Isolation and Characterization of Salivary Stem Cells

唾液腺在人类生物学中的主要作用是产生一种外分泌，即唾液，它为上胃肠道提供了大部分天然的宿主防御。其他功能包括形成获得的釉质膜，影响釉质表面的矿化，参与粘膜的维护和修复，并作用于溶解味觉呈现给味蕾。唾液分泌减少的情况会给患者带来重要的负面后遗症。临床上，接受放射治疗的头颈癌患者和自身免疫性疾病干燥S综合征患者，由于腺体实质的破坏，即分泌液体和蛋白质的腺泡细胞的丧失，导致唾液功能低下。目前，对这种不可逆性腺体损伤的患者还没有适当的治疗方法。 这项研究的一个主要目标是开发新的方法来再生成人腺体中的唾液腺泡和导管成分，并确定一个长期的基因治疗靶点。人的唾液腺被酶处理以产生单细胞悬液，并在低钙角质形成细胞无血清培养液中种植在层粘连蛋白1或IV型胶原涂层的组织培养塑料上，以促进上皮细胞的附着。收获细胞突起，以克隆密度复制14-21天，分离出几个克隆。在低钙条件下，细胞迅速增殖(直到第10代)，并呈现间充质形态，而不是上皮形态。当转移到正常的钙介质中时，它们恢复了上皮细胞的形态。来自单个克隆的细胞同时表达泛细胞角蛋白(一种上皮标记物)和波形蛋白(一种间质标记物)。这些结果表明，低钙培养条件促进了上皮细胞向间充质细胞的转变，这种转变可以被正常的钙水平逆转。 在其他实验中，将在低钙介质中扩张的细胞放入含有50%Matrigel、正常水平的钙和成纤维细胞生长因子-7或成纤维细胞生长因子-10的孔中，这已被证明可以诱导发育中的唾液腺的形态变化和分化。在这些条件下，细胞在有成纤维细胞生长因子-7或成纤维细胞生长因子-10的存在下聚集。然而，成纤维细胞生长因子-10诱导了细胞聚集的分枝和球状突起。目前正在分析这些聚集体中导管标志物(claudin-1和细胞角蛋白19)和腺泡标志物(淀粉酶、claudin-3和NKCC1)的表达。 除了这些体外研究，这些体外扩增的细胞的分化潜力正在通过体内移植进行分析，这是对潜在干细胞-S重塑组织能力的真正考验。以Matrigel为载体，将人细胞移植到免疫缺陷小鼠体内，1个月后取材，免疫组织化学检测。导管和腺泡样结构都可以被识别，然而，很明显Matrigel没有提供细胞适当组织所需的3D结构类型。我们将继续对这一有趣的细胞群体进行表征，以确定可能更好地鼓励它们分化为唾液腺的生长因子和三维支架，并开发可以测试其功能的动物模型。

The primary role of salivary glands in human biology is to produce an exocrine secretion, saliva, which provides much of the innate host defense for the upper gastrointestinal tract. Other functions include forming the acquired enamel pellicle, influencing enamel surface mineralization, participating in mucosal maintenance and repair, and acting to dissolve tastants for presentation to the taste buds. Conditions that decrease saliva production have important, negative adverse sequelae for a patient. Clinically, patients with head and neck cancer who undergo radiation treatment and patients with the autoimmune disease, Sjogren?s syndrome, develop salivary hypofunction as a result of destruction of glandular parenchyma; i.e., the loss of the fluid- and protein-secreting acinar cells. At present there is no adequate treatment for patients with such irreversible gland damage. A major goal of this study is to develop new methodologies for regeneration of salivary acinar and ductal elements in adult glands, and also to identify a long-lasting target for gene therapies. Human salivary glands were treated enzymatically to produce a single-cell suspension and plated in low-calcium Keratinocyte Serum-Free Medium on Laminin 1 or collagen Type IV-coated tissue culture plastic, to encourage attachment of epithelial cells. Cellular outgrowths were harvested and replated at clonal densities for 14-21 days, and several clones were isolated. In low-calcium conditions, the cells proliferated rapidly (until passage 10), and displayed a mesenchymal, rather than an epithelial, morphology. When transferred to normal calcium medium, they regained an epithelial morphology. Cells that were derived from a single clone expressed both pan-cytokeratin (an epithelial marker) and vimentin (a mesenchymal marker). These results suggest that low-calcium culture conditions promote an epithelial-mesenchymal transition that can be reversed by normal levels of calcium. In other experiments, cells expanded in low-calcium medium were placed into wells containing 50% Matrigel, normal levels of calcium and FGF-7 or FGF-10, which have been shown to induce morphological changes and differentiation in developing salivary glands. Under these conditions, the cells aggregated in the presence of either FGF-7 or FGF-10. However, FGF-10 induced what appears to be branching and globular outgrowth from the cell aggregate. These aggregates are currently being analyzed for the expression of ductal markers (claudin-1 and cytokeratin 19) and acinar markers (amylase, claudin-3 and NKCC1). In addition to these in vitro studies, the differentiation potential of these ex vivo expanded cells is being analyzed by in vivo transplantation, which is the true test of a potential stem cell?s ability to recapitulate a tissue. Human cells were transplanted into immunocompromised mice with Matrigel as a carrier, harvested after one month, and characterized by immunohistochemistry. Both ductal- and acinar-like structures could be identified, however, it is clear that Matrigel does not provide the type of 3D structure that is required for the cells to organize appropriately. We will continue to characterize this interesting population of cells to determine the growth factors and three-dimensional scaffolds that may better encourage them to differentiate into salivary glands, and to develop animal models in which their functionality can be tested.