Transplantation Of Engineered Cardiac Tissue For Myocardial Infarction Treatment

Ischemic heart disease was one of the top threatens to human health.Great amount of cardiomyocytes were lost and the functional cardiac tissue was finally replaced by scar tissue,resulting heart failure . As the adult human heart has a limited capability of self-regeneration, the replacement of the myocardium after infarction with a functional cardiac tissue was a fundamental approach. Recently,the transplantation of exogenous stem cell to regenerate heart tissue has aroused great attention.The embryonic stem cell ,which has a capability of self-proliferation and multi-directional differentiation,became a promising cell source.However,the problem of immunal rejection and teratoma formation has severly hampered the application of ESC in treatment of organ damage.The strategy of therapeutic cloning is expected to solve the immunal problem after ESC transplantation.With the development of tissue engineering, the construction of engineered heart tissue brought new hope for the treatment of heart ischemic disease.Recently,there are two main strategies:1.delievering of cardiomyoctes with injectable material as vehicle into the infarcted area;2.invitro construction of engineered cardiac tissue with certain size,shape and mechanical property for transplantation.This article was comprised of three parts:Part I: culturation,differentiation and enrichment of NT-mESC and its derivatives.Derivation of sufficient amount of seeding cells of robust quality is the premise and fundamental of cardiac tissue engineering. The purpose of this part is to derive sufficient seeding cells through NT-mESC culturation and differentiation. Feeder cells and LIF was prepared in order to maintain the pluripotential of NT-mESC.We applied STLV bioreactor for a large scale production of embryoid bodies with the removal of LIF and addition of ascorbic acid to induce a differentiation of NT-mESC towards cardiomyocytes.After culturation in STLV for 5 days , embroid bodies with certain size were transferred to gelatin coated dishes for further differentiation and the embroid bodies were observed daily. At day 14±,the amount of beating areas reached a peak. cTnT immunofluorescent staining showed existence of a good quantity of positive cells in EB.The NT-mESC derived cardiomyocytes were enriched by percoll density gradient centrifugation.Percoll was an effective approach in the enrichment of cardiomyocytes and avoied the safety problems caused by geneticalmodification,which made a great advantegous in the application of tissue engineering. PartⅡ:Invitro construction of engineered cardiac tissue using NT-mESC derived cardiomyocytes as seeding cellsIn this part,we constructed 3D engineered cardiac tissue by mixing the NT-mESC derived cardiomyocytes with collogen I and Matrigel,casting the mixure into self-prepared tooting.Histology and immunohistochemical results showed that the cells survive well in the scaffold.Spindle shaped cardiomyocytes could be observed in most parts of the engineered tissue ,but were as immature as neonatal rat cardiomyocytes .Besides cardiomyocytes,fibroblast cells and endothelial cells were also observed.Those non-cardiomyocytes were likely to promote the survival and differentiation of cardiomyocytes.In this part ,we constructed EHTs using NT-mESC derived cardiomyocytes as seeding cells ,there aren't relative reports both home and abroad.PartⅢ:Transplantation of engineered cardiac tissue to repaire cardiac infarction In order to study the function of EHTs for the repairment of infarcted area. In this part,we successfully established MI model and transplanted the EHTs 2 weeks later.We established four groups : healthy,MI only,EHT grafted and non-contractile material group in order to evaluate the improvement of heart function.The result showed that the graft survived well,integrated to host and heavily vascularized.Immunolabeling of connexin43 demonstrated electrical and mechanical intercellular connections inside the EHTs and between the host.The contract and dialation function was significantly improved in EHTs group.No report about the transplantation of engineered tissue based on NT-mESC was ever seen.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 typeⅠcollagen supplemented with EHM gel to construct engineered cardiac muscle tissues (EHTs). After in vitro stretching, the EHTs were implanted on myocardial infarcts in rats. Four weeks after implantation, the rats were subjected to echocardiography to determine the improvement of heart function.Then the rats were sacrificed and the grafted EHTs were extracted for histological examination to determine the survival and vascularization. The result showed that the grafted EHTs integrated with the host.This is the first attemptation for the combination of therapeutic cloning and tissue engineering technique and this study provide great clinical significance for the future treatment of ischemic heart diseases.