Abstract
Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.
Highlights
Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined
Superficial epithelial cell exuviation necessitates renewal by epithelial stem cells located in the corneal limbus (LESC) that differentiate into early progenitor and transient amplifying cells which continually differentiate and migrate to replenish the epithelium
These 16 clusters correspond to 11 subtypes of epithelial cells, keratocytes, Langerhans cells, melanocytes, vascular endothelial cells and corneal endothelial cells (CenC) (Fig. 1b,c)
Summary
Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches. The corneal endothelium (CenC) is a monolayer that unlike the epithelium does not divide This layer separates the cornea from the aqueous humor with extensive tight junctions to maintain barrier functions to pump fluids from the stroma in order to maintain proper hydration and corneal clarity.
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