Defining the Limbal Stem Cell Niche: Role of Cell–Cell– and Cell–Matrix Adhesion Molecules

Abstract

Abstract: : Purpose: To characterize the role of cell surface receptors involved in cell–cell and cell–matrix interaction as well as anchoring junctions in the generation of a microenvironmental niche for human corneal epithelial stem cells. Methods: Cryosections of corneoscleral specimens from 20 fresh human donor eyes were stained by indirect immunofluorescence using commercially available antibodies against various cell–cell and cell–matrix adhesion molecules including integrins, cadherins, catenins, syndecans, dystroglycan, CD44, and plectin. In double labeling experiments, antibodies against putative stem cell and differentiation markers, e.g. p63, ABCG2, connexin 43 (Cx43), were additionally used. Results: Basal cells of ocular surface epithelia including the limbus showed preferential immunoreactivity for the cell surface receptors cadherin–P, CD44, and integrins α3, αv, ß1, and ß5; dystroglycan, plectin, and integrins α6 and ß4 were restricted to their basal cell membranes. In contrast, cadherin–E, ß–catenin, and syndecan–1 and –4 were predominantly localized to suprabasal cells. Immunostaining for cadherin–P and –E, syndecan–1 and –4, and integrin α2, which are involved in cell–cell adhesion, revealed small clusters of negative cells in the basal limbal epithelium and appeared to co–localize with p63/ABCG2–positive and Cx43–negative cells. These cell clusters were also associated with distinct gaps in the basal cell membrane staining for dystroglycan, plectin, integrins α6 and ß4, which are involved in hemidesmosome formation. Reduced numbers of hemidesmosomes were also observed along small immature cells with a high nuclear:cytoplasmic ratio in the basal limbal epithelium by electron microscopy. Instead, integrin α3ß1 was more abundantly present along the basal cell membranes at the limbus. Conclusions: The absence of intercellular adhesion molecules may be an inherent feature of limbal stem cells reflecting the need for the uniqueness of their microenvironment. Adherence to their extracellular niche appears not to be primarily mediated by hemidesmosomes, but rather by increased expression of the basement membrane receptor integrin α3ß1, enabling individual stem cells to exit rapidly from their niche for self–renewal.

Keywords: cornea: epithelium • cornea: basic science • cell adhesions/cell junctions