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ResearchArticleExosomalMicroRNAsDerivedfromHumanAmnioticEpithelialCellsAccelerateWoundHealingbyPromotingtheProliferationandMigrationofFibroblastsBinZhao XiaodongLi ,3 XueqinShi WeiZhang GaofengWu XujieWang , andDahaiHu DepartmentofBurnsandCutaneousSurgery,XijingHospital,FourthMilitaryMedicalUniversity,Xian,Shannxi710032,China �JOEawJVP�VNF 2018 A�UJ��F *% 5420463 10 pa�FT migrationofbroblastsandfurtheracceleratedwoundheal-ing.ThesedatapresentedstrongevidencethathAEC-Exos-derivedmiRNAswouldhavepromisingclinicalapplicationonwoundhealing.2.Methods2.1.PatientsandEthicalApproval.Humanamniontissueswereobtainedfromvepregnantwomen(meanageof26years)intheDepartmentofObstetrics,XijingHospitalan,China).Allexperimentsincludinghumansandani-malswereconductedundertheprotocolsreviewedandapprovedbytheXijingHospitalEthicsCommittees.Thewritteninformedconsentswereobtainedfromallpatientsortheirlegalguardians.2.2.InVivoWoundHealing.MaleBALB/cmiceweighing25gwerepurchasedfromtheAnimalCenterofFourthMilitaryMedicalUniversityandhousedunderstandardlab-oratoryconditions.Micewererandomlydividedintofourgroups:(1)thephosphatebuersaline(PBS)controlgroup);(2)theExos-RNasetreatmentgroup();(3)theExos-PROsetreatmentgroup();and(4)theexosometreatmentgroup().Micewereanesthetizedbyintra-peritonealinjectionofpentobarbitalsodium(20mg/kgbw).Thehaironthebackandankwereclipped;1cm1cmfull-thicknesswoundsweregeneratedoneachmouseasdescribed[8].Wepreviouslyreportedthat50g/mLexo-somesderivedfromhumanamnioticepithelialcellsacceler-atedwoundhealing[7].Herein,100Lof50exosomesorequalamountofexosomestogetherwitheitherPROseorRNaseAwereinjectedintothesurroundingtissuesofthewoundsatfoursitesonday1andday3.Inthecontrolgroup,equalamountofPBSwasinjected.ThewoundsizewasmeasuredandanalyzedinatimelymannerwiththeImageProPlus6.0software(MedicalCybernetics,USA)accordingtotheprotocol[9].Skintissuesampleswerecol-lectedforfurtherhistologicalanalysis.Woundclosurerate(%)wascalculatedas((originalwoundareaonday0areaonday)/originalwoundareaonday0)2.3.HistologicalExamination.Skinsampleswereharvested,xed,dehydrated,paran-embedded,andthenslicedintomthicksections,followedbyhematoxylin-eosinstaining(H&E)andimmunohistochemistry(IHC)asdescribedpre-viously[10,11].TheimagesofstainedsectionswereobtainedbytheFSX100microscope(Olympus,Japan).2.4.IsolationofhAECsandhAEC-Exos.hAECswereisolatedandculturedaspreviouslydescribed[12].Briey,humanamnionlayerwasmechanicallypeeledofromtheplacentaandrinsedwithsterilizedPBS.Amnioticmembranesweresubjectedto0.25%trypsinandincubatedat37Cwithcon-stantagitationfor1h.Trypsinwasinactivatedbytheaddi-tionofDMEMsupplementedwith10%exosome-depletedFBS(SystemBiosciences,USA,number022515).CollectedhAECswerelteredthrougha200mcellstrainerandcen-trifugedat1000gfor5min;isolatedhAECswereculturedinDMEMsupplementedwith10%exosome-depletedFBSand1%antibiotic-antimycoticinahumidiedincubatoratCwith5%COFortheisolationofhAEC-Exos,1hAECswereseededintotheT75CultureFlask(Corning,USA)inDMEMsupplementedwith10%exosome-depletedFBSfor48h;theculturemediumofhAECswascollectedandcentrifugedatgfor5min.Aftercentrifugation,thesupernatantwascollectedandlteredthrougha0.22lter(Millipore,USA)toremovecelldebris.Theremainingsupernatantwasthenultracentrifugedat100,000gwiththerotorTi70(BeckmanCoulter,USA)for12h.Theexosome-enrichedpelletswereobtainedandresuspendedinasmallvolumeofPBSandthenmeasuredforproteincontentusingtheBCAproteinassaykit(BOSTERBiologicalTechnology,China)andadjustedtheconcentrationto50g/mL,storedC.hAEC-Exoswereexaminedtoconrmtheircharacteristicswithnanoparticletrackinganalyzer,trans-missionelectronmicroscope,owcytometry,andwesternblot,respectively.2.5.SizeDistributionandZeta-PotentialofhAEC-Exos.sizeandzeta-potentialofexosomeswereexaminedbytheNanoparticleTrackingAnalyzer(ParticleMetrixGmbH,Germany)withthecorrespondingZetaView®software[13].ThisinstrumenttrackedtheBrownianmotionofparticlesovertimeandsubsequentlycalculatedparticlesizes.2.6.TransmissionElectronMicroscope(TEM).ForTEMdetection,exosomeswerexedwith2%phosphotungsticacid,droppedontoaformvar/carbon-coatedcoppermeshgrids,andthenlefttodryatroomtemperature.ExosomeswereexaminedwithaHitachi7100transmissionelectronmicroscope(Hitachi,Japan).2.7.FlowCytometry.Theexosomeswereconrmedbyexo-somalsurfacemarkerCD63usingowcytometry[14].y,exosomeswereattachedonto4maldehyde/sulfatelatexbeadsandincubatedwithexosome-CD63antibody(SystemBiosciences,USA).ThepercentofpositivebeadswascalculatedbytheFACSAriaIIIinstrument(BD,USA).2.8.CellTreatment.Accordingtopreviousstudies,exosomesmainlycontainproteinsandRNAs[15].TodeterminewhichmoleculescarriedwithinhAEC-Exoswereresponsi-bleforwoundhealing,puriedhAEC-Exoswerepretreatedwith0.04%TritonX-100andthentreatedwithRNaseA(100units/mL)for60mintodepleteRNAsorPROseg/mL)for60mintodegradeexosomalproteinsaccordingtotheprotocoldescribedpreviously[16,17].ThecacyofPROseorRNaseenzymetreatmentswasdetectedbygelelectrophoresisorCoomassieblueG250staining,respectively.Furthermore,wealsotestedtheproteinconcen-trationbytheBCAproteinassaykit(BOSTERBiologicalTechnology,China)andRNAcontentbyspectrophotomet-ricratiousingabsorbancemeasurementsatwavelengthsof260nmand280nm.Adulthumandermal(ATCC,USA)weredividedintofourgroups.Cellsinexper-imentalgroupsweretreatedwithequalamountsofpurihAEC-ExostogetherwitheitherPROseorRNaseA.EqualStemCellsInternational amountsofhAEC-Exostreatedwith0.04%TritonX-100serveascontrol.2.9.PKH26LabelingofExosomes.ExosomeswerelabeledwithPKH26(Sigma,Germany)accordingtothemanufac-sprotocol.Briey,exosomeswereresuspendedindilu-entC(Sigma,Germany),mixedwithPKH26intoaconcentrationof1M,andthenincubatedat37Cfor5min.Excessdyewasremovedbycentrifugation.PKH26-labeledexosomeswerecoculturedwithbroblastsinFBS-freemediumfor12h.TheinternalizationofhAEC-Exosbybroblastswascounterstainedwith4,6-diamidino-2-