ROLE EPIGENETICS DIFFERENTIAT OF HUMAN EMBRYONIC STEM CELLS INTO VASCUL LINEAGES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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 去乙酰化信号，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促分化基因的抑制 血管平滑肌细胞(VSMC)的表型去分化，以及去甲基化和/或组蛋白乙酰化诱导的这些基因的激活，介导了VSMC和血管内皮细胞(EC)从胚胎干细胞(ESC)来源的血管前体细胞(VPC)分化。为了评估这一假设，我们将： 目的1.明确Dnmt1介导的分化基因调控在肿瘤细胞分化中的作用 VSMC与ESC来源的VPC的分化及其在VSMC表型去分化中的作用 VSMC。我们对我们的初步数据感到兴奋，这些数据证明了生成HES派生的可行性 类胚体(EB)和诱导向心肌细胞系的承诺。这个 目标1和2实验将扩展我们的初步研究，包括对该模型的修改，如下所示 由Sone等人描述。(2003)诱导HES来源的EB向多种血管分化 血统掌握在我们手中。流式细胞仪将用于分离表达VSMC标记的细胞。这个 隔离这些VSMC的能力将有助于关联动态时间 VPC和VSMC之间DNA甲基化/组蛋白乙酰化和基因表达谱变化。预计这些研究将是第一次记录VSMC和ESC特异性的DNA甲基化和基因表达特征。 目标1，将有3个子目标： A.确定Dnmt1介导的DNA甲基化/组蛋白去乙酰化在表型中的作用 VSMC去分化。这一分目标将回答以下问题： -Dnmt1在VSMC中过表达能否诱导VSMC表型去分化过程 没有有丝分裂原？ -RNAi能否通过抑制Dnmt1逆转VSMC的表型去分化？ -VSMC的表型去分化能否被脱甲基剂Aza-CDR或 HDAC抑制剂曲古抑素A(TSA)？这两种药剂有没有协同效应？这些问题的答案 最后一组问题将确定Dnmt1诱导的DNA甲基化和组蛋白的相对作用。 在这一过程中进行脱乙酰化。 B.确定Dnmt1介导的DNA甲基化/组蛋白去乙酰化在 VSMC与ESC来源的VPC的区别。这一分目标将回答以下问题： -能否通过RNAi抑制Dnmt1来诱导VSMC与VPC分化？ -能否通过去甲基化试剂aza抑制Dnmt1诱导VSMC与VPC分化？ CDR或用HDAC抑制剂TSA处理？这两种药剂有没有协同效应？ 对最后一组问题的回答将决定DNMT1诱导的DNA的相对作用 在这个过程中甲基化和组蛋白去乙酰化。 C.确定参与DNMT1介导的VSMC分化调控的基因 来源于ESC来源的VPC和VSMC的表型去分化。为此，我们会： -确定去分化的VSMC与缺失的正常VSMC之间的差异表达基因 阵列分析显示存在RNAi介导的Dnmt1抑制作用； -用MS-RDA检测去分化VSMC和正常VSMC之间差异甲基化的基因； -确定VPC与正常VSMC在缺失和存在的情况下的差异表达基因 阵列分析RNAi介导的Dnmt1抑制； -用MS-RDA检测VPC和正常VSMC之间的差异甲基化基因； 综上所述，这些研究将是第一次证明DNA所起的核心作用 与VSMC分化相关的基因表达重编程中的甲基化/组蛋白乙酰化可能提供一种抑制VSMC分化和表型的新机制 疾病中的去分化。 目的2.明确Dnmt1介导的分化基因调控在ESC来源的VPC向EC分化中的作用。VSMC分化的详细情况如上文目的I所示。

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.