Diverse epithelial cell populations contribute to the regeneration of secretory units in injured salivary glands.

Salivary glands exert exocrine secretory function to provide saliva for lubrication and protection of the oral cavity. Its epithelium consists of several differentiated cell types, including acinar, ductal and myoepithelial cells, that are maintained in a lineage-restricted manner during homeostasis or after mild injuries. Glandular regeneration following a near complete loss of secretory cells, however, may involve cellular plasticity, although the mechanism and extent of such plasticity remain unclear. Here, by combining lineage-tracing experiments with a model of severe glandular injury in the mouse submandibular gland, we show that de novo formation of acini involves induction of cellular plasticity in multiple non-acinar cell populations. Fate-mapping analysis revealed that, although ductal stem cells marked by cytokeratin K14 and Axin2 undergo a multipotency switch, they do not make a significant contribution to acinar regeneration. Intriguingly, more than 80% of regenerated acini derive from differentiated cells, including myoepithelial and ductal cells, that appear to dedifferentiate to a progenitor-like state before re-differentiation into acinar cells. The potential of diverse cell populations serving as a reserve source for acini widens the therapeutic options for hyposalivation.

c-Kit+ Cells in Adult Salivary Glands do not Function as Tissue Stem Cells.

A rare population of salivary gland cells isolated based on c-Kit immunoreactivity are thought to represent tissue stem cells since they exhibit the most robust proliferative and differentiation capacity ex vivo. Despite their high promise for cell-based therapies aimed at restoring salivary function, the precise location and in vivo function of c-Kit+ stem cells remain unclear. Here, by combining immunostaining with c-KitCreERT2-based genetic labeling and lineage tracing in the adult mouse salivary glands, we show that c-Kit is expressed in a relatively large and heterogeneous cell population that consists mostly of differentiated cells. Moreover, c-Kit does not mark ductal stem cells that are known to express cytokeratin K14. Tracking the fate of in vivo-labeled c-Kit+ or that of K14+ cells in spheroid cultures reveals a limited proliferative potential for c-Kit+ cells and identifies K14+ cells as the major source of salispheres in these cultures. Long-term in vivo lineage tracing studies indicate that although c-Kit marks at least two discrete ductal cell lineages, c-Kit+ cells do not contribute to the normal maintenance of any other cell lineages. Our results indicate that c-Kit is not a reliable marker for salivary gland stem cells, which has important implications for salivary gland regenerative therapies.

Analysis of histone H2BGFP retention in mouse submandibular gland reveals actively dividing stem cell populations.

The purpose of this study was to use histone 2B-green fluorescent protein (H2BGFP) pulse-chase experiments to provide a broad view of population dynamics in salivary gland and to identify the quiescent stem cells that had previously been suggested to reside in the gland. Two transgenic mouse models in which inducible H2BGFP expression was regulated either by keratin (K)14 promoter or by a ubiquitous promoter were generated. The level of fluorescent label in the submandibular gland induced by a pulse of H2BGFP expression was monitored over a period of 18 weeks of chase. Efficient targeting of H2BGFP label to the relatively undifferentiated ductal cells by K14 promoter did not identify a quiescent population of stem cells. Ubiquitous targeting of all ductal cells identified label-retaining cells but these cells were mapped to the more differentiating ductal compartments. Furthermore, they did not display the major characteristics of quiescent stem cells including the undifferentiated phenotype, mobilization in response to injury, and the clonogenicity in culture. Quantitative assessment of H2BGFP loss in various ductal compartments and short-term lineage tracing of K14(+) ductal cells were consistent with the presence of actively dividing pools of stem/progenitor cells in the intercalated ducts and the basal layer of excretory ducts functioning independently during homeostasis.