Table 1.

Assays used for the identification of potential salivary gland stem cells are listed with expected outcomes, the caveats associated with each assay, and possible alternative explanations

Stem cell assay	Expected outcome	Caveat	Alternative outcomes
Expression of stem cell markers	Cells expressing stem cell markers found in other tissues are considered stem cells	No universal marker for stem cells is known	Stem cell marker expression is not always limited to stem cells
Morphology and localization	Proliferating intercalated duct cells located in appropriate proximity to acini are designated as stem cells	Static analysis of cell proximity does not demonstrate a lineage relationship	Rapid division of duct cells generates only duct cells
Label-retaining assay	LRCs are slow cycling stem cells	Label can also be retained by postmitotic cells	LRCs can be long-lived terminally differentiated cells
In vitro proliferation	In vitro proliferation for more than one passage is an exclusive capability of stem cells	Removal of cells from in vivo niche can change properties Many differentiated cell types can continue to proliferate in vitro	Rapidly dividing and expanding cells may be an in vitro artifact
In vitro differentiation potential	Evidence of acinar, ductal, and myoepithelial markers or morphology indicates differentiation of a stem cell	Most assays are performed on dissociated cells of whole gland which includes differentiated cell types Differentiation into adipocyte, chondrocyte, and osteogenic cells is characteristic for mesenchymal stem cells, but does not demonstrate differentiation into acinar, duct, or myoepithelial cells of salivary gland	Presence of differentiated cells in starting culture confounds analysis Stress may cause cultured cells to transition to intermediate, dedifferentiated, or alternate cell types
Sphere formation	The formation of spheres in culture is evidence of stem cell activity	If not plated singly, cells can aggregate, a property of many cell types Use of dissociated glands for sphere formation includes large numbers of differentiated cells	Cells aggregated into spheres can include differentiated cells from original dissociation, and dividing cells that may not be stem cells
Transplantation	Rescue of salivary gland function by transplanted cells is due to regeneration by stem cells	Little evidence for cell engraftment or contribution to repair Injected cells may stimulate repair through paracrine signaling	Rescue of salivary gland function may be due to paracrine activity of injected cells
In vivo lineage tracing	Tracing a multipotent stem cell lineage should produce acinar, duct, and even myoepithelial cells	Cell labeling is based on the promoter chosen to drive Cre, which may be dynamically regulated or expressed in multiple cell types	Lineage tracing shows that acinar and duct cells are lineage restricted, except under conditions of extreme injury

Abbreviation: LRCs, label retaining cells.