Tissue Engineering Of Vascularized Myocardium From Genetically Modified Mouse Embryonic Stem Cells

Ischemic heart disease with high morbidity and mortality is one of the leading causes of death in industrialized countries. In the worldwide, thousands of people suffered from acute myocardial infarction each year resulting in the substantial loss of cardiomyocytes. Because cardiac myocytes can not replicate, patients who survive the acute myocardial infarction may eventually develop heart failure despite optimal medical therapy. Currently, the therapeutic options available to treat patients with end-stage heart failure are left ventricular assist devices (VAD) or heart transplantation. VAD is mainly used to prevent remodelling and dilation of the left ventricle, and can not fundamentally improve the heart performance. Unfortunately, Heart transplantation is limited by the inadequate source of donor organs and immunological rejection after transplantation. Myocardial tissue engineering is a rapidly growing discipline, which aims to repair or regenerate damaged myocardium with engineered heart tissue constructed by combination of cells and scaffolds in vitro. It is providing promising new cues for the treatment of ischemic heart disease. However, this strategy has been hampered by the lack of cardiomyocytes and the significant cell death following transplantation in vivo.Pluripotent embryonic stem cells (ESC) can not only proliferate indefinitely in vitro, but also differentiate into cardiomyocytes. ESC is one of the most promising sources for construction of engineered heart tissue. One of the biggest obstacles of clinical application of ESC is the tumorigenicity. Not only the transplantation of ESC but also the transplantation of ESC-derived differentiated cells would result in the tumor formation. Although it is reported a variety of methods for induction of ESC differentiation into cardiomyoctyes, the heterogenous population still contained a large number of non-myocardial cells and some of the undifferentiated ESC. How to effectively obtain ESC-derived cariomyocytes and eliminate the undifferentiated ESC is the problem to be solved for construction of engineered heart tissue with ESC-derived cardiomyocytes. In addition, the size of engineered heart tissue may be limited by lack of blood vessel network of the tissue construct. Most of the cardiac grafts are dependent on the host for vascularization. For thicker tissue, the blood vessels of host need a very long time to grow into the graft. In this period, insufficient graft vasculariztion is considered as the main factor responsible for cell death or apoptosis due to lack of nutrition, thereby affecting the function of cardiac graft. In this study, to face the above problems of myocardial tissue engineering, mouse ESC were genetically modified so as to confer the ESC and its derived cardiomyocytes with the specific selection markers, allowing enrichment of ESC-derived cardiomyocytes and elimination of the undifferentiated ESC more easily and effectively. On this basis, liquid collagen was used as scaffold to construct highly vascularized cardiac tissue in vitro. It is explored that genetically modified ESC was used as seed cells to engineer vascularized myocardial tissue, which has not been reported at home and abroad.Using mouse genomic DNA as template, the cardiacα-MHC promoter was amplified by polymerase chain reaction (PCR). The CMV promoter in pcDNA3.1(+)-EGFP-hygro vector was replaced by the cardiacα-MHC promoter to successfully constructα-MHC-EGFP expression vector.α-MHC-EGFP was transfected into primary mouse heart cells by electroporation. After 72 hours, green fluorescence could be observed in cardiomyocytes transfected positively under the fluorescence microscope, but not in transfected non-cardiomyocytes. It is demonstrated thatα-MHC-EGFP expression vector has the characteristic of cardiac-specific expression.In order to more easily select ESC-derived cardiomyocytes, EGFP was replaced by neomycin resistance gene to successfully construct cardiac-specific expression vector pMHC-neo/SV40-hygro which was transfected into mouse ESC to obtain six transgenic mouse ESC cell lines. One line, named MN6, not only stably expressed transgene, but also maintained undifferentiated state and proliferated rapidly. Expression of the pMHC-neo/SV40-hygro transgene also did not impact negatively on cardiogenic differentiation, as evidenced by their ability to differentiate into spontaneously beating cardiomyocytes when compared to the wild-type lines. An excellent spatial correlation was found between the contracting areas within the embryoid body (EB) and cTnT-positive areas. After 5 days of suspension culture, EB were formed from genetically modified ESC, and then were induced by ascorbic acid for 12 days. After G418 selection for 6 days, more than 99% of the cells showed immunoreactivity toα-sarcomeric actin. RT-PCR showed that Oct-4 expression was not detected in the selected cells indicating no undifferentiated ESC. Both laser scanning confocal microscopy and ultrastructural analysis demonstrated that G418-selected cardiomyocytes expressed cardiac-specific markers and had well developed myofibrils.For potential applications, a method of controllable large-scale production of ESC-derived cardiomyocytes needed to be developed. Genetically modified mouse ESC were cultured in stirred bioreactor. The different initial cell concentrations of ESC and the initial stirring speed of bioreactor were investigated to determine the optimal condition for EB formation. Induced by ascorbic acid, the differentiation of EB formed in stirred bioreactor into cariomyocytes was compared with EB formed in Petri dishes. Stirred bioreactor could effectively prevent extensive agglomeration among the EBs. EB formation was more efficient in stirred bioreactor. When ESC were seeded initially with 2×105 cells/ml and stirred speed was set to 25 rpm, there were about 482 EBs/ml in stirred bioreactor after 5 days of suspension culture. EBs were relatively uniform in size, and the diameter was about 496±20 m. AO/PI and H&E staining showed that most of cells in EBs formed in stirred bioreactor were viable and no necrotic centers were seen. The gene expression of cardiac-specific gene was increased within EBs fomed in stirred bioreactor. Stirred suspension culture also yielded a greater percentage of EBs containing beating cardiomyocytes, and increased the average percentage ofα-sarcomeric actinin-positive cells detected via flow cytometry. Furthermore, stirred suspension culture improved relative yield of ESC-derived cardiomyocytes after G418 selection.Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the cardiac graft, the interactions between endothelial and myocardial cells may also play an important role in enhancing cell survival and proliferation. Genetically modified mouse ESC were used as seed cells and cultured in stirred bioreactor for mass production of EBs. The EBs were induced into cardiomyocytes with ascorbic acid, and then ESC-derived caridomyocytes were selected by G418. The enriched ESC-derived cardiomyocytes with human umbilical vein endothelial cells (HUVEC) and mouse embryonic fibroblasts (MEF) were mixed with rat tail liquid collagen to construct highly vascularized cardiac tissue in vitro. A preliminary transplantation in vivo was also conducted. Cells were reconstituted compactly and uniformly in vascularized engineered cardiac tissue, in which apoptosis also decreased significantly. Four weeks after subcutaneous transplantation in nude mice, all vascularized cardiac tissue could survive and no teratoma formation was ovserved. Importantly, HUVEC could promote the vascularization of myocardial tissue, and MEF further supported the organization of endothelial cells into vessel networks. The number and area of lumens were increased so that thicker myocardium was formed.In summary, the results showed that genetically modified mouse ESC could be used as seed cells in myocardial tissue engineering. Combination of genetically modified ESC and stirred bioreactor cultivation not only benefited in large-scale production of pure ESC-derived cardiomyocytes, but also effectively control the potential risk of undifferentiated ESC. Based on liquid collagen as scaffold, the enriched cardiomyocytes derived from genetically modified ESC mixed with HUVEC and MEF in three-dimensional culture could construct a highly vascularized cardiac tissue.