Differentiation Status of Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane (AM), a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under air-lifting conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting, and quantitative polymerase chain reaction. Limbal epithelial cells expanded upon AM formed 4-6 stratified layers, both on intact AM (iAM) and denuded AM (dAM). On dAM the proportion of differentiated cells remained unaltered after air-lifting. Within cells grown on iAM, however, the number of differentiated cells increased significantly after air-lifting. These results have important implications for both basic and clinical research. First, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Second, these findings serve as a warning to clinicians that the effect of AM on transplantable cells is not fully understood; the use of iAM or dAM can produce different results in terms of the amount of differentiation, once cells are exposed to the air.