Abstract
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.
Highlights
Salivary acinar cells are responsible for the secretion of water, electrolytes, mucus, glycoproteins, enzymes, and anti-bacterial compounds including salivary peroxidase and lysozyme [1,2]
DMEM/F12-glutamax has been used for Mouse MSCs (mMSCs) and hepato-STIM media were specific for human primary salivary gland cells (pSGCs) [15]
As for co-culture, mMSCs were plated on the collagencoated glass slide in the culture wells for 12 hours before co-culture and the upper transwell membrane was seeded with pSGCs (Fig. S1B)
Summary
Salivary acinar cells are responsible for the secretion of water, electrolytes, mucus, glycoproteins, enzymes, and anti-bacterial compounds including salivary peroxidase and lysozyme [1,2]. Stem cell-based therapies have been applied to repair damaged tissues in various organs. Control of cell differentiation and specific linage development needs to be closely monitored to prevent the formation of teratomas by these cells. Adult stem cells, such as mesenchymal stem cells (MSCs), not as pluripotent as embryonic stem cells, offer many advantages for the development of restorative treatments. These advantages include but are not limited to their relative accessibility, stable phenotype, tissue compatibility, and immunosuppressive properties
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