Abstract
Purpose :
Limbal stem cells (LSCs) attribute to the maintenance of the corneal epithelium and are endowed with a capacity for self-renewal and proliferative potential. However, specific markers for limbal stem/progenitor cells have not been identified. LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5), a target of Wnt signaling, has been found in other organs and used as an adult stem cell marker. In this study, we investigated whether LGR5 can be used as a limbal stem cell marker.
Methods :
Post-keratoplasty discards of human corneal-limbal tissues from unidentifiable cadavers obtained were divided into 10 equal segments, cut into 0.5-mm-wide limbal strips, and cultured in air–liquid interface conditions on Transwell plates. Limbal epithelial cells were separated and analyzed for LGR5-positive cells using FACS. FACS were used to fractionate positive/negative cells and to confirm the expression of markers related to colony formation and stemness. Over-expressed genes in LGR5-positive cells were confirmed by PCR analysis.
Results :
The expression of LGR5 was confirmed by immunofluorescence staining from the corneal center to the limbus, as well as in the basal layer of the limbus. LGR5 was observed where known other markers p63α, CK15, ABCG2, and ABCB5 were positive. After 12-14 days of limbal explant culture and isolation, the ratio of LGR5 positive cells by FACS was 7.3 ± 2.7%. Colony-forming efficiency (CFE) of LGR5 positive/negative cells were significantly higher in LGR5 positive cells than negative cells. Additionally, PCR analysis demonstrated that fractionated LGR5 positive cells showed significant increases of other stemness-related markers p63α, ABCG2, and ABCB5 and proliferation marker Ki67.
Conclusions :
The expression of LGR5 in human limbal tissues was confirmed, as well as increases in CFE and stemness of LGR5 positive cells in limbal explant cultures. Thus, LGR5 demonstrates potential to be utilized as a new limbal stem/progenitor cell marker in the future.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.