Study On The Effect Of Biomimetic Electrical Stimulation To Myocardial Cell Inducing Differentiation Of ESCs Into Cardiomyogrnic Cells

Objective: To research the effect of biomimetic electrical stimulation to mature myocardial cell inducing the differentiation of ESCs into cardiomyogrnic cells, to provide the theoretical basis about establishing a simple and applied technique of cellular transplanting of ESCs.Methods: Primary myocardial cell was isolated from Sprague-Dawley neonatal rats, and marked the cell nuclear with DAPI . The blastocysts of 3.5 day and 4 day from Kunming species mouse were collected,cultured on the bone mesenchymal stem cells 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. ESCs of passage 3 to 4 then cocultured with neonatal cardiomyocyte,electrical stimulation,cardiomyocyte-lysate and so on. ESCs in group 2 and group 3 were cocultured respectively with cardiomyocytes and electrical stimulation,those in group 4 were cultured with electrical stimulation and cardiomyocytes conditioned medium,those in group 5 were cultured with electrical stimulation and cardiomyocytes. ESCs in group 1 were cultured with differentiated medium as control group.Expression of cTnT in the differentiated cells from ESCs were examined at 5 d,7 d,14 d by immunofluoresence.Results: (1) The embryonic stem cell colonies by cocculture with mesenchymal stem cells as feeder cells were identified. All the ES cells had its typical morphological characteristics and after the AKP and Oct-4 staining, all of the ES cells showed strong AKP and Oct-4 activity. (2)There are no cardiomyogrnic cells rhythmic beating all the time in group 1 and 3 and no cTnT expressed . (3) ESCs of passage 3 to 4 could be induced to differentiate into beating cardiomyocyte in group 4 and 5 on the 7th day, and beating with the frequency about 30-50/min on the 14th day. (4) To compare with group 4, the myocardial cell growth better and cardiomyogrnic cells differentiated from ESCs beat faster and at more equal pace. In the group cocultured with myocardial cell and electrical stimulation, the differentiating ratio of cardiomyocytes derived from ESCs expressed cardiac-specific proteins for cTnT was 40±2.39%, it was the highest differentiating ratio compared with the group cocultured with myocardial cell 28.7% (P<0.05) and the group cocultured with electrical stimulation and cardiomyocytes conditioned medium 17.1% (P<0.05).Conclusions: (1) By cocculture with mesenchymal stem cells as cells, we can isolate and culture embryonic stem cells (ES cells) which maintain undifferentiated and multipotential . (2) ESCs which not be established into the cell system could be induced to differentiate into beating cardiomyocyte. ESCs might be induced to differentiate into cardiomyocytes when cocultured with myocardial cell. And it could be at a high rate of differentiation than the natural differentiated group. It's mean that mature myocardial cell might be the important factor on directly inducing. (3)Only electrical stimulation can not induce the differentiation of ESCs into cardiomyogrnic cells yet. (4)Electrical stimulation can enhancement mature myocardial cell to induce the differentiation of ESCs into cardiomyogrnic cells, elevate the differentiating ratio of cardiomyocytes derived from ESCs, but can not make the process of differentiation of ESCs into cardiomyogrnic cells ahead of schedule.