Late-Breaking Basic Science: Stem Cells and Cardiac Cell Replacement 5000 Adult Mouse Spermatogonial Stem Cells Differentiate into Cardiovascular Lineages and Generate Functional Cardiomyocytes

Late-Breaking Basic Science Abstracts From the American Heart Association Scientific Sessions 2005. November 13–16, 2005 Dallas Convention Center Dallas, TX Late-Breaking Basic Science: Stem Cells and Cardiac Cell Replacement 5000 Adult Mouse Spermatogonial Stem Cells Differentiate into Cardiovascular Lineages and Generate Functional Cardiomyocytes Kaomei Guan, Department of Cardiology and Pneumology, Heart Center, Georg-August University of Goettingen, Goettingen, Germany; Karim Nayernia, Institute of Human Genetics, Georg-August University of Goettingen, Goettingen, Germany; Lars S Maier, Stefan Wagner, Frieder Wolf, Department of Cardiology and Pneumology, Heart Center, Georg-August University of Goettingen, Goettingen, Germany; Manyu Li, Wolfgang Engel, Institute of Human Genetics, Georg-August University of Goettingen, Goettingen, Germany; Gerd P Hasenfuss, Department of Cardiology and Pneumology, Heart Center, Georg-August University of Goettingen, Goettingen, Germany Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be ideal for organ regeneration strategies. Previous studies in neonatal mice suggest that the germline lineage may retain the ability to generate pluripotent cells. We aimed to evaluate the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Using a genetic approach we established the culture condition for isolation of SSCs from adult mouse testis (success rate 27% of mice). These isolated SSCs respond to the culture conditions and acquire ESC properties. They are able to spontaneously (without co-culture) differentiate into functional cardiomyocytes expressing cardiac-specific genes (isl1, GATA4, Nkx2.5, MEF2C, -MHC, MLC2v, ANF, SERCA2a and NCX) and proteins (sarcomeric MHC, cardiac troponin T and connexin 43). Four major types of action potentials (APs) characteristic for specialized cells of the ventricle, sinus node, Purkinje fibers and atrium are found in these cardiomyocytes. Functional maturity of ventricle-like cells (n 11 from 3 independent experiments) is shown by AP characteristics: upstroke velocity (dV/dtmax) being 55.8 4.8 V/s, amplitude (APA) being 115.8 3.3 mV, duration at 90% of repolarisation (APD90) being 288.9 55.5 ms and maximum diastolic potential (MDP) being -48.3 3.0 mV. The amplitudes of calcium transients (fluo4) measured in SSC-derived ventricle-like cardiomyocytes (n 10) are similar to those measured in ESC-derived cardiomyocytes (n 6; 464 77 nM vs. 287 69 nM , respectively). This is also comparable to adult cardiac myocytes. Besides cardiomyocytes, SSCs give rise to vascular smooth muscle (expressing smooth muscle-actin, vascular smooth muscle MHC) and endothelial cells (expressing PECAM-1, VEGFR2, and vWF). SSCs also differentiate into other somatic cell types in vitro. Furthermore, we show that SSCs are able to differentiate into cardiac cells and most other somatic cells in vivo after blastocyst microinjection. Thus, adult spermatogonal stem cells exhibit pluripotency like embryonic stem cells and may offer new possibilities in cell based cardiac regeneration strategies. 5001 Local Activation or Implantation of Cardiac Progenitor Cells Rescues Scarred Infarcted Myocardium Improving Cardiac Function Jan Kajstura, Marcello Rota, Stefano Chimenti, Federica Limana, Angelo Nascimbene, Claudia Bearzi, Toru Hosoda, Katsuya Amano, Thomas H Mitchell, Giuseppe Fiore, Francesco Rotatori, Stefano Cascapera, Antonella De Angelis, Edmund H Sonnenblick, Konrad Urbanek, New York Medical College, Valhalla, NY; Roberto Bolli, University of Louisville, Louisville, KY; Annarosa Leri, Piero Anversa, New York Medical College, Valhalla, NY Ischemic heart disease is characterized chronically by healed infarct and multiple foci of scarring that together contribute to the impairment of ventricular function. Although cardiac progenitor cells (CPCs) regenerate acutely infarcted myocardium, their effect on chronic infarcts is unknown. Thus, we tested whether CPCs implanted in proximity of healed infarcts one month after coronary occlusion in rats, invade the scar and generate myocytes and coronary vessels improving cardiac performance. As an alternative strategy, resident CPCs were stimulated locally with HGF and IGF-1 (GFs). HGF is a chemoattractant of CPCs and IGF-1 promotes their proliferation and survival. The effects of these interventions were evaluated echocardiographically, hemodynamically and structurally one month later, and the extent of repair of the infarcted area and the properties of the regenerated myocardium were determined. Injection of CPCs or activation of resident CPCs by GFs resulted in the replacement of 40–44% of the scarred infarct with new myocardium, prevented the chronic decline in function of the infarcted heart, and attenuated cavitary dilation and wall thinning. Myocardial repair was positively correlated with infarct size; that is, it was more pronounced with larger infarcts. Cardiac repair was mediated by the ability of CPCs to synthesize MMPs that degraded collagen, forming tunnels within the fibrotic tissue during their migration across the scarred myocardium. Injection of clonogenic c-kit-positive CPCs led to a more prominent generation of coronary arterioles and capillaries, while administration of GFs resulted in a greater number of myocytes. New myocytes had a 2C karyotype and possessed two sex chromosomes, excluding cell fusion. These data demonstrate, for the first time, that CPCs can repair scarred infarcts. CPCs may represent an ideal candidate cell for cardiac repair in patients with chronic heart failure. Potentially, CPCs may be isolated from myocardial biopsies and, following their expansion in vitro, administered back to patients avoiding the adverse effects of rejection with non-autologous transplantation. Alternatively, GFs may be delivered locally to stimulate resident CPCs and promote regeneration of lost myocardium. 5002 Engineered Cardiac Tissue Chambers Demonstrate Functional Frank-Starling Mechanism and Positive Stroke Work Eun Jung Lee, Do Eun Kim, Evren U Azeloglu, Columbia University, New York, NY; Cristina Alexandrescu, City College, New York, NY; Kevin D Costa, Columbia University, New York, NY Introduction: Before replacing damaged myocardium, engineered cardiac tissues have potential as model systems for high-throughput evaluation of therapeutic interventions. However, none have reproduced classical indices of physiologic myocardial pump function. We hypothesize that engineered cardiac tissue chambers (ETCH) can exhibit a functional Frank-Starling mechanism and generate positive stroke work. Methods: Living ETCH (n 3) were created from neonatal rat cardiac myocytes and fibroblasts suspended in a 3-D collagen and Matrigel scaffold, and cultured in a custom concentric mold with an inflatable central core. After 7–10 days of culture, the spontaneously beating ETCH was removed from the mold and attached to a high-sensitivity Langendorff setup at 37 C. Chamber pressure and area were acquired at 200 Hz and 50 fps, respectively. Results: ETCHs (9 mm diam, 0.3 mm thick) spontaneously contracted at 4–5 Hz. Fluorescence microscopy revealed a homogeneous cell distribution with randomly oriented myocytes having registered and striated sarcomeres. Developed pressure amplitude increased linearly with mean chamber pressure or preload (Fig). Cyclic changes in pressure and area resulted in a counter-clockwise pressure-area loop with a mean stroke work of 0.013 0.004 Pa (Fig). Conclusions: Thus, ETCHs exhibited a functional Frank-Starling mechanism and generated positive stroke work in an isolated pressure-volume measurement system. 5003 Combined Transplantation of Skeletal Myoblasts And Angiopoietic Progenitor Cells Reduce Infarct Size, Apoptosis And Improve Cardiac Function In A Model Of Chronic Myocardial Ischemia Nikolaos Bonaros, Rauend Rauf, Innsbruck Medical University, Department of Cardiac Surgery, Innsbruck, Austria; Dominik Wolf, Innsbruck Medical University, Department of Hematology, Innsbruck, Austria; Harald Ott, University of Minessota, Department of Physiology, Minneapolis, MN; Bernhard Schlechta, Alfred Kocher, Vienna Medical University, Department of Cardiothoracic Surgery, Vienna, Austria; Thomas Schachner, Innsbruck Medical University, Department of Cardiac Surgery, Innsbruck, Austria; Eva Margreiter, Innovacell Biotechnologies, Innsbruck, Austria; Rainer Marksteiner, IInnovacell Biotechnologies, Innsbruck, Austria; Steffen Hering, University of Vienna, Department of Pharmacology, Vienna, Austria; Guenther Laufer, Johannes Bonatti, Innsbruck Medical University, Department of Cardiac Surgery, Innsbruck, Austria Background. Cellular cardiomyoplasty using skeletal myoblasts or angiopoietic progenitor cells offers a promising approach for the treatment of ischemic heart failure. Although several studies have shown encouraging results in settings of acute and semi-chronic myocardial infarction, the efficacy of cell therapy on the chronic ischemic heart remains undetermined. Methods. A model of chronic ischemia was created using LAD-ligation in nude rats. Culture medium (C), homologous skeletal myoblasts (SM), human AC-133 cells (SC) and both homologous skeletal myoblasts and human AC-133 cells (SM-SC) were injected in the infarct (SM) and peri-infarct area (SC) four weeks after infarction. Assessment of myocardial function was performed using echocardiography 8 weeks after injections. Infarct size, collagen deposits and cardiomyocyte apoptosis were quantified to evaluate the effect cell injections using histology. Results. Echocardiographic studies revealed an amelioration of left ventricular dilatation in animals receiving combined cell transplantation (LVEDD: SM, SC, SM-SC, C: 7.5 1.5, 7.1 1.6, 5.7 0.8 and 7.7 0.09, p 0.003). Left ventricular ejection fraction improved significantly in all three cell therapy groups but no additional benefit was observed in the SM-SC group (SM, SC, SM-SC, C: 63.5 13.8, 63.3 7.8, 68.2 5.6 vs. control: 48.6 8.9, p 0.0017). Quantification of scar tissue showed a significant reduction of infarct 1200 Circulation Research Vol 97, No 11 November 25, 2005 by gest on Sptem er 4, 2017 http://circhajournals.org/ D ow nladed from area in the SM-SC group (SM, SC, SM-SC, C: 22.3 9.1%, 19.8 7.6%, 13.2 5.8%, 36.5 8.2%, p 0.008). Improvement of myocardial function was associated with reduced apoptotic index in animals after cellular cardiomyoplasty (SM, SC, SM-SC, C: 3.2 0.9, 3.1 0.6, 1.8 0.8, 10.3 1.6, p 0.0002). Conclusions. Combined tranplantation of skeletal myoblasts and AC-133 angiopoietic progenitor cells leads to improvement of diastolic and systolic left ventricular function, and reduction of scar size and myocardial apoptosis in a model of chronic ischemia. 5004 Transplanted Human Cord Blood Derived Unrestricted Somatic Stem Cells (ussc) Improve Left-ventricular Function And Prevent Left-ventricular Dilation And Scar Formation After Acute Myocardial Infarction Arjang Ruhparwar, Hannover Medical School, Hannover, Germany; Alireza Ghodsizad, Heinrich-Heine-University, Duesseldorf, Germany; Michael Niehaus, Hannover Medical School, Hannover, Germany; Gesine Koegler, Peter Wernet, Heinrich-Heine-University, Duesseldorf, Germany; Christoph Bara, Matthias Karck, Theo Kofidis, Mike Makoui, Ulrich Martin, Nawid Khaladj, Michael Mengel, Hannover Medical School, Hannover, Germany; Emmeran Gams, Hans Michael Klein, Heinrich-Heine-University, Duesseldorf, Germany; Axel Haverich, Hannover Medical School, Hannover, Germany Objectives: Intra-myocardial transplantation of adult stem cells as a therapy of heart failure is increasingly being discussed and performed in animal and human studies. Human unrestricted somatic stem cells [USSC] are newly discovered cord blood-derived cells with pluripotent differentiation potential. We aimed to assess whether intramyocardial transfer of USSC would improve global leftventricular function in a porcine model of myocardial infarction [MI]. Methods: USSC were isolated from cord blood [CB] of the umbilical cord vein. 13 x 10 6 cells were transplanted into the acutely ischemic lateral wall of the left ventricle (n 5). Control animals (n 5) received medium injection. LV dimension and function were assessed by transesophageal echocardiography [TEE] immediately before MI, after MI followed by cell transplantation and after 8 weeks. The hearts were examined for cell survival, cardiac differentiation and scar formation. Results: Global LVEF after MI was 32 8 % in controls and 36 9 % in the cell treated group. After 2 months, mean global LVEF had decreased to 27 5 % in the control group and increased to 52 2 % in the cell treated group (p 0.01). Leftventricular end-diastolic volume [LVEDV] after two months was 77 4 ml in the control group compared to 26 2 ml in the cord blood group (p 0.01). Myocardial scar was present only in the control group. Conclusion: Transplantation of USSC after myocardial infarction significantly improves LV function, reduces infarct size and prevents LV dilation. Late-Breaking Basic Science Poster Abstracts 5005 Reduction Of Fibulin-4 Expression Affects Elastogenesis And Results In Aortic Aneurysm And Dissection Katsuhiro Hanada, Marcel Vermeij, George A Garinis, Monique C de Waard, Maurice G Kunen, Dirk J Duncker, Carel Meijers, Roland Kanaar, Jeroen Essers, Erasmus MC, Rotterdam, The Netherlands The Fibulins are a six-member protein family, prominently expressed in blood vessels and hypothesized to function as intermolecular bridges that stabilize the organization of extracellular matrix structures such as elastic fibers and basement membranes. To examine a potential role of Fibulin-4/EFEMP2 in elastic fiber assembly and cardio-vascular disease we generated a mouse model underexpressing Fibulin-4. We decreased the mouse Fibulin-4/ EFEMP2 expression through transcriptional interference by targeted integration of the neomycin selectable marker. A reduction in Fibulin-4 RNA expression levels was confirmed by micro-array expression analysis and real-time quantitative PCR. Fibulin-4 heterozygous (Fibulin ) and homozygous (Fibulin) mice are viable, born at Mendelian frequencies and appeared indistinguishable from wild-type littermates during the first two weeks. Fibulin /R mice (with a 2-fold reduction in Fibulin-4 RNA expression levels) show abnormalities in the elastic fiber network of the aorta after Elastin staining of cross sections of the ascending aorta but no increased mortality or abnormal appearance during the first year was observed in these mice. In contrast, more than 80% of the Fibulin mice (with a 4-fold reduction in Fibulin-4 RNA expression levels) died suddenly after 2 weeks. Pathological analysis showed that these mice died from aortic dissection resulting from severe abnormalities in the elastic fiber network of the aorta. We pathologically analyzed heart and aorta of newborn, 2-week old and 10–20 week old surviving Fibulin mice and determined hemodynamic parameters. We found multiple heart and aortic defects, including a tortuous aorta and a 2-fold dilation of the left ventricle and ascending aorta. Furthermore, the 10–20 week old surviving Fibulin mice had a thickened and stiffened aorta. Heart rate and mean aortic pressure were not different between Fibulin-4 wild-type, Fibulin-4 /R and Fibulin-4 mice. However, in three surviving Fibulin-4 mice aortic pulse pressure was increased more than 2-fold consistent with a stiffening of the thickened and widened aorta compared to wild-type and Fibulin-4 /R mice. Also, Fibulin-4 mice had thickened aortic valvular leaflets that were associated with an increased systolic pressure-gradient between left ventricular pressure (134 4 mmHg) and aortic pressure (117 4 mmHg) and transvalvular blood flow velocity suggestive for the presence of a mild aortic valve stenosis. Altogether Fibulin-4 heterozygous and homozygous mice provide a unique model to follow the pathogenetic sequence for aneurysm. In addition, we implemented a functional genomics approach and determined the aorta transcriptome of wild type, Fibulin /R and Fibulin animals using full mouse genome Affymetrix arrays. This approach enabled us to identify those biological processes that were significantly over represented including apoptosis, blood pressure and coagulation and extracellular matrix organization as well as to identify several gene targets implicated in the response to aortic failure. 5006 Elevated Plasma Transforming Growth Factor-Beta 1 (TGF1) Reduces Aortic Atherocsclerosis, Aortic Root Dilation and Pseudoaneurysm Formation in Apolipoprotein E-null (Apoe) Mice Andrew D Frutkin, Goro Otsuka, Ming Xiao, University of Washington, Seattle, WA; Sunyoung Lee, UCLA, Los Angeles, CA; Helen L Dichek, David A Dichek, University of Washington, Seattle, WA INTRODUCTION The “protective cytokine” hypothesis predicts that elevated plasma TGF1 would limit atherogenesis. But, in humans high plasma TGF1 has been associated with both protection against and predisposition to severe atherosclerosis. An animal model of regulable TGF1 expression would permit a direct test of the hypothesis. METHODS We generated Apoe mice that express active TGF1 in a “tet-off” system ( MHC-tTA x tetO-TGF1). Dietary doxycycline (doxy) suppresses transgene expression. Removal of doxy from the diet at 6 weeks of age activates cardiac-specific expression of the TGF1 transgene and yields elevated plasma TGF1 within two weeks. We compared atherosclerosis in doubly transgenic mice off doxy (DT-Off) to doubly transgenic mice on doxy (DT-On) and singly transgenic mice off doxy (ST-Off). All mice were littermates, Apoe, and fat-fed from age 6 18 weeks. TGF1 transgene expression was evaluated by Northern analysis and by ELISA of plasma (n 7–14). Plaque size and histology were analyzed in aortic root sections (n 14–17). Plasma lipid measurements and FPLC were performed (n 8–13). RESULTS Only the hearts of DT-Off mice expressed TGF1 transgene mRNA. Hearts of DT-Off mice secreted more total TGF1 to yield a 10-fold elevation of plasma total TGF1. DT-Off mice had significant reductions in aortic root plaque area ( 20%) and lipid content ( 15%), aortic root circumference ( 10%), and fewer pseudoaneurysms ( 80%). CONCLUSIONS Elevated plasma TGF1 retards atherogenesis, limits aortic root dilation, and inhibits pseudoaneurysm formation. Elucidation of the mechanisms of these effects may suggest novel therapies for atherosclerosis and aneurysms.