysis.
Conclusions :
High-throughput single-cell 5’mRNA sequencing and phylogenetic analysis showed that Gpha2 is a potential marker for limbal stem cells, which was verified using RNA in situ sequencing. Further animal models are structuring to understand the changes that account for Gpha2 knockdown. High-throughput single-cell VDJ sequencing is not suitable for analyzing immune repertoires due to the rarity of immune cells in the cornea; however, preliminary VDJ sequencing showed that each individual has a unique T-cell and B-cell repertoire formed via VDJ rearrangement, which is consistent with previous reports for other tissues.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.