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letterstonatureNATURE|VOL433|17FEBRUARY2005|www.nature.com/nature © 2005 N G © 2005 N G © 2005 N G almostexclusivelytotheactivationofresident,agedprogenitorcells,nottotheengraftmentofcirculatingprogenitorcellsfromtheyoungpartner.Lessthan0.1%ofregeneratedmyotubesinagedmusclewereGFP-positive(SupplementaryFig.S4a–c),consistentwithcomparablestudiestestingforthecontributionofcirculatingcellstomuscleregeneration.TheGFPtransgenehasbeenshownnotonlytobeexpressedrobustlyinmaturemyobresofthetransgenicstrain,butcellsisolatedfromthisstrainexpressGFPafterincorporationintoaGFP-negativerecipient.Therefore,thisisclearlyasensitiveandaccuratemeasureofengraftment.NearlyallGFP-positivecellspresentatthesiteoftheinjurywerelocatedintheinterstitialspaces(SupplementaryFig.S4b),andthesecellswereprobablyinltratingleukocytes,ashasbeendescribed.Theseresultsindicatethattheimpairedregenerativepotentialofagedsatellitecellscanbeimprovedbyamodicationofthesystemicenvironment,bymeansofanincreaseofpositivefactorsinyoungmouseserum,adecreaseordilutionofinhibitoryfactorspresentinoldmouseserum,orboth.Thelossofmuscleregenerationwithageisdueatleastinparttoanage-relatedimpairmentintheupregulationoftheNotchligandDeltaaftermuscleinjury.Therefore,wetestedwhetherhetero-chronicparabiosisrestoredDeltaupregulationinagedsatellitecellsandthusenhancedtheiractivationandproliferation.Usingmyo-breexplantationtoassesssatellitecellactivation,weanalysedsatellitecellsfortheexpressionofDelta.InyoungisochronicandheterochronicparabiontstherewasamarkedupregulationofDeltainsatellitecells,whereasDeltainductionwaslackingintheold,isochronicparabionts(Fig.1c),typicaloftheresponseofaged.Notably,satellitecellsfromtheagedpartnersofhetero-chronicparabiontsshowedamarkedupregulationofDelta,com-parabletothatfoundintheiryoungpartners(Fig.1c)andinyoungmicenotsubjectedtoparabioticpairings.TherewasaslightinhibitionofDeltaupregulationinsatellitecellsfromyoungmiceinheterochronicparabiosescomparedwithyoungisochronicparabionts(Fig.1c).Thus,heterochronicparabiosisnotonlyenhancestheproliferativeresponseoftheresidentagedprogenitorcells,italsorestoresthekeymolecularsignallinginthesecellsthatisnecessaryformuscleregenerationTodeterminewhethertherejuvenatingeffectsofheterochronicparabiosiscouldbereplicatedinaninvitrosystem,satellitecells,culturedeitheraloneorinassociationwithmyobreexplants,werepreparedfromyoungandoldmiceandwereculturedinthepresenceofyoungoroldmouseserum,thusrecapitulatingthehumoralaspectsofheterochronicparabioses.Comparedwithyoungsatellitecellsculturedineitheryoungoroldserum,therewasmuchlessupregulationofDeltainoldsatellitecellsculturedwitholdmouseserum(Fig.2a,b);however,youngmouseserumrestoredtheupregulationofDelta(Fig.2a,b)andtheactivationofNotch(Figs2c,d)inagedsatellitecells.TherewasaninhibitoryeffectoftheoldmouseserumonyoungsatellitecellsintermsoftheupregulationofDelta(Fig.2b).Serumfromyoungmicealsosignicantlyenhancedtheproliferationofmyobre-associatedsatellitecellsintheoldculturescomparedwithserumfromoldmice(SupplementaryFig.S3).TherewasreducedproliferationofbothyoungandoldsatellitecellsculturedinyoungmouseserumwhenNotchsignallingwasinhibited(SupplementaryFig.S3).Therefore,enhancedactivationandproliferationofagedmyogenicprogenitorcellsbyserumfromyoungmiceisNotchdependent.Together,theseinvivoinvitrodatademonstratethatcom-ponentsinyoungserumalonearecapableofreversingthemolecu-larandcellularaspectsofage-relateddeclineinmusclestemcellactivation.Inaddition,italsoseemsthatfactorsinoldserumnegativelyaffecttheprocessesrequiredforsatellitecellactivationandmusclerepair.Encouragedbythesendings,wethenexaminedliverfromagedmicesubjectedtoheterochronicparabiosistotestforevidenceofamoregeneralcapabilitytorejuvenateaged,residentprogenitorcells.Inliver,therearemultipletypesofprogenitorcellsthatparticipateinregenerationandhomeostasis,dependingonthephysiologicalorpathologicalcondition.Here,westudiedproliferatinghepatocytesinvolvedinnormaltissueturnover,becausethereisawelldocu-menteddeclineinhepatocyteproliferationwithage.Hetero-chronicandisochronicparabiontswereexaminedforhepatocyteproliferationbytwoindependentcriteria:theincorporationofBrdUorexpressionoftheproliferationmarkerKi67inalbumin-positivecells.Inyoungisochronicparabionts(Fig.3a),thelevelsofbasalhepatocyteproliferationweretwo-tothreefoldgreaterthaninnon-parabiosedcontrols(seeSupplementaryFig.S5a),consistentwithpreviousreportsofeffectsofparabiosisonhepatocytesAlso,clustersofBrdU-positiveproliferatingcellsthatwerenothepatocyteswereobservedinagedlivers(SupplementaryFig.S5b).Proliferationofalbumin-positivecellsinoldisochronicparabiontswaslessthanthatobservedinyoungisochronicparabionts(Fig.3a),consistentwithpreviousreports4,7andourobservation(Sup-plementaryFig.S5a)thatthereisanage-relateddeclineinthebasalrateofhepatocyteproliferationinnon-parabiosedanimals.However,parabiosistoayoungpartnersignicantlyincreased Figure3Heterochronicparabiosisenhancesproliferationofagedliverprogenitorcellsandrestoresmoleculardeterminantsofyoungliverregeneration.,After5weeksofparabioticpairing,miceweregivenBrdUinjections5,3and1daybeforetheywerekilled.Liversectionswereanalysedforhepatocyteproliferationbasedonco-stainingwithalbuminandeitherBrdUorKi67(arrowspointtonucleiofdouble-labelledcells).Hoechstdye(blue)labelsallnuclei.Quanticationofhepatocyteproliferation(seeMethods)isshowntotherightofeachpanel(asterisk,0.05;doubleasterisk,,ExtractsfromliversectionsofparabiontswereimmunoprecipitatedwithanantibodyandthelevelsofBrmandcEBP-wereassessedbywesternblotanalysis.SimilaramountsofcEBP-weredetectedafterimmunoprecipitationandsimilarlevelsoftotalBrmwerepresentinallliverextracts(notshown),conrmingthattheformationofthecomplex,notthelevelsoftheindividualcomponents,wasaffectedbyageandmodiedbyheterochronicparabiosis.Similarresultswereobtainedinfourindependentexperiments.Errorbarsindicatestandarddeviation. letterstonatureNATURE|VOL433|17FEBRUARY2005|www.nature.com/nature hepatocyteproliferationinagedmice(Fig.3a).Asinmuscle,areproduciblereductionofprogenitorcellproliferationwasdetectedinliversofyoungmiceafterparabioticpairingwitholdmice(Fig.3a).Alsoasinmuscle,theenhancementofhepatocyteproliferationinagedmicewasduetoresidentcellsandnottheengraftmentofcirculatingcellsfromtheyoungpartner.Consistentwithpreviousobservations,lessthan0.1%ofhepatocytesexpressedGFPinthenon-transgenic,parabioticpartners.Theage-relateddeclineinhepatocyteproliferationisduetotheformationofanage-speciccomplexofcellcycleregulatorsassociatedwithcEBP-thatinhibitsE2F-drivengeneexpressionOneoftheproteinsdetectedinthiscomplexinold,butnotyoung,liveristhechromatinremodellingfactorBrm.LevelsofthecEBP-Brmcomplexincreaseintheliversofoldrodents,leadingtoadeclineofhepatocyteproliferation.WetestedwhethertheenhancementofagedhepatocyteproliferationcorrelatedwithreducedlevelsofcEBP-–Brmcomplexformationintheagedmicesubjectedtoheterochronicparabiosis.ThecEBP-proteincomplexwasdetectedinliverextractsfromoldisochronicparabiontsbutnotfromyoungisochronicparabionts(Fig.3b),similartopreviousreportsinnon-parabiosedrodents.Notably,theformationofthecEBP-–Brmcomplexwasdiminishedinliverextractsfromoldheterochronicparabionts(Fig.3b).Thus,themoleculardeterminanthadrevertedtothe‘youthful’phenotype,consistentwiththeenhancedproliferativeactivityofhepatocytesinthesemice.Thecomplexwaspresentatelevatedlevelsinyoungheterochronicparabiontscomparedwithyoungcontrols,alsoconsistentwiththemodestinhibitionofhepatocyteproliferationinyoungheterochronicparabionts.Thesedatashowthattheyoungsystemicenvironmentrestoresayoungerproleofmolecularsignallingtotheagedprogenitorcellsintheliverandalsoenhancestheirproliferation.Ourexperimentssuggestthattherearesystemicfactorsthatcanmodulatethemolecularsignallingpathwayscriticaltotheacti-vationoftissue-specicprogenitorcells,andthatthesystemicenvironmentofayounganimalisonethatpromotessuccessfulregeneration,whereasthatofanolderanimaleitherfailstopromoteoractivelyinhibitssuccessfultissueregeneration.Itwillbeofgreatinteresttoidentifythefactorsthathavesuchacriticalinuenceontissue-specicprogenitorcells.Ourstudiesalsodemonstratethatthedeclineoftissueregenerativepotentialwithagecanbereversedthroughthemodulationofsystemicfactors,suggestingthattissue-specicstemandprogenitorcellsretainmuchoftheirintrinsicproliferativepotentialevenwhenold,butthatage-relatedchangesinthesystemicenvironmentandnicheinwhichprogenitorcellsresideprecludefullactivationofthesecellsforproductivetissueMethodsAnimalstrainsandparabiosisYoungC57Bl/Ka-Ly5.2and-actin–eGFPtransgenicmicewerebredandmaintainedbytheWeissmanLaboratoryattheStanfordUniversityResearchAnimalFacility.GFP-transgenicmiceweregeneratedasdescribed,andwerebackcrossedforatleasttengenerationstoC57BL/Ka-Thy1.1mice.OldC57Bl/6micewereobtainedfromtheNationalInstitutesofAgingoragedonsite;youngC57Bl/6micewereobtainedfromJacksonLaboratories.AnimalswerehousedandhandledinaccordancewiththeguidelinesofVeterinaryMedicalUnitoftheVAPaloAltoHealthCareSystemandtheAdministrativePanelonLaboratoryAnimalCareofStanfordUniversity.Parabiosiswasperformedaspublished;theviabilityofthepairswassimilartopublisheddataAntibodiestoDeltaandcEBP-werepurchasedfromSantaCruzBiotechnology,toKi67fromDakoCytomation,toCD34andBrmfromBeckton-Dickinson,toalbuminandlamininfromSigma,andtoactiveNotch-1fromNovusBiologicals/ABCAM.TheantibodytoeMHCwasprovidedbyG.Pavlath.Theanti-BrdUstainingkitwasfromBeckton-DickinsonandtheBrdU-labellingreagentwasfromZymedandwasusedatadoseof50.SolubleDelta-4andJagged-1werefromR&Dsystems.Fluorophore-conjugatedsecondaryantibodiesforowcytometryandimmunouorescencewereobtainedfromCaltagandusedaspreviouslydescribedMuscleinjuryInordertoanalysesatellitecellactivationandmuscleregenerationinvivo,asmallfocalinjurywasmadebyanapplicationofdryicefor5sdirectlytothetibialisanteriormuscleThisgeneratesareproducible‘wedge’injuryinthemusclewithadiscreteborderbetweenuninjuredandinjuredmuscle,andthisborderremainsclearanddistinctduringtheregenerationoftheinjuredtissue.ImmunouorescenceandhistologicalanalysisMusclesweredissected,embedded,cryo-sectionedandimmunostainedaspreviously.Liverwassimilarlypreparedforcryo-sectioningandimmunohistologicalanalysis.LiversthatshowedsignsofgrosspathologicalabnormalitieswereexcludedbeforeTheeffectivenessofthemuscleregenerationwasassessedbycountingthenumberofeMHC-positivebresattheinjurysite,andnormalizingthistothetotalnumberofnucleiintheeldofviewin7–12randomlyselectedsections.Thisispresentedasthe‘regenerationindex’andreectsboththepositive(myotubeformation)andnegative(persistentinammatorycellinltrationandbroblastproliferation)aspectsofregenerationthatarepresentinyoungandagedmice.Toquantifyhepatocyteproliferation,thetotalnumberofalbumin-positivecellsthatwerealsoeitherBrdU-positiveorKi67-positivewerecountedandexpressedasapercentageoftotalalbumin-positivecellsinrandomelds.Ki67-positiveorBrdU-positivecellsthatwerenegativeforalbuminwerenotincluded(seeSupplementaryFig.S5b).SatellitecellactivationandisolationInordertoinduceactivationofsatellitecellsreproduciblyandrobustly,hindlimbmuscles(tibialisanterior,gastrocnemius,bicepsfemorisandquadriceps)weredissected,digestedintobulkbres,andculturedovernightexvivo.Satellitecellswerepuriedfrombulkbresasdescribed.Theresultingpreparationcontainedmononucleatedcellsthatwere95%CD34/M-cadherinsatellitecells,whichcouldbedistinguishedfromremainingdebrisusingowcytometrybythecharacteristicforwardscatterandsidescatterpropertiesofcellsSatellitecellcultureinvitroinvitrostudiesofsatellitecellactivationinthepresenceofmouseserum,bulkbreswereculturedataratioofonevolumeoflooselysettledbrestonolessthan20volumesofOptiMem(pluspenicillin/streptomycin(Gibco/Invitrogen)),supplementedwith5%serumfrommiceoftheindicatedage.Noadditionalmatrixorgrowthfactorswereaddedtothebres.FordetectionofactivatedNotch,satellitecellsweredigestedfrommusclebresasandplatedontoECM-coated(ECMgel,Sigma;diluted1:1,000inice-coldPBS,appliedat1gcm)chamberslides(Permanoxorglass8-well,Nalge-Nunc),andculturedatlowdensityinOptiMEMwith5%mouseserumasaboveuntilxationin4%paraformaldehyde/PBSfor5minforanalysis.Forsatellitecellproliferation,breswereculturedasabovewithone-halfofthemediareplacedeveryday.AfteradditionofBrdUtothemediumfor2h,thepercentagesofBrdU-positivecellsweredeterminedasthetotalnumberofBrdU-positivenucleiperthetotalnumberofnucleiinbreswithassociatedBrdU-positivecells.FlowcytometryCellswereanalysedeitherliveorafterxationwith4%paraformaldehyde.Cellswerestainedwithprimaryantibodiesorwithisotype-matchedcontrolantibodies(1ginl),followedbyincubationwithuorochrome-labelledsecondaryantibodies(1:200–1:500dilutions).Incubationswereperformedfor30minonice(livecells)orfor1hatroomtemperature(xedcells).Cellswereanalysedbyowcytometry(FACScan,BecktonDickinson)usingcompensationforgreenandreduorescenceanddoubletdiscriminationparameters.ImmunoprecipitationandwesternblottingImmunoprecipitationofcEBP-wasfromcollectedliversectionsasdescribed.WesternanalysiswasperformedaspreviouslydescribedStatisticalanalysisDataarepresentedasmeansandstandarddeviations.ComparisonsbetweengroupsweredoneusingStudent’s-testassumingtwo-taileddistributionandunequalvariances.Received14June;accepted9December2004;doi:10.1038/nature03260. 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letterstonatureNATURE|VOL433|17FEBRUARY2005|www.nature.com/nature763 © 2005 N G