flk-1+ progenitor cells derived from mouse induced pluripotent stem 的flk-1 +祖细胞来自小鼠诱导多能干细胞

《flk-1+ progenitor cells derived from mouse induced pluripotent stem 的flk-1 +祖细胞来自小鼠诱导多能干细胞》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、Mauritz–Flk-1progenitorcellsfrommurineiPSCsSUPPLEMENTARYMATERIALSupplementarymethodsCultivationofiPSCsTheiPSCcloneB4wasroutinelycultivatedandexpandedonmitoticallyinactivatedmurineembryonicfibroblasts(MEFs).TheculturemediumwascomposedofDulbecco’sModifiedEagle’sMedium(Invitrogen,Karlsruhe,Germany)

2、supplementedwith15%FCS(ThermoScientific,Bonn,Germany),0.2mML-glutamine(Invitrogen),0.1mMb–mercaptoethanol(Invitrogen),0.1mMnon-essentialaminoacidstock(Invitrogen),and0.1%huLIF(humanleukemiainhibitoryfactor)conditionedmedium.ThelatterhadbeenproducedbytransienttransfectionofahuLIFexpressionplasmid

3、intohumanembryonickidney293Tcells.Everythreetofourdayscoloniesweredetachedwith0.2%collagenaseIV(Invitrogen),dissociatedintosinglecellswith0.025%trypsin(Sigma-Aldrich,Taufkirchen,Germany)and0.1%chickenserum(Invitrogen)inphosphate-bufferedsaline(PBS)andplatedagainontoMEFs.Feeder-freeiPSCculturefor

4、initiationofdifferentiationandforcolonyassayswasperformedon6-wellculturedishes(ThermoScientific)coatedwith0.1%gelatin(FigureS1).Themediumwassupplementedwith1µMinhibitorofglycogensynthasekinase-3(Merck,Darmstadt,Germany)1.TeratomaassayofiPSCs2.5x105viableiPSCsfromtheB4clonewereinjectedintolowerli

5、mbskeletalmuscleofSCIDbeigemice(n=4).Fourweekslatertumoursweredissectedandparaffinsectionswerestainedwithhaematoxylinandeosin(H&E).Allsectionswereanalysedbyanexperiencedpathologistwithregardtoteratomaformation.14Mauritz–Flk-1progenitorcellsfrommurineiPSCsInvitrodifferentiationofiPSCsBeforestarti

6、ngdifferentiationiPSCswerecultivatedforseveralpassageswithoutfeedercellstoeliminatecontaminatingMEFs.Coloniesweredetachedwith0.2%collagenaseIVanddissociatedintosinglecellswith0.025%trypsinand0.1%chickenserum.Toinitiateembryoidbody(EB)formationinsuspensioniPSCswereculturedat0.5x105cells/mlindiffe

7、rentiationmediumconsistingofIscove’sModifiedDulbecco’sMedium(Invitrogen)supplementedwith15%FCS,0.2mML-glutamine,0.1mMb–mercaptoethanoland0.1mMnon-essentialaminoacidstockandkeptonanorbitalshaker.Fortheformationof“hangingdrop”