CD90 determined two subpopulations of gliomaassociated mesenchymal stem cells with different roles in tumour progression | Cell Death & Disease

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Abstract

Humanglioma-associatedmesenchymalstemcells(gbMSCs)arethestromalcellcomponentsthatcontributetothetumourigenesisofmalignantgliomas.RecentstudieshaveshownthatgbMSCsconsistoftwodistinctsubpopulations(CD90+andCD90鈭?/sup>gbMSCs).However,thedifferentrolesingliomaprogressionhavenotbeenexpounded.Inthisstudy,wefoundthatthedifferentrolesofgbMSCsingliomaprogressionwereassociatedwithCD90expression.CD90highgbMSCssignificantlydrovegliomaprogressionmainlybyincreasingproliferation,migrationandadhesion,whereasCD90lowgbMSCscontributedtogliomaprogressionchieflythroughthetransitiontopericytesandstimulationofvascularformationviavascularendothelialcells.FurThermore,discrepanciesinlongnon-codingRNAsandmRNAsexpressionwereverifiedinthesetwogbMSCsubpopulations,andthepotentialunderlyingmolecularmechanismwasdiscussed.OurdataconfirmforthefirsttimethatCD90highandCD90lowgbMSCsplaydifferentrolesinhumangliomaprogression.TheseresultsprovidenewinsightsintothepossIBLefutureuseofstrategiestargetinggbMSCsubpopulationsingliomapatients.

Introduction

Gliomasaretheprimarycentralnervoussystemtumourswiththehighestincidencedespiteprogressmadeincombinationtreatmentusingsurgical,rADIotherapyandchemotherapyapproaches1,2.Betterunderstandingofthetumourmicroenvironmentwillenablepursuitanddevelopmentofapromisingtherapeuticstrategyforgliomas3,4.

Generally,thetumourmicroenvironmentconsistsoftumourcells,fibroblasts,endothelialcells,mesenchymalstemcell(MSCs),andinflammatorycellsaswellascytokinesandchemokinessecretedbytumourandstromalcells3.Ingliomas,MSCscanberecruitedbysomefactorsintothetumourmicroenvironmentandmodulatetumourdevelopment5.Ourteamreportedthatglioma-associatedMSCs(gbMSCs)hadclassicalMSCsurfaceMarkers(CD105,CD73,CD90andCD44)butlackedexpressionofCD14,CD34andCD45.Gb-MSCsshowplasticadherentmorphologyandhavethecapacitytodifferentiateintoosteoblasts,adipocytesandchondroblastsinvitro6,7.ThepercentageofgbMSCsinhigh-gradegliomasamplesiscloselyrelatedtotheirsurvivalwithinGBMpatients8.Furthermore,wefoundthathumangbMSCswereintegralcomponentsinthepericytetransitionandtumourvascularformation.6SomereportshavedemonstratedthatgbMSCscanincreasegliomastemcellself-renewalandproliferationviasecretionofexosomesandfactors.9

RecentreportsfoundthatgbMSCscouldbedividedintotwosubtypesaccordingtoCD90expression(CD90+gbMSCsandCD90鈭?/b>gbMSCs).CD90鈭?/b>cellsareregardedasmoreactiveingliomavascularizationandimmunosuppressionthantheirCD90+counterparts,andCD90鈭?/b>andCD90+gbMSCsdiffergreatlyintheirmRNAexpressionpatterns10.However,theBIOLOGicalpropertiesofthesetwodistinctsubpopulationsandtheireffectsongliomahavenotbeenfullyelaborated.

Inthisstudy,weelaboratelysortedtwodistinctMSC-likecellpopulationsfromgbMSCsaccordingtodifferencesinCD90surfacemarkerexpressionandinvestigatedthedifferentrolesofthesetwogbMSCsubpopulationsingliomaprogression.

MaterialsandmethodsTumoursamples

HumanbraintumoursampleswereobtainedfromtheNeurosurgeryDepartmentatUnionHospitalinWuhan,China,afterpatientswithgliomaprovidedinformedconsent.Thespecimenswerereviewedbyaneuropathologisttoassessthegradeandtumourtypebeforetheassayswereperformed(Table1).Typically,cellseparationwasperformedwithin1鈥塰oftumourresection.

Table1Characteristicsof14patientswithgliomasusedforgbMSCisolationinthecurrentstudyFullsizetableIsolationandcultureofhumangbMSCs

ThesamplewastransferredtoaPetridish,washedthreetimesinphosphate-bufferedsaline(PBS,HyClone,USA),andcutinto1-mm3pieces.Next,0.25%trypsin(BIYUNTIAN,China)wasaddedtothetumourspecimens.Then,thesinglepiecesweredigestedfor20鈥塵in,filteredwitha70-碌mnylonmesh(Corning,USA)andcentrifugedat1000鈥塺pmfor15鈥塵in.ThemononuclearcellswerecollectedbyFicoll(2:1Genview,USA)densitygradientcentrifugationat1500鈥塺pmfor20鈥塵inwithoutbraking.Singlecellswerere-sUSPendedinDMEM(HyClone,USA)containing10%foetalbovineserum(BI,Israel)and100鈥塙/mlofpenicillin/streptomycin(GibcoBRL,GrandIsland,NY,USA),seededintoa25-cm2cultureflask(Corning,USA)andincubatedat37鈥壜癈andhumiditywith5%CO2.Themediumwaschanged1鈥?timeseveryweek.Cellsat70鈥?0%confluencywerepassagedusingAccutase(StemCell,Canada)andusedforexperimentsatpassages2to3.