pheny-lindole(DAPI)(Sigma,Germany)andobservedundertheZEISSinverteduorescencemicroscope(ZEISS,Germany).2.10.ImmunouorescentStaining.Todetecttheinternaliza-tionofPKH26-labeledexosomesbybroblasts,cellswerexedwith4%formaldehydeandwashedwithPBS.NucleiwerecounterstainedwithDAPI(Sigma,Germany).ImagesweretakenbyusingtheFSX100microscope(Olympus,Japan).Ki67wasstainedandusedforevaluatingcellprolifer-ation.Briebroblastswereseededatadensityof1,incubatedinserum-depletedmediumfor12h,stimulatedwithhAEC-Exosforanother48h,andthenincu-batedwithanti-ki67antibody(Abcam,USA)andcounter-stainedwithDAPI.2.11.WesternBlot.Exosomeswerelysedinlysisbucontainingacompleteproteaseinhibitortablet(Roche,Swiss).Fivemicrogramsofproteinswasloadedontopolyacrylamidegel,separatedbyelectrophoresis,andthentransferredtothepolyvinylidenediuoridemembrane(PVDF).Afterblockingwith5%nonfatmilk,PVDFmem-branewasincubatedwithrabbitmonoclonalanti-humanCD63,CD9,andCD81antibodies(Abcam,USA)over-nightat4C,followedbytheincubationwithhorseradishperoxidase-conjugatedgoatanti-rabbitsecondaryantibody(BOSTERBiologicalTechnology,China).Proteinswerevisualizedbytheenhancedchemiluminescencesystem(AlphaInnotech,USA).2.12.Real-TimeCellProliferationAnalysis.Fibroblastswereseededinto16E-plates(ACEA,USA)atthedensityofcells/wellandculturedinahumidied5%COincubator.Toavoidtheinterferenceofbackground,intherststepofanRTCAassay,cellmediumisaddedtothewellsandabackgroundvalueistaken,afterthat,ditreatmentswereappliedtoeachwellfor24h().ThecellindexineachE-platewellwascalculatedbytheRTCAsoftware1.2(Roche,France).Thegraphswerereal-timeoutputsgeneratedfromtheiCELLigencesystem(ACEA,USA)[18]. TEM 50 nm 50 nm (a) Exosome-CD63FACS (b) CellsGAPDHWestern blot exosomes (c) ZetaView (d) DiameterNumber relative1010010002000Mean diameter=103 nm 1 1:CharacterizationofhAEC-Exos.(a)TEMimageofhAEC-Exos().(b)FlowcytometryanalysisofexosomalmarkerCD63).(c)WesternblotanalysisofexosomalmarkersCD63,CD9,andCD81().(d)ZetaViewanalysisofhAEC-Exos().(e)SizedistributionofhAEC-Exos(meandiameter=103nm,StemCellsInternational 2.13.MigrationAssay.Fibroblastsweresubjectedtodienttreatmentsinconventionalscratchwoundassay[19].broblastswereseededatadensityof1ina35mmdishandstarvedinserum-freeDMEMfor12h.Gapswerecreatedinthemiddleofeachwellincrossshapewithapipettetip.CellswerethenwashedgentlywithPBSandappliedwithdierenttreatments,followedbyincubationat37Cin5%COinairatmospherefor24h.Imagesweretakeninatimelymanner.Gapareasweremeasuredandrecordedandthencomparedtotheinitialgapsizeat0hbytheImageProPlus6.0software.Theareaofmigrationwascalculatedas(%)=((originalblankareablankareaonh)/originalblankarea)100%.Themigrationofbroblastswasalsodeterminedbytranswellassayusing8mporeltersaccordingtothemanufac-turersrecommendation.Approximately1wereseededintotheuppercompartment,whileexosomeswithdierenttreatmentswereaddedintothelowercom-partment.Cellswerecoculturedfor24h;nonmigratedcellsintheupperchamberwerewipedandstainedwith0.4%crystalviolet.2.14.StatisticalAnalysis.Alldatawerepresentedasthemeanstandarddeviation(SD).StatisticalanalyseswereperformedusingtheSPSS11.0software(SPSSInc.,USA).One-wayANOVAwasusedtocompareamonggroups,followedbytheBonferroniposthoctest. wascon-sideredstatisticallysigni 