Novel Genetic Approach To Isolate Stem Cells Of The Oral Cavity

描述(申请人提供)：口腔复层鳞状上皮是自我更新的，有一个常驻的角质形成细胞干细胞群，需要产生新的细胞来补充从口腔表面丢失的终末分化细胞。这些干细胞及其派生的祖细胞被认为散布在最内侧的基底层，在那里它们以一种典型的慢循环状态休眠。干细胞在很长一段时间内保留标记的DNA的固有能力使其不仅可以标记它们在基底层中的位置并使其可视化，而且还可以预期地对它们进行纯化。在类似于BrdU脉冲追逐实验的策略中，已利用可调节的组蛋白2B-GFP转基因小鼠模型在体内对静止的干细胞进行荧光标记。作为一种补充方法，特定的标志物，如果有的话，也可以用来鉴定干细胞。然而，缺乏理想的表面标记一直是上皮干细胞分离和随后的生物学和生化特征的障碍，特别是在口腔中。最近的研究提供了令人信服的证据支持转录因子p63，特别是？Np63亚型，在复层鳞状上皮的干细胞室高表达。此外，对p63缺陷小鼠的广泛分析揭示了该因子在角质形成细胞自我更新中的重要作用。我们已经建立了一种新的？Np63-GFP报告小鼠模型系统，它允许标记高水平表达？Np63的细胞，我们假设这可能代表干细胞。历史2B-GFP和？NP63-GFP动物的可获得性将使我们能够从不同解剖位置的小鼠口腔上皮中鉴定和分类预期的干细胞，如颊粘膜、腭部和牙龈。重要的是，我们的策略将允许分离可存活的干细胞，随后可以在克隆形成和分子图谱研究中进行分析。最后，我们希望找到更多和长期寻找的口腔干细胞标记物，这对于口腔上皮细胞培养在细胞治疗中的适当临床应用以及上皮癌干细胞和肿瘤发生的研究无疑将是至关重要的。更重要的是，从我们的小鼠研究中获得的数据将对利用人类样本计划和执行类似的干细胞研究具有无价的价值。

DESCRIPTION (provided by applicant): The stratified squamous epithelium of the oral cavity is self-renewing and has a resident keratinocyte stem cell population necessary for generating new cells to replenish the terminally differentiated cells lost from the oral surface. These stem cells and their derivative progenitors are thought to be scattered along the inner most basal layer, where they lie dormant in a characteristic slow-cycling state. The inherent ability of the stem cells to retain labeled DNA for a prolonged time period makes it possible not only to mark their location within the basal layer and visualize them, but also to prospectively purify them. In a strategy analogous to a BrdU pulse- chase experiment, a regulatable Histone2B-GFP transgenic mouse model has been utilized to fluorescently label quiescent stem cells in vivo. As a complementary approach, specific markers, if available can also be utilized to identify stem cells. However, the lack of ideal surface markers has been an impediment for the isolation and subsequent biological and biochemical characterization of epithelial stem cells, particularly in the oral cavity. Recent studies have offered compelling evidence in support of the transcription factor p63, particularly the ?Np63 isoform, to be highly expressed in the stem cell compartment of the stratified squamous epithelium. In addition, extensive analysis of p63-deficient mice has revealed the essential role of this factor in the self-renewal of keratinocytes. We have generated a novel ?Np63-GFP reporter mouse model system that allows to mark the cells that express high levels of ?Np63, which we hypothesize are likely to represent stem cells. The availability of the Histone2B-GFP and ?Np63-GFP animals will allow us to identify and sort the prospective stem cells from the mouse oral epithelium at discrete anatomical sites such as buccal mucosa, palate and gingiva. Importantly, our strategy will permit the isolation of viable stem cells that can be subsequently analyzed in clonogenicity and molecular profiling studies. Finally, we hope to identify additional and long-sought markers of oral stem cells, which will undoubtedly be of crucial importance for proper clinical application of oral epithelial cultures in cell therapy as well as for studies on epithelial cancer stem cells and tumorigenesis. More importantly, the data obtained from our murine studies will be invaluable for planning and executing similar stem cell studies using human samples.