简介：Rats(Rattusnorvegicus)havemanyadvantagesovermiceinscientificstudies,forexample,theyaremorerelevanttohumaninphysiologicalandpharmacologicalresponses.Therefore,ratsarebroadlyusedinexperimentalstudies.Therecentbreakthroughinthegenerationofratembryonicstemcells(rESCs)opensthedoortoapplicationofgenetargetingtocreatemodelsforthestudyofhumandiseases.Inaddition,theinvitrodifferentiationofrESCsintoderivativesofthreegermlineswillserveasapowerfultoolandresourcefortheinvestigationofmammaliandevelopment,cellfunction,tissuerepair,anddrugdiscovery.However,thedistinctcultureconditionandsignalinhibitor-dependedmaintenanceofrESCsstandasaconsiderablechallengeforitsinvitrodifferentiation.Toaddressit,weinvestigatedwhetherrESCsarecapableofformingterminaldifferentiatedcardiomyocytes.Wefoundthattheembryoidbodies(EBs)-basedmethodusedinmouseESC(mESC)differentiationfailedtoworkinthecultivationofrESCs.WethenmodifiedthedifferentiationprotocolandsuccessfullydevelopedaninvitrodifferentiationsystemtodifferentiaterESCsintothreeembryonicgermlayers.Byusingthismethod,therESCsformspontaneousbeatingcardiomyocyteswiththepropertiessimilartothosederivedfromfetalratheartsandmESCs.Thisuniquecellularsystemwillprovideanewapproachtostudytheearlydevelopmentandcardiacfunctionaswellastoperformpharmacologicaltestandcelltherapystudy(Grants:theStateMajorResearchProgramofChina(2009ZX09503-024,2010CB945603)andCAS(XDA01030000).

简介：Itiswellestablishedthatstemcellscandifferentiateintocelltypesoftheorganinwhichthesearetransplanted.However,theprocessisveryslowduetolackofunderstandingofsignalsimportantfortheirsurvivalanddifferentiation,mostoptimalstemcellsandtheirplasticity.Limitationsandadvantagesofvariouscellsubtypeswillbedescribed.Therateofstemcellsmobilizationandtheirsurvivalintheischemicenvironmentaremajorobstaclesinengraftmentanddifferentiationofstemcellsformeaningfulrepairoftheinfarctedmyocardium.Manipulationofstemcellswithischemicpreconditioning,combinedgeneandcelltherapytogetherwithsimultaneousactivationofdiversesignalingpathwaysformassivestemcellmobilization®enerationhassignificantimpactontherepairprocessbystemcells.Theseandotherdifficultiesencounteredinefficientuseofvariousstemcellshaveresultedininventionofinducedpluripotentstemcellswhichcouldrevolutionizethestemcellbasedtherapyandtheirapplicationsforunderstandingofhumandiseaseanddrugscreeninginthenearfuture.ReprogrammingofadultcellsintoiPScellswithouttheuseofviralvectorsisamajorchallengetowardsgettingiPScellswithoutviralintegrationintocells.Tomeetthischallengewehaverepro-grammedskeletalmyoblastsintoiPScellswithhighefficiencyusingepigeneticmodifiers.TransplantationofiPScellsderivedpurecardiacprogenitorsintoinfarctedmyocardiumledtoextensiverepopulationofscarareawithfullydevelopedmyocyteswithouttumorformationandresultinginmarkedimprovementincardiacfunction.Reprogrammingwithpurechemicalmeanswillmaketherapeuticuseofthesecellsmoresafer.Targetingtheinducedpluripotentstemcellstowardscardiacprogenitorsandtheirapplicationtowardstransplantationisamajorstepforwardinenhancingthemyocardialrepaircapacitybythesecells.

简介：backgroundBonemarrowmesenchymalstemcells(BMSCs)canbeisolatedandculturedtomanypassages.However,StemcellsincludingBMSCsquicklyundergosenescenceinculture.Thecellsenescenceandmulti-directionaldifferentiationhavehamperedproducingBMSCsinquantitywiththeirundifferentiatedstate.Inthisstudywereportanaturalcompound,vitaminC(Vc),maintainsBMSCsstemproperty.MethodsHumanBMSCswereisolatedfrombonemarrowandpurifiedby1.073g/mLdensitygradientcentrifugation.50ng/mLVcwereaddedtoBMSCsfordifferenttimepoint.FlowcytometrywasusedtodetectcellsurfacemarkersofBMSCswithorwithoutVctreatment.BMSCsproliferationwasanalyzedbyMTTassay.PCR(polymerasechainreaction)andreal-timePCRwereusedfordetectingc-kit,nanog,andOct-4genesexpressionlevels.DNAmethyltransferase(Dnmt)1andDnmt3blevelswerealsodetectedbyreal-timePCR.ResultsFlowcytometryshowedthatafterVctreatmentfor6h,thesurfacemarkersofBMSCswerealmostunchanged.VcincreasedtheproliferationactivityofBMSCsfrom6hto24h.PCRshowedtheexpressionofc-kit,nanog,andoct-4geneswereobviouslyincreasedinVctreatedgroupthancontrolgroupat12h.Real-timePCRshowedthatthelevelofc-kit,nanog,andoct-4geneswereunregulatedfrom6hto12hcomparedwithcontrolgroup.VcalsoincreasedDnmt3bbutnotDnmt1geneexpression.ConclusionsOurresultsshowedVcactsatleastacceleratesBMSCsproliferationandmaintainsstemcellproperty.Inourstudy,wehighlightedamethodofimprovingthespeedofBMSCsgenerationandprovidedadditionalinsightsintothemechanisticbasisofpreventingBMSCssenescence.

