JournalofBiomedicineandBiotechnologyVolume2010,ArticleID105940,10pagesdoi:10.1155/2010/105940
MethodologyReport
AnImprovedHarvestandinVitroExpansionProtocolforMurineBoneMarrow-DerivedMesenchymalStemCells
SongXu,1,2,3AnnDeBecker,1,2BenVanCamp,2KarinVanderkerken,2andIvanVanRiet1,2
1Stem
CellLaboratory-DivisionClinicalHematology,UniversitairZiekenhuisBrussel(UZBrussel),Laarbeeklaan101,1090Brussels,Belgium
2DepartmentofHematologyandImmunology,VrijeUniversiteitBrussel(VUB)—MyelomaCenter,Laarbeeklaan103,1090Brussels,Belgium
3DepartmentofLungCancerSurgery,LungCancerInstitute,TianjinMedicalUniversityGeneralHospital,Tianjin300052,ChinaCorrespondenceshouldbeaddressedtoIvanVanRiet,ivan.vanriet@uzbrussel.beReceived9July2010;Revised6October2010;Accepted25October2010AcademicEditor:BarryJ.Byrne
Copyright©2010SongXuetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Comparedtobonemarrow(BM)derivedmesenchymalstemcells(MSCs)fromhumanoriginorfromotherspecies,theinvitroexpansionandpurificationofmurineMSCs(mMSCs)ismuchmoredifficultbecauseofthelowMSCyieldandtheunwantedgrowthofnon-MSCsintheinvitroexpansioncultures.WedescribeamodifiedprotocoltoisolateandexpandmurineBMderivedMSCsbasedonthecombinationofmechanicalcrushingandcollagenasedigestionatthemomentofharvest,followedbyanimmunodepletionstepusingmicrobeadscoatedwithCD11b,CD45andCD34antibodies.ThenumberofisolatedmMSCsasestimatedbycolonyformingunit-fibroblast(CFU-F)assayshowedthatthismodifiedisolationmethodcouldyield70.0%moreprimarycolonies.Afterimmunodepletion,ahomogenousmMSCpopulationcouldalreadybeobtainedaftertwopassages.ImmunodepletedmMSCs(ID-mMSCs)areuniformlypositiveforstemcellantigen-1(Sca-1),CD90,CD105andCD73cellsurfacemarkers,butnegativeforthehematopoieticsurfacemarkersCD14,CD34andCD45.Moreovertheimmunodepletedcellpopulationexhibitsmoredifferentiationpotentialintoadipogenic,osteogenicandchondrogeniclineages.OurdataillustratethedevelopmentofanefficientandreliableexpansionprotocolincreasingtheyieldandpurityofmMSCsandreducingtheoverallexpansiontime.
1.Introduction
Mesenchymalstemcellsareself-renewingandmultipotentprogenitorsthatcandifferentiateintoavarietyofcelltypes,includingadipocytes,osteoblasts,chondrocytes,myocytes,hepatocytes,cardiomyocytes,neurons,andbeta-pancreaticisletscells[1–4].BMisthemostcommonsourceofMSCs.However,MSCshavealsobeenisolatedfromvariousothersources,namely,placenta,amnioticfluid,cordblood,fetalliver,andadiposetissue[5–9].MSCshavebeenreferredbyotherterminologysuchascolony-formingfibroblasticcells,marrowstromalstemcells,andmesenchymalprogenitorcells[10–12].Atpresent,MSCsarereportedtopossess,besidestheirmultipotentdifferentiationcapacity,alsootherpropertiessuchaslowimmunogenicityandtissue-homingabilitymakingthemanattractivetoolforcell-mediated
therapyinseveraldiseasesprocesses,includingtissueinjuryandtissuedegenerationaswellasgraft-versus-hostdisease[13–16].
