Experiments Studys On The Differentiation Of Embryonic Stem Cell Into Cardiomyogrnic Cells In Simulative Cardiomyocytes Microenvironments In Vitro

Object: To explore the effect and strength of embryonic stem cells (ESCs) differentiating into cardiomyocytes in simulative cardiomyocytes microenvironment or cardiomyocyte-lysate in vitro.Methods:The blastocysts of 3.5 day and 4 day from Kunming species mouse were collected,cultured on the fibroblast cell feeder layers for 4-5 days,and the cells from inner cell mass(ICM)were isolated and subsequently cultured in vitro.The stem cells were identified bycharacterized morphology,AKP staining and Oct-4 staining. Day 2～3 embryoid bodies(EBs) were derived from ESCs of passage 3 to 5 and then cocultured with neonatal cardiomyocyte,cardiomyocyte-lysate and so on.EBs in group 1 and group 2 were cocultured respectively with cardiomyocytes and cardiomyocytes conditioned medium,those in group 3 were cultured with cardiomyocyte-lysate.EBs in group 4 were cultured with differentiated medium and were cultured on the mouse embryonic fibroblasts(MEFs) as control group.The expression of cardiac specific cardiac troponin-T(TnT) andа-Actin were detected by using immunofluoresence.Result: (1) It is good effect to trypsinize ICM colonies by 0.1%trypsine-0.02%EDTA with mechanical disperse for 2-4 min at room temperature.(2) The rhythmic beating embryoid bodies were observed on 3 day and reached 93% of rhythmic beating embryoid bodies on 12 day in group 1.The differentiating ratio of cardiomyocytes derived from ESCs expressed cardiac-specific proteins for TnT andа-Actin was 56.5±13.44%, it was the highest differentiating ratio compared with other groups (P<0.05).(3) The rhythmic beating embryoid bodies reached 8% on 12 day in group 2. The differentiating ratio of cardiomyocytes derived from ESCs was 5.2±2.01% , it was lower than group 1(P<0.05) and no statistics significant compared with control group(P>0.05).(4) The rhythmic beating embryoid bodies reached 36% on 12 day in group 3. The ratio of cardiomyocytes derived from ESCs was 8.2±2.45% , it was lower than group 1(P<0.05)and higher than control group(P<0.05).(5) The rhythmic beating embryoid bodies reached 7% on 12 day in group 4.The ratio of cardiomyocytes derived from ESCs was5.08±1.38%,it was lower than group 1(P<0.05)and no significant compared with control group(P>0.05).Conclusion: (1) The method of using 4day embryos and trypsinizing ICM colonies by 0.1%trypsine-0.02%EDTA with mechanical disperse for 2-4 min at room temperature are helpful to improve the capacity of establishment of ES cell lines. (2)Both myocardial cell coculturing system and cardiomyocyte-lysate system can induce mouse ESCs to differentiate into cardiomyocyte-like cells and the induced effect of direct cell-to-cell interaction with neonatal cardiomyocytes was higher than cardiomyocyte-lysate.