1000 bp700 bp500 bp400 bp300 bp200 bp100 bpExos-PROseExos-RNaseExosomesMarker (a) 170 KD130 KD100 KD70 KD55 KD40 KD25 KD15 KD Exos-PROseExos-RNaseExosomesMarker (b)  Protein concentration (30 min60 minExos without treatmentExos-RNaseExos-PROse (c)  15 min30 min60 minExos without treatmentExos-RNaseExos-PROse RNA concentration ( 2:CharacterizationofpuriedhAEC-ExostogetherwitheitherPROseorRNasetreatment.(a)GelelectrophoresshowthatexosomesweredepletedofRNAsafterRNasedigestion.(b)CoomassiebluestainingshowsthatproteinswerethoroughlydegradedafterPROsetreatment().(c)DetectionofproteinconcentrationbytheBCAproteinassaykitandRNAcontent(d)bythespectrophotometricratio( StemCellsInternational 3.Results3.1.IsolationandCharacterizationofhAEC-Exos.withroundorovalmorphologywereobservedbyTEM;thediametersrangedfrom30to150nm(Figure1(a)).FlowcytometrywasusedtoanalyzeexosomalsurfacemarkerCD63,andresultsshowedthathAEC-ExoswerepositiveforCD63(85.2%4.8%)(Figure1(b)).WesternblotresultsfurtherconrmedthatexosomalmarkersCD9,CD63,andCD81wereexpressedinhAEC-Exos(Figure1(c)).Dynamictrackingcapture(Figure1(d))andparticlesizedistribution(Figure1(e))weremeasuredbynanoparticletrackingana-lyzer;resultsshowedthatthesizeof90%particlesdistributedbetween30and150nm(theaveragediameter=103nm).3.2.EvaluationofhAEC-ExosTogetherwithEitherPROseOrRNaseA.edhAEC-Exoswerepretreatedwith0.04%TritonX-100andthentreatedwithPROseorRNaseA.Resultsfromgelelectrophoresis(Figure2(a))showedthatRNAcomponentscarriedwithinhAEC-ExosweremainlysmallRNAswiththesizebelow100base-pair,whileproteincomponentsinhAEC-ExoswerealmostcompletelydegradedbyPROsetreatment(Figure2(b)).Furthermore,wetestedtheproteinconcentration(Figure2(c))bytheBCAproteinassaykitandRNAcontent(Figure2(d))bythespectrophotometricratiousingabsorbancemeasure-mentsatwavelengthsof260nmand280nm.AsshowninFigure2(c),after60minofPROseincubation,theproteinscontainedinhAEC-Exoswerebarelydetectable.However, Merge 20 m (a) Exos-PROseControlExos-RNaseExosomes Cell indexTime (h) (b) Control Exo-RNase Exo-PROse Exosomes  Cell index1224 (c) Ki67DAPIKi67DAPIKi67DAPIKi67DAPI 3:EectsofhAEC-Exosontheproliferationofbroblasts.(a)FluorescentmicroscopyanalysisshowsthatPKH26-labeledhAEC-Exoswereinternalizedbybroblasts.NucleiwerecounterstainedwithDAPI.Scalebar=20m.(b)TheevaluationofproliferationafterdierenthAEC-ExostreatmentsbytheiCELLigencesystem.Eachgroupwasassessedintriplicate.Fibroblastswithoutanytreatmentserveascontrol.(c)Quantitativeanalysisofbroblastproliferationat12hand24h.(d)Fluorescentmicroscopyanalysisofki67immunostaining(red).NucleiwerecounterstainedwithDAPI.Scalebar=10m.( StemCellsInternational