Celllines

U87-MGswerepurchasedfromtheAmericanTypeCultureCollection(ATCC,Gaithersburg,MD,USA),andhumanumbilicalveinendothelialcells(HUVECs)werepurchasedfromLonza(MD,USA).TheU87-MGsandHUVECswereculturedinDulbecco鈥檚modifiedEagle鈥檚medium(DMEM)(HyClone,USA)andRPMI1640medium(Gibco,USA),respectively,supplementedwith10%foetalbovineserum(FBS;BI,Israel)and100鈥塙/mlofpenicillin/streptomycin(GibcoBRL,GrandIsland,NY,USA)inhumidifiedatmosphereat37鈥壜癈with5%CO2.

Conditionedmedia

Forallexperiments,U87-MGs,CD90highgbMSCsandCD90lowgbMSCswereseededintoT25tissuecultureflasksinDMEMwith10%FBScontainingpenicillin(100鈥塙/mL)/streptomycin(100鈥塵g/mL)(GibcoBRL,GrandIsland,NY,USA).Whenthecelldensityreached50鈥?0%confluency,themediumoftheU87-MGswasreplacedwithserum-freemedium(DMEM)andDMEMsupplementedwith10%FBS,themediumoftheCD90highandCD90lowgbMSCswasreplacedwithserum-freemedium(DMEM),andthecellswereculturedfor72鈥塰.Conditionedmediawerecollectedfromtheflasksandcentrifugedat1000鈥壝椻€?i>gfor10鈥塵intoremovecellsandcellulardebris.Afterward,thecollectedconditionedmedia(CD90highCM,CD90lowCM,0%gb-CM,andS-gb-CM)werestoredat鈭?0鈥壜癈priortouse.

DifferentiationofgbMSCs

ThegbMSCswereAdipogenically,osteogenicallyandchondrogenicallyinducedusingready-to-usedifferentiationmedia(allfromStemcell,Canada)followingthemanufacturer鈥檚instructions.AdipogenicdifferentiationwasevaluatedbyoilredOstaining,osteogenicdifferentiationwasevaluatedbyAlizarinredstainingandchondrogenicdifferentiationwasevaluatedbyAlcianbluestaining(allfromSigma,USA).Thespecificstepswereasfollows.

Forosteoblastdifferentiation,thecellswereculturedingrowthmediuminasix-wellplateandincubatedat37鈥壜癈with5%CO2untiltheywereapproximately70鈥?0%confluent.Next,themediumwasreplacedbycompleteosteogenicstimulatorymedium,thecellswereincubatedat37鈥壜癈andthemediumwaschangedevery3days.Thedifferentiationassaytookapproximately3weeks.OsteogenicdifferentiationwasvisualizedbyAlizarinredSstaining.

Foradipocytedifferentiation,thecellswereculturedinstandardmediuminasix-wellplateat37鈥壜癈and5%CO2untiltheywereapproximately90鈥?00%confluent.Then,themediumwasaspiratedandreplacedwithcompleteadipogenicdifferentiationmedium,whichwaschangedevery3days.Thedifferentiationassaytookapproximately14days.AdipogenicdifferentiationwasvisualizedbyoilredOstaining.

Forchondrocytedifferentiation,cellpelletsweregrowninchondrogenesisinductionmediumfor21days,andhalfofthemediumwaschangedduringdifferentiation.Histologicalsectionsofthepelletweregeneratedbyfixingthepelletsin10%formalinfor30鈥塵inatroomtemperature(15鈥?5鈥壜癈),followedbysubsequentstandardparaffinembeddingmethodsandstainingof6-碌m-thicksectionswithAlcianblue.

Magneticactivatedcellsorting(MACS)ofthegbMSCs

gbMSCsgrowningoodconditionwereusedfortheMACSexperiment.First,thecellswereimmuno-labelledwithCD90microbeads(Miltenyi,Germany).Magneticlabellingwasperformedstrictlyaccordingtothemanufacturer鈥檚instructions.Briefly,thegbMSCsweredigestedusingAccutase(Stemcell,Canada)andcentrifugedat1500鈥塺pmfor6鈥塵in.Thecellpelletwasre-suspendedin80鈥壜礚ofprecooledsortingbufferper107totalcells,and20鈥壜礚ofCD90MicroBeadswasaddedper107totalcells.Then,thesolutionwasmixedwellandincubatedfor15鈥塵ininthedarkintherefrigerator(2鈭?鈥壜癈).Thecellswerewashedbyadding2鈥塵Lofbufferper107cellsandcentrifugedat1500鈥塺pmfor6鈥塵in.Thesupernatantwasaspiratedcompletely.Thecellswerere-suspendedin500鈥壩糒ofsortingbuffer.MagneticseparationwasperformedusingtheautoMACSProSeparator(Miltenyi,Germany).Ultimately,weobtainedCD90highgbMSCsandCD90lowgbMSCs.

Flowcytometry

FlowcytometryanalysiswasperformedusingFluorochrome-conjugatedantibodies.Briefly,differentpassagesofgbMSCs,CD90highgbMSCsandCD90lowgbMSCsweretrypsinizedandwashedinPBS,andthenthepelletswerere-suspendedinfluorescent-activatedcellsorting(FACS)buffer.Thesesingle-cellsuspensionswereincubatedinthedarkat4鈥壜癈for30鈥塵inwithPE-,FITC-,PE-Cy7-,APC-Cy7-,PerCP-andAPC-conjugatedantibodiesagainsthumanCD73,CD105,CD90,CD44,CD13,CD34,CD31,Desmin,伪-SMA(fromeBioscience,USA),NG2andPDGFR-尾(fromRDSystems,USA).Then,thecellswerecentrifuged,re-suspendedinPBSandanalysedusingaflowcytometer(BDBiosciences).ThedatawerecollectedandanalysedusingtheFlowJo(TreeStar,Ashland,OR,USA)software.

