Isolation of limbal epithelial and mesenchymal stem cells from human corneal donor tissue using magnetic-activated cell sorting.

This study investigates the isolation and expansion of limbal epithelial stem cells (LESC) and limbal mesenchymal stem cells (LMSC) from human corneal donor tissue using magnetic-activated cell sorting (MACS) with ABCG2 and CD90 microbeads, respectively. The tissue was enzymatically digested, and the resulting cell populations were characterized based on their marker expression and growth properties. A weak negative correlation between storage duration and cell yield was observed, while donor age had minimal influence. Laminin-111 coating was used to investigate its effect on cell growth and stem cell marker expression. Immunofluorescence analysis confirmed the enrichment of ABCG2+ LESC and CD90+ LMSCs by using magnetic-activated cell sorting (MACS). ABCG2+ cells exhibited typical stem cell morphology and high expression of p63α and CK15, while LMSC showed mesenchymal characteristics, including vimentin expression. Expansion of ABCG2+ LESC revealed a shift from epithelial to mesenchymal-like cells over several passages. While CD90-based MACS enables a robust and reproducible isolation of LMSC, ABCG2 alone is not sufficient for the isolation of a pure and stable LESC population. These findings highlight the need for combinatorial or sequential sorting strategies, such as CD90 followed by ABCG2, to achieve pure LESC populations. This study underscores the potential of discarded corneoscleral tissue as a valuable source for regenerative therapies and the need for optimized protocols to improve LESC isolation by MACS.