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Single cell transcriptomics reveal the heterogeneity of the human cornea to identify novel markers of the limbus and stroma

ABSTRACT: The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified that the expression of CAV1, CXCL14, HOMER3 and CPVL were exclusive to the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE186433 | GEO | 2021/10/25

REPOSITORIES: GEO

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High throughput gene expression analysis identifies reliable expression markers of human corneal endothelial cells.

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Project description:Corneal organoids are useful tools for disease modeling and tissue transplantation however they have not yet been well studied during maturation. We characterized human iPSC derived corneal organoids at 1, 2, 3 and 4 months of development using single-cell RNA sequencing to determine the cellular heterogeneity at each stage. We found pluripotent cell clusters committed to epithelial cell lineage at 1 month early corneal epithelial, endothelial and stromal cells markers at 2 months, keratocytes as the largest cell population at 3 months, and a large epithelial cell population at 4 months. We compared organoid to fetal corneal development at different stages and found that 4 month organoids closely resemble the corneal cellular complexity of the fetal (16 post conception week) and adult cornea. Using RNA velocity pseudotime trajectory analysis, we found that eye field primordial cells and limbal stem cells appear to give rise to corneal epithelial cells during development.

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RNA sequencing analysis for comparing gene expression of the corneal endothelium of Slc4a11-/- vs Slc4a11+/+ mice

Project description:Purpose: Slc4a11 KO mice show significant edema and altered corneal endothelial morphology at an early age with concomitant increased mitochondrial ROS and oxidative damage relative to wild type. Here we used RNA-Seq with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function alterations. Methods: Corneal endothelium-Descemet’s membrane (CEDM) samples were from WT and Slc4a11 KO mice at 12 weeks of age. RNA sequencing data was subjected to Ingenuity Pathway Analysis and QPCR for validation. Results: The RNA sequencing IPA analysis predicted activation, inhibition or differential regulation of several pathways. We validated downregulation of Glycolytic enzymes, Mitochondrial complex components and Ion transporters. We observed upregulation of genes of cholesterol biosynthesis, GSH metabolism and tight and adherens junctions. Conclusions: Slc4a11 KO induces a coordinated decrease in glycolysis, glutaminolysis, oxygen consumption, lactate transporters and Na-K-ATPase. These changes together with the altered barrier function cause an accumulation of stromal lactate in Slc4a11 KO mice leading to chronic corneal edema.

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