Non-adherent spheres formed in vitro
from dissociated salivary gland tissue have been shown to include progenitor and/or stem cells that contribute to the restoration of secretory function in damaged glands [5
]. Here we demonstrate that cells expressing the transcription factor Ascl3 are adult progenitor cells present in all three major salivary glands.
In the adult salivary gland, Ascl3+ cells are found exclusively in the ducts, from the smallest intercalated, to the striated, granular, and large excretory ducts, but represent less than 5% of total duct cells [1
]. In developing glands, Ascl3+ cells are mitotically active [6
], and lineage tracing has established that they are precursors of both duct and acinar cells [1
]. To establish that Ascl3+ cells continue to act as progenitors in the adult gland, we have cultured spheres from adult gland tissue and demonstrated that they generate differentiated progeny over time in culture. Furthermore, the Ascl3+ cells isolated from adult glands retain their multilineage potential, generating both duct and acinar cell types in vitro
within the spheres. Notably, no sphere was found in which all cells were generated from Ascl3+ progenitors. Furthermore, the co-localization experiments indicate that Ascl3+ cells are not capable of generating all cell types in the salivary gland, and there was no evidence of self-renewal within the spheres. Thus, the Ascl3+ cells are clearly not stem cells, but represent an intermediate lineage-restricted progenitor population of the adult glands.
In this report, co-localization studies using lineage tracing and immunohistochemistry revealed that Ascl3+ progenitors in the spheres are precursors of duct cells expressing the differentiation marker Keratin 8. In addition, some Ascl3+ descendants express the stem/progenitor cell marker Sca1. Although Sca1 was identified as a progenitor cell marker in the salivary gland, it is widely expressed in differentiated epithelia of several tissues [26
]. We conclude that the Sca1+/EYFP+ cells are likely differentiated duct cells derived from Ascl3+ progenitors. Co-localization was also found between Ascl3+ descendants and secretory cells expressing salivary androgen binding protein (Sabpa), a member of the secretoglobin protein family. Sabpa, for which at least six paralogues have been detected in the mouse genome [27
], is expressed by a subpopulation of acinar cells in the submandibular gland, which are presumed to be seromucous cells [23
]. In contrast, we saw no co-localization of the Mist1 transcription factor with EYFP+ progeny of Ascl3+ cells. In the salivary glands, Mist1 expression is found in serous-secreting acinar cells, but not in serous demilune or mucous-secreting cells [18
]. We have previously demonstrated that Ascl3+ cells are progenitors of serous demilune cells in the sublingual glands [1
]. Thus, it is clear that secretory acinar cells are a heterogeneous population, and may arise from separate progenitors. We propose that Ascl3+ progenitors generate ductal, mucous-secreting, and possibly, seromucous type cells, but that they are not precursors of serous acinar cells. Expression of a closely related transcription factor, Ascl1
, was recently shown to define one of several competence-restricted progenitor populations in the retina [28
]. In an analogous manner, Ascl3 expression may define a restricted set of progenitor cells in the salivary gland.
A number of progenitor cell populations have been identified in the salivary gland [1
]. In addition to Ascl3, c-Kit and Keratin 5 also mark progenitor cells [4
]. To begin to establish the relationship between these cell populations, we analyzed Ascl3+ descendants in the spheres for Krt5 expression. We found no co-localization of EYFP-labeled progeny with cells expressing Krt5. Thus, Krt5+ cells are not lineally derived from Ascl3+ progenitors. We recently generated a targeted cell ablation model in the salivary glands in order to establish the role of the Ascl3+ progenitor cells. In the absence of Ascl3+ cells, the salivary glands appeared to develop normally, and were able to efficiently regenerate after ductal ligation [6
]. Consistent with the lineage tracing results reported here, the Krt5+ basal cells are still present in the Ascl3-cell ablation model [6
]. These results clearly indicate that adult salivary glands harbor more than a single population with progenitor cell capacity. Observations in other tissues suggest that rather than being redundant, these cell populations may contribute differently to maintenance and repair [29
We observed a distinct spatial organization of cell types within the spheres. Distinct cellular organization has also been reported in spheres derived from mammary gland [11
] and tracheal basal cells [33
]. In the salivary gland spheres, cells expressing differentiation-specific markers, including the acinar cell markers Mist1 and Sabpa, as well as the duct cell-specific marker Krt8, were located on the outer periphery. In contrast, proliferating cells incorporating BrdU were localized in the center of the spheres. Furthermore Ascl3+ cells were found within the spheres, while differentiated descendants marked by EYFP were predominantly found at the periphery. Absence of Ascl3 expression in peripheral cells is consistent with our earlier report that Ascl3 expression is down regulated in differentiated progeny [1
]. However, in contrast to the internal localization of Ascl3+ progenitor cells, immunostaining demonstrated that Krt5+ progenitor cells are localized at the outside of the spheres. This was also observed of Sca1+ cells, another proposed marker of progenitor cells [5
]. Both Krt5+ and Sca1+ are expressed by ductal basal cells in vivo
. Thus their external localization on the spheres may reflect a similarity in interaction of their cell membranes with neighboring cells. The biological relevance of cellular organization within the spheres is not yet clear, and further characterization of each progenitor type is required to resolve their individual roles.
The use of stem or progenitor cells for therapeutic treatment requires precise knowledge of their differentiation potential. Using lineage tracing to definitively establish the progenitor-progeny relationship, we have shown that Ascl3+ cells are multipotent lineage-restricted progenitors of the adult salivary glands. The characterization of such progenitor cells is a critical step in the process of developing cell replacement therapies.