Characterization of LGR5 as a Definitive and Functional Marker for Human Limbal Epithelial Stem Cells

To investigate the potential of LGR5, a Wnt signaling target and known adult stem cell marker in other organs, as a specific marker for limbal epithelial stem cells (LSCs), which are crucial for corneal epithelium maintenance.

Post-keratoplasty discards of human corneal-limbal tissues obtained from unidentifiable cadavers were cut into limbal strips and stained for tissue immunofluorescence and cultured in air-liquid interface conditions. Limbal epithelial cells were separated and analyzed for LGR5+ cells using fluorescence-activated cell sorting (FACS). Immunofluorescence, flow cytometry, quantitative PCR, and western blot were used to analyze markers such as LGR5, ABCB5, ABCG2, p63α, and Ki67. Colony-forming efficiency (CFE) was assessed by seeding cells on collagen-coated plates and analyzing colonies after 14 days.

Immunofluorescence analysis confirmed LGR5 expression in the human limbus, co-expressing with ABCB5 and ABCG2 but partially overlapping with p63α and Ki67. Flow cytometry showed LGR5+ cells of cultured limbal epithelial cells, fully inclusive of ABCB5+ and ABCG2+ cells but distinct from p63α+ cells. LGR5+ cells exhibited higher gene and protein expression of LGR5, ABCB5, and ABCG2, with no significant changes in p63α. CFE and Ki67 expression were significantly elevated in LGR5+ cells compared to negative cells and ABCB5+ cells.

LGR5 expression in human limbal tissues and its association with increased CFE and stemness in cultured limbal epithelial cells suggest its potential as a new definitive marker for limbal epithelial stem/progenitor cells.