LONG TERM GENE EXPRESSION BY EPIDERMAL STEM CELLS

所有更新的上皮组织都通过持续的分裂来维持 增殖群体中的细胞来取代 进入环境中的分化隔间。这 细胞分裂率与细胞分裂率的平衡机制 丢失是上皮细胞动态平衡的关键，必须维持 生活。在这样的组织中，增殖的群体由一个 终生保持细胞分裂的层次结构。在这样的组织中， 增殖群体由分裂细胞的层次结构组成 由一小部分干细胞维持。干细胞是 通过细胞分裂进行自我更新，产生干细胞和 增殖的子代，运输放大细胞。凌日 放大细胞分裂以维持组织，但它们仅限于 在分化之前有限数量的细胞分裂，并且 最终还是输了。只有干细胞在组织的生命周期内存活。 因此，在设计针对基因的基因治疗方案时，这是至关重要的。 我们针对干细胞群体的遗传性疾病。不幸的是， 从数据来看，分离的上皮干细胞一直存在问题。先前 使用氚胸腺嘧啶核苷，我们能够标记一个慢速亚群 循环小鼠皮肤和口腔粘膜中的基底细胞，我们称之为标记- 再培养细胞(LRC)，表现出干细胞的特性。 这种标记方法在过去的十年里一直被用来识别茎 其他几个上皮细胞中的细胞。最近，我们设计了一种方法， 使小鼠耳组织中的LRC数量从2%增加到90%以上 培养皿，进一步我们已经显示了选定的细胞显示 预计表皮干细胞的生长和再繁殖潜力。在……里面 这个可行性项目，我们将测试使用这些技术的潜力 选择干细胞进行长期重组基因表达。虽然 在表皮组织中表达重组基因的几种技术 到目前为止，只尝试过直接或在培养的表皮细胞中进行 有两项研究报道了DNA的长期表达。他们用的是体外培养的 他们在培养中转导角质形成细胞的方法 逆转录病毒构建，然后将角质形成细胞移植回 主持人。这两项研究都报告了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。这表明被转导的干细胞非常少。 在这两项研究中，研究人员之前都没有尝试浓缩干细胞 转染法。在这里，我们建议使用我们的新富集法来 首先选择表皮干细胞，然后测试只有 干细胞将通过转基因获得长期的重组基因表达 这些预先选择的细胞带有携带LacZ的逆转录病毒载体 报告基因或带有绿色荧光的腺相关病毒 蛋白质，然后使用转基因细胞来生物工程表皮和 器官培养中长期表达的测试 移植的皮肤移植。

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.