Molecular mechanisms of transdifferentiation

摘要 转分化已成为体干细胞的一个共同主张，但如何实现这一点还没有 被好好调查了。虽然有很多证据表明，体干细胞可以改变其蛋白质 表型，目前尚不清楚涉及的机制，以及干细胞是否真的改变了它们的表型。 并保持这种变化。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EpiSC表达中间丝角蛋白5和14，并形成连接的细胞片层， 通过粘附和桥粒连接。相反，B淋巴细胞不表达角蛋白中间体 细丝。它们表达一组确定的细胞表面蛋白，具有充分研究的表达动力学。为了 B淋巴细胞要产生特异性免疫球蛋白，就必须发生永久性的遗传变化。这 涉及其免疫球蛋白重链基因座中VDJ区段的缺失和重排。这 重排由RAG 1和RAG 2重组酶介导，并由Pax 5编码的 转录因子BSAP（B细胞特异性激活蛋白）。EpiSC不表现VDJ重排;表达 RAG 1、RAG 2或Pax 5基因;或产生免疫球蛋白。我们的初步数据表明，EpiSCs可以被 定向表达B淋巴细胞标志物和基因，并显示VDJ片段的重排。我们 假设EpiSC在发育上是灵活的，即它们可以被引导以产生以下细胞： 交替的血统为了验证这一假设，在目标1中，我们建议将EpiSC（标记为<$gal）定向至 转分化为B淋巴细胞。我们将通过检测表皮干细胞中的 B细胞表面标记和重组酶基因，并通过PCR分析VDJ重排在半乳糖苷酶基因， 表达细胞。由于转分化事件的持久性对于未来的治疗是重要的，我们将测试 当从B细胞中取出时，改变其细胞谱系的EpiSC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EpiSC 转分化我们的初步研究结果表明，EpiSCs需要与S17基质细胞直接接触， 才能转分化。我们假设S17细胞通过受体介导诱导EpiSC改变其谱系。 配体相互作用。我们将通过检查EpiSCs的膜来验证这一假设，在细胞增殖之前和期间， 与S17细胞共培养，用于候选受体-配体对的表达。因为皮肤是最大的 干细胞数量最多的器官，了解其背后的机制 这些细胞的转分化是它们用于组织再生的一个步骤。

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.