The Bone Marrow Mesenchymal Stem Cells Of SD Rat Were Induced In Vitro To Myocardial Cells By Transfected Nkx2.5, GATA4, TBX5Gene

Objective: Cardiovascular disease, with a high incidence, is greatlyharmful to human health. In acute myocardial infarction (AMI), the loss offunctional myocardial cells and the formation of scar tissue lead to ventricularreconstruction, even heart failure and life-threatening. It is a very promisingtreatment to transplant exogenous functional cells into infarcted myocardiumto improve cardiac function. In the past decades, many kinds of stem cellswere tried as cell sources for cell transplantation therapy. Bone marrowmesenchymal stem cell (BMSC) is one of the best candidates. BMSCs,classified in adult stem cells (ASCs), have obvious advantages compared withother stem cells:①They can be used for autologous transplantation, withoutinvolving ethical controversy.②They can be isolated easily, then amplifyhighly in vitro. They are genetic stable for several passages in vitro.③Theyhave multilineage differentiation potential.④They have low-immunogenicityand portability. So, BMSCs are ideal seed cells for stem cell transplantationtherapy for cardiovascular disease.There are three methods to induce BMSCs to differentiate intomyocardial cells in vitro:①be induced by drug;②be co-cultured withmyocardial cells;③be modified genetically. However, the differentiationefficiency is still low and how to improve it is becomming the research focus.With the development of molecular biology techniques, it is a newmethod to modify BMSCs genetically to promote the cardiomyocytedifferentiation. It is aimed to start one or some of the key genes and activategene regulatory networks of cardiac differentiation. Cardiac-specific earlytranscription factors, such as Nkx2.5, GATA4, TBX5, regulate geneexpression of cardiac structure and play an important role in normal development of the heart. They have been transfected into embryonic stemcells (ESCs) to promote the cardiomyocyte differentiation.The drug inducers for MSCs cardiomyocyte differentiation include5-azacytidine (5-aza), dimethylsulfoxide (DMSO), the combination of insulin,dexamethasone, ascorbic acid. The commonly used agent,5-aza, is notsuitable for clinical treatment because of the cell toxicity. In recent years, wehave researched the possibility of oxytocin (OT) as a drug inducer of stemcell's cardiomyocyte differentiation. Researches show that in addition of theability to regulate blood pressure, OT plays a role on heart development. OThas been used to induce P19cells, P19CL6cells, ESCs, adult cardiacSca-1-positive cells and cardiac side population cells into cardiomyocytes.Therefore, in this study we try to transfect BMSCs with Nkx2.5, GATA4,TBX5, or use OT as the inducer, or use the combination of two methods. Theaim is to explore the relationship between Nkx2.5, GATA4, TBX5andcardiomyocyte differentiation of BMSCs, and to provide effective strategy fortransforming various BMSCs into myocardial cells.Methods:1The isolation, culture, and characterization of BMSCsSD rat BMSCs were isolated by the whole bone marrow culture,amplified by serial subcultivation and purified by digestion time control andsubcultivation. We characterized BMSCs in three aspects:①the adheredcultured cells were observed;②the expression of CD90, CD29, CD45weredetected by flow cytometry;③the multilineage differentiation potential wasdetected by adipogenic, osteoblasts and chondrogenic differentiation in vitro.2Exogenous genes of Nkx2.5, GATA4, TBX5induced BMSCs todifferentiate into myocardial cells.Experimental groups: A1group (transfected with pEGFP-N1-Nkx2.5),A2group (transfected with pEGFP-N1empty plasmid), A3group (blankgroup); B1group (transfected with pVP22-GATA-4/myc-His), B2group(transfected with pcDNA3.1empty plasmid), B3group (blank group); C1group (transfected with pcDNA3.1-TBX5), C2group (transfected with pcDNA3.1empty plasmid), C3group (blank group). Before transfection,preliminary experiment was used to determine the suitable cell seeding densityand the best transfection system. Using the cationic liposome reagent,Lipofectamine2000, plasmid was transfected into BMSCs. Exogenous geneexpression were detected with western blot after48hours of transfection.After4weeks