Limbal explant cultures on amniotic membrane: The effects of passaging the explants on cell phenotype

In vitro expansion of limbal epithelial stem cells (LESCs) while maintaining their characteristics has the potential to address the urgent need in ophthalmology clinics for the treatment of limbal stem cell deficiency (LSCD). Herein, we investigated the impact of explant passaging on the phenotype of LESCs cultured on human amniotic membrane (hAM). Following initial coverage of the hAM surface by cells (passage 0), the rabbit limbal explants underwent two additional passages. Expanded cells were then counted using a hemocytometer and examined by immunocytochemistry and RT-qPCR to assess markers associated with LESCs (ABCG2, P63, CK14, CXCR4, BMI-1, and vimentin) and differentiated LESCs (CK3 and connexin 43). The cell yield of passage 1 was the highest among all passages. Immunocytochemistry analysis revealed that the number of CK14-positive cells was similar across all passages; vimentin-positive cells were the lowest in passage 0, while vimentin-positive cells were the highest in passage 1; and CK3-positive cells were the highest in passage 0. RT-qPCR analysis revealed that CK3 and connexin 43 expression was significantly higher in passage 0 cells than in passage 2 cells; and CXCR4 and BMI-1 expressions were significantly higher in passage 1 cells than in passage 0 cells. Our data highlight that the passaging of limbal explant on hAM results in varying cell characteristics. The decrease in CK3 and increase in ABCG2 expression in cells obtained by passaging the limbal explant suggest that passaging could potentially enhance the stem cell population within the in vitro limbal explant culture on hAM.