ABSTRACT: PURPOSE. Building on identification of ABCB5 as a limbal stem cell (LSC) marker, this study evaluates CD63, a newly identified LSC molecule co-expressed with ABCB5, in human limbal epithelial cells to elucidate its role in maintaining corneal epithelial identity. METHODS. RNA sequencing (RNA-seq) was performed on sorted Abcb5-positive and Abcb5-negative murine corneal epithelial cells. CD63 expression in human corneal tissue was assessed by flow cytometry and immunostaining. siRNA-mediated knockdown (KD) of CD63 was conducted in cultured human limbal epithelial cells. Subsequent analyses included qRT-PCR, flow cytometry, RNA-seq, and western blotting. RESULTS. RNA-seq analysis of mouse corneal epithelial cells revealed high expression of LSC markers, including Krt15, Krt6b, Fgfr1, Gpha2, Ifitm3, and Cd63, as well as low expression of differentiation-associated markers, such as Krt12 and Gja1, in Abcb5-positive cells. In human corneal tissue, flow cytometry and immunostaining confirmed robust CD63 expression in the limbal region. siRNA-mediated KD of CD63 in cultured human limbal epithelial cells resulted in a marked reduction in the expression of corneal epithelium-enriched genes, including KRT12, CLU, ALDH1A1, ALDH3A1, TGFBI and MYEOV. Notably, CD63 KD resulted in a ~50 % decrease in expression of a key transcriptional regulator of corneal epithelial identity, PAX6. CONCLUSIONS. CD63 is abundantly expressed in the human limbus and plays a critical role in maintaining the expression of corneal epithelium-specific proteins, likely through modulation of PAX6. These findings underscore the functional significance of CD63 in limbal stem cell biology and its essential contribution to corneal epithelial homeostasis.