of transfection and the following culture, the expression ofcardiac troponins T (cTnT) and connexin43(Cx43) were detected withimmunocytochemical staining and Western blot. Real Time PCR was used todetect the expression of Cx43, β-myosin heavy chain (β-MHC), myosin lightchain-2(MLC-2) in P3BMSCs and experimental groups.3Combination of exogenous genes of Nkx2.5, GATA4, TBX5and oxytocininduced BMSCs into myocardial cells.Experimental groups:①transfection groups: Nkx2.5group (transfectedwith pEGFP-N1-Nkx2.5), GATA4group (transfected withpVP22-GATA-4/myc-His), TBX5group (transfected withpcDNA3.1-TBX5);②transfection+induced drug groups: Nkx2.5+OT group(transfected with pEGFP-N1-Nkx2.5+OT induction), GATA4+OT group(transfected with pVP22-GATA-4/myc-His+OT induction), TBX5+OT group(transfected with pcDNA3.1-TBX5+OT induction);③OT group (OTinduction);④blank group. Using Lipofectamine2000, plasmid wastransfected into BMSCs. After4weeks of transfection and the followingculture, the expression of cTnT and Cx43were detected withimmunocytochemical staining and Western blot, and the ultrastructuralchanges of the cells were observed.Results:1The isolation, culture, and characterization of BMSCsThe first time to exchange the medium of primary cells was at48hoursafter islation. The adherent cells were round or polygonal or spindle-shaped.After3to4days, primary cells began to proliferate rapidly. After9to11days,cells arranged swirlingly or radially, reaching80%confluence. Passage cellsgrew rapidly. With medium exchange and passage, the morphology of adherent cells was becoming identical. The third passage cells werespindle-shaped.Flow cytometry results showed that CD90+/CD29+/CD45-cells were upto99%of P3BMSCs.During adipogenic induction, small lipid droplets accumulated andbecame larger gradually in some cells. The induced cells were positive for oilred O staining after2weeks. During osteogenic induction, BMSCs graduallytransformed to polygonal or irregular-shaped cells and grew intensively. Theinduced cells were positive for alizarin red staining after2weeks. Duringcartilage induction, the local part of BMSCs gradually changed into round oroval and grew rapidly and intensively to form nodular structures. After2weeks, the nodular structures were positive for alcian blue staining.2Exogenous genes of Nkx2.5, GATA4, TBX5induced BMSCs todifferentiate into myocardial cells.The experiment confirmed that the appropriate cell seeding density is4×104/cm2, the best transfection system is the plasmid DNA(μg) andLipofectamine2000(μl) at a ratio of1:2.5.Results of Western blot at48hours after transfection showed thatNkx2.5-EGFP fusion protein expressed in A1group, whereas none in groupA2or A3. The myc protein expressed in B1group, while none in group B2orB3. The TBX5protein expressed in group C1, C2and C3, but that in C1groupwas the highest (P<0.05). The results showed that plasmid pEGFP-N1-Nkx2.5,pVP22-GATA-4/myc-His and pcDNA3.1-TBX5can transfect BMSCs bycationic liposome reagent.Four weeks after the exogenous gene transfection and the followingculture, some cells of group A1, B1and C1elongated and widened, and thelocal cells grew intensively. Few cells in the other groups changed similarly.Four weeks after the exogenous gene transfection and the followingculture, immunocytochemistry results showed that cTnT and Cx43expressedin group A1, B1and C1. There were brown filament-like or granular structuresin the cytoplasm of cTnT positive cells, and tan particles in the cytoplasm of Cx43ones. Few positive cells were in the other groups. The cTnT and Cx43expressed in group A1, A2and A3, but these in A1group were the highest(P<0.05). The cTnT and Cx43expressed in group B1, B2and B3, but these inB1group were the highest (P<0.05). The cTnT and Cx43expressed in groupC1, C2and C3, but these in C1group were the highest (P<0.05).Four weeks after the exogenous gene transfection and the followingculture, Western blot results showed that cTnT expressions of transfectedgroups were significinatly higher than those of empty plasmid groups andblank ones. The results were identical with immunocytochemistry results.Real Time PCR results showed that Cx43expression level trends of A1,B1and C1group were similar. They increased gradually at1^stweek, reached