ROLE EPIGENETICS DIFFERENTIAT OF HUMAN EMBRYONIC STEM CELLS INTO VASCUL LINEAGES

人胚胎干细胞分化为血管谱系的表观遗传学作用

• 批准号: 7561420
• 负责人: Methode Bacanamwo
• 金额: $ 17.1万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7561420
• 关键词: Blood Vessels; Cardiac Myocytes; Cell Differentiation process; Cells; Computer Retrieval of Information on Scientific Projects Database; DNA Methylation; Data; Disease; Endothelial Cells; Epigenetic Process; Funding; Gene Activation; Gene Expression; Gene Expression Profile; Genes; Grant; Hand; Histone Acetylation; Histone Deacetylase; Histone Deacetylation; Institution; Mediating; Mitogens; Modeling; Modification; Molecular Profiling; Myoblasts; Process; RNA Interference; Regulation; Relative (related person); Repression; Research; Research Personnel; Resources; Role; Role playing therapy; Smooth Muscle Myocytes; Source; Stem cells; Trichostatin A; United States National Institutes of Health; demethylation; embryonic stem cell; human embryonic stem cell; inhibitor/antagonist; novel; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SPECIFIC AIMS Our central hypothesis postulates the existence of lineage specific DNA methylation/histone deacetylation signatures that regulate the expression of lineage-specific genes and mediate the differentiation into various lineages. Specifically, we hypothesize that epigenetic mechanisms involving DNA methylation and/or histone deacetylation mediate the repression of pro-differentiation genes in phenotypic dedifferentiation of vascular smooth muscle cells (VSMC), while demethylation and/or histone acetylation-induced activation of these genes mediate the differentiation of VSMC and endothelial cells (EC) from embryonic stem cell (ESC)-derived vascular progenitor cells (VPC). To assess this hypothesis we will: Aim 1. Define the role of Dnmt1-mediated regulation of differentiation genes in the differentiation of VSMC from ESC-derived VPC and in the phenotypic dedifferentiation of VSMC. We are excited by our preliminary data that documents the feasibility of generating hES-derived embryroid bodies (EB) and inducing commitment to a cardiomyocyte lineage. The Aim 1 and 2 experiments will extend our preliminary studies by including modifications to this model as described by Sone et al. (2003) to induce the differentiation of hES-derived EB into various vascular lineages in our hands. FACS will be used to isolate cells expressing VSMC markers. The capacity to isolate these VSMC will facilitate the process of correlating dynamic temporal changes in the DNA methylation/histone acetylation and gene expression profiles between VPC and VSMC. It is anticipated that these studies will be the first to document DNA methylation and gene expression signatures specific to differentiation of VSMC from ESC. Aim 1, will have 3 sub-aims: A. Define the role of Dnmt1-mediated DNA methylation/histone deacetylation in the phenotypic dedifferentiation of VSMC. This sub-aim will answer these questions: -Can the process of phenotypic dedifferentiation of VSMC be induced by over-expressing Dnmt1 in the absence of a mitogen? -Can the phenotypic dedifferentiation of VSMC be reversed by inhibiting Dnmt1 by RNAi? -Can the phenotypic dedifferentiation of VSMC be reversed by the demethylating agent aza-CdR or the HDAC inhibitor trichostatin A (TSA)? Do the two agents have a synergistic