Background: The corneal epithelium is renewed by stem cells located at the limbus, the so-called limbal stem cells (LSCs). Absence, damage or loss of the LSC population leads to the painful and blinding condition of LSC deficiency (LSCD). Ex vivo expansion of LSCs is an increasingly well recognized treatment modality for LSCD. One method of ex vivo expansion of LSCs involves the culture of limbal explants on amniotic membrane (AM). The purpose of this study was to analyze the outgrowths from human cadaveric limbal explants cultured on AM for properties associated with LSCs. In particular, the expression of putative stem cell markers and the colony-forming efficiency of the different zones of the outgrowth were studied.
Methods: The limbal explants were expanded in the standard way used for clinical transplantation and the outgrowths were divided into three zones depending on proximity to the explant (inner, middle and outer zones). The colony-forming efficiencies (CFEs) of cells from each zone were calculated. In addition, the expression of DeltaNp63, ABCG2 (both putative positive LSC markers) and cytokeratin K3 (marker of corneal differentiation) were assessed using quantitative reverse transcription PCR (RT-PCR). Immunohistochemistry on paraffin-embedded sections was also performed to demonstrate protein localization and allow further confirmation of the quantitative RT-PCR results.
Results: Successful cultures for both the explant outgrowths and the CFE calculations were obtained in every case. CFE showed a successive decline in zones further away from the explant (p < 0.00005). Quantitative RT-PCR revealed that the expression of the positive putative LSC markers DeltaNp63 and ABCG2 also showed a steady decrease in the zones furthest from the explant (p < 0.05 and p < 0.005, respectively). The expression of cytokeratin K3 was increased in zones furthest from the explant (p < 0.005). Immunohistochemistry on paraffin-embedded sections of intact ex vivo-expanded limbal epithelium for the putative positive marker p63 and cytokeratin K3 confirmed the findings of the quantitative RT-PCR and CFE results.
Conclusions: We demonstrate for the first time that outgrowths from human limbal explants, a widely used technique in ex vivo expansion of LSCs for clinical transplantation, show a steady decline in a wide range of stem cell properties with distance from the central explant. These findings support the importance of proximity of stem cells to their niche environment in maintaining their undifferentiated state. These findings suggest the need for modifications of existing techniques to ensure maximum numbers of LSCs following ex vivo expansion protocols, which will then ensure the success of subsequent engraftment.