Cellproliferationassay

CellviABIlitywasanalysedusingtheCellCountingkit-8(CCK-8Kit,DojindoLaboratories,Japan).U87cells(3000/well)wasseededintoa96-wellplateandculturedovernight.Then,themediumwasreplacedwith100鈥壜祃ofserum-freemedium(0%DMEM),serum-freeCD90highgbMSC-conditionedmedium(CD90highCM),orserum-freeCD90lowgbMSC-conditionedmedium(CD90lowCM)andculturedfor1,2,3,or4days.CD90lowandCD90highgbMSCswereseededinto100鈥壜祃ofserum-freemediumsupplementedwith10%FBS(10%DMEM)andculturedfor1,2,3,or4鈥塰.Atvarioustimepoints,10鈥壜祃ofCCK-8wasaddedtoeachwellandincubatedfor2鈥塰at37鈥壜癈with5%CO2.Then,theabsorbanceofeachwellwasmeasuredat450鈥塶musingamicroplatereader(Perkinelmer,USA).Atleastthreewellswereusedforeachsampleindifferentmedia.Theassayswererepeatedatleastthreetimes.

Adhesionassay

U87cellsweretreatedwithCD90highCMandCD90lowCMfor48鈥塰.Then,thecellswereseededatadensityof1鈥壝椻€?04cells/wellin96-wellplatespre-coatedwithmatrixadhesiveandincubatedwith0%DMEM,CD90highCMandCD90lowCMfor1鈥塰at37鈥壜癈inanatmosphereenrichedwith5%CO2.Thenon-adherentcellswereremovedbywashingcarefullythreetimeswithPBS,andthecultureswereincubatedin100鈥壜祃ofmediumwith10鈥壜祃ofCCK8(DojindoLaboratories,Japan)for2鈥塰.Celladhesionwasanalysedbymeasuringtheopticaldensity(OD)at450鈥塶minamicroplatereader(PerkinElmer,USA).Atleastthreewellswereusedforeachsampleindifferentmedia.Theassayswererepeatedatleastthreetimes.

MigrationassaysTranswellchamberassay

ThemigrationcapacityoftheU87cellswasevaluatedin24-wellplateswithTranswellinsertswithan8-碌mporesize(BDFALCON,USA).U87cells(5鈥壝椻€?04/ml)in100鈥壜祃ofserum-freeDMEMwereaddedtotheupperchamber,and800鈥壜祃ofthetestedsamples(0%DMEM,CD90highCMandCD90lowCM)wasplacedinthelowerchambers.Cellmigrationwasallowedfor24鈥塰at37鈥壜癈with5%CO2.Followingincubation,themediawereaspirated,andthecellsremainingontheuppersurfaceofthepolycarbonatemembranewereremovedwithacottonswab.ThecellsthatmigratedtothelowersurfacewerestainedwithGiemsafor20鈥塵in.Cellcountingwasperformedunderaninvertedmicroscopebytworesearchersindependently.Theaveragenumbersofmigratedcellsweredeterminedbycountingthecellsin5randomhigh-powerfields(x200).

Wound-healingassay

Awound-healingassaywasusedtoevaluatethemigrationabilityoftheCD90highandCD90lowgbMSCsindifferentmediainvitro.Thecellswereincubatedin6-wellplatesuntiltheyreached90鈥?00%confluence.Then,crosslineswerecarefullymadeusinga10-碌lPipettetip,andthedebriswaswashedawaywithPBS.Themediumwasreplacedwithserum-freemedium(0%DMEM),standardmedium(10%DMEM),serum-freeglioblastoma-conditionedmedium(0%gb-CM)andstandardglioblastoma-conditionedmedium(S-gb-CM).TheareasofthescratchwoundswerephotographedwithanOlympusmicroscopeat0and8鈥塰.Theassayswereperformedintriplicateatleastthreetimes,andthedatawereanalysedusingtheImageJsoftware(NIH,USA).

ImmunochemistryandImmunofluorescence

Forimmunochemistry,tumourtissuespecimenswerefixedin4%paraformaldehydeandembeddedinparaffinaftercollectionfromsacrificedmice.Thetissuesectionswerecutanddewaxed,andthenantigenswereretrieval.TheslideswererinsedinPBS,incubatedovernightat4鈥壜癈withdilutedanti-CD31(1:50)andanti-Ki67(1:800)antibodies(ProteinTech,WuHan,China),andthenincubatedwithanHRP-conjugatedsecondaryantibody(1:1,Boster,WuHan,China).BindingwasdetectedusingaDABsolution(Boster,WuHan,China).Thetissueswerecounterstainedusinghaematoxylin(Boster,WuHan,China).ImagesofthestainedtissuesampleswereobtainedusinganOlympusmicroscope.

Forimmunofluorescence,humanGBMsurgicalspecimenswereharvestedandfixedwith4%paraformaldehyde,andsectionswerepreparedforimmunostaining.Nonspecificstainingwasblockedbypre-incubationofthesesectionsingoatserumdilutedwithPBSfor30鈥塵inatroomtemperature.Theprimaryantibodiesusedwereasfollows:goatanti-CD105polyclonalantibody(1:25,RDSystems,USA)andrabbitanti-CD90monoclonalantibody(1:25,Boster,WuHan,China).Afterincubationwiththeprimaryantibodyovernightat4鈥壜癈,thesectionswererinsedseveraltimeswithPBSandincubatedwiththeappropriatesecondaryantibodiesatroomtemperaturefor1鈥塰.Thesecondaryantibodiesusedwereasfollows:Cy3-conjugatedgoatanti-rabbitandFITC-conjugatedgoatanti-goatantibodies(1:100,allfromBoster,WuHan,China).AfterwashinginPBS,thesectionswerecounterstainedwithDAPI(Beyotime,WuHan,China)andmountedwithanti-fademountingmedium.ImmunofluorescencemicroscopywasperformedwithanOlympusmicroscope.

