Cell-Cell Signaling Through NOTCH Regulates Human Embryonic Stem Cell Proliferation

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716particularmightplayaroleintheetiologyofhumanteratocar-cinomas[22,23].WenowreportfunctionalevidenceindicatingthatNOTCHsignalingdoesplayacrucialroleinthemainte-nanceofundifferentiatedhumanECandEScells.

CellCultureandTreatmentwith?-SecretaseInhibitor

HumanEScellsH1andH7[24]wereculturedinknockoutDul-becco’smodifiedEagle’smedium(DMEM)(Invitrogen,Carlsbad,CA,http://wendang.chazidian.com),supplementedwith20%SerumReplacer(Invitrogen)and4ng/mlbasicfibroblastgrowthfactor(Peprotech,RockyHill,NJ,http://wendang.chazidian.com),onmouseembryonicfibroblastfeederlayers,mitoticallyinactivatedwithmi-tomycinC(Sigma-Aldrich,St.Louis,http://wendang.chazidian.com)asdescribedpreviously[24].HumanECcellsNTERA2cl.D1(NTERA2)and2102Epcl.2A6(2102Ep)[9,25]wereculturedinDMEMcontaining10%heat-inactivatedfetalcalfserum(FCS)(Invitrogen)asdescribedpreviously.FortreatmentwithL-685,485,humanEScellswereharvestedbytreatmentwithcollagenaseIVfollowedbyscrapingwithsterile3-mmglassbeadsandseededassmallcoloniescomposedofapproximately5–20cellsataconcen-trationof1.3?104coloniesperwellofasix-wellplate;humanECcellswereseededassinglecellsobtainedviatreatmentwith0.25%(wt/vol)trypsinin2mMEDTAataconcentrationof2?105cellsperwellofasix-wellplate.HumanESandhumanECcellsweretreated24hoursafterplatingandeverydaythereafterwith20?ML-685,485(Sigma-Aldrich)dilutedinprewarmedmediumfroma10mMstockpreparedindimethylsulfoxide(DMSO).

RNAInterference

Double-strandedsmallinterferingRNAs(siRNAs)correspondingto?2-microglobulin(B2M),Ok(a),CBF-1,NOTCH1,andNOTCH2wereselectedonthebasisofclosenesstoanidealG/Ccontentof50%andsequencespecificityasassessedbypublicsequenceinterrogationusingBLASTandwerechemicallysynthe-sizedbyXeragonInc.(nowQiagen,Valencia,CA,http://wendang.chazidian.com),withthefollowingsenseandantisensesequences:B2M:sense,5?-GAUUCAGGUUUACUCACGUdTdT-3?;anti-sense,ACGUGAGUAAACCUGAAUCdTdTstartingfromnu-cleotide91ofB2Msequence(GenBankaccessionno.AB021288);

OK(a):sense,5?-GAGCAGGTTCTTCGTGAGTTC-3?;antisense,

5?-GAACCACGAATAACTGCTC-3?startingfromnucleotide911ofhumanBSG(Okbloodgroup)sequence(GenBankaccessionno.NM_2001728);

CBF-1:sense,5?-UCGACUACGAUCCCAGACAdTdT-3?;anti-sense,UGUCUGGGAUCGUAGUCGAdTdTstartingfromnu-cleotide564ofCBF-1sequence(GenBankaccessionno.XM_517135);

NOTCH1:sense,5?-AACAUCAACGAGUGCUCCAGCdTdT-3?;

antisense,5?-GCUGGAGCACUCCUUGAUGUU-3?startingfromnucleotide1,807ofNOTCH1sequence(GenBankacces-sionno.NM_17617);

NOTCH2:sense,5?-GCAACACGGUCGAGUGCCUdTdT-3?;an-tisense,5?-AGGCACUCGACCGUGUUGCdTdT-3?startingfromnucleotide4,707ofNOTCH2sequence(GenBankacces-sionno.NM_024408).FortreatmentwithsiRNA,NTERA2and2102Epcellsweretrypsinizedtoproducesinglecellsandplatedintosix-wellplatesatadensityof2?105cellsperwell.TreatmentwithsiRNAwascarriedout24hoursafterplating.TopreparesiRNAfortransfec-tion,10?lofa20-?MsolutionofsiRNAwasincubatedwith4?lofOligofectamine(Invitrogen)in190?lofOpti-MEM(Invitrogen)for20minutesatroomtemperature.Duringthistime,thecellswerefedwith1mloffreshmedium.Cellswereincubatedwith200?loftheOligofectamine/siRNAmixturefor24hoursat37°Candthenfedthenextdaywithfreshmedium.

