The primary role of salivary glands in human biology is to produce an exocrine secretion, saliva, which provides much of the innate host defense for the upper gastrointestinal tract. Other functions include forming the acquired enamel pellicle, influencing enamel surface mineralization, participating in mucosal maintenance and repair, and acting to dissolve tastants for presentation to the taste buds. Conditions that decrease saliva production have important, negative adverse sequelae for a patient. Clinically, patients with head and neck cancer who undergo radiation treatment and patients with the autoimmune disease, Sjogren?s syndrome, develop salivary hypofunction as a result of destruction of glandular parenchyma; i.e., the loss of the fluid- and protein-secreting acinar cells. At present there is no adequate treatment for patients with such irreversible gland damage. A major goal of this study is to develop new methodologies for regeneration of salivary acinar and ductal elements in adult glands, and also to identify a long-lasting target for gene therapies. Human salivary glands were treated enzymatically to produce a single-cell suspension and plated in low-calcium Keratinocyte Serum-Free Medium on Laminin 1 or collagen Type IV-coated tissue culture plastic, to encourage attachment of epithelial cells. Cellular outgrowths were harvested and replated at clonal densities for 14-21 days, and several clones were isolated. In low-calcium conditions, the cells proliferated rapidly (until passage 10), and displayed a mesenchymal, rather than an epithelial, morphology. When transferred to normal calcium medium, they regained an epithelial morphology. Cells that were derived from a single clone expressed both pan-cytokeratin (an epithelial marker) and vimentin (a mesenchymal marker). These results suggest that low-calcium culture conditions promote an epithelial-mesenchymal transition that can be reversed by normal levels of calcium. In other experiments, cells expanded in low-calcium medium were placed into wells containing 50% Matrigel, normal levels of calcium and FGF-7 or FGF-10, which have been shown to induce morphological changes and differentiation in developing salivary glands. Under these conditions, the cells aggregated in the presence of either FGF-7 or FGF-10. However, FGF-10 induced what appears to be branching and globular outgrowth from the cell aggregate. These aggregates are currently being analyzed for the expression of ductal markers (claudin-1 and cytokeratin 19) and acinar markers (amylase, claudin-3 and NKCC1). In addition to these in vitro studies, the differentiation potential of these ex vivo expanded cells is being analyzed by in vivo transplantation, which is the true test of a potential stem cell?s ability to recapitulate a tissue. Human cells were transplanted into immunocompromised mice with Matrigel as a carrier, harvested after one month, and characterized by immunohistochemistry. Both ductal- and acinar-like structures could be identified, however, it is clear that Matrigel does not provide the type of 3D structure that is required for the cells to organize appropriately. We will continue to characterize this interesting population of cells to determine the growth factors and three-dimensional scaffolds that may better encourage them to differentiate into salivary glands, and to develop animal models in which their functionality can be tested.