Tubeformationassay

Growthfactor-reducedMatrigel(BD,USA)wasaddedtoaflat-bottomed,pre-cooled,96-wellplate.Afterincubationat37鈥夆剝with5%CO2for1鈥塰.CD90highandCD90lowgbMSCswerelabelledusingCalceinAM(Tocris,USA)andDiO(Yeasen,Shanghai,China).CalceinAM-labelledCD90highandCD90lowgbMSCswereseeded(2鈥壝椻€?04/well)intowellscontainingserum-freemedium(0%DMEM),standardmedium(10%DMEM),serum-freeglioblastoma-conditionedmedium(0%gb-CM)orstandardglioblastoma-conditionedmedium(S-gb-CM).HUVECs(2鈥壝椻€?04/well)wereseededintowellscontaining0%DMEM,CD90highCMandCD90lowCM.TheHUVECs(2鈥壝椻€?04/well)wereco-culturedwithDiO-labelledCD90highandCD90lowgbMSCs(1鈥壝椻€?04/well)inwellscontainingserum-freeglioblastoma-conditionedmedium(0%gb-CM).Then,the96-wellplatewasincubatedat37鈥壜癈in5%CO2.Threewellswereusedforeachmediumsample.After6鈥塰,tubeformationwasphotodocumentedwithanOlympusmicroscope.Capillary-liketubeformationandtheattachmentofCD90highandCD90lowgbMSCsontotheHUVECswereanalysedinthreerandomfieldsofviewperwellusingtheImageJsoftware(NIH,USA).

ELISAassay

TheVEGF,bFGF,SDF-1伪,CCL5,MMP9andIL-6levelsinthesupernatantsofeachmediumsample(0%DMEM,CD90highCMandCD90lowCM)weremeasuredusingtheirrespectiveELISAkits(Neobioscience,China).Allprocedureswereperformedasdescribedinthemanufacturer鈥檚instructions.Theabsorbancewasmeasuredat450鈥塶m.Threewellswereusedforeachmediumsample.Theassayswererepeatedthreetimes.

RNAextractionandClariomDmicroarray

CD90highandCD90lowgbMSCswereculturedinstandardmediumuntilapproximately90%confluent.TotalRNAwasextractedfromthreedifferentbatchesofCD90highgbMSCsandthreecorrespondingCD90lowgbMSCsculturedinstandardmediumusingtheTRIzolreagent(Invitrogen,USA).AfterthetotalRNAqualitywasanalysedwithaNanodrop(ThomasFisherScientific),CDNAwaspreparedusingtheGeneChipWTPLUSKitandhybridizedontoAffymetrixGeneChip庐ClariomDarrays(Affymetrix,SantaClara,CA,USA),whichwerewashedandscannedaccordingtoAffymetrix鈥檚instructions(FluidicsProtocolFS450_0003).ThemicroarraydataweremeasuredandsummarizedusingtheClariomDQCtoolsoftware(Affymetrix,SantaClara,CA,USA).

Invivoexperiment

MaleBALB/c-numice(4鈥?weeksold;BeijingVitalRiverLaboratoryAnimal)werekeptintheanimalfacilitiesatHuazhongUniversityofScienceandTechnologyandmaintainedunderspecificpathogen-freeconditions.Intraperitoneal(IP)injectionsofchloralhydrate(2.5鈥塵l/kg)wereusedtoanaesthetizetheanimalsinallexperiments.Allanimalprocedureswereconductedinaccordancewithinstitutionalguidelinesunderapprovedprotocols.FortheintracranialimplantationofsyngeneicU87gliomacellsinthebrainsoftheBALB/c-numice,theanimalswerestereotacticallyinoculatedwithaCD90highCMorCD90lowCMsuspensioncontaining5鈥壝椻€?05U87cellsintotherightfrontallobe(2鈥塵mlateraland1鈥塵manteriortothebregmAATa3.5-mmdepthfromtheskullbase)usingaHamiltonsyringe(HamiltonCompany,USA).Themiceweresacrificed28daysafterinjection.ThetumourvolumesofthenudemiceineachgroupwerecalculatedasV鈥?鈥?/2LW2(L鈥?鈥塼umourlength,W鈥?鈥塼umourwidth).

Additionally,wetookadvantageofthefulllistofavailabledatasetspresentedontheGlioVishomepage(http://gliovis.bioinfo.cnio.es/)toanalysetheoverallsurvivalofpatientswithgliomasingroupswithdifferingCD90expressionlevelsfromTCGAGBMdataset.

Statisticalanalysis

ThestatisticalanalyseswereperformedwithGraphPadPrism.Unlessspecificallynoted,alldatawererepresentativeofatleastthreeseparateexperiments.Errorbarsrepresentthestandarddeviations(SDs)andwerecalculatedusingPrism.ThespecificstatisticaltestsusedwereattestforsinglecomparisonsorANOVAfollowedbyTukey鈥檚testformultiplecomparisons,andallPvalues鈥?lt;鈥?.05wereconsideredstatisticallysignificant.GraphPadPrismwasusedtocomparetwosurvivalcurveswiththelog-ranktest.

ResultsCharacteristicsofCD90highandCD90lowgbMSCs

gbMSCsweresuccessfullyisolatedfromfreshtumourtissuesfrompatientswithdifferentgradegliomas,asshowninTable1.ThesegbMSCsfromastrocytomasandglioblastomasshowedsimilarclassicalMSCcharacteristicsinvitro.Theydisplayedafibroblasticmorphologyandgrewadherenttoflasksinstandardmedium(Fig.聽1a).TheflowcytometricanalysisshowedthatthegbMSCsexpressedMSCmarkers,includingCD73,CD105,CD44,andCD90,butnotCD31,CD34,CD14,NG2andPDGF尾-R;moreover,slight伪-SMAanddesminexpressionwasobserved(Fig.聽1b).ThepotentialofgbMSCstodifferentiateintoadipocytes,osteoblastsandchondrocyteswastestedinvitrousingspecificstimulitopromoteadipogenesis,osteogenesisandchondrogenesis,respectively.AdipogenicdifferentiationwasobservedwithoilredOstaining,osteogenicdifferentiationwasobservedwithAlizarinredstainingandchondrogenicdifferentiationwasobservedwithAlcianbluestaining(Fig.聽1c).ThegbMSCswereabletodifferentiateintoadipocytes,osteoblastsandchondrocytes.

