Genetical modification of cardiac Sca-1+ stem cells towards prolonged survival and cardiac differentiation
Final Report Abstract
Initially, the characterization of endogenous cardiac Sca-1+ progenitors was proposed with respect to their cardiomyogenic differentiation potential. However, over the time period the initial proposal was evaluated, ongoing research in the Field lab revealed that adult heart-derived Sca-1+ cells lacked cardiomyogenic activity following engraftment into normal or injured adult hearts. Consequently, the myogenic activity and / or engraftability of the literally more considered population of putative resident cardiac c-kit+ stem cells were evaluated. Magnetic activated cell sorting was utilized to isolate c-kit+ cells from the hearts of neonatal and adult ACT-EGFP/ MHC-nLAC double transgenic mice. These animals exhibit widespread enhanced green fluorescent protein (EGFP) expression and cardiomyocyterestricted nuclear β-galactosidase activity, thus permitting simultaneous tracking of cell survival and differentiation into cardiomyocytes. A subpopulation of the neonatal c-kit+ cells acquired a robust cardiomyogenic phenotype when cocultured with fetal cardiomyocytes (2.4% of all EGFP+ cells screened). The rate of cell fusion was calculated to be 0.05%. In contrast, c-kit+ cells derived from normal adult double transgenic hearts failed to undergo cardiomyogenic differentiation when cocultured with nontransgenic fetal cardiomyocytes (> 18 000 EGFP+ cells screened) or when transplanted into normal or infarcted adult mouse hearts (14 EGFP+ grafts examined). Co-cultures of c-kit+ cells from infarcted adult hearts revealed a very low cardiomyogenic phenotype (< 0.01%). These data suggest that the cardiomyogenic potential of cardiac resident c-kit+ cells is subject to temporal limitations or, alternatively, that the cardiomyogenic population is lost. Elucidation of the underlying molecular basis may permit robust cardiomyogenic induction in adult-derived cardiac c-kit+ cells. Since the cardiomyogenic potential of adult c-kit+ cells was almost absent the proposed strategies to enhance cardioyogenic differentiation and proliferation would have been of limited value. However, own experiments suggest an important role of cardiac SDF-1/CXCR4 interactions in stem cell homing and cardiac repair after ischemia. In particular, I could show that stem cell mobilization with G-CSF and facilitating CXCR4 dependent cell homing by inhibition of the SDF-1 degrading enzyme dipeptidylpeptidase IV (CD26) attenuates ischemic cardiomyopathy mainly by increased angiogenesis and prevention of myocardial remodeling and apoptosis. Accordingly, aim2 (increase cardiomyogenic proliferation by MHC-cyclin D overexpression) and aim3 (increase survival of CMs by MHC-bcl2 overexpression) were modified: An experimental study was conducted to investigate whether DPP-IV inhibition and G-CSF treatment in combination with targeted expression of the cell cycle activator cyclin D2 driven by the α-MHC promoter results in increased cardiomyocyte regeneration following myocardial infarction (MI). MI was induced by ligation of the LAD in wild type (wt) or MHC-cyclin D2 animals, treated with G-CSF (100µg/kg/d) and the DPP-IV inhibitor DiprotinA (70mg/kg/BID) (treatment) or saline for 7 days. 60 days after MI, infarct size was similar in mice with stem cell mobilization and/or cell cycle induction. However, infarct thickness and the total cardiomyocyte content per infarct were significantly improved in MHC-cycD2 mice with G-CSF plus DPP-IV inhibition as compared to the other groups. Quantification of cardiomyocytes within the area of infarction revealed a 10.0 fold increase in MHC-cyclin D2 + G-CSF + DipA, a 5.3 fold increase in MHC-cyclin D2 + saline, and a 3.9 fold increase in wt + G-CSF + DipA animals as compared to and wt + saline (normalized to 1) treated mice. Echocardiography revealed that stem cell mobilization/homing and cardiomyocyte cell cycle activation had an additive effect on functional recovery. In conclusion, my data suggest that stem cell mobilization/homing combined with cell cycle activation in cardiomyocytes additively increase myocardial regeneration. Finally, a recently published study suggests sustainable cardiomyogenic regeneration in human hearts at a low rate of ca. 1% per year. The cellular source for this regeneration remained unclear. Since c-kit+/CXCR4+ cell populations have been linked to cardiovascular regeneration a novel conditional Cre/loxP based CXCR4 transgene for direct lineage tracing experiments was establish exploiting homologous recombination in E. coli (recombineering).
Publications
- ‘‘Remedies for Ischemia’’ (EP2007/003272 and US 60/792,943), Ludwig- Maximilians-University, Munich, Germany
- 64-year old patient with tachycardia and signs of heart failure. Internist (Berl). 2009 Apr;50(4):489-92
Zaruba MM, Waggershauser T, Weckbach S, Gerbes A, Mühling O, Kääb S
- Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4/SDF-1 axis. FASEB J. 2009 Feb;23(2):351-61
Brunner S, Winogradow J, Huber BC, Zaruba MM, Fischer R, David R, Assmann G, Herbach N, Wanke R, Mueller- Hoecker J, Franz WM
- Synergy between CD26/DPP-IV inhibition and G-CSF improves cardiac function after acute myocardial infarction. Cell Stem Cell. 2009 Apr 3;4(4):313-23
Zaruba MM, Theiss HD, Vallaster M, Mehl U, Brunner S, David R, Fischer R, Krieg L, Hirsch E, Huber B, Nathan P, Israel L, Imhof A, Herbach N, Assmann G, Wanke R, Mueller-Hoecker J, Steinbeck G, Franz WM
- The mouse as a model system to study cardiac regeneration. Drug Discovery Today: Disease Models. 2009;5(3):165-171
Zaruba MM, Field LJ
- Assessment of human MAPCs for stem cell transplantation and cardiac regeneration after myocardial infarction in SCID mice. Exp Hematol. 2010 Jul 8. [Epub ahead of print]
Dimomeletis I, Deindl E, Zaruba MM, Groebner M, Zahler S, Laslo SM, David R, Kostin S, Deutsch MA, Assmann G, Mueller-Hoecker J, Feuring-Buske M M, Franz WM
- Cardiomyogenic potential of c-kit+ expressing cells derived from neonatal and adult mouse hearts. Circulation. 2010; May 11;121(18):1992-2000
Zaruba MM, Soonpaa M, Reuter S, Field LJ
- Comparison of Parathyroid hormone and G-CSF treatment after myocardial infarction on perfusion and stem cell homing. Am J Physiol Heart Circ Physiol. 2010; Mar 5 [Epub ahead of print]
Huber BC, Fischer R, Brunner S, Groebner M, Rischpler C, Segeth A, Zaruba MM, Wollenweber T, Hacker M, Franz WM
- Role of the SDF-1-CXCR4 axis in stem cell-based therapies for ischemic cardiomyopathy. Expert Opin Biol Ther. 2010;10(3):321-335
Zaruba MM, Franz WM
- Role of the SDF-1-CXCR4 axis in stem cell-based therapies for ischemic cardiomyopathy. Expert Opin Biol Ther. 2010;10(3):321-335
Zaruba MM, Franz WM
- Safety and efficacy of SITAgliptin plus GRanulocyte-colony-stimulating factor in patients suffering from Acute Myocardial Infarction (SITAGRAMI-Trial) - Rationale, design and first interim analysis. Int J Cardiol. 2010; Jan 3 [Epub ahead of print]
Theiss HD, Brenner C, Engelmann MG, Zaruba MM, Huber B, Henschel V, Mansmann U, Wintersperger B, Reiser M, Steinbeck G, Franz WM
