Differention Of P19 Cells Into Cardiomyocytes In Cardiac Microenvionments In Vitro

Cardiomyocytes of adult mammal are thought to be terminally differentiated cell which have no growth and proliferation capacity. The myocardial tissue damaged by myocardial infarction can not be restored, and the loss of numerous cardiomyocytes leaded by myocardial infarction has been known as the main reason of causing heart failure and other heart disease.Currently cell transplantation provides a new therapeutic scheme for cardiomyocyte repair. Using skeletal muscle cells to replace the cardiomyocyte have been discussed in some studies, but the skeletal muscle cell transplantation might lead some patients suffering from serious cardiac arrhythmia even to death. The successed of isolation of the embryonic stem cells give people lots of hope of repairing myocardial necrosis.Embryonic stem cells are 'totipotent cells ' which can differentiate into various cells of adult. Thus, in theory, under the suitable conditions, embryonic stem cells can develop into cardiomyocytes . In recent years, many studies are working at finding suitable conditions that induce the embryonic stem cells differentiation toward cardiomyocytes.Now it is agreed that when they are in the microenvironment, the stem cells would give some responses to the various suitable physical or chemical signals, which also named environmental induction of differentiation.Variety of cell differentiation in vivo studies have shown that,cardiac environment or "cardiac-like"environment is more important than chemical induction.In view of it, the study try to induce P19 cells differentiating toward cardiomyocytes through the P19 cells and neonatal rat myocardial cells co-culture or in "cardiac-like" conditions culture,to research P19 cells' growth and differentiation state in the cardiac environment, to discuss the role of environmental factors on cell differentiation and differentiation mechanism ,afford groundwork for the study of the differentiation of P19 cells in vivo and cell transplantation research.Contents are as follows1 P19 cells' passage and suspension cultureP19 cells resuscitated and culture generally. After passageed more than 4 generation, the cells were seeded into petri dishes containing a thin layer of soft agar in a concentration of 5×10~5 cells per mL. Then kept in suspension for4 days to form cell aggregates or EBs(embryonic bodies,EBs). The cell growth, aggregations and differentiation of P19 cells were observed by inverted phase contrast microscope.The results show that after passage of about 2h, P19 cells adherent grew and the cells are round. After 24h the cells covered about 50% of bottom, which were flat and irregular-shaped. The cells intensive grew after 48h. In the dishes with low melting point agaric after 48h, the cells aggregated and formed cell mass.They are uniform and growing as suspension,until the fourth day to form EBs.2 The neonatal rat cardiomyocytes' isolation , culture and identification.1-3-days-old SD neonatal rat were disinfected the skin with 75% alcohol,then operated the chest to take the ventricle out and cut into small pieces aboout 1mm3,dispersed the tissue by trypsin-EDTA until it was digested completely.Collected the digestive juice and then filtrated, centrifugaed and resuspended the cells,purified cardiomyocytes through differentially adherent and add to 0.1mmol/L BrdU. After 3 days, observed with inverted microscope : cardiomyocytes were fusiform,star,or irregular shape and so on. They touched with each other by extented processes, which linking together make a network,emerged into a piece and beated synchronously at a frequency mostly 60-120 times / min.The expression of cardiomyocytes specific cTnT was detected by immunocytochemical stain to identify the cardiomyocytes differentiation. The cardiomyocytes expressed cTnT, and the positive cells contain brown and filamentous structures in cytoplasm. The purity of neonatal rat cardiomyocytes in the process of cultured for 72 hours reach up to 70%.3 Differentiation of P19 cells toward cardiomyocytes induced by myocardial microenvironment in vitro.3.1 P19 cells and cardiomyocytes in co-cultureP19 cells were labeled with BrdU of 15μmol/L and marked the cells for 48 hours. Immunocytochemical staining showed: the nucleus of P19 cells appeared brown. The labeling efficiency is 50%.The P19 cells and cardiomyocytes were co-cultured to 7 and 10 days, the immunocytochemical staining against cTnT showed that cytoplasm of cardiomyocytes appear brown filamentous structures, parts of the P19 cells labeled with BrdU had brown nuclei and brown filamentous structures in cytoplasm. With culture time, the expression of cTnT increased. These results suggested that part of P19 cells differentiation to myocardial cell, and the number of positive cells increased with the culture time.Western Blotting results show that the expression of cTnT and Cx43 in co-culture group were significantly higher than that in the comparison group of cultured cardiomyocytes (P<0.01), suggesting that the increase parts mainly from the differentiated P19 cells .3.2 Myocardial cell lysate and conditioned medium cultured EBsP19 cells kept in suspension for 4 days to form EBs.The EBs cultured 7,10 days by cardiomyocytes lysate and cardiomyocytes conditioned medium, compared to the EBs cultured by complete medium.Western Blotting results show that the expression of cTnT and Connexin 43 (Cx43) in myocardial cell lysate group and the myocardial cell conditioned medium group were higher than the control group (P<0.05). The expression of cTnT and Cx43 in cardiomyocytes lysate group were significantly higher than that in the cardiomyocytes conditioned medium (P<0.01) ,there was a significant difference between the results.3.3 The expression of cTnT in co-cultured group, cardiomyocytes lysate group and cardiomyocytes conditioned medium group were dectected with immunocytochemical staining The cells cultured to 10th day,the expression of cTnT in co-cultured group was significantly higher than cardiomyocytes lysate group and cardiomyocytes conditioned medium group (P<0.05),and the expression of cTnT in cardiomyocytes lysate group was higher than cardiomyocytes conditioned medium group (P<0.05).Conclusion:1 P19 cells co-cultured with neonatal rat cardiomyocytes or with cardiomyocytes conditioned medium or with cardiomyocytes lysate can simulate myocardial microenvironment in vitro ,they can induct P19 cells to differentiate toward cardiomyocytes,and express cTnT and Cx43. With culture time, the expression of cTnT and Cx43 increased.2 The expression of cTnT in P19 cells co-cultured with neonatal rat cardiomyocytes was significantly higher than that in the cardiomyocytes lysate and cardiomyocytes conditioned medium. Some of differentiated P19 cells connected with cardiomyocytes and beat together, show that the role of mechanical traction can effectively promote the differentiation of P19 cells into cardiomyocytes.