Fig.1:IsolationandcharacterizationofgbMSCsderivedfromhumanhigh-gradegliomatissues.

aAdherentgrowthpatternofgbMSCsculturedin10%DMEM(脳40,scalebars鈥?鈥?00鈥壩糾).bFACSanalysisoftypicalgbMSCs(n鈥夆墺鈥?)inculture.cTri-lineagedifferentiationofgbMSCs:gbMSCsweretreatedwithspecificconditionsforosteogenicdifferentiation(upperleft),adipogenicdifferentiation(uppermiddle),andchondrogenicdifferentiation(upperright)(脳200,scalebars鈥?鈥?0鈥壩糾).ThelowerpanelsshowstainingofcellsgrowninMSCmediumasacontrol

Fullsizeimage

Similarly,CD90highandCD90lowgbMSCsallshowedtypicaladherentgrowthinvitro(Fig.聽2a,b).gbMSCsassociatedwithCD90expressionwereverifiedbyflowcytometricanalysis(Fig.聽2c,d).Additionally,wefoundthattheirgrowthwassignificantlydifferentandthatCD90lowgbMSCsculturedin10%DMEMgrewfasterthanCD90highgbMSCsinvitro(Fig.聽2e).Furthermore,themigrationcapacityoftheCD90lowgbMSCsincubatedindifferentconditionedmediawassignificantlystrongerthanthatofCD90highgbMSCs(Fig.聽2f).

Fig.2:CharacteristicsofCD90highandCD90lowgbMSCsculturedinvitro.

a,bAdherentgrowthpatternsofCD90highandCD90lowgbMSCsculturedin10%DMEM(脳40,scalebars鈥?鈥?00鈥壜祄).cFACSanalysisofsortedCD90highgbMSCs(n鈥夆墺鈥?).dFACSanalysisofsortedCD90highgbMSCs(n鈥夆墺鈥?).eGrowthofCD90highandCD90lowgbMSCsculturedin10%DMEM(n鈥夆墺鈥?).*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01.fWound-healingassayofCD90highandCD90lowgbMSCs(n鈥夆墺鈥?)for8鈥塰withdifferentmedia(脳40,scalebars鈥?鈥?00鈥壜祄).*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,***P鈥?lt;鈥?.001and****P鈥?lt;鈥?.0001

FullsizeimageCD90highgbMSCssignificantlypromotegliomaprogressioninvitro

TodemonstratethatCD90highgbMSCssignificantlypromotedglioblastomagrowth,migrationandadhesioninvitro,experimentswereperformedusingCCK-8,Transwellchamberandadhesionassays,respectively.U87cellsculturedinCD90highCMexhibitedasignificantlygreatermigrationabilitythancellsculturedinCD90lowCMand0%DMEM(Fig.聽3a).Furthermore,U87cellswereincubatedwithserum-freemedium,CD90lowgbMSCsconditionedmediumandCD90highgbMSCsconditionedmedium(0%DMEM,CD90lowCM,andCD90highCM,respectively).WefoundthattheproliferationabilityoftheU87cellswassignificantlyincreasedwhenincubatedinCD90highCMcomparedtothatofthecellsincubatedinCD90lowCMand0%DMEM(Fig.聽3b).Inaddition,theadhesionassayrevealedthattheCD90highCMsignificantlyenhancedtheadhesivecapacityoftheU87cellscomparedtothatoftheCD90lowCMand0%DMEM(Fig.聽3c).

Fig.3:Themigration,proliferationandadhesioncapacitiesofU87cellsincubatedwithdifferentmediainvitro.

aTranswellassayofU87cellsculturedfor24鈥塰indifferentmedia(n鈥夆墺鈥?)(serum-freemedium,CD90lowCMandCD90highCM).*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,***P鈥?lt;鈥?.001and****P鈥?lt;鈥?.0001.Serum-freemediumwasusedasacontrol.bCCK8assayofU87cells(n鈥夆墺鈥?)toevaluateproliferationindifferentmediainvitro.U87cellswereincubatedin0%DMEM,CD90highCMandCD90lowCM.*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,***P鈥?lt;鈥?.001.Serum-freemediumwasusedasacontrol.cAdhesionassaytoestimatetheeffectof0%DMEM,CD90highCMandCD90lowCMonU87celladhesion.(n鈥夆墺鈥?)*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,***P鈥?lt;鈥?.001.Serum-freemediumwereusedasacontrol

FullsizeimageCD90lowgbMSCsshowstrongangiogenesisandcontributetonewtubeformationinvitro

TheinvolvementofMSC-derivedhumangliomainneovascularizationiswellestablished6.Thus,wefocusedonstudiesofthevascularformationabilityofthegbMSCsubtypesinglioblastoma-conditionedmedia.Wefoundthattubeformationat6鈥塰wassignificantlyincreasedintheCD90lowgbMSCsincubatedindifferentmedia(0%DMEM,10%DMEM,0%gb-CM,andS-gb-CM)comparedtothatoftheirCD90highcounterparts.Thetotaltubesegmentlengthswerealsoquantified;thetubesegmentlengthsoftheCD90lowgbMSCsweresignificantlylongerthanthoseoftheirCD90highcounterparts(Fig.聽4a).

