Limbal Epithelial Stem Cell Sheets From Young Donors Have Better Regenerative Potential For Clinical Applications

Purpose: To investigate the stemness of limbal epithelial stem cell sheets in relation to donor’s age. Methods: : Human limbal explants from cadaveric donors were set on human amniotic membranes with xenofree medium. We evaluated limbal epithelial sheet size, expression of stem/progenitor cell markers, and colony formation efficiency from donors of different age groups (age ≤ 45, age 45-65, and age > 65). Expression of the proliferation marker Ki67, stem/progenitor cell markers p63α and ABCG2, cornea specific marker PANCK, and differentiation marker CK12 were evaluated. To determine the effect of donor age on the storage period of limbal explant sheets, the limbal explant outgrowth sheets were stored in 4℃ for 2 days and analyzed for JC-1, p63a, and PANCK with FACS on each day. Results: : From days 6 to 12, the outgrowth area of the limbal epithelial stem cell sheet was significantly larger in the age ≤ 45 group compared to the other two age groups. In terms of stemness, outgrowth cells from aged donors (age > 65) showed lower expression of stem/progenitor cell markers p63α and ABCG2 and decreased CFE compared to the other two groups. Conclusion: Our results imply that donors younger than 65 years of age are a better source of limbal epithelial stem cell sheet generation with high regeneration potential.