NOTCHSignalinginHumanEmbryonicStemCells

ReverseTranscription-PolymeraseChainReaction

?-Actin:GCTGCG-3forward,?;5?reverse,-ATCTGGCACCACACCTTCTACAATGA-5?-CGTCATACTCCTGCTTGCT-GATCCACATCTGC-3?;annealingtemperature(Ta)?60;NOTCH-2:forward,5?-TCGTGCAAGAGCCAGTTACCC-3?;re-verse,5?-AATGTCATGGCCGCTTCAGAG-3?;Ta?60;CBF-1:forward,5?-TCCTGTGCCTGTGGTAGAGA-3?;reverse,

5?-ACTGTGGCTGTAGATGATGTGA-3?;Ta?60;

HES1:forward,5?-CAGGCTGGAGAGGCGGCTAAGGTG-3?;

reverse,5?-ATAATACAAAGGCGCAATCCAATA-3?;Ta?60;

TLE-1:forward,5?-CTCCAGCCATAGACCCCCTCGTTA-3?;re-verse,5?-CACTATGAGAGTGCAGCCATCGGGT-3?;Ta?60;

TLE-4:forward,5?-TGGCTGCTTTCTTCCCCCTTTCTC-3?;re-verse,5?-CCCGCCGGCAGCTCCTCTCG-3?;Ta?53;

Id1:forward,5?-CACCCTCAACGGCGAGATC-3?;reverse,5?-CCACAGAGCACGTAATTCCTC-3?;Ta?61;

Id3:forward,5?-GCGGCTGCTACGAGGCGGTGT-3?;reverse,

5?-AAGTGGGCAGGGCGAAGTTGG-3?;Ta?63;

Hey2:forward,5?-CGACGTGGGGAGCGAGAACAA-3?;re-verse,5?-GTGGCGCAAGTGCTGAGATGAGAC-3?;Ta?57.

Antibodies

ThefollowingprimarymonoclonalantibodieswereusedinWesternblottingandimmunostainingaftertitration:bTAN20(ratIgGanti-NOTCH1)[26],DevelopmentalStudiesHybridomaBank,IowaCity,IA,http://www.uiowa.edu/?dshbwww;C651.6DbHn(ratIgGanti-NOTCH2)[26],DevelopmentalStudiesHybridomaBank;andT6719(ratIgGanti-CBF-1)[27],agiftfromtheInstituteofImmunology,Tokyo.ThemonoclonalantibodyTRA-1–60(mouseIgM)waspreparedandusedasdescribedpreviously[28].

Immunocytochemistry

Forimmunocytochemistryallcellswerefixedin4%paraformal-dehydefor15minutesatroomtemperature.Cellswereblockedandstainedin1%goatserum(Sigma-Aldrich);thiswassupplementedwith0.01%TritonX-100duringstainingofintracellularproteins(NOTCH1andNOTCH2).Incubationswithallantibodieswerefor30minutesatroomtemperaturewhileshaking.Fordetectionofnuclearantigens,cellswerestainedonglasscoverslipsandcostainedwiththenucleardyeHoechst33342(Sigma-Aldrich)ataconcentrationof5?g/mlfor10minutespriortomountinginCFPVOHsemipermanentmountantwithAF100antifadent(Citi-fluor,London,http://www.citifluor.co.uk).ThestainingwasimagedusinganOlympusIX70invertedmicroscopewithmercuryarcillumination(OlympusUKLtd.,Watford,U.K.,http://wendang.chazidian.com)withaHamamatsuORCAERblackandwhitecooledcharge-coupleddevicecamera(Hamamatsu,HamamatsuCity,Japan,http://wendang.chazidian.com)controlledviaSimplePCIsoftware(Hamamatsu).Overlaysweregeneratedfromsinglechannelimagesforeachfluorochromeused.AllimageswereadjustedtosamecontrastlevelusingthelevelstoolandoverlaidinPhotoshop(AdobeSystemsInc.,SanJose,CA,http://wendang.chazidian.com).

