There is great interest in therapies for heart disease employing adult and embryonic stem (ES) cells. However, their clinical application requires that a number of fundamental issues be resolved in order to reach the goal of regeneration of physiologically significant amounts of new myocardium following cell transplantation. These issues include understanding the molecular mechanisms of cardiac regeneration, identifying appropriate growth factor(s) to enhance regeneration and devising ways to modulate and quantitatively analyze cell survival, apoptosis, and engraftment and differentiation. Accordingly, this proposal is designed to understand molecular mechanisms of pro/anti-apoptotic and cell signaling genes in undifferentiated or differentiated ES cells. We also propose to develop strategies to enhance cell survival, engraftment and differentiation of transplanted ES cells following myocardial infarction (MI). Our preliminary data suggest that TGF?2, an essential growth factor for cardiomyogenesis, enhances ES cell-derived embryoid body proliferation and cardiac myocyte differentiation and that TGF?2 primed or unprimed ES cell conditioned medium inhibits H9C2 cell apoptosis in vitro. To build on these findings, we propose the following Specific Aims: 1) Determine if transplantation of TGF?2 primed ES cells attenuates apoptosis and enhances engraftment and cell proliferation of donor cells following MI, 2) Determine the long-term fate of TGF?2 primed ES cells after transplantation and 3) Determine the effects of TGF?2 on transcriptional profiles and post-translational changes occurring in undifferentiated and differentiated ES cells. Our general approach is to transplant TGF?2 primed or unprimed ES cells expressing yellow or green fluorescence protein at 3-5 hrs post-MI in mice. Histology and a biochemical assay will be employed to determine extent of fibrosis and MI size. Ex-vivo immunohistochemistry will be performed to detect apoptosis, engraftment, cell proliferation and differentiation at short (0, 12, 24, 48 and 72hrs) and long (2, 4, 6 and 8 weeks) time points post-MI. Left ventricular function will be assessed using high-resolution echocardiography. For transcriptional profiles and associated translational changes, undifferentiated or cardiomyocyte committed differentiated ES cells (i.e., embryoid body cells) primed with or without TGF?2 will be studied using the mouse genome, DNA microarray, real time-PCR and Western blotting. These studies will help elucidate how ES cells may be utilized to repair injured myocardium. If successful, they should constitute a significant step toward to clinically practical cell therapy. Project Narrative The most common cause of heart disease in the U.S. is coronary artery disease and associated myocardial infarction, which results in the irreversible loss of functional heart muscle. The pharmacological agents (drugs) are unable to completely cure the disease. The results from the proposed projects will have long-term implications for future cell-based therapy to treat heart disease (s) using embryonic stem cells or potentially other donor cells. ? ? ?
|Singla, Dinender K; Singla, Reetu D; Lamm, Stephanie et al. (2011) TGF-?2 treatment enhances cytoprotective factors released from embryonic stem cells and inhibits apoptosis in infarcted myocardium. Am J Physiol Heart Circ Physiol 300:H1442-50|
|Singla, Dinender K (2010) Stem cells in the infarcted heart. J Cardiovasc Transl Res 3:73-8|
|Fatma, Sumbul; Selby, Donald E; Singla, Reetu D et al. (2010) Factors Released from Embryonic Stem Cells Stimulate c-kit-FLK-1(+ve) Progenitor Cells and Enhance Neovascularization. Antioxid Redox Signal 13:1857-65|
|Glass, Carley E; Singal, Pawan K; Singla, Dinender K (2010) Stem cells in the diabetic infarcted heart. Heart Fail Rev 15:581-8|
|Singla, Dinender K (2009) Embryonic stem cells in cardiac repair and regeneration. Antioxid Redox Signal 11:1857-63|
|Singla, Dinender K; Singla, Reetu D; McDonald, Debbie E (2008) Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway. Am J Physiol Heart Circ Physiol 295:H907-13|
|Singla, Dinender K; McDonald, Debbie E (2007) Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells. Am J Physiol Heart Circ Physiol 293:H1590-5|