Fig.4:TubeformationcapacityofgbMSCsandHUVECsincubatedindifferentmedia.

aAngiogeniccapacityofCD90highandCD90lowgbMSCsculturedin0%DMEM,10%DMEM,0%gb-CMandS-gb-CMfor6鈥塰onMatrigel(脳100,scalebars鈥?鈥?00鈥壜祄).(n鈥夆墺鈥?)*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,***P鈥?lt;鈥?.001.bAngiogeniccapacityofHUVECsculturedin0%DMEM,CD90highCMandCD90lowCMfor6鈥塰onMatrigel(脳100,scalebars鈥?鈥?00鈥壜祄).(n鈥夆墺鈥?)*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01.cAttachmentcapacityofDiO-labelledCD90lowandCD90highgbMSCsontovascularstructuresformedbyHUVECsin0%gb-CM(脳100,scalebars鈥?鈥?00鈥壜祄).(n鈥夆墺鈥?)*P鈥?lt;鈥?.05and**P鈥?lt;鈥?.01

Fullsizeimage

TostudythecapacityforangiogenesisgeneratedbyHUVECsculturedinthedifferentmedia,HUVECswereseededintowellsandincubatedwith0%DMEM,CD90lowCMandCD90highCM.Thetubenetworkswerephotographedat6鈥塰andanalysed.TheresultsshowedthethatangiogeniccapacityofHUVECsculturedinCD90lowCMwassignificantlygreaterthanthatofthecellsculturedinCD90highCMand0%DMEM(Fig.聽4b).

AttachmentofpericytestoECshelpsmaintainandstabilizecapillary-likestructures.Therefore,wesoughttoelucidatetheattachmentcapacityofCD90lowandCD90highgbMSC-derivedpericytes.CalceinAM-labelledCD90lowgbMSCsandHUVECs(1:2)andCalceinAM-labelledCD90highgbMSCsandHUVECs(1:2)wereco-seededintothesamemediumandphotographedat6鈥塰.ThetubeformationanalysesshowedthattheattachmentcapacityoftheCD90lowgbMSC-derivedpericyteswasbetterthanthatofCD90highgbMSC-derivedpericytes(Fig.聽4c).

DifferentfunctionsofCD90highandCD90lowgbMSCsinvivo

TodemonstratethatCD90highCMandCD90lowCMhaddifferentfunctionsinvivo,U87cellswithCD90highCMandCD90lowCMwereimplantedsimultaneouslyintothebrainsofmice(N鈥?鈥?鈥塵ice/group).ThetumoursizewassignificantlylargerwithCD90highCMthanwithCD90lowCM(Fig.聽5a),indicatingthatCD90highCMincreasedtheproliferationand/ortumourigenesisofU87cellsinvivo.CD31expressionintheCD90lowCMtumourspecimenswassignificantlyhigherthanthatoftheirCD90highCMcounterparts,whereasKi-67expressionwassignificantlyhigherintheCD90highCMtumourspecimensthanCD90lowCMcounterparts(Fig.聽5b).However,thesurvivaltimeofthemiceimplantedwithU87cellsco-culturedwithCD90highCMwasnotsignificantlyshorterthanthatofmiceimplantedwithU87cellsculturedinCD90lowCM(Fig.聽5c).Additionally,wefoundthatCD90-CD105+cellsweremainlylocatedinthevesselwalls,whereasCD105+CD90+gbMSCswerelocatedaroundthetumourparenchymaspecimensinhumanglioblastomas(Fig.聽5d).Moreover,theoverallsurvivalofpatientswithgliomasfromTCGAdatabasewasnotsignificantlydifferentwhenthepatientsweregroupbasedontheirCD90expressionlevels(Fig.聽5e).

Fig.5:ConditionedmediafromCD90highandCD90lowgbMSCshavedifferentfunctionsinvivo.

aRepresentativemicefromintracranialxenograftexperimentsinwhichU87cellswithCD90highCM(left)orU87cellswithCD90lowCM(right)wereinjectedintotherightfrontallobesofnudemice.Obviously,thesizesoftheCD90highCMgrouptumoursweregreaterthanthoseoftheirCD90lowCMcounterparts.*P鈥?lt;鈥?.05.bBoththeCD90highCMandCD90lowCMtumoursectionswerestainedwithHE(脳200,scalebars鈥?鈥?0鈥壩糾).IntheCD90highCMandCD90lowCMtumourtissues,IHCwasemployedtodetectCD31andKi-67expression(脳400,scalebars鈥?鈥?5鈥壜祄).(n鈥夆墺鈥?)*P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01.cSurvivalcurvesofglioma-bearingmice.ThesurvivaltimesofmiceimplantedwithU87cellsculturedwithCD90highCMwerenotsignificantlyshorterthanthoseofmiceimplantedwithU87cellsculturedinCD90lowCM.dDoublestainingforCD105(green)andCD90(red)revealedthatCD105+CD90鈭?/sup>cellswerelocatedinthevesselwalls,whereasCD105+CD90+cellswerelocatedaroundthetumourparenchyma.(脳400,scalebars鈥?鈥?5鈥壜祄).eKaplan鈥揗eiersurvivalcurvesforpatientswithlowandhighCD90expression.ThesurvivalofgliomapatientswithdifferentCD90expressionlevelsinTCGAdatabasewasnotsignificantlydifferent

FullsizeimageSecretionoffactorsrelatedtoCD90highandCD90lowgbMSCs

Toanalysethesecretionofseveralfactorsindifferentmedia,supernatantswerecollectedandevaluatedbyELISA.WefoundthattheVEGF,FGF-2,andIL-6levelswerehigherintheCD90lowCMthanintheCD90highCMand0%DMEM(Fig.聽6a-c).Conversely,theSDF-1伪,CCL5,andMMP9levelswerehigherintheCD90highCMthanintheCD90lowCMand0%DMEM(Fig.聽6d-f).

