Title : Single-Cell Transcriptomics Discovers Unique Markers for Human Corneal Epithelial Stem cells
Abstract:
Purpose: Human corneal epithelial stem cells, also referred to as limbal stem cells (LSCs), have been recognized to locate in corneal limbus for three decades. However, the molecular identity and definitive markers of LSCs are still elusive. This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC population at single cell resolution.
Methods: Single cells of human limbal basal epithelium were isolated from young donor corneas. Singlecell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method Uniform Manifold Approximation and Projection (UMAP) and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using ex vivo donor corneal tissues and in vitro culture models of primary human limbal epithelial cells (HLECs).
Results: Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched top expressed genes known as markers of putative epithelial stem cells. TSPAN7 gene coding Tetraspanin 7 protein was discovered and validated as a unique LSC marker based on its exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunofluorescent staining, as well as the functional role in cell growth and tissue regeneration models in cultures with or without RNA interference. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness.
Conclusions: This human corneal single-cell transcriptomics uncovers unique markers that identifies the LSC population, and reveals novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and benefit stem cell expansion for clinical use. This study lays the foundation for understanding the corneal homeostasis and diseases.
