Isolation and Characterization of Salivary Stem Cells

基于唾液腺经历连续替换的事实，假设在成人唾液腺中发现的所有细胞类型都被干细胞或祖细胞改造，并且这些细胞可用于在体内移植时改革唾液组织。 今年启动的研究旨在确定是否有可能从唾液腺中分离出克隆性自我更新细胞，以及是否存在能够改革唾液腺内所有细胞类型的共同干细胞，或者更倾向于一种特定表型或另一种表型的干细胞或祖细胞的层次结构。其他研究正在进行中，以设计体内移植的方法，这是鉴定干细胞的关键测定法，也是潜在的临床应用。 作为第一近似，最近建立的从胰腺导管和腺泡成分分离干细胞的技术正在被使用。对鼠和人唾液腺进行酶处理以产生细胞悬浮液，并将其接种在含有角质形成细胞生长因子的无血清培养基或肝细胞生长培养基中。从这些程序中衍生的细胞被表征为表达各种标记蛋白，包括细胞角蛋白7、8、18和19，以检查细胞的一般唾液和上皮性质，并使用可用于区分腺泡和导管细胞表型的多种抗体。（淀粉酶和密蛋白）。 虽然体外分化的特征很重要，但干细胞研究的金标准是体内移植。雄性鼠细胞和人类细胞被移植到雌性免疫功能低下的小鼠体内。产生的组织的供体来源可以通过FISH（小鼠中的Y染色体到小鼠移植物中）和通过与人特异性alu序列的原位杂交（人到小鼠移植物中）来确定。迄今为止，细胞已与基质胶组合并皮下放置到免疫受损动物中。组织学上分析了所得的移植物，并鉴定了导管样结构。 目前的研究旨在改善来自小鼠和人唾液腺的克隆的生长（其非常缓慢），并开发更好的体内移植测定以支持腺泡组织的形成。

Based on the fact that the salivary gland undergoes continuous replacement, it is hypothesized that all cell types that are found in the adult salivary gland are reformed by stem or progenitor cells, and that these cells can be used to reform salivary tissues upon in vivo transplantation. Studies were initiated this year that are aimed at determining whether it is possible to isolate a clonogenic, self-renewing cell from salivary glands, and if there is a common stem cell, able to reform all cell types within a salivary gland, or a hierarchy of stem or progenitor cells that are more committed to one particular phenotype or another. Other studies are under way to devise methods of in vivo transplantation, a critical assay for the identification of a stem cell, and also for potential clinical applications. As a first approximation, techniques that have recently been established to isolate stem cells from pancreatic ductal and acinar elements are being used. Murine and human salivary glands are treated enzymatically to produce a cell suspension and plated in serum-free medium containing keratinocyte growth factor or hepatocyte growth medium. Cells derived from these procedures are being characterized for expression of various marker proteins, including cytokeratins 7, 8, 18, and 19 to examine the general salivary and epithelial nature of the cells, and with multiple antibodies that can be used to distinguish acinar and ductal cellular phenotypes. (amylase and claudin). While it is important to characterize in vitro differentiation, the gold standard for stem cell studies is in vivo transplantation. Male murine cells and human cells are being transplanted as into female immunocompromised mice. The donor origin of the tissue that is generated can be determined by FISH (Y chromosome in mouse into mouse transplants) and by in situ hybridization with human specific alu sequences (human into mouse transplants). To date, cells have been combined with Matrigel and placed into immunocompromised animals either subcutaneously. The resulting transplants have been analyzed histologically, and duct-like structures have been identified. Current studies are aimed at improving the growth of clones derived from murine and human salivary glands (which is exceedingly slow), and to develop better in vivo transplantation assays to support the formation of acinar tissues.