Immunofluorescence tomography of mouse ocular surface epithelial stem cells and their niche microenvironment

Purpose: Currently, there are no definitive immunomarkers for epithelial stem cells (corneal and conjunctival) or their poorly understood niche microenvironment. The H2B-GFP/K5tTA mouse enables visualization of label-retaining cells (LRCs), which exhibit the functional marker of stem cell quiescence. We used immunofluorescence tomography to evaluate putative stem cell markers and LRCs of the mouse ocular surface. Methods: H2B-GFP/K5tTA mice were pulsed for 56 days and then chased with doxycycline to label LRCs. Limbus and eyelid tissue was 3-dimensionally (3-D) reconstructed using immunofluorescence tomography to identify and characterize LRCs using the putative stem cell markers sox9, keratin 19, lrig1, blimp1, and abcb5. Results: After 28 days of chase, LRCs were localized to the entire limbus epithelium and, infrequently, the anterior limbal stroma. Label-retaining cells comprised 3% of limbal epithelial cells after 56 days of chase. Conjunctival LRCs were localized to the fornix and comprised 4% of the total fornix epithelial cells. No stem cell immunomarker was specific for ocular surface LRCs; however, blimp1 enriched for limbal basal epithelial cells and 100% of green fluorescent protein-positive (GFP+) cells at the limbus and fornix were found to be lrig1-positive. Conclusions: Label-retaining cells represent a larger population of the mouse limbus than previously thought. They decrease in number with increased doxycycline chase, suggesting that LRC populations with different cell cycle lengths exist at the limbus. We conclude that current immunomarkers are unable to colocalize with the functional marker of epithelial stem cell quiescence; however, blimp1 may enrich for limbal epithelial basal cells