Novel Genetic Approach To Isolate Stem Cells Of The Oral Cavity

描述（由申请人提供）：口腔的复层鳞状上皮是自我更新的，并且具有产生新细胞以补充从口腔表面丢失的终末分化细胞所必需的常驻角质形成细胞干细胞群。这些干细胞和它们的衍生祖细胞被认为沿着最内层的基底层分散，在那里它们以特征性的慢循环状态休眠。干细胞保留标记DNA的固有能力延长了时间段，使得不仅可以标记它们在基底层内的位置并使它们可视化，而且可以前瞻性地纯化它们。在类似于BrdU脉冲追踪实验的策略中，可调节的组蛋白2B-GFP转基因小鼠模型已被用于在体内荧光标记静止干细胞。作为一种补充方法，如果可用的话，也可以利用特异性标志物来鉴定干细胞。然而，缺乏理想的表面标记物一直是上皮干细胞的分离和随后的生物学和生化表征的障碍，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？Np 63亚型，在复层鳞状上皮的干细胞区室中高度表达。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。我们创造了一部小说？Np 63-GFP报告小鼠模型系统，允许标记的细胞表达高水平的？Np 63，我们假设它可能代表干细胞。Histone 2B-GFP和？Np 63-GFP动物将使我们能够识别和分选来自小鼠口腔上皮的潜在干细胞，这些干细胞位于离散的解剖部位，如颊粘膜、腭和牙龈。重要的是，我们的策略将允许分离活的干细胞，随后可以在克隆形成和分子谱研究中进行分析。最后，我们希望确定额外的和长期寻求的标记的口腔干细胞，这无疑将是至关重要的适当的临床应用口腔上皮细胞培养的细胞治疗，以及上皮癌干细胞和肿瘤发生的研究。更重要的是，从我们的小鼠研究中获得的数据将是非常宝贵的计划和执行类似的干细胞研究使用人类样本。 公共卫生相关性：口腔复层上皮不断从干细胞（SC）中产生新的细胞，以补充从表面脱落的分化细胞。与其他器官和组织不同，我们对口腔粘膜中SC生物学和功能的了解有限-因此，越来越需要鉴定，分离和表征口腔上皮中的SC。通过使用两种互补的遗传策略，我们计划分离口腔SC，测试其独特的自我更新特性并生成其独特的分子指纹，以更好地了解口腔复层上皮的生物学并利用SC的再生能力。

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. PUBLIC HEALTH RELEVANCE: Oral stratified epithelia continuously generate new cells from stem cells (SCs) to replenish the differentiated cells that are sloughed off from the surface. Unlike other organs and tissues, our knowledge about SC biology and function in the oral mucosa is limited - therefore, there is a growing need to identify, isolate, and characterize SCs of the oral epithelia. By using two complementary genetic strategies, we plan to isolate the oral SCs, test their distinctive self- renewing properties and generate their distinctive molecular fingerprints to better understand the biology of oral stratified epithelia and harness the regenerative power of SCs.