Fig.6:TheVEGF,IL-6,bFGF,MMP9,CCL5andSDF-1伪levelsindifferenttreatmentmediabyELISA.

aTheVEGF(n鈥夆墺鈥?)levelsweresignificantlyhigherinCD90lowCMcomparedtothoseinCD90highCMand0%DMEM.bThebFGF(n鈥夆墺鈥?)levelsweresignificantlyhigherinCD90lowCMcomparedtothoseinCD90highCMand0%DMEM.cTheIL-6(n鈥夆墺鈥?)levelsweresignificantlyhigherinCD90lowCMcomparedtothoseinCD90highCMand0%DMEM.*dTheSDF-1伪(n鈥夆墺鈥?)levelswerehigherinCD90highCMcomparedtothoseinCD90lowCMand0%DMEM.eTheCCL5(n鈥夆墺鈥?)levelswerehigherinCD90highCMcomparedtothoseinCD90lowCMand0%DMEM.fTheMMP9(n鈥夆墺鈥?)levelsweresignificantlyhigherinCD90highCMcomparedtothoseinCD90lowCMand0%DMEM.P鈥?lt;鈥?.05,**P鈥?lt;鈥?.01,***P鈥?lt;鈥?.001and****P鈥?lt;鈥?.0001

FullsizeimageDifferentiallncRNAandmRNAexpressionintheCD90highandCD90lowgbMSCs

ClariomDanalysisoftheCD90highandCD90lowgbMSCsculturedinstandardmediumrevealeddifferentlncRNAandmRNAexpressionprofiles.Subsequently,atestwasperformedtoidentifydifferentiallyexpressedgenesbetweentheCD90highandCD90lowgbMSCs,andatotalof4977genes(2055upregulatedand2922downregulatedintheCD90highgbMSCs)wereidentified(Fig.聽7a).DifferentiallyexpressedmRNAtargetgeneswerepredictedusingTargetScan,lncRNA.organdtheLnRDBAdatabase,andtheresultswereappliedtoageneontology(GO)termanalysis(Fig.聽7b),includingthebiologicalprocess(BP),molecularfunction(MF)andcellularcomponent(CC)categories.Thepredictedtargetgeneswereenrichedincellmigration,proliferation,adhesionandangiogenesis.

Fig.7:ClariomDexpressionprofilesofCD90highandCD90lowgbMSCs(n鈥?鈥?).

aHeatmapofdifferentiallyexpressedlncRNAsfromamicroarrayassayperformedonCD90highandCD90lowgbMSCs.鈥楻ed鈥?indicateshighrelativeexpression,and鈥榞reen鈥?indicateslowrelativeexpression.bGOtermsforthepredictedtargetedgenes.P鈥?lt;鈥?.05usingthetwo-sidedFisher鈥檚exacttestwasdefinedasstatisticallysignificant

FullsizeimageDiscussion

In1966,theexistenceofMSCsinbonemarrowwasfirstreportedbyFriendensteinetal.11.Thereafter,MSCswerewidelyandgraduallyresearchedinseveralfields,butstudieswerelimitedbythelackofadefinitivemarkerforMSCs,whichmeantthatthecelltypecouldonlybedefinedintermsofcellularmorphology,surfacemarkersanddifferentiationpotential.In2006,theInternationalSocietyforCellularTherapy(ISCT)definedtheminimalcriteriaforMSCsaccordingtotheirbiologicalfeatures12.PositiveCD90expressionisoneoftheminimalcriteriausedtodefineMSCs,andMSCsexpressinghighCD90levelscanbefoundintissuesincludingbonemarrowandadiposetissue13.Ingliomas,tumourcellsrecruitMSCsfromdifferenttissuesandcorruptthemintogbMSCstopromotetumourprogression14.ComparedtotheirmainBM-MSCresources,gbMSCsharbourgeneticalterations,ofwhichCD90expressionisonedifference6.Inthecurrentstudy,wefoundtwosubpopulations(CD90highgbMSCsandCD90lowgbMSCs)thatcouldbesortedfromallfreshgliomatissues(WHOII鈥揑Vgliomas).CD90lowgbMSCsweremoreabundantthanCD90highgbMSCsinthesamegliomatissues.WiththeexceptionofCD90expression,thesetwosubpopulationsshowedsimilarcellularmorphologies.TheCD90lowgbMSCshadstrongerproliferationandmigrationabilitiesthantheCD90highgbMSCs.PreviousliteraturereportedthatangiogenicstimuliandmechanicalstressledtoalossofCD90expressioninMSCs15,16.OurpreviousstudyreportedthatCD90lowgbMSCscoulddifferentiateintopericytesandcontributetoangiogenesisinthemalignantgliomamicroenvironment6.Thus,tumourcellsmayinfluenceCD90expressiononMSCsrecruitedtoagliomaforangiogenesis,buttumourcellsmayrecruitdifferentgbMSCsubpopulationsfromdifferentresourcesfordifferenthallmarksofmalignantgliomas.

