Experimental Study On Differentiation Of Human Embryonic Germ Cells Into Cardiomyocytes In Vitro

Objective:1.To evaluate the feasibility of retinoic acid, shen mai, ascorbic acid on the myocardiac differentiation of human embryonic germ cells (hEGCs) in vitro. 2.To observe the differentiation of hEGCs on different times of induction by three inductors separately. 3.To find out the more safe and efficient inductor of differentiation of hEGCs towards cardiomyocytes.Methods:We obtained the human embryonic germ cells through tissue culturing, hEGCs were suspended in the Petri dishes at a density of 1×105cells/ml. After 5 days, embryoid bodies(EBs) formed from hEGCs were seeded in 24-well plates and incubated with differentiation medium containing retinoic acid, ascorbic acid and shenmai separately in various concentration. The cells were collected at 1 week, 2 weeks, 3 weeks, 4 weeks of induction. Immunocytochemistrical staining was used to identify the expression of primordial myocardium-specific transcription factor GATA-4 and cardiac troponin T(cTnT). Then the differentiation ratios were calculated to find out the optimal concentration of the three inductors and to compare their effect for myocardiac differentiation.Results:By the treatment of retinoic acid, shen mai and ascorbic acid, the spindle-shaped and irregular cells increased at the periphery of EBs in one week. The connections between adjacent cells became apparent and formed a intercalated-like structure in three weeks. Immunocytochemistry showed that, the primordial myocardium-specific transcription factor GATA-4 were stained positively in the differentiated cells just after one week of induction, and up to the maximum after three weeks. The cTnT were not stained positively until two weeks and the expression increased gradually in three weeks and four weeks. We also found out that the maximum cardiac differentiation rate of hEGCs was reached at a concentration of 10-7 or 10-8mol/l retinoic acid, 1g/l shenmai and 0.1mg/ml ascorbic acid.Conclusions: Retinoic acid, shen mai and ascorbic acid all can enhance the differentiation of hEGCs into cardiomyocytes. Ascorbic acid, as much as RA, is a high efficient inductor and yet safer than RA. Although the differentiation rate of shen mai is lower than RA, it is safer than RA too. So shen mai can be also served as a valuable inductor for cardiac differentiation of hEGCs in vitro.