Differentiation Of Mouse Embryonic Stem Cells Into Cardiomyocytes In Vitro And Study On The Expression Of Nesprins Gene During This Process

PART ONE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES IN VITROObjective To investigate a method of inducing embryonic stem cells (ESCs) of mouse high differentiation into cardiomyocytes in vitro. Methods1. Prepared mouse embryo fibroblasts (MEF) as feeder layer.2. Thawed ESCs were cultured on feeder layer.When the cells grew up to 80% of 6-well culture dish,they could were passaged or freezed.3. There were 6 groups according to inducers added in the culture system of hanging drop culture and suspension culture respectively: groupⅠ(salvia miltorrhiza and 5-azacytidine as inducer),groupⅡ(salvia miltorrhiza and retinoic acid as inducer),groupⅢ(5-azacytidine as inducer),groupⅣ(salvia miltorrhiza as inducer),groupⅤ(retinoic acid as inducer),groupⅥ(no inducer).The rates of differentiation of cardiomyocyte from ESCs were compared between 6 groups,2 culture systems respectively.4. The leves ofα-MHC gene and MLC-2v gene mRNA expression at ESCs time,the cells cultured for day7 and day16 respectively were analysed by RT-PCR.5. The expression of cardiomyocyte marker proteins ofα-actinin, myosin,α-actin were detected by immunocytochemistry .Results1. MEF cells displayed monolayer growth and arranged tightly as short spindle shape.2. ESCs were plated on feeder layer of MEF,within one day,groups of small,tightly packed cells had began to proliferate and formed flat and compact colonies as bird nests.The refraction of colonise was very intensive,observation under phase contrast microscope.Undifferentiated ESCs formed embryoid bodies(EBs) which had three-dimensional structures like globe by hanging drop-suspension culture or suspension culture. Spontaneously contracting cells appeared and were identified at day 9.One EB had one or more contraction focis.3. The rates of differentiation of groupⅠ,Ⅲ,Ⅳ,ⅤandⅥwere (77.78±5.09)%,(66.67±8.82)%, (51.11±1.92)% , (44.44±5.09)% , (23.33±3.34)% in hanging drop culture, and (42.22±6.94)%(,36.67±8.82)%(,25.55±10.71)%(,17.78±5.09)%(,10.00±2.84)% in suspension culture, they were statistically diferent in these two methods(P<0.05);The rate of differentiation of the groupⅠ(up to 78%) was higher than other groups by hanging drop and the difference was significant(P<0.05).4. RT-PCR analyses demonstrated that the cardiomyocyte marker geneα-MHC began to express from day 7 and the ventricular myocardium gene MLC-2v had expressed at day 16.5. The immunocytochemistry shew the expression of cardiomyocyte marker proteins ofα-actinin, myosin,α-actin were positive. Conculsion Traditional chinese drug salvia miltorrhiza and 5-azacytidine as inducers in culture system of hanging drop cultures can be a theoretical reference for differentiation of ESCs into cardiomyocytes in vitro. PART TWO STUDY ON EXPRESSION OF NESPRINS GENE DUIRING MOUSE EMBRYONIC STEM CELLS DIFFERENTIATE INTO CARDIOMYOCYTES IN VITROObjective To investigate expression of both nesprin-1 and nesprin-2 gene during mouse embryonic stem cells (ESCs) differentiate into cardiomyocytes in vitro , and the roles of nesprin-1 and nesprin-2 gene during this process.Methods1. Mouse ESCs were cultivated in vitro.Used hanging drop-suspension culture and 5-azacytidine as inducer to stimulate mouse ESCs differentiate into cardiomyocytes.2. Identifing the differentiated cells were cardiomyocytes by RT-PCR and immunocytochemistry.3. The leves of nesprin-1 gene and nesprin-2 gene mRNA expression at ESCs time,the cells cultured for day7,day10,day13,day16 and day27 respectively were analysed by RT-PCR.4. The distribution of nesprin-1 protein at ESCs time,the cells cultured for day7,day10,day13,day16 and day27 respectively were determined by immunofluorescence. Results1. ESCs were plated onto a feeder layer of MEF(mouse embryo fibroblasts),within one day,groups of small,tightly packed cells had began to proliferate and formed flat and compact colonies as bird nests. The refraction of colonise is very intensive,observation under phase contrast microscope.Undifferentiated ESCs formed EBs which had three-dimensional structures like globe in hanging drop-suspension culture.Spontaneously contracting cells appeared and were identified at differentiation day9 by using 5-azacytidine as inducer.One EB had one or more contraction focis.2. RT-PCR analyses demonstrated that the cardiomyocyte marker geneα-MHC began to express from day 7 and the ventricular myocardium gene MLC-2v had expressed at day 16.The immunocytochemistry shew the expression of cardiomyocyte marker proteins ofα-actinin, myosin,α-actin were positive.3. There were dynamic expression pattern of nesprin-1 and nesprin-2 during ESCs differentiate into cardiomyocyte by RT-PCR.The expression of nesprin-1 mRNA gradually enhanced from ESC stage to cultured for day13,and at day7,10,13 was higher levels then subsequently decreased.It was express very lowly in day27,there was significant difference(P<0.05)compared with other stage.The expression of nesprin-2 mRNA was highest at day7,there was significant difference(P<0.05),and decreased gradually in later stage and was lowest at day27.4. The immunofluorescence shew that nesprin-1 protein was detected at nuclear nuvelop and perinuclear space during ESCs differentiate into cardiomyocytes.Conculsion The data shew nesprin-1 and nesprin-2 expressed dynamicly during ESCs differentiate into cardiomyocytes.The peaks of expression of the both genes were differentiated earlier period and intermediate period associated with cardiac muscle proliferation and cardiac conduction system developing stage.So,we can be presumed that nesprins could maintain cardiac muscle cell development and play an important role in urging ESCs'differentiation into cardiomyocytes.