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 7
In vitro differentiation of Col1a1-GFP derived submandibular salivary gland cells on matrigel treated with TGF-1 or TGF-1 plus TGF-R1 inhibitor, SB525334 (TGF-1 + SB525334). Mixed cells were plated on matrigel with DMEM plus serum medium alone (left column) or supplemented with TGF-1 (middle column) or TGF-1 with SB525334 inhibitor (right column). Specific staining for salivary gland epithelial cells demonstrated the presence of differentiated salivary gland cells at day 5. (a)–(c) CD44 staining (in red) showed the formation of acinar-like structures (round structures with polarized nuclei) in all three groups. Large acini-like structures with branching were found in the TGF-1 + SB525334 (c). (d)–(o) Salivary gland epithelium and acinar formation were identified by E-cadherin (E-cad), LAMP-1, AMY-1, and AQP-5 staining (in red). Salivary gland acini-like structures (in red) closely located to mesenchymal cells (in green) were found in the matrigel (d, g, j, and m) and TGF-1 + SB525334 (f, i, l, and o) groups whereas no acinar formation was observed in close proximity of mesenchymal cells in the TGF-1 group (e and h). Acinar-like structures were also found in areas free of mesenchymal cells in all three groups. Cells in the TGF-1 + SB525334 (c, f, i insets, l, and o) formed larger acini-like structures than those in the matrigel alone (a, d, g insets, j, and m) and TGF-1 (b, e, h insets, k, and n). Salivary gland mesenchymal cells were polarized and elongated in the TGF-1 (e and h) whereas mesenchymal cells in the matrigel (d, g, and m) and TGF-1 + SB525334 (f, i, l, and o) demonstrated cobblestone appearance. Some GFP-positive cells were found to integrate or locate peripherally to acini-like structures in the matrigel (a, d, and g insets) and TGF-1 + SB525334 (c, f, and i insets), but not in the TGF-1 (b, e, and h insets). (j)–(p) The expression of salivary gland epithelial genes, Amylase-1, Aquaporin-5 (Aqp-5), Zonula occludens (ZO-1), Occludin, and salivary gland mesenchymal genes, Fgf-7, Fgf-10, and Collagen type I, was determined after 3- and 5-day treatments. At day 3 (D3), cells treated with TGF-1 + SB525334 significantly upregulated some of salivary gland epithelial genes, Amylase-1 and Occludin, and all salivary gland mesenchymal genes except Collagen type I, compared to cells on matrigel alone and/or cells treated with TGF-1. The TGF-1-treated cells significantly expressed lower level of Amylase-1 expression (p), but higher level of Collagen type I (v), compared to untreated cells, and TGF-1 + SB525334-treated cells. At day 5 (D5), cells treated with TGF-1 + SB525334 expressed the highest levels of both salivary gland epithelial and mesenchymal genes and were significantly different compared to untreated and TGF-1 treated cells. The ZO-1 expression was comparable but insignificantly different between TGF-1 and TGF-1 + SB525334 treated cells (r). The expression of Fgf-7 and Fgf-10 was remarkably increased in the TGF-1 + SB525334 group (t and u). The highest level of Collagen type I was observed and shown a significantly statistical difference in TGF-1 treated cells at both day 3 and 5 compared to other groups (v). 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.
