Studies On Transformation Of Human Embryonic Pluripotent Stem Cells Into Cardiomyocyte-like Cells

Myocardial infarction is the main cause of cardiac death.Patients who have the occurrence of myocardial infarction get acute and chronic necrosis on a large number of myocardial cells,the absolute number of myocardial cells will be reduced only by fibroblasts to fill to form scar tissue,resulting in partial myocardial contractility weakening to disorder,myocardial function decreasing,and heart failure and sudden cardiac death at last.Although the survival rate of patients with myocardial infarction can be increased by medication,percutaneous coronary intervention,coronary artery bypass surgery and other methods to restrict the transportation condition of coronary blood,heart failure caused by the non-reversible loss of myocardial cells is still the main problem which impacts long-term prognosis.Medical and surgical treatment existing can not solve this problem effectively.In recent years,myocardial cells plasty, that by transplanting cells with a contractile function at the site of necrosis,has provided new research directions for the treatment of ischemic heart disease.Most of the cells chosen in this technology are various sources of stem cells with multiple differential potential.To investigate using 5-Azacytidine to induce human fetal liver tissue-derived PSC cultured in vitro to cardiomyocyte-like role in cell differentiation,the establishment of the system that in vitro source of human fetal liver tissue PSC to cardiomyocyte-like cell differentiation provides a preliminary basis for studying of early heart development,understanding of the pathogenesis of congenital heart disease,studying of cardiac myocytes' electrophysiological and stem cell transplantation in the treatment of heart disease.In this paper,human fetal liver tissue-derived PSC are extracted from human fetal liver tissues which are from the human embryo abortion,and cultured in the high glucose DMEM medium with 10%FBS,that is in a incubator with 37℃and 5%CO₂ After subculturing 3 generations,they are randomly divided into induced group and control group.Control group is cultured in high-glucose DMEM medium with 10% FBS;Induced group is added into high-glucose DMEM medium with 10%FBS which contained the final concentration of 10pmmol/L 5-aza;After 24 hours,the medium is changed into the high glucose DMEM medium with 10%FBS.Dynamically observing these cells under a microscope and photographic recording the morphological changes.Collecting the cells subcultured after 21-28 days,and identifying the expression of cardiac troponinâ… and connexin 43 with the use of immunohistochemical techniques.Further,we will go on with PT-PCR,and analyze the expression of MLC-2A,MLC-2V and GATA4 of cells induced,after extracting mRNA from Ttizol.The observation of human fetal liver tissue-derived PSC morphology:the cells of the original generation grows slowly,while the sizes of the new cells are not uniform, showing polygonal.Most of the nucleus are round or oval,and cytoplasm is less. Change the liquid after culturing the cells in the Petri dishes 24 hours later,at the same time,we can observe that the majority of cells are suspending and some cells affix to the bottom and extend,that are oval.After 2～3 days,the majority of cells can be seen affixed to the bottom of petri dishes,cell volume has increased,cells become polygonal,and the gaps of the cells gradually become narrow.The cells look like vortex or flame,showing the characteristics of fibroblast growth.After 5～7 days,the cells contact with each other and are full of vision.After subculturing,the cells grow rapidly and gradually purified.They have less and uniform cytoplasm and no particles. The shape of the cells are fusiform or rhomboid.Under the microscope,we can see that these cells has a high ratio of plasma/nuclear,and the nuclear are large.Some have dual-core.All of these indicate that the cells has a very strong capacity for splitting,which is the typical characteristics of stem cells.Before induction,human fetal liver tissue-derived PSC grow gradually,are spindle-shaped,have obvious nucleus and loose cytoplasm.After induction,the cells grow slowly;cell shrinkage becomes shorter,and gather gradually.Little particles come out in the cytoplasm. Induced after a week,the cells of induced group turn to rod-shaped,showing a close parallel with the growth.After two weeks,the size of the cells increases obviously.It can be seen that cell fusion-like situation has emerged.A large number of actin filament-like structure turns out after three weeks.While the cells of control group have no changes.Doing immunohistochemical staining to the two groups separately to detect the expression of troponinâ… and Connectin43 of the cells.Parts of the cells of induced group show the expression of these two kinds of protein;while The test results of control group is negative without stained.The result of PT-PCR shows that control group does not have the expression of MLC-2A,MLC-2V and GATA4,and induced group has.It is initially indicated that human fetal liver tissue-derived MSCs can be induced to differentiate into cardiomyocyte-like cells in vitro in certain conditions.However, the cells after induction are myocardial cell-like cells for naive,not entirely on the meaning of the myocardial cells.The cultured environment for the differentiation plays a stimulative role.It provides a preliminary basis for clinical application of stem cells.