WesternBlotting

Toprepareproteinextracts,cellswererecoveredbytrypsinization(5minutesforEScellsand2minutesforECcells),counted,andresuspendedinlysisbuffer(1%[wt/vol]NonidetP40,1%[vol/vol]sodiumdeoxycholate,0.1mMphenylmethylsulfonylfluorideinphosphate-bufferedsaline[PBS])ataconcentrationof2.5?107cellspermilliliter.GenomicDNAwassheeredandremovedbypassingthelysatethrougha21-gaugeneedlefollowedbycentrif-ugationat10,000gfor10minutesat4°C.TopreparesamplesforSDS-polyacrylamidegelelectrophoresis,proteinextractswereheatedin1?sampleloadingbufferdiluted1:6from6?loadingbuffer?(0.125MTris-HCl,pH6.8,4%SDS,3%glycerol,0.02%minutes.-mercaptoethanol,Samples5were0.02%runbromphenolon6%or10%bluegelsinatwater)aconcentrationat60°Cforof51.25?10cellsperlaneandtransferredtonitrocellulosemem-

Fox,Gokhale,Walshetal.717

FlowCytometry

Cellsurfaceantigenexpressionwasassayedbyimmunofluores-cenceandflowcytometryasdescribedpreviouslyusingthefollow-ingmonoclonalantibodies:MC631,anti-SSEA3;MC813-70,anti-SSEA4;TRA-1–60;andTRA-1–81.ImmunofluorescenceofspecificantibodieswascomparedwiththatofanegativecontrolantibodyobtainedfromtheparentmyelomacelllineP3X63Ag8asdescribedpreviously.

ApoptosisAnalysis

ApoptoticanalysisofhumanESandhumanECcellswascarriedoutbycostaininglivecellswithrecombinanthumanfluoresceinisothiocyanate(FITC)-conjugatedAnnexinV(R&DSystemsInc.,Minneapolis,http://wendang.chazidian.com)andpropidiumiodide(PI)(Sigma-Aldrich).StainingwithAnnexinV/PIwascarriedoutonfloatingandattachedcellsasfollows.Floatingcellsinthemediumwerecollectedandretained.Attachedcellswerethenharvestedviatreatmentwithtrypsin/EDTAfor2minutesat37°Candaddedtothefloatingcellsuspension.FloatingandattachedcellswerethenpelletedviacentrifugationandresuspendedinAnnexinbuffer(10mMHEPES,140mMNaCl,2.5mMCaCl2,pH7.4)ataconcentrationof1?106cellspermilliliter.Atotalof1?105cells(100?lofthecellsuspension)wereincubatedwith10?lofAnnexinV-FITCfor30minutesatroomtemperatureinthedark.CostainingwithPIwascarriedoutataconcentrationof50?g/mlfor2minutespriortoanalysisbyflowcytometry.DuringapoptoticanalysisofhumanEScells,costainingwiththestemcellmarkerSSEA3wascarriedoutpriortostainingwithAnnexinV/PItofacilitatetheanalysisofcelldeathintheundifferentiatedstemcellcompartment.StainingforSSEA3wascarriedoutasdescribedabovewiththeexceptionthatantibodyincubationswerecarriedoutfor5minutesatroomtemperature.

Figure1.CleavageofNOTCHproteinswasdetectedinavarietyofhumanembryonicstemandhumanembryonalcarcinomacelllinesusingmonoclonalantibodiesspecificfortheintracellulardomainofNOTCH1andNOTCH2.BothreceptorsappeartoundergoS1andS3cleavage.ProductionofS4cleavageproductswasconsistentlyseenonlyinthecaseofNOTCH2.NeitherantibodyreactssignificantlywithproteinsamplesisolatedfromMefs.Abbreviations:Mef,mouseembry-onicfibroblast;MW,molecularweight;NEXT,c-terminalintracellularfragmentoftheNOTCHreceptor;NICD,transcriptionallyactiveformofNotch;TIMC,functionallymatureheterodimericNOTCH

receptor.

