Experimental Study On Mouse Embryonic Stem Cells Derived By Nuclear Transplantation Differentiation Into Cardiomyocytes

Objective:In the end stage of ischemic heart diseases, 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 and immunological rejections after transplantation. 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. We established a convenient and high-efficiency system of differentiation by various inductors.Methods:⑴With the feeder layer , which was made up of mouse embryonic fibroblasts ,and leukaemia inhibitory factor,NT-mESC kept undifferentiated in vitro.⑵NT-mESC 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 mediums supplemented with various inductors and we identified them with cardiomyocytes character.⑶The NT-mESC-derived cardiomyocytes were enriched by Percoll density gradient centrifugation. The enriched cardiomyocytes were mixed with liquid typeⅠcollagen supplemented with ECM gel to construct engineered cardiac muscle tissues(cardiac muscle lamellar) and we detected them in vitro.Results:⑴We established a culture system of NT-mESC in the condition of mouse embryonic fibroblasts and leukaemia inhibitory factor .We optimized culture conditions.⑵EBs could be efficiently formed in the STLV bioreactor and didn't show extensive agglomeration and necrotic areas in the center. After differentiation culture, EBs quickly attached to the substratum and grew outgrowth. The percentage of EBs containing beating cardiomyocytes induced by combination of ascorbic acid , dimethyl sulfoxide and ratinoic acid was 87.5±3.4%.⑶The ESC-derived cardiomyocytes were enriched in fractionⅢand fractionⅣby 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 fractionⅢand fractionⅣwith liquid typeⅠcollagen and cardiac muscle lamellar resembled immature myocardium of neonatal mouse. The cardiomyocytes reconstituted longitudinally oriented in the ECTs, but the myofilaments were sparse.Conclusion: NT-mESCs can be used as a source of seed cells for cardiac tissue engineering. We established a convenient and high-efficiency system of differentiation by combination of ascorbic acid, dimethyl sulfoxide and ratinoic acid .The structures of ECTs construction based on NT-mESCs resembled the native myocardium of newborn mice.