干口是唾液腺功能障碍，对患者的生活质量产生负面影响。然而，对于干口目前尚无有效治疗方法。通过使用从新生儿唾液腺获得的干细胞和结扎损伤模型生成的生物工程器官，是一种可行的干口治疗新策略。通过用人类小唾液腺获得的种子细胞重建唾液腺植物体，为临床应用和器官再生研究提供理论基础和技术支持。在本文中，我们旨在提出一种通过Wnt信号激活的三维环境中体外培养和富集成年人小唾液腺干细胞(hMSGSCs)的新方法。获得并鉴定了具有自主组织形成能力的hMSGSCs，并通过三维培养生成了器官体。我们使用MTT(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物)实验评估hMSGSCs的增殖和集落形成情况。通过端粒酶反转录染色、流式细胞术、免疫荧光检测、RNA提取、逆转录PCR和qPCR等方法评估了hMSGSCs的结构和体外重建器官的功能。hMSGSCs表现出典型的上皮样特征，如CD49f和细胞KRT表达阳性。hMSGSCs在唾液腺中作为成年干细胞，并能分化为腺泡细胞和导管细胞。将Noggin、CHIR99021和Wnt3A添加到三维培养系统中后，hMSGSCs显示出更高的LGR5表达，并减少AMY1B和MUC5B的表达。因此，Wnt和骨形态发生蛋白(BMP)通路在调控hMSGSCs的自主组织形成和分化中起重要作用。我们展示了在三维培养系统中通过激活Wnt信号通路和抑制BMP信号通路，可以维持hMSGSCs的干细胞特性。我们的发现为体外生成唾液腺器官体提供了新的见解。©2023 BioMed Central Ltd., 汤森路透旗下一部分。

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.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.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.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.© 2023. BioMed Central Ltd., part of Springer Nature.