MSCshavebeensuccessfullyisolatedandcharacterizedfromBMsamplesofmanyspeciesincludinghuman,rabbit,rat,sheep,goat,rhesusmonkeys,dog,andpigthroughtheirpreferentialattachmenttotissuecultureplastic[17–25].Incontrast,theisolationandpurificationofmMSCsfromBMhasbeenmoredifficultthanthatfromhumanandotherspecieswhosemarrowadherentcellsarerelativelyhomogenousandcontainahighpercentageofMSCs.ThefutureuseofMSCsforhumandiseasetherapiesdependsontheestablishmentofrepresentativeandefficientpreclinicalanimalmodels.Inmanyrespects,amurinemodelisanidealmodeltostudythecellbiologyandthetherapeuticpotentialofMSCs.Thestandardmethodofplasticadherencehas,
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however,provenineffectivetoyieldrelativelypuremMSCspopulationssincevarioushematopoieticcelllineagessurviveand/orproliferateonstromallayers,evenintheabsenceofexogenouscytokines[26].Themajorityofthecontaminatedhematopoieticcellscanberemovedafteraprolongedexpansiontimebyfrequentsubculturing.TheMSCsdiffer-entiationpotentialsmight,however,decreaseduringlong-terminvitroculture.Inaddition,severalreportssuggestthatmMSCscanundergospontaneoustransformationfollowinglongterminvitroculture[27–29].Sofar,severaltechniqueshavebeendescribedtoreduceoreliminatenon-MSCsfromplasticadherentmurineBMcultures,includingtheuseoflow-densityculture,frequentmediumchange,andpositiveandnegativeselection[30–37].However,noneofthesetechniqueshasgainedwidespreadacceptancesofar.Itis,therefore,stillnecessarytodevelopastandardized,reliable,andeasy-to-performmethodtoobtainhighamountsofpurifiedmMSCs,butwithreducedexpansiontimetoavoidpossibletransformationandtoretainthedifferentiationpotentialoftheexpandedstemcells.Ourpresentstudyaimedtodevelopanimprovedmethodtoincreasetheyieldandpurityofculture-expandedmMSCs.
2.MaterialsandMethods
2.1.mMSCsModifiedHarvestandPrimaryCulture.C57BL/KaLwRijmice(HarlanCPB,TheNetherlands),6–8weeksold,weresacrificedbycervicaldislocation.Thehindlegsandvertebraeweredissectedandcarefullycleanedfromadherenttissues.ThestandardacceptedisolationforBM-derivedMSCswastoflushBMcellsfromhindlegs[38].Here,wemodifiedtheharvestprotocol.AfterthetipsofeachbonewereremovedandtheBMwascollectedbyflushingoutthecontentoffemursandtibiaswithRPMI1640(Lonza,Verviers,Belgium),wefurtherchoppedthefemurs,tibiasandvertebraeintofinepieces(1-2mm)withascalpelandcrushedgentlywiththebacksideofa5-mlsyringeinRPMI1640.Next,thebonefragmentsweretransferredintoa50mlpolypropylenetubewith10ml0.25%CollagenaseAsolution(RocheDiagnosticsGmbH,Mannheim,Germany).ThetubewithbonefragmentsandCollagenaseAsolutionwasplacedin37◦Cwaterbathfor30minutes.Nextphosphatebufferedsaline(PBS)wasaddedtoafinalvolumeof30ml.Whenthefragmentsweresettled,supernatantwascollected,mixedwiththepreviouslyharvestedcells,andfilteredthrough70-μmnylonmeshfilter.CellswerethenwashedtwicewithPBS,andsubsequentlynucleatedandviablecellswerecountedinahemocytometerusing3%AceticacidwithCrystalVioletandTrypan2Blue,respectively.Cellswereplatedat1×106cells/cminMcCoy’s5Amedia(Lonza,Verviers,Belgium)containing20%mesenchymalstemcellstimulatorysupplementsformouse(StemCellTechnologies,Vancouver,BC,Canada),1%L-glutamine(Lonza,Verviers,Belgium),and1%penicillin/streptomycin(Lonza,Verviers,Belgium).Theculturewaskeptinahumidified5%CO2incubatorat37◦Cfor24–48hours,followedbyremovalofnonadherentcellswithPBSandreplacementwithfreshcompletemedium.Alloftheproceduresinvolvingmicewere
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approvedbytheEthicalCommitteeforAnimalExperiments,VUB(licenseno.LA1230281).
