Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, TX, USA.
Katz Department of Oral and Maxillofacial Surgery, The University of Texas Health Science Center at Houston, Houston, TX, USA.
Methods Mol Biol. 2024;2749:25-38. doi: 10.1007/978-1-0716-3609-1_3.
Resident stem/progenitor cells within the secretory salivary glands offer a potential therapeutic resource for use in the regeneration of salivary glands needed to restore saliva production in patients with chronic xerostomia, or dry mouth. Methods were developed previously to isolate human stem/progenitor cells (hS/PCs) from major salivary glands (parotid/submandibular). Abundant minor salivary glands located in readily accessible locations in the oral cavity and lip could provide an additional valuable therapeutic resource. An advantage of this cell resource is that these minor glands about the size of grape seeds can be harvested from healthy donors using minimally invasive surgical procedures. The disadvantage of using minor glands is that they contain many fewer cells than do major glands, and thus harvested cells need to be expanded in the lab to create a therapeutic resource. While earlier work has described isolation of proliferative cell populations from minor salivary glands that could be used in regenerative medicine, most of these expanded cells possess properties of mesenchymal cells rather than the epithelial population that secretes salivary products.Here, we describe in detail our recently established methods to isolate and expand hS/PCs isolated from human labial minor salivary glands. Expanded hS/PC populations are epithelial assessed by their expression of epithelial progenitor markers K5 and K14. Like expandable cell populations previously isolated from the major salivary glands, these cells also express nuclear p63, consistent with their ability to be expanded after explant culture. When hS/PCs with these properties are encapsulated into a customized 3D biomimetic hyaluronic acid-based hydrogel, they will assemble into microstructures that retain some progenitor markers while also beginning to differentiate. The increased expression of secreted mucin MUC-7 was used to demonstrate differentiation and secretory potential in assembled hS/PC microstructures. Compared to hS/PCs from major glands, those from minor salivary glands tend to be more heterogeneous in early passage; thus, use of K5/K14/p63 as an early quality assessment tool is highly recommended. Additionally, hS/PCs from minor glands are sensitive to stress and if mishandled will demonstrate a stress response that leads to their transitioning to a flat, squamous cell-like appearance that is of limited utility in regenerative medicine applications. We conclude that properly handled hS/PCs from minor salivary glands represent a powerful new source of therapeutic cells for applications including treating patients with chronic xerostomia.
分泌性唾液腺中的常驻干细胞/祖细胞为治疗慢性口干症患者的唾液腺再生提供了一种有潜力的治疗资源，这种疾病需要恢复唾液分泌。先前已经开发出了从主要唾液腺（腮腺/颌下腺）中分离人干细胞/祖细胞（hS/PCs）的方法。位于口腔和唇部易于接近位置的大量小唾液腺可以提供另一种有价值的治疗资源。这种细胞资源的一个优势是，这些大小如葡萄籽的小腺体可以通过微创外科手术从健康供体中收获。使用小腺体的缺点是，与主要腺体相比，它们所含的细胞要少得多，因此需要在实验室中扩增收获的细胞，以创建治疗资源。虽然早期的工作已经描述了从小唾液腺中分离增殖细胞群体用于再生医学，但这些扩增的细胞大多具有间充质细胞的特性，而不是分泌唾液产物的上皮细胞群体。在这里，我们详细描述了我们最近建立的从人类唇小唾液腺中分离和扩增 hS/PCs 的方法。通过表达上皮祖细胞标志物 K5 和 K14 来评估扩增的 hS/PC 群体是否为上皮细胞。与先前从主要唾液腺中分离的可扩增细胞群体一样，这些细胞也表达核 p63，这与其在离体培养后能够扩增一致。当具有这些特性的 hS/PC 被包封在定制的 3D 仿生透明质酸基水凝胶中时，它们将组装成保留一些祖细胞标志物的微结构，同时开始分化。组装的 hS/PC 微结构中分泌黏蛋白 MUC-7 的表达增加用于证明分化和分泌潜力。与来自主要腺体的 hS/PC 相比，来自小唾液腺的 hS/PC 在早期传代时往往更具异质性；因此，强烈建议将 K5/K14/p63 用作早期质量评估工具。此外，来自小唾液腺的 hS/PC 对压力敏感，如果处理不当，它们会表现出应激反应，导致它们向扁平的、鳞状细胞样外观转变，这在再生医学应用中用途有限。我们得出结论，适当处理的来自小唾液腺的 hS/PC 代表了一种强大的新型治疗细胞来源，可用于治疗慢性口干症等患者。
