Essential Role of CD63 in Maintaining Corneal Epithelial Identity in the Human Limbus.

Purpose: Building on the identification of ABCB5 as a marker of limbal stem cells (LSCs), this study examines CD63, a newly identified molecule co-expressed with ABCB5 in limbal epithelial cells, to define its role in maintaining corneal epithelial cell identity. Methods: RNA sequencing (RNA-seq) was performed on flow cytometry-sorted Abcb5-positive and Abcb5-negative murine corneal epithelial cells. CD63 expression in human corneal tissue was assessed by immunostaining. CD63 was silenced in cultured human limbal epithelial cells using siRNA-mediated knockdown and resulting molecular and cellular changes were analyzed by qRT-PCR, flow cytometry, RNA-seq, Western blotting, and cell proliferation assays. Results: RNA-seq analysis revealed increased expression of LSC markers, including Krt15, Krt6b, Fgfr1, Gpha2, Ifitm3, Ifitm1, and Cd63, and decreased expression of differentiation-associated markers, such as Krt12, Gja1, and Ovol1 in Abcb5-positive cells. Immunostaining of human corneal tissue demonstrated strong CD63 expression localized to the limbal region. Knockdown of CD63 in cultured human limbal epithelial cells resulted in reduced cell proliferation and significantly decreased expression of corneal epithelium-enriched genes, including KRT12, CLU, ALDH1A1, ALDH3A1, TGFBI, and MYEOV. Notably, CD63 knockdown led to an approximately 50% reduction in expression of PAX6, a key transcriptional regulator of corneal epithelial identity. Conclusions: CD63 is highly expressed in the human limbus and is required for maintaining cell proliferation and the expression of corneal epithelium-specific proteins, likely through regulation of PAX6. These findings establish CD63 as a functionally important component of limbal stem cell biology and a key contributor to corneal epithelial homeostasis.