Abstract 14966: Small Molecule Induced Global DNA Hypomethylation Upregulates Nkx2.5 in IPS Derived Cardiac Progenitors by Targeting GPCR Signaling Pathway
Background The use of induced pluripotent stem cells (IPS) is limited due to their ability to form tumors and low efficiency of differentiation into cardiac progenitors. We investigated the use of a small molecule in upregulating cardiac factors in IPS to generate cardiac progenitors cell for repair of the infarcted myocardium
Methods and Results IPs were treated with a small molecule (ZP-G;20uM) for three to five days and analyzed for DNA methyltransferase (Dnmt) activity, cell proliferation, and cardiac genes expression. DNMT activity was completely abolished with 95% reduction in global DNA methylation in small molecule treated IPS. Increased proliferative activity (p<0.01 vs non treated IPS) evaluated by cell proliferation assay and cytochrome c transloaction to cytosol was observed. Amongst cardiac genes determined by RT PCR analysis showed a significant upregulation of Nkx2.5 (p<0.01 vs non treated IPS). Given that posttranscriptional regulation is crucial for gene expression and cell survival, molecular phenotypic analysis was performed. Affymetrix array-based gene expression profiling further confirmed 2-3 folds downregulation of Dnmt1,Dnmt3b and Max gene associated protein which were associated with global DNA hypomethylation and myc dependent cell transformation. In addition, there was 2-3 folds concomitant upregulation of CCL7, CXCR2, CXCR5, integral membrane protein 2A, and EphrinA3. miR Microarray analysis showed upregulation of cardiac specific mir -133, mir-762 and down regulation of pluripotency associated miR-290 cluster, miR-574-5p and let-7 family. Western blot analysis displayed significant upregulation of Gα protein levels as compared to non treated IPS cells. Small molecule mediated effect was abolished by concomitant treatment of IPS cells with GPCR blocker.
Conclusion: This study provides the first evidence that use of small molecule to differentiate IPS to cardiac progenitors by targeting GPCR pathway which would be a useful strategy to achieve patient specific cell types.
- © 2012 by American Heart Association, Inc.