Expression Characteristics Of APJ In The Process Of Differentiating Human Embryonic Stem Cells Into Cardiomyocytes

Part 1 Differentiation of Human Embryonic Stem Cells into Cardiomyocytes with Chemically Defined and Feeder Free Culturing Medium Part 1Objective: To investigate the role of chemically defined CDM3 medium in differentiating human ESCs(embryonic stem cells)into cardiomyocytes and the differentiation efficiency of different seeding desity.Methods: Before differentiation,culturing human ESCs at least 3 passages.Culturing ESCs on reduced growth factor basement membrane matrix,when they reached 90% confluence,medium was changed to CDM3.For d0-d2,the medium was consisting of CDM3 and 6ummol/L CHIR99021,d3-d4 CDM3 and 2 ummol/L Wnt-59,d5-CDM3 only.Contracting cells were noted from d7 to d16.Then detect the differentiation efficiency of different seeding desity of 0.5×10~4?1×10~4?1.5×10~4?2×10~4cells/cm~2.Results: Contracting cells were noted from d7,and its number was increasing till d16.Results of cell immunofluorescence show the expression of a-Actinin?cTNNT2 of contracting cells which presenting sarcomere and intercalated discs clearly.Human ESCs with the seeding desity of 0.5×10~4cells/cm~2 were not be differentiated to cardiomyocytes,the differentiation efficiency of 2×10~4 cells/cm~2 desity was lower than 1×10~4 and 1.5×10~4 cells/cm~2 desity.Conclusion: Human ESCs can be differentiated into myocardiacytes successfully using CDM3 medium.The best seeding desity was between 1 to 1.5 cells/cm~2.Part 2 Expression Characteristics of APJ in the Process of Differentiating Human Embryonic Stem Cells into Cardiomyocytes Part 2Objective: To investigate the expression characteristics of APJ in process of cardiac differentiation of human embryonic stem cells according to the three stage markers of mesoderm?earliest cardiac morphogenesis?later cardiac morphogenesis.Methods: After culturing human ESCs on monolayer-based medium.,differentiate ESCs to myocardiocytes with chemically defined medium.Then detecting the protein expression of Brachyury T?mesp1?Nkx2.5 and APJ with confocal microscope,the m RNA expression with real-time PCR..Results: Beating cardiac cells began at d7 and their number were increasing till d16.Cyto-immunofluorescence staining showed that APJ was co-expression with stage-specific markers of Brachyury T?mesp1?Nkx2.5 on d2?d3?d7?The m RNA expression of APJ was persistent in all three stages,highest in mesoderm and then decrease gradually.Conclusion:APJ was co-expression with stage-specific markers of Brachyury T?mesp1 ? Nkx2.5 and APJ was persistently expressed in the process of human myocardial development.