Molecular mechanisms of transdifferentiation

转分化的分子机制

• 批准号: 7496173
• 负责人: JACKIE R BICKENBACH
• 金额: $ 15.8万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-10 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7496173
• 关键词: Alzheimer' s Disease; Antibodies; Antigens; Autologous; B-Lymphocytes; Bone Marrow; Cause of Death; Cell Aging; Cell Line; Cell Lineage; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Characteristics; Coculture Techniques; Complement; Complement Factor B; DNA; DNA Sequence Rearrangement; Data; Detection; Diabetes Mellitus; Disease; Ensure; Environment; Enzyme Gene; Enzymes; Erinaceidae; Event; Exhibits; Exposure to; Future; Genes; Green Fluorescent Proteins; Hand; Heart Diseases; IGH@ gene cluster; Immunoglobulins; Individual; Intermediate Filaments; Keratin; Kinetics; Label; Ligands; Lymphocyte; Mediating; Membrane; Mesenchymal; Molecular; Monitor; Mus; Mutation; Numbers; Organ; Personal Satisfaction; Phenotype; Polymerase Chain Reaction; Population; Process; Proteins; Proto-Oncogene Protein c-kit; Skin; Staging; Stem cells; Stromal Cells; Testing; Time; Tissues; V(D)J Recombination; adult stem cell; cell age; cell type; clinically significant; cytokine; embryonic stem cell; epidermis cell; keratin 14, K14; keratin 5; keratinocyte; notch protein; paired box 5 protein (B-cell lineage specific activator); panacea; protein B; receptor; recombinase; response; tissue regeneration; transcription factor; transdifferentiation

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.

描述（由申请人提供）：转分化已成为体细胞干细胞的一种常见说法，但如何实现这一目标尚未得到很好的研究。尽管有很多证据表明体细胞干细胞可以改变其蛋白质表型，但尚不清楚涉及的机制，以及干细胞是否实际上改变了它们的功能并维持这种变化。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。EpiSCs表达中间丝角蛋白5和14，并形成由粘附体和桥粒连接连接的细胞片。相反，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，表达动力学研究得很好。为了使B淋巴细胞产生特定的免疫球蛋白，它必须引起永久性的遗传变化。这涉及免疫球蛋白重链位点的VDJ片段的删除和重排。这种重排是由RAG1和RAG2重组酶以及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；表达RAG1、RAG2或Pax5基因；或者产生免疫球蛋白。我们的初步数据表明EpiSCs可以定向表达B淋巴细胞标记物和基因，并显示VDJ片段的重排。我们假设EpiSCs在发育上是灵活的，即它们可以被引导产生不同谱系的细胞。为了验证这一假设，在Aim 1中，我们建议引导EpiSCs（标记为2gal）转分化为B淋巴细胞。我们将通过检测B细胞表面标记物和重组酶基因，以及在表达2gal的细胞中进行VDJ重排的PCR分析来验证单个EpiSCs是否存在转分化。由于转分化事件的持久性对未来的治疗非常重要，我们将测试当从B细胞诱导环境中移除时，改变其细胞谱系的EpiSCs是否仍保持转分化。在目标2中，我们将尝试确定单个EpiSCs转分化的机制。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-配体相互作用诱导EpiSCs改变其谱系。我们将通过检测EpiSCs与S17细胞共培养前和共培养期间的膜来检验这一假设，以检测候选受体-配体对的表达。由于皮肤是可能拥有最多干细胞的最大器官，了解这些细胞转分化背后的机制是将其用于组织再生的一步。许多疾病，包括糖尿病、老年痴呆症和心脏病，都是由主要器官细胞死亡引起的。为了治疗这些疾病，丢失的细胞必须被替换，最好是自体细胞。这些细胞必须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。来自皮肤外层表皮的干细胞比其他类型的成体干细胞有优势，因为它们可以被分离出来，对个体几乎没有伤害，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背后的机制及其组织再生潜力具有临床意义。