Isolation and characterization of salivary stem cells

唾液腺的主要作用是产生唾液，唾液在口腔中提供大部分先天宿主防御。在临床上，唾液腺功能减退会导致严重的发病率，并且对于不可逆的腺体损失没有常规的治疗方法。本研究的目的是鉴定和分离成人唾液腺干细胞。对人下颌下腺进行酶促处理以产生单细胞悬液，并以克隆密度接种在IV型胶原包被的组织培养塑料上以促进上皮细胞的附着，并在低钙（0.1 mM）无血清营养培养基中孵育。通过免疫细胞化学表征在低钙培养基中扩增的克隆细胞，随后将其置于含有50%基质胶、正常钙（1.2 mM）和FGF-7或FGF-10的威尔斯孔中。细胞也被移植到免疫功能低下的小鼠与聚乙醇酸支架与基质胶作为载体，两个月后收获，并通过免疫组织化学表征。 分离了几个克隆。在低钙条件下，细胞迅速增殖（直至第10代），并显示间充质而非上皮形态。来自单个克隆的细胞表达泛细胞角蛋白（上皮标记物）和波形蛋白（间充质标记物）。当在低钙培养基中生长时，细胞不表达腺泡细胞特异性mRNA，但在正常钙条件下，它们表达导管和腺泡mRNA标记物。在基质胶中，这些细胞聚集，FGF-10诱导从聚集体的分支和球状生长，以及导管细胞特异性蛋白的表达。移植的细胞产生导管样和腺泡样结构，并表达导管和腺泡标记物（mRNA和蛋白质）。从酶促分散的人下颌下腺获得的细胞似乎包括祖细胞，其中一个子集可能能够形成导管和腺泡表型。 为了在体内标记假定的干细胞（缓慢分裂，标记保留细胞），将BrdU腹膜内注射到成年小鼠中18天。需要这种延长的注射系列来增加很少分裂的干细胞被标记的机会。在注射的最后一天，然后在0.5，1，2，4，6和7个月后，收集下颌下腺，以观察标记细胞（分化细胞和瞬时分裂祖细胞）的消失，并确定标记保留细胞的位置，这将是一个假定的唾液干细胞。从7天到100天，标记保留细胞分别占细胞总数的70%到30%。但152天后，标记保留细胞不到总细胞的3%。标记保留细胞不仅是导管细胞，而且是腺泡细胞，并且少数标记保留细胞本质上是非导管、非腺泡和非肌上皮细胞。186天后，标记保留细胞已从唾液腺中消失。这些结果表明，一个子集的腺泡细胞，横纹和颗粒导管细胞，和一个未知身份的细胞类型正在缓慢分裂，标记保留细胞，作为储备细胞池，也许自我更新。损伤后，人们认为干细胞会立即分裂以启动再生。在未来的研究中，我们的目标是通过免疫组织化学染色来表征这种细胞类型，以确定这种细胞类型的一组标志物。 目前，我们正在确定表达的导管和腺泡标记的细胞聚集体和体内移植的RT-PCR和免疫组织化学。同样，我们特别关注可用于细胞分选实验的细胞表面标志物的表达。在这项研究完成之前，还有一些关键的实验必须进行，那就是确定细胞是否能够真正发挥作用。为了做到这一点，已经计划了两个实验。首先是确定细胞是否会并入受损的唾液腺并与残留的腺体形成适当的连接。第二种是通过辐射破坏小鼠产生唾液的腺泡细胞（这是接受放射治疗的头颈癌患者的主要问题），从而诱导口干。然后将细胞移植到残留的导管细胞（不产生唾液）周围，并确定移植的细胞是否可以重组腺体，以及是否产生唾液。

The primary role of salivary glands is to produce saliva, which provides much of the innate host defense in the oral cavity. Clinically, salivary hypofunction causes significant morbidity and there is no conventional treatment for irreversible gland loss. The purpose of this study is to identify and isolate stem cells from adult human salivary glands. Human submandibular glands were treated enzymatically to produce single cell suspensions and plated at clonal densities on Type IV collagen-coated tissue culture plastic to encourage attachment of epithelial cells, and incubated in low calcium (0.1 mM) serum-free nutrient medium. Clonal cells expanded in low calcium medium were characterized by immunocytochemistry, and subsequently were placed into wells containing 50% Matrigel, normal calcium (1.2 mM), and FGF-7 or FGF-10. Cells were also transplanted into immunocompromised mice with a polyglycolic acid scaffolding with Matrigel as a carrier, harvested after two months, and characterized by immunohistochemistry. Several clones were isolated. In low calcium, the cells proliferated rapidly (until passage 10), and displayed a mesenchymal rather than epithelial morphology. Cells that were derived from a single clone expressed both pan-cytokeratin (an epithelial marker) and vimentin (a mesenchymal marker). When grown in low calcium medium, cells did not express acinar cell-specific mRNAs, but in normal calcium conditions, they expressed both ductal and acinar mRNA markers. In Matrigel, these cells aggregated and FGF-10 induced branching and globular outgrowth from the aggregates, as well as expression of ductal cell-specific protein. Transplanted cells made both ductal- and acinar-like structures, and expressed both ductal and acinar markers (both mRNAs and proteins). Cells obtained from enzymatically dispersed human submandibular glands appear to include progenitors, a subset of which may be able to form both ductal and acinar phenotypes. To label a putative stem cell (slowly-dividing, label retaining cell) in vivo, BrdU was injected into young-adult mice intraperitoneally for 18 days. This extended series of injections is needed to increase the chance that the stem cell, which rarely divides, to be labeled. On the last day of injection, and then at 0.5, 1, 2, 4, 6, and 7 months later, submandibular salivary glands were harvested in order to watch the disappearance of labeled cells (differentiated cells and transiently dividing progenitor cells) and to identify the location of the label-retaining cell, which would be a putative salivary stem cell. From 7 days to 100 days, label-retaining cells range from 70% to 30% of the total number of cells respectively. But after 152 days, the label-retaining cells are less than 3% of the total cells. Label-retaining cells are not only ductal cells but also acinar cells, and a few label-retaining cells are non-ductal, non-acinar, and non-myoepithelial in nature. After 186 days, the label-retaining cells have disappeared from the salivary gland. These results suggest that a subset of acinar cells, striated and granular duct cells, and a cell type of unknown identity are slowly dividing, label-retaining cells that serve as reserve cell pools, perhaps by self-renewal. After injury, it is thought that a stem cell will divide immediately in order to initiate regeneration. In future studies, we will aim to characterize this cell type by immunohistochemical staining to identify a set of markers for this cell type. Currently, we are determining the expression of ductal and acinar markers of the cell aggregates and in vivo transplants by RT-PCR and immunohistochemistry. Again, we are paying particular attention to the expression of cell surface markers that could be used for the cell sorting experiments. There are still some critical experiments that must be performed before this study is complete, and that is to determine if the cells are able to actually function. In order to do this, two experiments have been planned. The first is to determine if the cells will incorporate into an injured salivary gland and form appropriate connections with the residual gland. The second is to destroy saliva-producing acinar cells in mice by irradiation (this is a major problem in patients undergoing radiation for the treatment of head and neck cancer), thereby inducing dry mouth. The cells will then be transplanted around the residual ductal cells (which do not produce saliva), and determine if gland can be reformed by transplanted cells, and if saliva is produced.