UNIVERSITY OF ILLINOIS
Grant: $196,250 - National Institutes of Health - Aug. 14, 2009
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Award Description: The major barrier to use of autologous EPCs in regenerative vascular medicine is the low number and limited trans-differentiation potential. It is necessary to develop an efficient method to increase the number and/or potency of autologous EPCs. As of now there were no efficient methods to increase the autologous cells and test its potential both in vitro and in vivo. Endothelial progenitor cells (EPCs) have been repeatedly shown to enhance neovascularization in ischemic myocardium and are being investigated in a number of ongoing clinical trials. Currently, the benefit of adult stem-cell therapy appears to be limited to vascular repair; myogenesis and/or myocardial regeneration seem to be unaffected. Although the potential for improved vascularization with EPC therapy will likely provide considerable clinical benefit to patients with acute myocardial infarction and chronic heart failure, techniques that can consistently regenerate cardiomyocytes are still of great interest. Thus, the development of autologous stem cells with enhanced lineage plasticity for subsequent testing in myocardial ischemia is an integral part of stem-cell research. We tested the hypothesis that the treatment of EPC with chromatin modifying agents Trichostatin A (TSA- inhibitor for histone deacetylase) and 5Aza-2-deoxycytidine (5Aza- inhibitor for DNA methylation) influence histone acetylation and DNA methylation respectively thereby modify the EPCs into cells with a more multipotent phenotype, and the de-differentiated EPCs can subsequently differentiate into multiple cell lineages, including cardiomyocytes that could be used as potential source of cells for treating degenerative diseases. Preliminary Results: In a series of preliminary experiments performed with EPCs that had been treated with 5Aza and TSA, we made the following observations: 1) 5Aza and TSA treatment induced expression of pluripotent genes (Oct4 and Nanog), and down regulated expression of endothelial-specific genes, 2) treated EPCs differentiated into ectodermal (neuronal) and mesodermal (cardiomyocyte) lineages, 3) aceH3K9 activity was higher and HDAC1 activity was lower in treated EPCs than in control cells, 4) functional and histological evidence from our preliminary studies in mouse AMI model also suggest that intracardiac transplantation of reprogrammed EPC improve left ventricular functions and differentiate into cardiomyocytes and endothelial cell phenotypes. Expected outcomes: We expect to generate the following results after 2 years: 1. Identify a potential chromatin modifying agents that could de-differentiate unipotent EPCs into multipotent stem cells. 2. Is the de-differentiated EPCs show higher degree of stable state over long period of time? 3. Possibility of generating single cell clones. 4. Understand the molecular mechanism of chromatin modifying agents induced de-differentiation of EPCs by evaluating the promoter methylation status of Oct4 and acetylation of histone H3/4 interaction with Oct4 5’ region. 5. Potential of re-differentiation into different cell lineages in vitro. 6. Therapeutic potential of treated EPCs in clinical relevant models. Since the preliminary experiments are encouraging and all the techniques are well established in our research group and we believe the above expected outcomes are deliverable.
Project Description: As of now there were no efficient methods to increase the autologous cells and test its potential both in vitro and in vivo. Preliminary experiments based on the hypothesis that epigenetic modifying agents can de-differentiate EPCs into cells with a more pluripotent phenotype, and the de-differentiated EPCs generate more number of cells and can subsequently differentiate into multiple cell lineages, including cardiomyocytes. While these preliminary experiments in transformed EPCs provide proof of concept and corroborate limited available data in the literature, many questions however remain to be answered to validate and extend these preliminary findings. First, it remains to establish if transformed drug treated EPCs may show higher degree of stable de-differentiated state, over long period of time? Second, is any possibility of single cell clone? Thirdly, is any specific HDAC might be responsible for the reprogramming? For this the cells were treated with various doses of chromatin modifying agents 5Aza 2-deoxycytidine, Trichostatin A (HDAC pan inhibitor) and HDAC specific inhibitors and compare and identify more potent specific inhibitor in reprogramming of EPCs. For this, department of Pharmacy at University of Illinois, we have a good state of art for synthesizing chemical inhibitors specific for HDACs. Dr. Kozikowski has agreed to provide HDACs inhibitors. Currently, I am performing the de-differentiation culture of mouse EPCs with above agents at various time points to address the above questions. We are also on the way of standardizing the Chip assay to see the interaction of various transcription factors at Oct4 and Nanog promoter regions. I received this grant from Sep 1 2009, and ordered antibodies, culture media for cell culture works and Chip assay kit from Millipore.
Jobs Summary: To fulfill the criteria of the recovery Act, this grant will allow us to retain Dr. Rajasingh Johnson to carry out this study. Without this support, we will not be able to retain this talented and dedicated individual and develop his careers. This study outcome will generate strong data for his R01 grant application. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
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