2.2.IncreasingthePurityofinVitroExpandedmMSCsbyImmunodepletionandmMSCsSubculture.Afterabout7–10days,whenprimarycultures(passage0)becamenearlyconfluent,thecellsweredetachedwith0.25%trypsin/0.02%EDTAfor5minutesat37◦C.Theresidualcellswhichwerenotdetachedwithin5minuteswerecollectedbygentlescrapingusingacellscraper.Thecellswereresuspendedin1ml0.1%BSA/PBS,followedbyanimmunodepletionusinganti-CD11b,CD34,CD45-conjugatedDynabeadsM-280Streptavidinsuperparamagneticpolystyrenebeads(InvitrogenDynalAS,Oslo,Norway)accordingtotheman-ufacturer’sinstruction.Inbrief,Dynabeadswerewashedwith0.1%BSA/PBSfor5timesandthenprecoatedwiththefollowingbiotinylatedantibodies,respectively,atappropriateconcentration(5–10μgantibodiespermgDynabeadsM-280streptavidin)byincubationfor30minutesatroomtemperatureusinggentlerotation:BiotinantimouseCD11b,CD34andCD45(eBioscience,SanDiego,USA).ThreeconsecutiveroundsofimmunodepletionusingantibodiesagainstCD11b,CD34andCD45,respectively,wereper-formedfordepletingcontaminatednon-MSCs.Ineachcase,cellsandantibodyconjugatedDynabeadswerethoroughlymixedataratioof1cell:5beadsandincubatedontherotatorfor30minutesat4◦C.Then,cellswerewashedusingaDynalMPCtoremoveunboundcells.Theimmunodepletedcellsweresuspendedincompletemedium,platedinoneT-25flaskat1000cells/cm2(Nunc,VWRInternational,Leuven,Belgium)followedbyincubationina37◦Cwith5%CO2humidifiedincubator.Culturemediumwaschangedevery3-4daysuntilcellculturereached80%–90%confluence.Atthatpointcellsweretrypsinized(passage1)andplatedinoneT-75flaskat1000cells/cm2.Subsequentpassageswereperformedsimilarly,butsplitratioswere1:2(T-75flask).2.3.CFU-FAssay.TheCFU-Fassaywas6performedasdescribedpreviously[39].Inbrief,1×10nucleatedcellsobtainedfromfreshBMwereplatedintoeachwellofasix-wellplateandincubatedfor10daysinhumidifiedatmosphere(37◦C,5%CO2).Culturemediumwaschangedondays3and8ofculture.Subsequently,cultureswerefixedandstainedwithGiemsa.ThenumberofcoloniesdisplayingfiveormorecellswithspindledmMSCsmorphologywasscoredunderaninvertedmicroscope.Colonieswhosemor-phologyclearlydifferedfromthemMSCmorphologywereexcludedfromtheresults.ThisassaywasperformedusingBMsamplesfrom10mice(for5mice,cellswereisolatedusingCollagenase,whilefor5othermicecellswereisolatedwithoutCollagenase).Eachsamplewasanalyzedintriplicate.2.4.GrowthCurveandDoublingTime.Passage1mMSCswereplatedin6-wellcultureplatesat2000cells/wellinmediumwith20%mesenchymalstemcellstimulatorysupplementsor10%fetalbovineserum(FBS).Thecellsfromeachwellweretrypsinizedandcountedinduplicatewithahemocytometereverytwodaysuntilday12.Thedoubling
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ThehindlegsandvertebraearedissectedandremovedfromadherenttissuesFlushBMCutbonesCrashbones(a)tionedigessanegCollaMixflushedBMandenzyme-treatedfragmentsforculture24hlater,washandrefreshmedium100μmT=24hRefreshmediumevery3days100μmT=7d100μmCellsarecharacterizedbymorphology,cell-surfaceantigensandtri-lineages