brane.Blockingandantibodyincubationswerecarriedoutin5%fat-freemilkpowderinPBSfor1houratroomtemperaturefol-lowedbyextensivewashing.Visualizationwasachievedusingenhancedchemiluminescence(GEHealthcare,LittleChalfont,U.K.,http://wendang.chazidian.com).Antibodiesandconcentrationswereasfollows:anti-NOTCH1antibody(bTAN20,DevelopmentalStudiesHybridomaBank),tissueculturesupernatant,diluted1:5;anti-NOTCH2antibody(C6576.DbHn,DevelopmentalStudiesHy-bridomaBank),ascitesfluid,diluted1:1,000;andanti-mCBF-1(RatIgG,agiftfromtheInstituteofImmunology),diluted1:2,500[27].Anti-mouseIgGhorseradishperoxidase-labeledsecondaryantibody(SantaCruzBiotechnologyInc.,SantaCruz,CA,http://wendang.chazidian.com)(diluted1:1,000)wasusedtodetecttheNOTCH2primary;anti-ratIgGhorseradishperoxidase-labeledsecondaryantibody(SantaCruzBiotechnology)(diluted1:5,000)wasusedtodetectNOTCH1andCBF-1.

CellCycleAnalysis

ToanalyzecellcyclephasedistributionbyPIstaining,humanECandhumanEScellswereharvestedassinglecellsusingtrypsin/EDTA,washedthreetimesinCa2?,Mg2?-freePBS,andthenfixedfor48hoursin70%ethanolat4°C.EnzymaticremovalofRNAwascarriedoutusing0.25unitsofRNaseper5?105cellsatroomtemperature.CellswerethenstainedwithPIatafinalconcentrationof50?g/mlat4°C.Flowcytometricanalysiswascarriedouton10,000gatedeventsusingaCyAnADPbenchtopflowcytometer(DakoCytomation,Glostrup,Denmark,http://wendang.chazidian.com).CellswerefirstgatedaccordingtosizeandthenaccordingtotheareaandintensityofPItoexcludedoublets.Cellcycle

phase

Figure2.PhotomicrographsillustratingtheexpressionoftheNOTCHreceptorsNOTCH1andNOTCH2inhumanpluripo-tentstemcells.(A):TRA-1–60andN1ex-pressioninNTERA2embryonalcarcinoma(EC)andH1embryonicstem(ES)cells(magnification,?60):TRA-1–60(Aa,Ae),N1(Ab,Af),Cy3secondary-onlycontrol(Ac,Ag),andHoechst(Ad,Ah).(B):TRA-1–60andN2expressioninNTERA2ECandH1EScells(magnification,?60):TRA-1–60(Ba,Be),N2(Bb,Bf),Cy3secondary-onlycontrol(Bc,Bg),Hoechst(Bd,Bh).TRA-1–60.Abbreviations:N1,NOTCH1;N2,NOTCH2.

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718NOTCHSignalinginHumanEmbryonicStemCells

Figure3.CanonicalNOTCHsignalingisimportantforthesurvivalofhumanembry-onalcarcinoma(EC)cells.(A):ReductionintheexpressionofNOTCH1,NOTCH2,andCBF-1inducedbytreatmentofthehumanECcelllineNTERA2withsmallinterferingRNA(siRNA),shownbyWesternblotanal-ysis,andtheconsequentdownregulationofputativedownstreamtargetgenesassessedbyreversetranscription-polymerasechainreaction.(B):ThemorphologyofNTERA2cells4daysaftertreatmentwithsiRNA.Scalebars?100?m.(C):GrowthcurvesofNTERA2cellstreatedwithsiRNAtoNOTCH1,NOTCH2,andCBF-1.(D):In-ductionofapoptosisasmeasuredbystainingforAnnexinVandPI.Abbreviations:MW,molecularweight;PI,propidiumiodide;RNAi,RNAinterference.

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Fox,Gokhale,Walshetal.719

Figure4.Humanembryonalcarcinoma(EC)cellsundergoapoptoticcelldeathwhentreatedwiththe?-secretaseinhibitorL-685,485.(A):ThemorphologyofthehumanECcelllineNTERA2following3dayscoculturewithL-685,485.NotethehighlevelsofcelldeathincomparisonwithDMSOanduntreatedcontrols.Scalebars?100?m.(B):Inductionofapoptosisfollowing3daysofcoculturewithinhibitorL-685,458,assessedbyAnnexinVandPIstainingandbycellcounting.Abbreviations:DMSO,dimethylsulfoxide;PI,propidiumiodide.

distributionwasanalyzedusingModfitFlowCytometryModelingSoftware,version3(VeritySoftwareHouse,Topsham,ME,http://wendang.chazidian.com).

