Reconstruction Of Embryo-derived Embryonic Stem Cells As Seed Cells To Construct The Experimental Study Of The Engineered Cardiac Tissue

Background: Heart disease is the number one cause of death in industrialized nations. Myocardial infarction and heart failure resemble the most prevalent pathologies. In end stage heart failure, heart transplantation remains the last treatment option with good long-term results. Unfortunately, heart transplantation is limited due to an inadequate supply with donor organs. The development of tissue engineering technology is providing promising new cues. Restoration of heart function by replacement of diseased myocardium with functional cardiomyocytes seeded onto a biodegradable scaffold is an intriguing strategy that may offer a potential cure. In this study, mouse embryonic stem cells derived by nuclear transplantation (NT-mESC), which could provide unlimited cells and resolve immune rejections, were used as seed cells for engineering cardiac muscle tissue.Methods: NT-mESCs were cultured in a slow turning lateral vessel (STLV) for mass production of embryoid bodies (EBs). The EBs were induced to differentiate into cardiomyocytes in differentiation medium supplemented with ascorbic acid. The ESC-derived cardiomyocytes were then enriched by Percoll density gradient centrifugation. The enriched cardiomyocytes were mixed with liquid type I collagen supplemented with ECM gel to construct engineered cardiac muscle tissues (ECTs). After in vitro stretching, the ECTs were implanted on myocardial infarcts in rats. Four weeks after implantation, ECTs were extracted for histological examination to determine the survival and vascularization.Results: EBs could be efficiently formed in the STLV bioreactor and didn't show extensive agglomeration and necrotic areas in the center. After differentiation culture, EBs could quickly attach to the substratum and grow outgrowth. The percentage of EBs containing beating cardiomyocytes induced by ascorbic acid was approximately 90%. The ESC-derived cardiomyocytes were enriched in fraction III and fraction IV by Percoll density gradient centrifugation, and the percentages of the cTnT positive cells were 35±2% and 73±4% respectively. ECTs were constructed by mixing the cells in fraction III and fraction IV with liquid type I collagen, and resembled immature myocardium of neonatal mouse after stretching. The cardiomyocytes reconstituted longitudinally oriented in the ECTs, but the myofilaments were sparse. Four weeks after implantation, ECTs not only could survive and retain more mature cardiac muscle structures, but also showed extensive vascularization. More importantly, no teratoma formation was observed.Conclusions: NT-mESCs can be used as a source of seed cells for cardiac tissue engineering. The structures of ECTs construction based on NT-mESCs resembled the native myocardium of newborn mice. After implantation in vivo, ECTs survived and formed cardiac muscle, moreover, were highly vascularized. Therefore. ECTs may be helpful in repairing diseased myocardium.