Todate,MSCsfromdifferentsourceshaveshowndifferentabilitiestopromoteorsuppressthegrowthofgliomacellsunderdifferentconditions17,18,19,20,21.Ingliomas,tumourcellsdonotrecruitMSCsintothetumourmicroenvironmentasbystandersorinhibitorsofprogression5,14.PreviousstudiesfoundthathighpercentagesofgbMSCsintumoursamplescorrelatedwithpooroutcomesofpatientswithhigh-gradegliomas.Additionally,gbMSCscouldincreasethetumourigenicityandmaintainthestemnessofgliomastemcells9.Inthecurrentstudy,weinvestigatedthedifferentrolesoftwosubpopulations(CD90highandCD90lowgbMSCs)inaggressiveprogressionofgliomas.ConditionedmediumfromCD90highgbMSCsinducedfasterproliferationandstrongermigrationandadherenceofU87cellsinvitro,andinjectionofconditionedmediumfromCD90highgbMSCsresultedinalargertumourvolumeandmoreKi-67-labelledtumourcellsinananimalmodel.Conversely,theCD90lowgbMSCshadastrongabilitytodifferentiateintopericytesandinducetubeformationinvitro,andconditionedmediumfromtheCD90lowgbMSCsinducedmorecapillary-likeendothelialcellstructures.Inanimalmodels,injectionofconditionedmediumfromCD90lowgbMSCsinducedmoreCD31-expressingvessels.Inhumanglioblastomatissues,CD105+CD90鈭?/sup>cellswerelocatedinthevesselwalls,andCD105+CD90+cellswerelocatedaroundthetumourparenchyma.ThesedatasuggestedthatCD90highgbMSCsmainlypromotedtumourcellproliferation,whereasCD90lowgbMSCsmainlydifferentiatedintopericytesandcontributedtoangiogenesis.However,gbMSCsarenotthesoleplayersinthetumourigenicityofmalignantgliomas,andtheoverallsurvivalofpatientswithgliomasinTCGAdatabaseandtumour-bearingmiceisnotsignificantlydifferentbetweengroupswithdifferentCD90expressionlevels.

Cytokinesareaveryimportantcomponentofthecross-talkbetweentumourcellsandthetumourmicroenvironment3,6.PreviousstudiesshowedthatcytokinesandsecretedexosomeswereimportantfortheeffectofgbMSCsonthetumourigenicityofmalignantgliomas9,22.Therefore,cytokinesandrelatedlnRNAswerecomparedbetweenCD90highandCD90lowgbMSCsinthecurrentstudy.CD90lowgbMSCsproducedhigherlevelsoftheangiogenesisfactorsVEGF,bFGFandIL-6,andCD90highgbMSCsproducedhigherlevelsofthegrowthfactorsSDF-1伪,CCL5,andMMP9.Theseresultsareconsistentwithpreviousfindings23,24,25,26,27,28.Toexploretheirgeneprofiles,weanalysedifferencesinexpressionbasedontheClariomDassayandthepredictedgeneontologiesoftheCD90highandCD90lowgbMSCs.WeidentifiedspecificlncRNA,mRNAandtargetgenepairsintheCD90highandCD90lowgbMSCs.PreviousreportsindicatedimportantlinksbetweentheGprotein-coupledreceptorpathwayandtumourproliferation29,30,31,32andangiogenesis33,34,35.Infuturestudies,wewillidentifythespecificlncRNA,mRNAandtargetgenepairsthataresignificantlydifferentbetweentheCD90lowandCD90highgbMSCsandpromotetumourproliferationandangiogenesis.

Inconclusion,weelaboratelysortedtwosubpopulations(CD90highandCD90lowgbMSCs)fromgbMSCs.BothsubpopulationshadcellularmorphologiesandsurfacemarkerssimilartothoseofclassicalMSCsbutshowedslightlydifferentbiologicalfeaturesandplayedenormouslydifferentrolesinthegliomamicroenvironment.Bothinvivoandinvitro,CD90highgbMSCssignificantlypromotedgliomacellgrowth,andtheCD90lowgbMSCspromotedangiogenesisviapericytetransition.Additionally,weprovidedevidencethattheexpressionpatternsofsecretedfactorsandrelatedlncRNAsinthesetwosubpopulationsmightaffecttheirrolesingliomaprogression.However,determiningtheunderlyingmechanismrequiresfurtherexplorationinmoredetail.Therefore,theseresultsrevealedthatfuturetherapiestargetingthetwodistinctgbMSCsubpopulationsshouldusedifferentstrategies.

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Acknowledgements

ThisworkwassupportedbyagrantfromtheNationalNaturalScienceFoundationofChina(No.81572488)andgrantsfromtheScienceandTechnologyDepartmentofHubeiProvince(2015CFB458and2017CFB268).

AuthorinformationAuthornotes

Theseauthorscontributedequally:QingZhang,Dong-YeYi

EditedbyY.Shi

AffiliationsDepartmentofNeurosurgery,UnionHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan,430022,ChinaQingZhang,聽Dong-YeYi,聽Bing-ZhouXue,聽AhmedAbdelmaksou,聽Hong-YangZhao,聽Nan-XiangXiong,聽WeiXiang聽聽PengFuDepartmentofCardiology,BeijingAnzhenHospital,CapitalMedicalUniversity,No.2,AnzhenRoad,ChaoyangDistrict,Beijing,100029,ChinaWan-WanWenInstituteofInfectionandImmunology,UnionHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan,430022,ChinaYin-PingLuDepartmentofNeurosurgery,FacultyofMedicine,HelwanUniversity,Cairo,11435,EgyptAhmedAbdelmaksouJiangsuKeyLabofMedicalOptics,SuzhouInstituteofBiomedicalEngineeringandTechnology,ChineseAcademyofSciences,Suzhou,215163,ChinaMin-xuanSun聽聽De-tianYuanAuthorsQingZhangViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarDong-YeYiViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarBing-ZhouXueViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarWan-WanWenViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarYin-PingLuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarAhmedAbdelmaksouViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarMin-xuanSunViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarDe-tianYuanViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarHong-YangZhaoViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarNan-XiangXiongViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarWeiXiangViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarPengFuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarCorrespondingauthors

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Zhang,Q.,Yi,D.,Xue,B.etal.CD90determinedtwosubpopulationsofglioma-associatedmesenchymalstemcellswithdifferentrolesintumourprogression.CellDeathDis9,1101(2018).https://doi.org/10.1038/s41419-018-1140-6

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Received:17July2018

Revised:08October2018

Accepted:11October2018

Published:27October2018

DOI:https://doi.org/10.1038/s41419-018-1140-6