The Role Of Brachyury In Cardiomyocyte Differentiation Of Mouse Embryonic Stem Cells

Mouse embryonic stem cells(m ESCs)which is derived from the embryonic pluripotent stem cells before implantation,can keep the ability of self-renewal and multi-directional differentiation potential and can differentiate into various cell types of the body in vitro culture.They can differentiate into various cell types of the body,They are already play an indispensable role in building differentiation model at present,at the same time,they are widely used in regenerative medicine research and the study of cell transplantation.Classic Wnt/beta-catenin signaling pathway play an important role in embryonic stem cells,hematopoietic stem cells and neural stem cells in the differentiation.Studies have shown that Wnt/beta-catenin signaling pathway play an indispensable role in heart development and the process of angiogenesis.We have known that in specific period of embryonic stem cells differentiation activate the signaling pathways induced directed differentiation of embryonic stem cells into cardiomyocytes.Brachyury(T)gene is one of Wnt/beta-catenin signaling pathways downstream target genes and also a sign of mesoderm gene which highly expressed in the differentiation process of mesoderm.In order to study T gene in the role of m ESCs differentiation process,we have by PB(Piggy Bac)plasmid overexpressing the gene it is.Then we transfect the overexpression plasmid into m ESCs using LIF/serum medium.We take cell count method to test cell proliferation and alkaline phosphatase staining(AP staining)todetection m ESCs differentiation after overexpression the T gene.Differentiation experiment adoptsThe method of differentiation experiment was that single cell suspension culture was adopted to form embryoid bodies(EBs).The EBs formed three layers,the mesoderm can be further differentiated to cardiac muscle cells.Research reports that used alone GSK3?inhibitor BIO can be induced embryonic stem cells differentiate into cardiomyocytes.Our research have shown that in the early period of m ESCs differentiation to join GSK3? inhibitor CHIR99021(CHIR)can promote embryonic stem cells to myocardial cell differentiation.So we join the CHIR as positive control group during the test.Research results showed that overexpression the T gene cell proliferation is slower than the control group,and cell differentiation is increased.The q RT-PCR results showed that the expression of myocardial marker genes c Tn T?Cx43?Nkx2.5 and?-MHC are increase gradually in three groups.T group produced higher gene expression compared with PB group and the CH group as positive control is higher than T group.Western blotting analysis suggested that the protein expression of ?-MHC and Cx43 in T group were higher than PB group,and T group was slightly lower than CH group on the 10 th day.On the 15 th day the protein expression have the same trends to 10 th day,but each of the protein expression is higher than the 10 th day.The immunofluorescence staining suggested that three groups of ?-MHC and Cx43 were positively stained,and T group produced higher lever versus PB group,while CH group had higher lever than T group.Therefore,overexpress T gene can promote m ESCs to myocardial cell differentiation and the differentiation efficiency is weaker than GSK3?inhibitor CHIR induction efficiency.In order to further study the T gene promote the differentiation of m ESCs to cardiomyocytes,we use slow viruses,p LKO 1 knockdown the gene.The recombinantplasmid transfection into m ESCs by using LIF/serum medium.Suspension culture was used to form EBs.The m RNA expression of c Tn T?Cx43?Nkx2.5 and?-MHC were detected by q RT-PCR at different time points.Protein of ?-MHC and Cx43 was detected by western blot.The experimental results analysis suggested that the m RNA expression levels of c Tn T?Cx43?Nkx2.5 and?-MHC increase gradually,showing a peak of expression on the fifteenth day during the differentiation.Knockdown group produced lower gene expression compared with control group.Western blot analysis suggested that the protein expression of ?-MHC and Cx43 in knockdown group were lower than control group on the 10 th day.On the 15 th day the protein expression have the same trends to 10 th day,but each of the protein expression is higher than the 10 th day.Therefore,differentiation efficiency of myocardial cells from embryonic stem cells were reduced after knockdown T gene.Our research shown that T gene promotes cardiomyocyte differentiation in mouse embryonic stem cells but the efficiency of differentiation lower than GSK3?inhibitor CHIR induction.