Comparison Between The ESC-derived Cardiomyocytes Differentiated From Oxytocin And The Mouse Cardiomyocyte

Embryonic Stem cells(ESC) derived from the inner cell mass (ICM),embryonic ectoderm,and primordial germ cells of the fetal genital ridge and represent pluripotent undifferentiated cells.In the particular environment,ESC can differentiate into different kinds of cell.,including cardiomyocyte.In the procedure of differentiation into cardiomyocyte,many cell factor and drug play an important role,such as HGF,EGF,bFGF,TGF2β,RA et,al.These cell factor promote the differentiation,and increase the ratio of ESC-derived cardiomyocytes in all cells.Oxytocin is a very abundant neuropeptide secreted mainly from the hypothalamic nuclei.Oxytocin and oxytocin receptors have long been recognized to be mainly involved in the regulation of reproductive functions,such as the induction of uterine contractions,milk ejection reflex and ovulation.Recent studies have indicated the role of oxytocin in cardiac development as the oxytocin receptor increases at the protein level in the murine heart from day 7 of gestation,when cardiac differentiation starts.OT/OTR are found in mESc and mouse cardiomyocyte.Furthermore,oxytocin has been found to be a potent inducer cardiomyocyte differentiation of P19.Recent experiment resarch pointed out the possible involvement of the oxytocin/oxytocin receptor system in cardiomyocyte differentiation of mESc were confirmed,these cardiomyocyte-like cells show spontaneous and rhythmical beating.But this result lacks the comparison between the cardiomyocyte-like cells and mouse cardiomyocyte in structure,fuction and physiological character.Methed:In our experiment,first,EScell morphological changes were observed under an inverted microscope and alkaline phosphatase staining was used.The mRNA expression of Oct-4 and Nanog was analyzed by RT-PCR. The proteins for Oct-4 and Nanog were determined by Western-blot.These methods were used to measure the pluripotency of mice ESCs.mESc(S19)were recovered and passaged,then treatment with oxytocin(experiment group).After 10 days,review ESC-derived cardiomyocytes beating under light microscope ,The proteins forα-sarcomericactinan,ANF and cTnT were determined by Western-blot and immunohisochemistry.ESCderived cardiomyocytes show ultramicrostructure under electron microscope.Cardiomyocytes-like cells were treated with 10^-5M concentration of isoprenaline and carbachol for Pharmacological responses of ESC-derived cardiomyocytes.All methed also useing in mouse cardiomycoyes(control group),and results compared with experiment group.Result:In the first experiment,After 14 days,in experimental group,almost 80%of ES cells to sustain good cell colony form and a well stereoscopic appearance,which showed strong AKP staining,in the control group,only 35% of ES cells hold undifferentiation mode,the cells were stained for alkaline phospha tase(AKP) weakly positive.The quantity of undifferentiation mES cells in experimental group(7.02±0.34) is higher than in control group(4.28±0.37, P＜0.05).RT-PCR results indicated that Nanog and Oct-4 mRNA expression were both higher in experimental group(204.95±11.24,160.69±11.52),with that in control group(122.89±9.25,88.21±4.69,P＜0.05).Western-blot results indicated that Nanog and Oct-4 expression were also both higher in experimental group(238.14±14.72,187.16±9.02) than in control(205.04±10.03, 132.41±15.78,P＜0.05).In the second experiment,cardiomyocytes and cardiomyocytes-like cells differentiation of mESC by oxytocin all stained positive for cTnT in immunohisochemistry,the ratio is 61.2%and 54.8% respectively.Western-blot results indicated that ANF,cTnT andα-sarcomeric actinan expression have no statistical significance(P＜0.05) between experiment group(198.31±2.68 169.29±8.55 190.13±19.15)and control group(197.97±2.00 171.98±8.31 189.95±16.24).Under electron microscope,we can saw some of the cardiomycoyes-like cells contained a higher number of myofibrils,clearly defined sarconmeres and Z lines were observed.Significan positive chronotropic effects on the cardiomyocytes were observed after the application of carbachol in both group,isoprenaline enhanced the rate of beating frequency in experiment group and control group(27.3±6.41,26.17±6.15,P＞0.5).In contrast,negtive chronotropic effects on cells appeared after treatment with carbachol(24.66±7.12 24.25±5.47,P＞0.5).Carbachol degraded the rate of beating frequency in experiment group and control group.Result of Pharmacological responses indicated that there are no statistical significance between experiment group and control group.Conclusion:1,Retinol showed a syner gistic effect in maintaining pluripotency of mESCs when combined with LIF factor of moderate concentration.2,Compared mouse cardiomycoyes,ESC-derived cardiomyocytes in autorhythmicity and conductibility have no difference.3,oxytocin induce the expression of cardiac genes,includingα-sarcomeric actinan,ANF and cTnT.Pharmacological responses of ESC-derived cardiomyocytes certifyied that function of adrenoceptor and muscarinic cholinoceptors is same like mouse cardiomyocytes.In brief,whatever in construction or in expression of cardiac genes,cardiomyocyte differentiation from mouse embryonic stem cells are similar to the mouse cardiomyocyte.It means mESC-derived cardiomyocytes have many special function just like mouse cardiomyocyte.mESC-derived cardiomyocytes induced by oxytocin will became batter cell resource for transpla ntation in vivo.