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1、EmbryoidBodiesFromEmbryonicStemCellsIn-vitroRobertChristensenBiologyDepartmentEasternConnecticutStateUniversityEndoderm–SpecificGeneExpressioninEmbryonicStem–CellsDifferentiatedToEmbryoidBodiesExperimentalCellResearch229(1999):pp27-34KoichiroAbe,HitoshiNiwa
2、,KatsuroIwase,MasakiTakiguchi,MasatakaMori,Shin–IchiAbe,KuniyaAbe,andKen-IchiYamamuraIntroductionCelllineagesariseduringdevelopmentEctodermaltissuesMesodermaltissuesEndodermaltissuesEBsresembletheembryooftheegg-cylinderstageNeuronalcellsCardiacmusclecellsHe
3、matopoieticcellsYolksaccellsEScellshavefulldevelopmentalpotentialEBsfromEScellsEBsconsistof:LaterstagesEBsarecomposedof:PaststudiesshowedspecificchangesinexpressionofendodermmarkergenesduringEBdevelopmentandsuggestedthatEBformationcouldbeconsideredanin-vitr
4、omodelforendodermdifferentiation.RNAblotReversetranscriptionpolymerasechainreaction(RT-PCR)In-situhybridizationThenatureofendodermdifferentiationin-vitroisdiscussedinrelationtonormalembryonicandextraembryonicdevelopmentin-vivo.Thispaperextendedtheiranalysis
5、andsystematicallycharacterizedtemporalexpressionpatternsofendodermmarkergenesduringEBformation.MaterialsandMethodsCellculturesEScellline,D3,culturedIncubated3daysinDulbecco’smodifiedeaglesmedium(DMEM)15%fetalcalfserum0.1mM2-mercaptoethanol110µg/mlsodiumpyru
6、vate4.5mg/mlD-glucoseIssupplementedwith1000units/mlrecombinantmurineleukemiainhibitoryfactor(LIF)LIFwaswithdrawnThecellswereallowedtoaggregateTheDMEMwaschangedeveryotherdaythroughoutthecultureNorthernBlotAnalysisTotalRNApreparedfromundifferentiatedEScells,p
7、reaggregationEScells,andEBsofvariousstages1wholedishofEBswasusedforisolationoftotalRNASampleswerecollectedfromundifferentiatedEScells,preaggregationphasecells,andEBsatdays0,1,2,3,4,5,7,9,11,13,15,and18RNAelectrophoresedandtransferredtoHybond-NmembranesHybri
8、dizationsperformedandsignalsdetectedRT-PCRTotalRNAfromvariousstages,andadultmouseliverwastreatedwithRNase-freeDNasecDNAsynthesisParallelreactionperformedwithoutreversetranscriptasetoassesspresenceofDNA
