Novel Genetic Approach To Isolate Stem Cells Of The Oral Cavity

描述（由申请人提供）：口腔的分层鳞状上皮是自我更新的，具有常驻的角质细胞干细胞群，需要产生新细胞来补充从口腔表面丢失的终末分化细胞。这些干细胞及其衍生祖细胞被认为分散在最内层的基底层，在那里它们处于休眠状态，处于典型的慢循环状态。干细胞具有长时间保留标记DNA的固有能力，这不仅可以标记它们在基底层的位置并使它们可视化，而且还可以对它们进行前瞻性纯化。在一个类似于BrdU脉冲追踪实验的策略中，一个可调节的Histone2B-GFP转基因小鼠模型被用于荧光标记体内静止干细胞。作为一种补充方法，特定标记物，如果可用，也可用于识别干细胞。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？Np63亚型，在层状鳞状上皮的干细胞室中高度表达。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。我们创作了一部小说？Np63-GFP报告小鼠模型系统，允许标记表达高水平？Np63，我们假设它可能代表干细胞。Histone2B-GFP和？Np63-GFP动物将使我们能够识别和分类来自小鼠口腔上皮离散解剖部位的干细胞，如口腔黏膜、腭和牙龈。重要的是，我们的策略将允许分离可行的干细胞，这些干细胞可以随后在克隆原性和分子谱研究中分析。最后，我们希望鉴定出口腔干细胞的额外和长期寻找的标记物，这无疑将对口腔上皮培养物在细胞治疗中的临床应用以及上皮癌干细胞和肿瘤发生的研究至关重要。更重要的是，从我们的小鼠研究中获得的数据对于计划和执行使用人类样本的类似干细胞研究将是无价的。

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.