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Investigation of corneal limbal stem cells and immune repertoires in human infant cornea by multi-omics solution

ABSTRACT: This project aims to study two key points related to keratoplasty resistance. LSCs (limbal stem cells), which are crucial for the growth and repair of the cornea, have not been identified by specific markers. Furthermore, the characterization of corneal T/B cells has been rarely studied, even though they play a vital role in transplant rejection. To optimize donated corneas, researchers have utilized single-cell multi-omics methods such as single-cell 5’ mRNA and single-cell V(D)J sequencing to explore both LSCs and T/B immune repertoires (IR) simultaneously. Potential LSCs and dominant V(D)J types were analyzed, and the location of the cell marker was determined by RNA in situ sequencing. From the single-cell transcriptomics of 17,218 whole corneal cells, 20 cell subtypes were observed. A subcluster (0.3% of total cells) was identified as putative epithelial LSCs based on the known markers stating stem cells in the G0 cell cycle. TCR/BCR were rarely found in the cornea. The results suggest that GPHB5 could be a potential marker for limbal stem cells, but high-throughput single-cell VDJ sequencing is not the ideal method for analyzing corneal immune repertoires due to the rare presence of T/B cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE230439 | GEO | 2024/06/25

REPOSITORIES: GEO

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Project description:Limbal stem cells (LSCs), known as corneal epithelial stem cells, are located at the basal epithelial layer of the corneal limbus and serve an important function in maintaining the homeostasis of the corneal epithelium. Several putative molecular markers of LSCs have been previously identified. However, the specificity of these markers remains largely controversial. To address this gap in the current understanding of LSCs, we performed a transcriptome profiling of heterogeneous corneal limbal basal cells using single-cell transcriptomics technology to identify LSCs and their exclusive markers. We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration using Seurat package (https://satijalab.org/seurat), about 16,400 cells were retained for further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization. We identified 11 unique clusters of cells assigned to a putative cell type based on published known biomarkers for differentiation, proliferation and putative epithelial stem cells. As a results, we found 2 Terminally Differentiated Cell (TDC), 3 Post-Mitotic Cell (PMC), 1 Transient Amplifying Cell (TAC), 2 Limbal Progenitor Cell (LPC), Putative Limbal Stem Cell (LSC) and 2 Melanocyte (MC) sub-cell type clusters. Furthermore, we confirmed the trajectory order of LSC differentiation for 9 clusters using Pseudotemporal and Functional PCA analysis. Lastly, we validated each assigned cell subtypes and determined their location in human corneal limbus tissue with 9 markers using RNAscope We were able to reveal the heterogeneity of corneal limbal basal epithelium by defining novel dynamic trajectories for cell types in a pseudotemporal manner. This approach allowed us to identify the distinct clusters of LSCs and progenitors with exclusively expressed markers, and might apply for translational research on regenerating a normal corneal epithelium and restoring vision.

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Investigation of corneal limbal stem cells and immune repertoires in human infant cornea by multi-omics solution

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