Stomatal movement in response to 1 mM HCO₃^- added at time 0 min.

<p>Data were obtained from 60 stomata in three independent experiments and presented as means ± SE. The asterisks indicate significantly different mean values at P <0.05.</p

STEM analysis showing metabolite accumulation patterns across the time-course of HCO₃^- treatment of (A) MC and (B) GC.

<p>Numbers at the bottom-left indicate the metabolites with similar trends, and the star indicates significance (P < 0.05).</p

Responses of major metabolite groups in MCs and GCs upon HCO₃^- treatment in the time-course study.

<p>Green and red squares indicate decreased (<0.8) and increased (>1.2) fold changes, respectively. The asterisks indicate significant changes (P <0.05).</p

Identification of Regulatory Factors for Mesenchymal Stem Cell-Derived Salivary Epithelial Cells in a Co-Culture System

<div><p>Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.</p></div

Summary of the increased and decreased metabolic pathways in (A) GCs and (B) MCs after HCO₃^- treatment.

<p>Summary of the increased and decreased metabolic pathways in (A) GCs and (B) MCs after HCO₃^- treatment.</p

Principal component analysis (PCA) and orthogonal partial least square discriminant (OPLS-DA) analysis of metabolite changes in GCs and MCs after HCO₃^- treatment.

<p>PCA was performed using four replicate data of relative metabolite abundances in the cell-types at 0, 5, 15, 30, 60, 120 mpi, and the generated PC1 and PC2 were plotted. PCA of two cell-types showing a clear separation of the two groups based on the 268 metabolites for the effect of treatments in (<b>A</b>) GCs, and (<b>B</b>) MCs. The effects of only <b>(C)</b> time and both (<b>D</b>) ‘treatment x time’ were displayed. In OPLS-DA, the metabolite changes as a result of interactions among ‘cell-type x treatment x time were displayed in <b>(E)</b> GCs and (<b>F</b>) MCs.</p

Hierarchical cluster analysis (HCA) of mean values of metabolite contents from four biological replicates showing 268 metabolites common to the two cell-types depicting the data structure dependent on the cell-types and time course (0–120 mpi) of HCO₃^- treatment.

<p>Red and green indicate high and low concentrations of metabolites, respectively. Values were subjected to average linkage clustering (Euclidean distance). Outlined blocks in yellow show grouped metabolites in the two cell-types.</p

Co-cultured mMSCs resemble primary salivary gland cell morphology and express salivary gland epithelial cell markers.

<p>A) Microscope images (at 20X and 40X magnifications) of pSGCs from C57BL/6 mice (first panel), control mMSCs (second panel), and co-cultured mMSCs with pSGCs were shown. Mouse pSGCs showed islet-like cell morphology whereas control mMSCs exhibit typical fibroblast-like appearance. Aggregated cell masses, which resemble islet-like pSGCs, at each time point were indicated by black arrowheads. Co-culture was carried out for 7 days without replacing media. B) Co-cultured mMSCs were positively stained for acinar cell markers, such as a-amylase, and M3R (green color in each column) in a time dependent manner and a ductal cell marker CK19 (red color). Control mMSCs (second row) were negative while cytospinned pSGCs (first row) from the submandibular glands were positive for these markers. The nuclei were stained with DAPI and the column of +DAPI indicates merged images. Scale Bar = 50 µm. C) Co-cultured mMSCs were counted from four independent biological replicates after staining using a fluorescent microscope. Y-axis represents a percentage of positively stained mMSCs for each marker protein at a given time point. Pictures were taken at a 20X magnification. Quantification of cell numbers over time was performed by one-way ANOVA with Bonferroni post-hoc test (*p<0.05, **p<0.01, NS: no significant).</p

Functional network of key transcription factors during development.

<p>Based on data analysis using STRING 9.1 and WikiPathway, numerous proteins appear to be functionally associated with TCF3 during developmental processes. A dotted line indicates a potential association in function between TCF3 and PTF1α.</p

Two-dimensional gel electrophoresis images and spot analysis revealed 58 differentially expressed proteins.

<p>A) Following the co-culture of mMSCs with pSGCs for 1, 3, 5 and 7 days, total cell lysates (200 µg) were separated on pH 3–10 linear IPG strips in the first dimension and 12.5% SDS-PAGE in the second dimension. The gels were stained with mass spectrophotometry-compatible silver staining kit. B) According to the data analyses, expression levels of 58 spots (circled) were significantly altered at least by 1.5 fold (p<0.05, one-way ANOVA with Bonferroni post-hoc test). Each of these spots has a specific spot number for database storage and further analysis. Data from five independent experiments (Five gels in duplicate for each time point) were analyzed and the gel figure presented here is from day 7.</p