An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-1 in Salivary Gland Epithelial and Mesenchymal Differentiation
Figure 8
The expression of TGF-1 ligand and receptor in Col1a1-GFP derived submandibular salivary gland cells after in vitro differentiation on matrigel treated with TGF-1 or TGF-1 and plus TGF-R1 inhibitor, SB525334 (TGF-1 + SB525334). (a)–(f) and (g)–(l) TGF-1 and TGF-1R staining (in red) showed the formation of acinar-like structures, respectively. Acinar formation was observed adjacent to mesenchymal cells in the matrigel only (a and g) and TGF-1 + SB525334 (c and i) groups, but not in the TGF-1 (b and h) group. In the areas free of mesenchymal cells, acinar-like structures were also found in all three groups. TGF-1 + SB525334 treated cells formed larger acinar-like structures (c, f, i, and l) compared to untreated (a, d, g, and j) and TGF-1 treated cells (e and k). Acini-like structures in the matrigel (d and j) and TGF-1 + SB525334 (f and l), but not in the TGF-1 (e and k) showed GFP-positive mesenchymal cells located peripherally or adjacent to acini-like structures. Elongated and polarized salivary gland mesenchymal cells were found in the TGF-1 treated group (b and h), but not in the untreated (a and g) and TGF-1 + SB525334 groups (c and i). (m) and (n) The expression of TGF- 1 ligand (Tgf-b1) and receptor (Tgf-br1) were also examined, respectively. The cells treated with TGF-1 significantly increased the level of Tgf-b1 and Tgf-br1 expression compared to untreated and TGF-1 + SB525334 treated cells both day 3 (D3) and similar pattern was observed at day 5 (D5). In the control, untreated cells on matrigel, the expression levels of both Tgf-b1 and receptor Tgf-br1 decreased at D5 compared to D3 but this pattern is not seen in the TGF-1 and TGF-1 + SB525344 groups. Relative expression was normalized to the expression of Gapdh which was used as the reference gene. Values were represented as mean ± SEM from three independent experiments (). Student’s -test was analyzed to compare between matrigel (untreated; black bar), TGF-1 treated (dark gray bar), and TGF-1 + SB525334 treated cells (light gray bar), , , or . Scale bars = 100 μm.
