An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-𝛽1 in Salivary Gland Epithelial and Mesenchymal Differentiation

Figure 4

Characterization of Col1a1-GFP derived submandibular salivary gland cells cultured in DMEM plus serum media. (a)–(l) Specific staining showed the presence of salivary gland epithelial and mesenchymal cells in DMEM media plus serum in early (passage 1; 1 week in culture) and late cultures (passage 9; 8 weeks in culture). GFP-positive spindle-shaped cells represented mesenchymal cell population. Cells in early culture were stained positively for von Willebrand Factor (vWF) (a), smooth muscle actin (SMA) (c) and S100 (e), CD44 (g), and amylase-1 (AMY-1) (i) (in red), which are markers for endothelial cells, myoepithelial cells, and salivary gland epithelial cells, respectively. In the late culture, an increased number of GFP-positive cells was observed. vWF (b), SMA (d), S100 (e), and CD44 (h) staining were seen. SMA, S100, and CD44 expression seem to be increased in the late passaged mesenchymal cells, which was illustrated by the costaining of SMA, S100, and CD44 with GFP. SMA staining demonstrated four cell populations in mixed salivary gland cultured cells, GFP+/SMA+, GFP+/SMA−, GFP−/SMA+, and GFP−/SMA− cells but a majority of cells were GFP+/SMA− cells, indicating some mesenchymal cells upregulated SMA expression in the late culture (d), compared to that in the early culture (c). AMY-1 (j), E-cadherin (E-cad) (k), and LAMP-1 (l) were specifically positive for salivary gland epithelium in red, but not mesenchymal cells. (f) RT-PCR analysis displayed a gene profile corresponding of a mixed salivary gland cell culture throughout long-term culture in DMEM plus serum medium from early through late passages. The gene expression of salivary gland epithelium, Aqp-5 (Aquaporin-5), ZO-1 (Zona occludens-1), and Amy-1 (Amylase-1), as well as salivary gland mesenchyme, Pdgfr-a, Fgf-7, Fgf-10, Col1a1 (Collagen type I), were detected in both early and late passages, indicating the existence of salivary gland epithelial and mesenchymal in these cultures. This gene expression profile was detected in three different lines of salivary gland cells which were derived from three different Col1a1-GFP mice. Early culture = passage 1 (P1), and late culture = passage 9 (P9). P1, 5, and 9 = passages 1, 5, and 9, respectively. Submandibular salivary gland (SMG) was used as positive control whereas no template was used as negative control (NCT). Scale bars = 100 μm.