Abstract
BACKGROUND: Xerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation. METHODS: Obtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro. RESULTS: hMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation. CONCLUSIONS: We showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.