apeak at2^ndor3^rdweek, followed by expression down. β-MHC expressionlevel of group A1from1^stto3^rdweek were significantly higher than that of P3BMSCs (P<0.05), then decreased at4^thweek, without significant differencewith that of P3BMSCs (P>0.05). β-MHC expression of group B1was highestat3rdweek (P<0.05), then decreased. There were no significant differenceamong1^st,2^nd,4^thweek and P3BMSCs (P>0.05). β-MHC expression of groupC1increased gradually and was highest at4^thweek (P<0.05). There were nosignificant difference among1^st,2^nd, week and P3BMSCs (P>0.05). MLC-2expression levels of group A1and C1at1^stweek had no significant differencewith that of P3BMSCs (P>0.05), then significantly increased at2ndweek(P<0.05), decreased gradually at3rdweek. The expression was weak at4^thweek, without significant difference with that of P3BMSCs (P>0.05). MLC-2expression of group C1was highest at3rdweek (P<0.05), then decreased.There were no significant difference among1^st,2^nd,4^thweek and P3BMSCs(P>0.05).3Combination of exogenous genes of Nkx2.5, GATA4, TBX5and oxytocininduced BMSCs into myocardial cells.Four weeks after the exogenous gene transfection and the followingculture, some cells of all the groups except blank one elongated and widened,and the local cells grew intensively. Few cells in blank group changed similarly.Four weeks after the exogenous gene transfection and the followingculture, immunocytochemistry results showed that cTnT and Cx43expressedin all the groups except blank one. There were brown filament-like or granularstructures in the cytoplasm of cTnT positive cells, and tan particles in thecytoplasm of Cx43ones. Few positive cells were in blank group. The cTnTexpressions of transfection groups and OT group were high, oftransfection+induced drug groups were the highest, of blank group were little.There were significant difference between induced groups and blank group(P<0.05). The Cx43expressions of transfection groups, transfection+induceddrug groups and OT group were high, of blank group were little. There weresignificant difference between induced groups and blank group (P<0.05).Western blot results showed that the cTnT expressions of transfectiongroups and OT group were high, of transfection+induced drug groups werethe highest, of blank group were little. There were significant differencebetween induced groups and blank group (P<0.05). The results were identicalwith immunocytochemistry results.Observation of the ultrastructure by transmission electron microscopyshowed P3BMSCs and blank group cells were spindle-shaped and riched withmany cell organelles such as ribosomes, mitochondria, rough endoplasmicreticulum, lysosomes in the cytoplast. Gap junctions were visible occasionallybetween a few of cells. The cells of transfection groups, transfection+OTgroups and OT group were spindle-shaped and riched with many cellorganelles such as mitochondria, ribosomes, rough endoplasmic reticulum,lysosomes in the cytoplast. The oval nucleus located in the cell central. Manyparalleled myofilament-like structures with dense zone appeared around thenucleus and more were in the side or end of the cytoplast. More gap junctionswere observed in the side or end of the cells. More myofilament-like structuresand gap junctions appeared in the cells of transfection groups andtransfection+OT groups than those in the cells of OT group. Conclusion:1SD rat BMSCs were isolated, amplified and characterized successfully.2The plasmids of pEGFP-N1-Nkx2.5, pVP22-GATA-4/myc-His andpcDNA3.1-TBX5transfected BMSCs by cationic liposome reagent,Lipofectamine2000.3SD rat BMSCs were transfected with the heart early transcription factorof Nkx2.5, GATA4and TBX5, and differentiated toward myocardial cells.The differentiated cells expressed myocardial specific markers, cTnT, Cx43,β-MHC and MLC-2, in both gene level and protein level, and had earlymyocardial cells' structural characteristics.4OT induced SD rat BMSCs to differentiate toward myocardial cells.The differentiated cells had early myocardial cells' structural characteristics.5There were superimposed effect of cardiomyocyte differentiationbetween Nkx2.5, GATA4or TBX5transfection and OT induction. Thecombination method gained the highest expression level of cTnT, but didn't ofCx43.