Single-Cell Transcriptomics Reveals Novel Cell Types in Human Limbus Where Corneal Epithelial Stem Cells Reside

Abstract

Purpose : Human corneal epithelial stem cells, also referred as to limbal stem cells (LSCs), have been recognized to locate in corneal limbus for three decades. However, the identity and unique markers of LSCs are still elusive. To address this gap, the single-cell transcriptomics technology was used to uncover novel cell types in heterogenous basal epithelium of human limbus.

Methods : Single cell populations of human limbal basal epithelium were isolated from young donor corneas. Using 10x Genomics platform, the transcriptome of 16,859 individual single-cells was obtained. Principal component analysis (PCA) was used to reduce the dimensionality of the dataset, followed by clustering unique cell types through the graph-based visualization method Uniform Manifold Approximation and Projection (UMAP). Tissue RNA in situ hybridization using RNAscope, RT-qPCR, antibody staining and functional models were used for validation.

Results : A total of 11 unique clusters of cells were identified. Two clusters of terminal differentiated cells (TDC) and 3 of post-mitotic cell (PMC) were defined by corneal differentiation markers KRT3, KRT12 and GJA1 in UMAP graph. An unique cluster was characterized as transient amplifying cells (TAC) by cell cycle and proliferation related genes. Two progenitor clusters and one putative limbal stem cell (LSC) cluster were identified by unique expression patterns of multiple stem-cell markers. In addition, two melanocyte clusters were found in limbal epithelium. Interestingly, novel unique markers for each cell types are uncovered as top-expressed genes by scRNAseq. RNAscope, antibody staining and culture models validated such exclusive genes as potential new markers for LSC and its progenies based on their spatial localization on fresh donor limbal tissues and functional behavior during corneal epithelial regeneration. Further investigations for exclusive LSC markers and signaling pathway driving LSC differentiation are underway.

Conclusions : High-resolution single-cell transcriptomics reveals the insight of heterogeneous basal epithelium of human limbus. Novel cell types with unique new markers have been uncovered for further identification of LSC. These novel findings suggest an important advance in adult stem cell concept and potential clinical implication for corneal tissue bioengineering and transplantation.

This is a 2020 ARVO Annual Meeting abstract.