effect? Answers to these last sets of questions will determine the relative roles of Dnmt1-induced DNA methylation and histone deacetylation in this process. B. Define the role of Dnmt1-mediated DNA methylation/histone deacetylation in the differentiation of VSMC from ESC-derived VPC. This sub-aim will answer these questions: -Can VSMC be induced to differentiate from VPC by inhibiting Dnmt1 by RNAi? - Can VSMC be induced to differentiate from VPC by inhibiting Dnmt1 by the demethylating agent aza- CdR or by treatment with the HDAC inhibitor TSA? Do the two agents have a synergistic effect? Answers to these last sets of questions will determine the relative roles of Dnmt1-induced DNA methylation and histone deacetylation in this process. C. Determine the genes involved in the Dnmt1-mediated regulation of differentiation of VSMC from ESC-derived VPC and in the phenotypic dedifferentiation of VSMC. To this end, we will: -Determine genes differentially expressed between dedifferentiated VSMC vs. normal VSMC in absence and presence of RNAi-mediated Dnmt1 inhibition by array analysis; -Determine genes differentially methylated between dedifferentiated and normal VSMC by MS-RDA; - Determine genes differentially expressed between VPC vs. normal VSMC in absence and presence of RNAi-mediated Dnmt1 inhibition by array analysis; -Determine genes differentially methylated between VPC and normal VSMC by MS-RDA; Taken together, these studies will be the first to document the central role played by DNA Methylation/histone acetylation in gene expression reprogramming associated with differentiation of VSMC and will potentially provide a novel mechanism to inhibit VSMC differentiation and phenotypic dedifferentiation in disease. Aim 2. Define the role of Dnmt1-mediated regulation of differentiation genes in the differentiation of EC from ESC-derived VPC. Details as in Aim I above for the differentiation of VSMC.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 具体目标 我们的中心假设假设存在谱系特异性DNA甲基化/组蛋白 去乙酰化标签，调节谱系特异性基因的表达，并介导 分化成不同的谱系。具体地说，我们假设，表观遗传机制涉及 DNA甲基化和/或组蛋白去乙酰化介导了促分化基因的抑制， 表型去分化的血管平滑肌细胞（VSMC），而去甲基化和/或组蛋白乙酰化诱导的激活这些基因介导的分化VSMC和内皮细胞（EC）从胚胎干细胞（ESC）衍生的血管祖细胞（VPC）。为了评估这一假设，我们将： 目标1。确定Dnmt 1介导的分化基因调控在肿瘤细胞中的作用。 VSMC从ESC衍生的VPC的分化和VSMC的表型去分化 VSMC。我们对我们的初步数据感到兴奋，这些数据记录了产生hES衍生的 胚状体（EB）和诱导定型为心肌细胞谱系。的 目标1和2实验将扩展我们的初步研究，包括修改这个模型， Sone等（2003）描述的方法诱导hES衍生的EB分化为各种血管内皮细胞， 血统在我们手中。将使用FACS分离表达VSMC标志物的细胞。的 隔离这些VSMC的能力将有助于将动态时间相关性 VPC和VSMC之间DNA甲基化/组蛋白乙酰化和基因表达谱的变化。预计这些研究将是第一个记录DNA甲基化和基因表达特征特异性VSMC从ESC分化。 目标1将有3个次级目标： A.确定Dnmt 1介导的DNA甲基化/组蛋白去乙酰化在表型中的作用 VSMC脱分化。这一次级目标将回答这些问题： - VSMC的表型去分化过程是否可以通过在VSMC中过表达Dnmt 1来诱导？ 没有丝裂原 - 通过RNAi抑制Dnmt 1可以逆转VSMC的表型去分化吗？ - VSMC的表型去分化是否可以通过去甲基化剂aza-CdR或 HDAC抑制剂阿司他丁A（TSA）？这两种药物是否有协同作用？回答这些 最后几组问题将确定Dnmt 1诱导的DNA甲基化和组蛋白 在这个过程中脱乙酰基。 B。定义Dnmt 1介导的DNA甲基化/组蛋白去乙酰化在 VSMC与ESC衍生的VPC的分化。这一次级目标将回答这些问题： - 通过RNAi抑制Dnmt 1可以诱导VSMC从VPC分化吗？ - 去甲基化剂aza-1抑制Dnmt 1能诱导VSMC向VPC分化吗？ CdR或HDAC抑制剂TSA治疗？这两种药物是否有协同作用？ 最后几组问题的答案将决定Dnmt 1诱导的DNA的相对作用。 甲基化和组蛋白去乙酰化。 C.确定参与Dnmt 1介导的VSMC分化调控的基因 从ESC衍生的VPC和VSMC的表型去分化。为此，我们将： - 确定去分化VSMC与不存在的正常VSMC之间差异表达的基因 和通过阵列分析的RNAi介导的Dnmt 1抑制的存在; - 通过MS-RDA确定去分化和正常VSMC之间差异甲基化的基因; - 确定VPC与正常VSMC之间在不存在和存在以下情况下差异表达的基因： 基因芯片分析RNAi介导的Dnmt 1抑制作用 - 通过MS-RDA确定VPC和正常VSMC之间差异甲基化的基因; 综合起来，这些研究将是第一个记录DNA所扮演的核心角色的研究。 甲基化/组蛋白乙酰化在与VSMC分化相关的基因表达重编程中的作用，将潜在地提供抑制VSMC分化和表型改变的新机制。 疾病中的去分化 目标二。确定Dnmt 1介导的分化基因调控在EC从ESC衍生的VPC分化中的作用。关于VSMC的分化，详见上述目标I。