differentiationability
CD11b/CD34/CD45microbeads-basedimmunodepletiontoremovecontaminatedhematopoieticcells
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Figure1:Flowchartforourmodifiedharvestandexpansionprotocol.(a)Miceweresacrificedbycervicaldislocation.Thehindlegsandvertebraeweredissectedandcarefullyremovedfromadherenttissues.TheBMwascollectedbyflushingoutthecontentoffemursandtibiaswithRPMI1640.Next,thefemurs,tibias,andvertebraewerecuttedintosmallpiecesandcrushedgently.Next,thebonefragmentswereincubatedwith0.25%CollagenaseAsolutionin37◦Cwaterbathfor30minutes.(b)Collagenase-treatedbonefragmentsweremixedwiththepreviouslyflushedBMcells,filteredthrough70-μmnylonmeshfilterandculturedat1×106cells/cm2.At24hafterinitialculture,thenonadherentcellswerewashedaway.Afterabout7–10days,whenprimaryculturesbecamenearlyconfluent,thecellsweretrypsinized,followedbyaCD11b/CD34/CD45negativeimmunodepletioninordertoremovecontaminatedhematopoieticcells.Theimmunodepletedcellswerecultivatedandcharacterizedattheleveloftheirmorphology,immunophenotype,anddifferentiationpotentials.Scalebar=100μm.
timewascalculatedaccordingtotheequation:TD=t×lg2/lg(Nt/N0),whereN0istheinitialcellnumber,Ntistheendpointcellnumber,andtistimeinterval.
2.5.FlowCytometryAnalysis.mMSCsweredetachedfromtheculturedishusing0.25%trypsin/0.02%EDTA,and1×
105mMSCswerewashedby4%HuAlb/PBSandpelletedbycentrifugationfor3minutesat400g.Thecellswerestainedwithratanti-mouseCD14,CD34,CD105,Sca-1,CD45,CD90(allpurchasedfromeBioscience,SanDiego,USA),andCD73(PharMingen,SanDiego,USA),ataconcentrationof2μg/mlat4◦C.Thecellsstainedwithcorrespondingrat
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Figure2:OptimizedisolationofmurineBMderivedMSCs.(a)IntheCFU-Fassay,70.0%morecoloniesareobservedwiththemodifiedisolationmethod,n=6/group.∗P<.05(b)OnerepresentativecolonyinCFU-Fassay(Giemsastaning,×40),scalebar=100μm.
×1042.6.DifferentiationAssays
2.6.1.AdipogenicDifferentiation.Foradipogenicdifferenti-ation,2×105P3mMSCswereseededintoawellofa6-wellplate(Nunc,VWRInternational,Leuven,Belgium)andfedevery2-3daysbyreplacingthecompletemediumuntilaconfluentcelllayerwasformed.Thencells,werestimulatedtodifferentiateintotheadipogeniclineagebysubmittingthemtothreecyclesofalternatingcultureinadipogenicinductionmedium(LONZA,Walkersville,USA)andadipogenicmaintenancemedium(LONZA,Walk-ersville,USA)accordingtothemanufacturer’sinstruc-tions.TheadipogenicinductionmediumcontainsaccordingtothemanufactureInsulin(recombinant),L-glutamine,MCGS,dexamethasone,indomethacin,IBMX(3-isobuty-l-methyl-xanthine),andPen/Strep,whilethetheAdipogenicMaintenanceMediumincludesinsulin(recombinant),L-Glutamine,MCGS,andpen/strep.Attheendofthesecycles,cellsweregrownforanother7daysinadipogenicmaintenancemedium.AsacontrolMSCweregrowninAdipogenicMaintenanceMediumonlytoexcludesponta-neousadipogenicdifferentiation.Tovisualizeadipocytes,cellswerestainedwithOilRedO(Sigma,Bornem,Belgium)[40].
