Summary To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA- and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders. Key findings scRNA-Seq of adult human cornea and conjunctiva reveals the signature of various ocular surface cell populations scRNA-Seq of human developing cornea identifies stage-specific definitions of corneal epithelial, stromal and endothelial layers scRNA-Seq analysis results in identification of novel markers for qLSCs and TA cells Combined scRNA- and ATAC-Seq analysis reveals key transcriptional networks in qLSCs and TA cells and close interactions with immune cells Expansion of limbal epithelium results in downregulation of qLSCs and acquisition of proliferative limbal epithelial progenitor markers scRNA-Seq of keratoconus corneas reveals activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium Graphical abstract Schematic presentation of main techniques and findings presented in this manuscript.

A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche