Introduction According to the American Heart Association, one in three deaths in the Western World is caused by heart disease [1]. Due to the inability of heart tissue to regenerate after it has been damaged and the severe shortage of heart donors, two alternative treatment options to facilitate cardiac repair have been gaining attention: the injection of cells into the diseased tissue, and the in vitro cultivation of cell-based cardiac grafts. The grafting of cells of various origins and developmental stages to the heart in vivo has shown partial restoration of myocardial function [2]; however, conflicting evidence is reported on the integration and localization of the cells upon injection, and the underlying mechanisms of functional improvement are still unknown. Improved understanding of the physiological repair processes between the native tissue and the implanted tissue is crucial to continued progress in developing both functional implantable cardiac tissue and cardiac cell injection therapies. We propose here to develop a model system to study cardiac cell injection, using engineered heart tissue (EHT) to mimic heart muscle. This approach will enable us to accurately track injected cells, and to determine survival upon injection and integration with the surrounding tissue. Currently, such precise tracking is not possible in conventional animal models and myocardial explants. In this work we specifically explored mouse embryonic stem cell (ESC) differentiation and integration upon injection into the engineered myocardium.

Materials and Methods Cardiomyocytes (CM) isolated from neonatal Sprague-Dawle rats were seeded onto collagen sponges (1.3x106 cells in 20µL Matrigel/scaffold, 10x20x0.3mm). The EHT was maintained in orbitally mixed dishes (25 rpm) in high glucose Dulbecco's Modified Eagle's Medium (DMEM) with 10% Fetal Bovine Serum (FBS). Once functional tissue had formed, a small controlled amount (1.3x105 cells, 5µL Matrigel) of fluorescently labeled (yellow fluorescent protein, YFP) undifferentiated R1 ESCs suspended in Matrigel was injected into the center of the upper layer of engineered tissue. (YFP ESCs were expanded on 0.2% gelatin-coated flasks and cultured in DMEM containing 15% FBS, 1% non-essential amino acid, 1% sodium pyruvate, 1% beta-marcaptoethanol, 0.5% penicillin and streptomycin and 1000U/mL leukemia inhibitory factor.) Injected ESCs were tracked using fluorescence microscopy every two days for eight days. Tissue function was established at the end of these eight days by measuring excitation threshold (ET) and maximum capture rate (MCR). Uninjected CM scaffolds and ESCs seeded on collagen sponges were used as controls. Results and Discussion The feasibility of consistent spatial and temporal mapping of YFP+ cells was established through fluorescent imaging and FACS. ESCs were observed to proliferate and migrate away from the injection site over nine days of cultivation. The injected EHT exhibited strong spontaneous contractions throughout the proposed experimental time points (indicating the absence of oxygen and nutrient limitations [3]), as well as the ability to respond to electrical field stimulation. Injected constructs exhibited lower ET (2.5V/cm vs 3.5V/cm) and comparable MCR values to uninjected constructs. Importantly, cell number was two-fold higher in the ESC injected constructs compared to non-injected controls (5x106 vs 0.2x106), demonstrating ESC survival and proliferation in EHT; these results are consistent with the observed superior functional properties of injected EHT.

Future studies will focus on cultivating EHT with electrical field stimulation to mimic the physiological contractions of the heart. Tissue morphology and localization of cardiac markers will be determined by immunofluorscence of paraffin embedded or frozen sections followed by staining for sacromeric proteins.

Conclusions Preliminary results show the suitability and validity of the proposed model system for cell injection studies in myocardium. The use of EHT will shed new light on the mechanisms of cell integration and processes of physiological reconstitution, thereby increasing the success of cell therapies in animal studies and clinical trials.

References 1. Pescovitz, D. The Heart of Tissue Engineering. Berkeley Engineering. www.coe.berkeley.edu, 2002. 2. Zimmerman, W.H., Melnychenko, I., Wasmeier, G., Didie, M., Naito, H., Nixdorff, U., Hess, A., Budinsky, L., Brune, K., Michaelis, B., Dhein, S., Schwoerer, A., Ehmke, H., and Eschenhagen, T. Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nature Medicine, 12, 4, 2006. 3. Radisic, M., Malda, J., Epping, E., Geng, W., Langer, R., and Vunjak-Novakovic, G. Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue. Biotechnology and Bioengineering, 93, 2, 2006.

Acknowledgements This work was supported by grants from NSERC (DG), CFI (LOF), NIH (R01HL076485), an OGSST and a UofT Open Fellowship. JD thanks the Zandstra lab for their continued guidance.