Molecular mechanisms of transdifferentiation

描述(由申请人提供)：转分化已成为躯体干细胞的一种常见说法，但这是如何实现的还没有得到很好的研究。虽然已有大量证据表明，体细胞可以改变其蛋白质表型，但目前尚不清楚其中涉及的机制，以及干细胞是否真的改变了其功能并保持了这种改变。为了研究转分化的机制，我们建议使用两种具有不同特征和功能的细胞类型：表皮干细胞(EpiSC)和B淋巴细胞。EpiSCs表达中间细丝角蛋白5和14，并形成由黏附连接和桥粒连接连接的细胞片。相比之下，B淋巴细胞不表达角蛋白中间丝。它们表达一组明确的细胞表面蛋白，其表达动力学已得到充分研究。为了让B淋巴细胞产生一种特定的免疫球蛋白，它必须引起永久性的遗传变化。这涉及免疫球蛋白重链基因中VDJ片段的缺失和重排。这种重排是由RAG1和RAG2重组酶以及由Pax5编码的转录因子BSAP(B细胞特异性激活蛋白)介导的。EpiSCs不表现VDJ重排，不表达RAG1、RAG2或Pax5基因，也不产生免疫球蛋白。我们的初步数据表明，EpiSCs可以被定向表达B淋巴细胞标志和基因，并显示VDJ片段的重排。我们假设EpiSCs在发育上是灵活的，也就是说，它们可以被引导产生不同血统的细胞。为了验证这一假设，在目标1中，我们建议引导EpiSCs(标记为2Gals)转分化为B淋巴细胞。我们将通过检测B细胞表面标志物和重组酶基因，以及在表达VDJ的细胞中进行VDJ重排的PCR分析，来验证单个EpiSCs确实发生了转分化。由于转分化事件的持久性对于未来的治疗很重要，我们将测试改变其细胞谱系的EpiSCs在被从B细胞诱导环境中移除时是否保持转分化。在目标2中，我们将尝试确定单个EpiSCs转分化的机制(S)。我们的初步发现表明，EpiSCs需要与S17基质细胞直接接触才能转分化。我们假设S17细胞通过受体-配体相互作用诱导EpiSCs改变其谱系(S)。我们将通过检测EpiSCs在与S17细胞共培养之前和过程中的膜上候选受体-配体对的表达来检验这一假说。由于皮肤是最大的器官，可能拥有最多的干细胞，了解这些细胞转分化背后的机制是将它们用于组织再生的一步。许多疾病，包括糖尿病、阿尔茨海默氏症和心脏病，都是由主要器官中的细胞死亡引起的。为了治疗这些疾病，丢失的细胞必须被替换，理想情况下是用自体细胞取代。这样的细胞将不得不被诱导改变它们的谱系并移植到一个新的环境中，这个过程统称为转分化。与其他类型的成人干细胞相比，来自皮肤外层的表皮干细胞具有优势，因为它们可以在对个体几乎没有伤害的情况下被分离出来，而且由于皮肤是人体最大的器官，它们大量存在。因此，确定表皮干细胞转分化的机制及其组织再生的潜能具有临床意义。

DESCRIPTION (provided by applicant): Transdifferentiation has become a common claim for somatic stem cells, yet how this is accomplished has not been well investigated. Although much evidence exists that somatic stem cells can change their protein phenotype, it is not clear what mechanisms are involved, and whether the stem cells actually change their function and maintain this change. To investigate the mechanisms of transdifferentiation, we propose to use two cell types with distinct characteristics and functions: the epidermal stem cell (EpiSC) and the B lymphocyte. EpiSCs express the intermediate filaments keratins 5 and 14 and form sheets of cells connected by adherens and desmosomal junctions. B lymphocytes, in contrast, do not express keratin intermediate filaments. They express a defined set of cell surface proteins with well-studied kinetics of expression. In order for a B lymphocyte to produce a specific immunoglobulin, it must incur a permanent genetic change. This involves deletion and rearrangement of VDJ segments in their immunoglobulin heavy chain locus. This rearrangement is mediated by the RAG1 and RAG2 recombinase enzymes, and by the Pax5-encoded transcription factor BSAP (B cell specific activator protein). EpiSCs do not exhibit VDJ rearrangement; express the RAG1, RAG2, or Pax5 gene; or produce immunoglobulin. Our preliminary data suggest that EpiSCs can be directed to express the B lymphocyte cell markers and genes, and show rearrangement of VDJ segments. We hypothesize that EpiSCs are developmentally flexible, i.e. that they can be directed to produce cells of alternate lineages. To test this hypothesis, in Aim 1, we propose to direct EpiSCs (marked with 2gal) to transdifferentiate into B lymphocytes. We will verify that individual EpiSCs did transdifferentiate by detection of B cell surface markers and recombinase enzyme genes, and by PCR analysis for VDJ rearrangement in 2gal-expressing cells. Since permanency of the transdifferentiation event is important for future therapy, we will test whether or not EpiSCs that alter their cell lineage remain transdifferentiated when removed from the B cell inductive environment. In Aim 2, we will attempt to determine the mechanism(s) by which individual EpiSCs transdifferentiate. Our preliminary findings indicate that EpiSCs require direct contact with the S17 stromal cells in order to transdifferentiate. We hypothesize that S17 cells induce EpiSCs to alter their lineage via receptor- ligand interaction(s). We will test this hypothesis by examining the membranes of EpiSCs, before and during co-culture with S17 cells, for the expression of candidate receptor-ligand pairs. Since the skin is the largest organ with potentially the greatest number of stem cells, understanding the mechanism behind transdifferentiation of these cells is a step toward their use in tissue regeneration. Many diseases, including diabetes, Alzheimer's, and heart disease, are caused by the death of cells in major organs. To treat these diseases, the lost cells must be replaced, ideally with autologous cells. Such cells will have to be induced to change their lineage and engraft into a new environment, a process collectively called transdifferentiation. Stem cells from the epidermis, the outer layer of the skin, offer advantages over other types of adult stem cells in that they can be isolated with little harm to the individual and, since the skin is the largest organ in the body, they are present in large quantities. Therefore, determining the mechanism behind transdifferentiating epidermal stem cells and their potential for tissue regeneration has clinical significance.