Salivary gland spheres include both differentiated and undifferentiated cells. (A, B) Submandibular spheres cultured for 5 days were labeled for 3 hours with BrdU, fixed and stained with antibody to BrdU. (A) Control, no primary BrdU antibody. (B) Staining with antibody to BrdU reveals that proliferating cells are concentrated in the center of the sphere. Scale bar = 100 μm. (C, D) The acinar cell-specific transcription factor Mist1 antibody labels cells localized at the periphery of the spheres. (E, F) Antibody to acinar cell-specific aquaporin 5 also labels cells at the surface of the spheres. Scale bars = 10 μm. (G, H) Antibody to Keratin 8, a marker of differentiated duct cells, labels cells located only at the periphery of the spheres. Scale bar = 5 μm. (I) The expression of several stem or progenitor cell markers in spheres was analyzed by RT-PCR at days 0, 2, 3, 4 and 7 of culture. Primers to LE32 detect a 60S ribosomal protein and were used as a control for RNA quality. The expression of most stem/progenitor cell markers remains constant over time in culture. In contrast, the transcription factor Ascl2 is upregulated as the spheres mature. EYFP expression is increased over time in culture, reflecting the increase in labeled descendants of Ascl3+ progenitor cells.

Ascl3-expressing progenitor cells are localized within salivary gland spheres. (A) Confocal slice image of a day 7 sphere stained with antibody to Sabpa (red). Ascl3-expressing cells are marked by EGFP expression (green) driven from the Ascl3 promoter, and are located only within the spheres (arrowheads). (B) Spheres were collected at days 0, 2, 3, 4 and 7 of culture, and RNA isolated. RT-PCR was performed using primers for Ascl3 and LE32, used as a control. Ascl3 expression is detected at all stages of sphere culture.

Ascl3-expressing cells from adult glands retain progenitor capacity in vitro within the spheres. (A–C) Day 4 spheres generated from single-cell suspensions of submandibular, sublingual or parotid glands isolated from Ascl3^EGFP-Cre/R26R^LacZ mice, and stained for beta-galactosidase (LacZ) activity. LacZ+ labeled progeny (blue), derived from Ascl3-expressing progenitor cells, are found in spheres derived from (A) submandibular, (B) sublingual, and (C) parotid glands. Scale bars = 200 μm. (D, E) Lineage tracing in spheres derived from the Ascl3^EGFP-Cre/R26R^EYFP reporter mouse strain, in which all descendants of Ascl3 progenitors are labeled by EYFP expression (green). An increased number of EYFP-labeled descendants is evident with increased time in culture, from day 4 (D) to day 10 (E). Insets show bright field image of each sphere. Scale bars are labeled. (F) Day 7 sphere from Ascl3^EGFP-Cre/R26R^EYFP reporter mouse, stained with DAPI to label nuclei. EYFP+ descendants are located at the periphery of the spheres. Scale bar = 20 μm.

Ascl3+ progenitors isolated from adult glands can generate more than one cell type in cultured spheres. (A) EYFP-labeled descendants of Ascl3+ progenitor cells (green; arrowheads). (B) Antibody to Sabpa labels distinct cells in the spheres (red; arrowheads). (C) Merged image of (A) and (B). EYFP-labeled descendants of Ascl3+ progenitors co-localize with cells labeled by Sabpa antibody (arrowheads). (D) EYFP-labeled descendants of Ascl3+ progenitor cells (green). Scale bars = 50 μm. (E) Antibody to serous acinar cell-specific transcription factor, Mist1, labels cells in the spheres (red). (F) Merged image of (D) and (E) shows that Ascl3+ descendants expressing EYFP do not co-localize with cells labeled by Mist1 antibody. Scale bar = 20 μm. (G) EYFP-labeled descendants of Ascl3+ progenitor cells (green; arrowhead). (H) Antibody to Sca1 labels the membranes of cells located at the periphery of spheres (red; arrowhead). (I) Merged image of (G) and (H). EYFP+ descendants of Ascl3+ progenitors co-localize with cells labeled by antibody to Sca1 at the surface of cultured spheres (arrowhead). Scale bars = 10 μm. (J) EYFP-labeled descendants of Ascl3+ progenitor cells (green; arrowhead). The sphere was stained with DAPI to label cell nuclei. (K) Antibody to Keratin 8, a duct cell-specific marker, labels cells located exclusively at the periphery of the spheres (red; arrowhead). (L) Merged image of (J) and (K). EYFP+ descendants of Ascl3+ progenitor cells express the ductal cell marker Keratin 8 (arrowhead). Scale bar = 20 μm.

Ascl3+ progenitors are not precursors of ductal Krt5+ progenitor cells. (A) EYFP-labeled descendants of Ascl3+ progenitor cells (green; gray arrowheads). (B) Sphere stained with antibody to keratin 5 (red; white arrowhead) and DAPI to label nuclei (blue). (C) Merged image of (A) and (B). Cells positive for Keratin 5 antibody (red; white arrowhead) show no overlap with EYFP+ descendants of Ascl3+ progenitor cells (gray arrowheads). Bright green punctate stain is non-specific background. Scale bars = 20 μm. Rugel-Stahl, Elliot and Ovitt