Single-cell RNA sequencing reveals novel regulatory pathways in maintaining limbal epithelial stem cell homeostasis

The epithelial stem cells for the corneal epithelium and their immediate progeny, the early TA cells, are preferentially located in the basal layer of the limbal epithelium and are key for maintaining corneal epithelial homeostasis. The early TA cells migrate toward central cornea and populate the peripheral corneal epithelial basal layer. The more mature or late TA cells are believed to reside in the central corneal epithelial basal layer. This paradigm of corneal epithelial stem/TA cell/post-mitotic cell is over 30 years old and some of the features of these three populations have been defined. However, due to the heterogeneity of the limbal and peripheral corneal epithelial basal layer, it has been extremely difficult to isolate the stem/early TA cell population. Thus, it was technically difficult for the characterization of stem vs early TA versus late TA cells. We have conducted single cell RNA sequencing and established a single cell transcriptome for discrete limbal/corneal epithelial cell populations. Unbiased clustering identified stem/early TA, late TA and differentiated epithelial cells. Using this single cell transcriptome data, we discovered that ID3 was one of the highest expressed genes in stem cell population. ID3 is a key regulator for maintaining stem cell homeostasis in several non-ocular tissues and not previously recognized in corneal/limbal epithelium. ID3 proteins were specifically detected in stem cell-enriched limbal epithelial basal layer and nearly undetectable in corneal epithelium. Depletion of ID3 in limbal epithelium reduced the numbers of cells expressing putative limbal epithelial stem cell (LESC) markers, suggesting that ID3 plays a positive role in maintaining LESCs, which will be investigated in Aim1. Our preliminary data also lead to the idea that LRRK1 is a positive regulator of ID3 in limbal epithelium via targeting CaMKIIb and/or CTCF (Aim2) and LRRK1/CaMKIIb/CTCF/ID3 axis may help maintain LESCs (Aims1 and 2). To accomplish these goals, we will capitalize on our ability to modulate LRRK1, ID3, CaMKIIb and CTCF levels in complimentary model systems that include submerge cultured primary cells, 3-D organotypic raft cultures, and mice. Dysregulation of ID3 is associated with the pathogenesis of various diseases (e.g., Sjögren’s Syndrome). Knowledge on how ID3 is regulated will translate into a better understanding of the pathogenesis of the diseases with dysregulation of ID3. Since abnormal LESC homeostasis is involved in various corneal epithelial disorders (e.g., diabetic cornea), the knowledge gained from this proposal may provide a rationale for developing novel therapeutics based on modulating the expression of ID3 for treating these cornea diseases associated with compromised LESCs.