Understanding corneal limbal stem cells during wound healing

Summary Corneal damage is a leading cause of blindness and visual impairment, affecting over 11 million individuals worldwide. Despite the prevalence, current treatment options for corneal disorders, such as abrasions, dry eye disease, keratitis, and corneal dystrophies, are largely limited to palliative care or, in severe cases, corneal transplantation with the latter being associated with complications including limited tissue availability, risk of rejection and cost. Thus, the development of effective restorative treatments that actively reestablish corneal tissue is necessary to meet patient needs. Critical to the development of regenerative treatments is understanding how injury impacts the limbal stem cell (LSC) niche. There are two distinct yet anatomically continuous epithelial cell populations: the limbus and peripheral cornea, with the limbus serving as a niche for LSCs. LSCs are crucial for maintaining corneal homeostasis and wound repair, with activation resulting in the replenishment of the differentiated epithelial layers. In cases where the limbus is compromised, such as limbal stem cell deficiency, the boundaries between cell populations are disrupted which can result in conjunctivalization, vascularization, chronic inflammation and vision loss. Previous studies have identified two distinct limbal stem cell populations residing in the limbal region: active inner limbal stem cells (iLSCs) and quiescent outer limbal stem cells (oLSCs). Active iLSCs continuously proliferate and contribute to corneal epithelial maintenance, while quiescent oLSCs are activated and migrate in response to corneal injury. Quiescence is a critical feature of adult stem cells, protecting them from genotoxic damage and environmental trauma. As such, oLSCs hold significant potential for clinical applications to regenerate the damaged cornea. Yet, there is very little understanding of the function of these cells during homeostasis and after injury, or the mechanisms by which they are regulated. Also unclear are the boundaries that separate oLSCs from conjunctival cells and iLSCs, and how they are impacted by injury. To begin to address these questions, we have developed a genetic lineage tracing mouse model that is specific to oLSCs and will test this model through 2 specific aims: 1) Characterize the response of the outer limbal stem cells to small- and large-scale limbal-sparing wounds using a novel lineage tracing mouse model, and 2) Determine the cellular/molecular identities and cell communication at boundaries adjacent to limbal stem cells in homeostatic and injured corneas. We envisage applying this model and the techniques utilized here (e.g., spatial-omics) in future studies to define limbal-sparing vs. limbal- compromising outcomes, to understand how these cells respond to multiple injuries mediated over time, and how chronic diseases such as dry eye impact oLSC function.