Aquaporin 1-positive stromal niche-like cells directly interact with N-cadherin-positive clusters in the basal limbal epithelium

Stem cells have a specialized microenvironment for maintaining self-renewal and multipotent capacities. It is believed that a cornea epithelial stem cell niche exists in the limbus. To characterize the niche of limbal epithelial stem cells, we observed the limbal basal epithelial layer by histological analysis.Cell clusters or cell suspensions from limbal tissue were prepared with collagenase or dispase II and fixed for cytospin sections. Adhesion assays were done to quantitate calcium-dependent cell adhesion. Limbal tissue and cytospin sections were analyzed by immunohistochemistry, transmission electron microscopy and confocal microscopy.AQP1 positive (AQP1⁺) cells were observed as non-epithelial cells in the subepithelial stroma. AQP1 expression did not co-localize with CD31, podoplanin, MART-1 positive cells, but were observed in vimentin positive stromal cells. When we made a thorough search of limbal basal cells by confocal microscopy, AQP1⁺ were observed in the proximity of N-cad, K15 and p63 positive limbal basal epithelial cells. Furthermore, electron microscope revealed stromal cells penetrating the epithelial basal membrane and forming calcium-dependent cellular adhesions with N-cad⁺ limbal basal epithelial cells.Although we could not clearly detect the expression of N-cad in the AQP1⁺ cells, AQP1⁺ cells immediately beneath the epithelial basement membrane may be stromal niche-like cells that directly interact with N-cad⁺ limbal basal epithelial progenitor cells.