ExpressionandLocalizationofNOTCHProteinsinUndifferentiatedHumanECandESCells

BycostainingfixedcellswiththeantibodiestotheNOTCHintracellulardomainandthestemcellmarkerTRA-1–60,weconfirmedthepresenceofcleavedNOTCHreceptorsinthepluripotentstemcellcompartment.TheintracellulardomainofNOTCH1wasdetectedinmostTRA-1–60-positivehumanECandEScells(Fig.2A),NuclearlocalizationofNOTCHwasevidentintheECcells,butitwasmuchmorediffuselyspread,withoutclearnuclearlocalization,intheEScells.Bycontrast,theexpressionpatternofNOTCH2wasalmostidenticalinESandECcells(Fig.2B),inwhichitwasreadilydetectedinthenucleiofallTRA-1–60-positivecells(supplementalonlineFig.2B).

StatisticalAnalysis

Single-time-pointdatawereanalyzedusingthettestprocedureusingSAS,version9.1(SASInstitute,Cary,NC,http://wendang.chazidian.com);equalityofvariancewasconformed.ForRNAinterferencedata,multipleregressionanalysiswascarriedoutusingthePROCMIXEDalgorithminSAS,version9.1.(SASInstitute).

NOTCHReceptorsUndergoProteolyticCleavageinHumanECandESCellCultures

ToassesswhethertheNOTCHreceptorsareexpressedinhu-manECandEScellsandundergothefourcleavageeventstoyieldtheS1,S2,S3,andS4fragmentsrequiredforcanonicalNOTCHsignaling,weusedWesternblotanalysiswithtwomonoclonalantibodiesthatrecognizetheintracellulardomainofNOTCH1andNOTCH2(Fig.1).BothantibodiesidentifiedabandcorrespondinginsizetotheS1andS3productfrombothNOTCH1andNOTCH2inextractsfromtwohumanECcelllines,2102EpandNTERA2;twokaryotypicallynormalhumanEScelllines,H1,H7.s14;andoneculture-adaptedhumanEScellline,H7.s6.Athirdband,mostlikelyrepresentingtheproductreleasedfollowingS4cleavage,wasalsovisibleinsamplesanalyzedforNOTCH2,butnoequivalentbandwaseverseenduringWesternblotanalysisforNOTCH1.Similarly,abandcorrespondingtoS2wasrarely,ifever,detectableforNOTCH1butcould,onoccasion,http://wendang.chazidian.com

NOTCHSignalingIsRequiredfortheProliferationofPluripotentHumanStemCells

WenextsoughttoinvestigatetheroleofNOTCHsignalinginhumanstemcellsusingtransientsiRNAtoknockdownexpres-sionofNOTCH1,NOTCH2,andCBF-1inthepluripotenthumanECcelllineNTERA2.Weobservedamarkedreductioninlevelsofthecorrespondingproteinswithin1–2dayspost-transfection(Fig.3A).Atthesametime,severalknowndown-streamtargetgenesofNOTCHwerealsodownregulated,in-cludingTLE1,TLE4,andHEY2,althoughHES1,atargetofNOTCHsignalinginothersystems,wasnotaffected.TheseknockdownsalsoresultedindownregulationofID1andID3,twomembersoftheIDfamilythatencodeshelix-loop-helixtranscriptionfactors.Notably,thesehavebeenimplicatedincontrolofmouseEScells,butastargetsofBMPratherthanNOTCHsignaling[4].

FollowingknockdownofNOTCH2andCBF-1,thegrowthofthecellswasreduced(Fig.3B,3C)incomparisonwithamocktransfectionandatransfectionofsiRNAagainstagene(Ok(a))whoseremovalhasnoconsequenceforpluripotentcells(p?.05andp??.001atdays4and6aftersiRNAtreatment,respectively).NOTCH2andCBF-1knockdownsalsoresultedinanincreaseincelldeathduetoapoptosis,whichwasevident

at