简介：ObjectivesToinvestigatetheanti-apoptoticeffectsofmesenchymalstemcells(MSCs)onhypoxicinjuredcardiacmyocytesinvitro.MethodsMSCswereisolatedfrombonemarrowofSprague-Dawley(SD)rats,andcardiacmyocytesfromneonatalrats.Theratcardiacmyocyteswereco-culturedwithMSCsorMSC-conditionedmediainanoxia(95%N2+5%CO2)for72hours.CellapoptosiswasmeasuredbyHoechst33258staining.TheexpressionofBcl-2andBaxincardiacmyocyteswastestedbyWesternBlot.ResultsTheapoptoticratewas51.6%±2.4%whencardiacmyocyteswereculturedincontinuoushypoxiaandwassignificantlydecreasedwhencardiacmyocyteswerecoculturedwithMSCsorMSC-conditionedmedia(15.1%±5.4%and24.0%±4.2%respectively,P<0.001).ThedecreasedexpressionofBaxinthecardiacmyocyteswasgreatlyrelatedtothedecreasingofapoptosis,buttherewasnodifferenceinBcl-2expressionamongthesegroups.ConclusionsCo-culturedMSCsshowedsignificantanti-apoptoticeffectsoncardiacmyocytesincontinuoushypoxia.ThemechanismmaybetheinteractofcelltocellandparacrineofcytokineswhicheffectedtheexpressionofBaxinthecardiacmyocytes.

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简介：ObjectivesToconstructarecombinantplasmidcarryingenhancedgreenfluorescentprotein(EGFP)andhumanvascularendothelialgrowthfactor(VEGF)121geneanddetectitsexpressioninratmesenchymalstemcells(MSCs).MethodsHumanVEGF121cDNAwasamplifiedwithpolymerasechainreaction(PCR)frompCD/hVEGF121andwasinsertedintotheeukaryoticexpressionvectorpEGFPC1.AfterbeingidentifiedwithPCR,doubleenzymedigestionandDNAsequencing.TherecombinantplasmidpEGFP/hVEGF121wastransferredintoratMSCswithlipofectamine.TheexpressionofEGFP/VEGF121fusionproteinweredetectedwithfluorescencemicroscopeandimmunocytochemicalstainingrespectively.ResultsTherecombinantplasmidwasconfirmedwithPCR,doubleenzymedigestionandDNAsequencing.ThefluorescencemicroscopeandimmunocytochemicalstainingresultsshowedthattheEGFPandVEGF121proteinwereexpressedinMSCs48haftertransfection.ConclusionsTherecombinantplasmidcarryingEGFPandhumanVEGFwassuccessfullyconstructedandexpressedpositivelyinratMSCs.ItoffersapromisetoolforfurtherresearchondifferentiationofMSCsandVEGFgenetherapyforischemialcardiovasculardisease.

简介：BackgroundOurpreviousstudyshowedthe150mg/mLfetalcardiacsupernatant(FCS)couldinducedifferentiationofBMSCsintocardiomyocye-likecellswithoutcardiomyocytetouch,butdifferentiationefficiencyisnothighenough.Inhibitionofglycogensynthasekinase-3enhancedtheproliferationandsurvivesofstemcells.Wetestedif6-bromoindirubin-3-oxime(BIO,glycogensynthasekinase-3inhibitor)enhancestheeffectsofFCSondifferentiationofBMSCsandexplorethegrowthfactorsinFCS.MethodsBMSCswereisolatedfromthefemurandtibiaoffour-week-oldmaleSprague-Dawleyratsandco-culturedwithFCS(150mg/mL)thatwasmadefromfetalheartsfromnineteen-daypregnantWistarrats.BIOwithdifferentconcentration(0,1,10,and100nM)wasintroducedinculturedishes.Transforminggrowthfactorbeta1(TGF-β1),bonemorphogeneticprotein2(BMP-2)andAktincardiacsupernatantandculturemediumwereassayedwithELISAmethods.ResultsAfterco-culturingwithFCS,beatingmyotubeswereobservedin25.9%BMSCsdishesafter1to2weeks’culture.ThelevelsofTGF-β1andBMP-2inFCSconcentrationswerenomorethanthatinyoungandadultcardiacsupernatant.AllBIOgroupssignificantlyenhancedtheeffectsofFCSondifferentiationofBMSCsintothecardiomyocyte-likecells(1nM,83%;10nM,73%;100nM,100%).AktlevelswerehigherinBMSCsculturalmediumwithFCS.ConclusionsFCScouldinducethedifferentiationofBMSCsintothecardiomyocyte-likecells.TGF-β1andBMP-2mightnotplayaroleinthedifferentiationofBMSCsinducedbyFCS.BIOenhancedtheeffectsofFCSonthedifferentiationofBMSCsintocardiomyocyte-likecells,whichmightinvolvetheAktpathway.