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.