2.6.2.OsteogenicDifferentiation.Osteogenicdifferentiationwasinducedbyexposing3×104P3mMSCstoosteogenicinductionmedium(LONZA,Walkersville,USA)intoawellofa6-wellplate.Themediumwaschangedevery3to4days.Asanegativecontrol,cellswereculturedincompletemediumandmediumwaschangedatthesamefrequencyasthatforthedifferentiatingMSC.Theosteogenicinductionmediumcontainsaccordingtothemanufacturerdexamethasone,L-Glutamine,ascorbate,Pen/Strep,MCGS,andglycerophosphate.Toverifyosteogenicdifferentiation,vonKossa’smethodwasusedtostaincalciumdeposits[41].
2520Cellnumber151050
0246Time(day)81012Supplements10%FBSFigure3:Comparisonproliferationrateinmediumwithfetalbovineserumversusmesenchymalstemcellstimulatorysupple-ments.Passage1cellsshowahighergrowthrateinmediumwithmesenchymalstemcellstimulatorysupplementsascomparedtotheproliferationrateobservedinmediumwiththe10%FBS.Thevaluesareexpressedasmeans±SDof3independentmeasurements.
anti-mouseIgGservedasnegativecontrols.After30minutes,unboundantibodywaswashedwith2ml1%HuAlb/PBS.Next,thecellswereincubatedwith10μLmouseantiratfluoresceinisothiocyanate(FITC)antibodyat4◦Cfor30minutesfollowedbyawashwith2ml1%HuAlb/PBS.Thecellpelletswereresuspendedin600μlPBSandexaminedbyflowcytometry(CoulterEpicsXL-MCL,Brussels,Belgium)with5,000eventsbeingrecordedforeachcondition.TheresultswereanalyzedbycellquestsoftwareandWinMDI2.8softwarewasusedtocreatethehistograms.
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2.6.3.ChondrogenicDifferentiation.Inordertoinducechon-drogenicdifferentiation2.5×105P3mMSCswerewashedtwiceinincompletechondrogenesisinductionmedium(LONZA,Walkersville,USA).Cellswerethensedimentedbycentrifugationat150gfor5minutesand0.5mLcom-pletechondrogenesisinductionmedium(LONZA,Walk-ersville,USA)wasaddedtothecellpellet.Incompletechondrogenesisinductionmediumcontainsaccordingtothemanufacturerdexamethasone,ascorbate,ITS+supple-ment,pen/strep,sodiumpyruvate,ProlineandL-Glutamine.Completemediumwasmadebyadding5μLTGFβ3(LONZA,Walkersville,USA)to1mLincompletemedium.Themediumwaschangedevery3to4days.After21daysofculture,thepelletswereembeddedinparaffin,cutinto4μmsectionsandimmunohistochemicallystainedbyArabbitpolyclonalanticollagenIIantibody(NCL-COLL-IIp,NovoCastra,Prosan,Merelbeke,Belgium).
2.7.StatisticalAnalysis.Statisticalsignificancewasassessedbycomparingmeanvalues(±SD)usingMannWhitneytest.P<.05wereconsideredstatisticallysignificant.
3.Results
Asshownintheflowchart(Figure1),theisolationandexpansionmethodofBM-derivedmMSCsincludesacombi-nationofmechanicalbonecrushing,enzymatictreatmentofbonefragments,andflushingoutBMcellsattheharveststepwithimmunodepletionofCD11b+,CD34+,andCD45+cellsinthefollowingculturestep.
