ROLE EPIGENETICS DIFFERENTIAT OF HUMAN EMBRYONIC STEM CELLS INTO VASCUL LINEAGES

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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 edifferentiated 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.