Transcriptomic analysis of stem cells and progenitors in mouse ocular epithelium

The ocular epithelium is sustained by resident stem cell and progenitor populations, which exhibit a compartmentalized spatial and functional organization. In the cornea, the limbus serves as a niche for long-lived stem cells responsible for tissue regeneration. Through the application of single-cell lineage tracing analysis and two-photon live imaging, we have identified two distinct stem cell populations characterized by their unique localization and cycling dynamics. Furthermore, our research reveals that stem cells situated in the inner limbus undergo symmetric division, producing progenitors that migrate centripetally within the basal layer. As these progenitors approach the central cornea, their likelihood of terminal differentiation and delamination increases. Conversely, stem cells located in the outer limbus, along with basal progenitors in the adjacent conjunctiva, demonstrate localized clonal dynamics. To investigate the molecular mechanisms underpinning stem cell organization within the ocular epithelium, we employed a reporter mouse model (Cyclin B1-GFP), which marks actively cycling cells. Given that only basally located stem and progenitor cells in stratified epithelia possess proliferative capability, this in vivo cell cycle reporter was anticipated to enable the selective capture of these cells for comprehensive gene expression analysis. Consequently, we utilized fluorescently activated cell sorting to isolate CB1-GFP cells from freshly dissected mouse corneas, followed by single-cell RNA sequencing analysis.