IFITM1 / OVOL1 Axis Is a Novel Regulator of the Expansion of the Limbal Epithelial Stem/Early Transient Amplifying Cell Population

ABSTRACT Limbal epithelial stem cells (LESCs), located in the basal layer of the limbal epithelium, rarely proliferate under normal conditions. Upon proliferation, LESCs give rise to early transient amplifying (eTA) cells, which are thought to be morphologically and phenotypically indistinguishable from LESCs. Following corneal epithelial wounding, LESCs are activated to repair the corneal epithelium via expansion of eTA cells, a process crucial for maintaining corneal epithelial homeostasis and tissue transparency as well as essential for clear vision. To understand how this process is regulated, we conducted a single cell RNA sequencing assay of mouse corneal rims with and without injury and observed an expansion of the stem/eTA cell cluster after corneal injury. Interestingly, we found that Interferon Induced Transmembrane Protein 1 (IFITM1) was predominantly expressed in stem/eTA cells and was positively associated with such stem/eTA cell expansion after corneal wounding. In vivo knockdown of IFITM1 using an AAV (adeno‐associated virus) vector significantly attenuated stem/eTA cell expansion and activation of stem/eTA cells to proliferate after mouse corneal wounding. In human limbal epithelial cell cultures, IFITM1 positively impacted the proliferation of stem/eTA cell‐enriched limbal epithelial cells, contributing to expansion of the stem/eTA cell population. Such expansion was due, in part, to inhibition of OVOL1 (Ovo like zinc finger 1), a negative regulator of epithelial cell proliferation. These results provide key molecular insights into how stem cell activation and eTA cell expansion are regulated. Elucidating the IFITM1/OVOL1 pathway that governs stem/eTA cell proliferation not only deepens our knowledge of tissue homeostasis but also opens avenues for developing novel regenerative therapies.