Molecular mechanisms of transdifferentiation

摘要 转分化已经成为躯体干细胞的普遍要求，但这是如何实现的还没有。 被调查得很清楚。尽管有很多证据表明，体细胞可以改变它们的蛋白质 表型，目前还不清楚涉及什么机制，以及干细胞是否真的改变了他们的 功能并维护此更改。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：表皮干细胞和B细胞 淋巴细胞。EpiSCs表达中间细丝角蛋白5和14，并形成连接的细胞片 由黏附和桥粒连接形成。相反，B淋巴细胞不表达角蛋白中间体。 细丝。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。按顺序 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 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)。EpiSCs不显示VDJ重排；Express RAG1、RAG2或Pax5基因；或产生免疫球蛋白。我们的初步数据表明，EpiSCs可以 目的是表达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，即它们可以被指导产生 另一种血统。为了验证这一假设，在目标1中，我们建议将EpiSCs(标记为GAL)定向到 转分化为B淋巴细胞。我们将通过检测单个EpiSCs是否确实发生了转分化来验证 B细胞表面标志物和重组酶基因，并通过聚合酶链式反应分析VDJ重排。 表达细胞。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在从B细胞中移除后是否保持转分化 感应环境。在目标2中，我们将尝试确定单个EpiSCs的机制(S) 转换差异化。我们的初步发现表明，EpiSCs需要与S17基质细胞直接接触 为了实现差异化。我们假设S17细胞通过受体诱导EpiSCs改变其谱系- 配基相互作用(S)。我们将通过检查EpiSCs的膜来检验这一假说 与S17细胞共培养，获得候选受体-配体对的表达。因为皮肤是最大的 可能拥有最多干细胞的器官，了解背后的机制 这些细胞的转分化是它们用于组织再生的一步。

ABSTRACT 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 ¿gal) 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 ¿gal- 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.