LONG TERM GENE EXPRESSION BY EPIDERMAL STEM CELLS

所有更新的上皮组织都是通过细胞的连续分裂来维持的。 增殖群体中的细胞，以取代增殖群体中的细胞。 分化区室脱落到环境中。这 平衡细胞分裂速度和细胞生长速度的机制 损失对于上皮稳态是必不可少的， 生活在这样的组织中，增殖群体由以下组成： 为生命维持的分裂细胞的等级。在这样的组织中， 增殖细胞群由一系列分裂细胞组成 由一小群干细胞维持。述干细胞是 通过细胞分裂产生干细胞和 增殖的子细胞，即转运放大细胞。过境 放大细胞分裂以维持组织，但它们仅限于一个细胞。 在它们分化之前有限数量的细胞分裂， 最终失去了。只有干细胞才能在组织的生命中保留下来。 因此，在设计基因治疗方案时， 遗传性疾病，我们针对干细胞群体。不幸的是， 根据数据，分离的上皮干细胞一直存在问题。先前 使用氚标记的胸苷，我们能够标记一个亚群， 在小鼠皮肤和口腔粘膜中的循环基底细胞，我们称之为标签， 再训练细胞（LRCs），表现出干细胞的特征。 这种标记方法在过去十年中一直用于识别茎 其他几种上皮细胞。最近，我们发明了一种方法， 将LRC群体从小鼠耳组织中的2%富集到小鼠耳组织中的90%以上。 培养皿，并且进一步我们已经表明，所选择的细胞显示出 预期表皮干细胞的生长和再增殖潜力。在 在这个可行性项目中，我们将测试使用这些 选择用于长期重组基因表达的干细胞。虽然 在表皮组织中表达重组基因的几种技术 直接或在培养的表皮细胞中进行了尝试， 两项研究报道了长期DNA表达。他们用了一种体外 他们将培养的角质形成细胞用一种 逆转录病毒构建体，然后将角质形成细胞移植回 主持人两项研究都报告了DNA表达的快速下降， 1%的初始转导细胞仍表达重组DNA 两个月后。这表明很少有干细胞被转导。 在这两项研究中，研究人员之前都没有尝试过富集干细胞 转染在这里，我们建议使用我们的新的富集方法， 首先选择表皮干细胞，然后测试假设， 干细胞将通过转染给予长期的重组基因表达 这些预先选定的细胞带有携带LacZ的逆转录病毒载体 报告基因或腺相关病毒带绿色荧光 蛋白质，然后使用转染的细胞来生物工程表皮， 测试在器官型培养物中和 移植的皮肤

All renewing epithelial tissues are maintained by continuous division of cells in the proliferative population to replace cells in the differentiation compartment that are sloughed into the environment. This mechanism of balancing the rate of cell division with the rate of cell loss is essential for epithelial homeostasis and must be maintained for life. In such tissues, the proliferative population consists of a hierarchy of dividing cells maintained for life. In such tissues, the proliferative population consists of a hierarchy of dividing cells maintained by a small subpopulation of stem cells. The stem cells are self-renewing with cell division producing both stem cells and proliferating daughters, the transit amplifying cells. The transit amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are ultimately lost. Only the stem cells remain for the life of the tissue. Thus, it is critical when designing gene therapy regimes for genetically inherited diseases that we target the stem cell population. Unfortunately, to data, isolated epithelial stem cells has been problematic. Previously using tritiated thymidine, we were able to label a subpopulation of slowly cycling basal cells in mouse skin and oral mucosa that we called label- retraining cells (LRCs), which showed the characteristics of stem cells. This tagging method has been used for the last decade to identify stem cells in several other epithelia. Recently, we devised a method that enriches the LRC population from 2% in mouse ear tissue to over 90% in the culture dish, and further we have shown that the selected cells show the growth and repopulation potential expected for epidermal stem cells. In this feasibility project, we will test the potential of using these selected stem cells for long term recombinant gene expression. Although several techniques for expressing recombinant genes in epidermal tissues directly or in epidermal cells in culture have been tried, to date only two studies have reported long term DNA expression. They used an ex vivo approach in which they transduced keratinocytes in culture with a retroviral construct, then transplanted the keratinocytes back to the host. Both studies reported a rapid drop in DNA expression, with less than 1% of the initially transduced cells still expressing the recombinant DNA after two months. This suggests that very few stem cells were transduced. In neither study did the researchers try to enrich for stem cells before transfection. Here, we propose to use our novel enrichment method to first select epidermal stem cells, then to test the hypothesis that only stem cells will give long term recombinant gene expression by transfected these preselected cells with a retroviral vector carrying the LacZ reporter gene or with adeno-associated virus with green fluorescent protein, then use the transfected cells to bioengineer an epidermis and test for long term expression both in organotypic culture and in transplanted skin grafts.