ThenumberofBM-nucleatedcellsharvestedwiththemodifiedmethodwas19.4%higherthanthenumberofBMnucleatedcellsobtainedbythestandardmethodalthoughthisdifferencewasnotsignificant(datanotshown).How-ever,withthemodifiedharvestmethod,themeannumberofcolonieswas70.0%morethanthemeannumberofcoloniesobtainedbythestandardmethod(P<.05)(Figure2(a)).Thenumberofcoloniesdisplayingfiveormorecellswasscoredunderaninvertedmicroscope(Figure2(c)).Colonieswithfourcellswerecountedwhenoneofthempresentedtwonuclei.ColonieswhosemorphologyclearlydifferedfromthemMSCmorphologywereexcludedfromtheresults.Wecomparedmediumwithfetalbovineserum(FBS)tomediumwithmesenchymalstemcellstimulatorysupplements(mouse)fromStemcelltechnologiesandfoundthatcellsculturedwithMesenchymalStemCellStimulatorySupplementsshowedahigherproliferationrate(Figure3).
Usingthestandardexpansionconditions,culturesusu-allyremainedmorphologicallyheterogeneousatpassage2,presentinground,polygonal,spindle-shaped,andflattenedcells(Figure4).Somedegreeofmorphologicalheterogene-itycouldstillbeobserveduptopassage6–8(datanotshown).However,usingtheimmunodepletionstep,muchlessheterogeneitycouldbeobserved,andatpassage2,ahomogeneouspopulationofspindle-shapedcellscouldbeobserved.ThedoublingtimeofID-mMSCswasfoundtobe46.9±1.78h.
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P0100μmControlIDP2P2100μm100μmFigure4:MorphologyofculturedmouseBMcells.SevendayspostharvestCD11b/CD34/CD45immunodepletionwasperformed.Culturedcellsalreadyappearedhomogeneousalreadyatpassage2whilecontrolsremainheterogeneous(×100).Representativepicturesareshown,n=6/group,scalebar=100μm.
WefurtherinvestigatedwhetherthetwodifferentharvestmethodshadaneffectontheyieldsofID-mMSCs.Theresultshowedthatusingthesameimmunodepletionprocedure,theID-mMSCyieldwiththestandardisolationmethodwas3.7%,whiletheyieldwiththemodifiedisolationmethodcouldreachupto6.6%(Figure5(a)),confirmingthatourmodifiedisolationmethodresultsinahighermMSCharvest.
Thecells(culturedwithandwithoutimmunodepletion)werefurtheranalyzedforcellsurfaceantigensatpassage3.ResultsshowedthatID-mMSCswerestronglypositiveforCD90,CD73andSca-1,buttotallynegativeforCD14,CD34,andCD45(Figure5(b)).Thecellpopulationcul-turedwithoutimmunodepletionshowedsignificantlylessexpressionofCD90,CD73,andSca-1butmoreexpressionofCD45comparedwithID-mMSCs(Figure5(c)).WithoutimmunodepletionmMSCsstillseemedtobecontaminatedwithhematopoieticcellsafter5-6passages,whichwasconsistentwiththemorphologicalobservations.
Tri-lineagedifferentiationabilitiesofcellsculturedwithandwithoutimmunodepletionweretestedatpassage3.Whenculturedinadipogenic,osteogenic,andchondro-genicmedia,cellswereabletodifferentiateexclusivelyintoadipocytes,osteoblasts,andchondrocytesasdeterminedby
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Figure5:Cell-surfaceantigensonmMSCs.(a)TheyieldofID-mMSCswithmodifiedisolationmethodis78.3%higherascomparedtothestandardisolationmethold,n=6/group,∗P<.05.(b)ArepresentativephenotypeofID-mMSCsfrom6donorsatpassage3.PlotsshowisotypecontrolIgGstainingprofile(red)versusspecificAbstainingprofile(white).(c)ContaminatedheamatopoieticcellsstillcanbedetectedinthenonimmunodepletedMSCfractionshowingCD14,CD34,andCD45expressionatpassage5,n=6/group,∗P<.05.
OilRedO,vonKossaandtypeIIcollagenstaining,respec-tively.However,thedifferentiationabilitiesweresignificantlydifferent.Theextentofadipogenicdifferentiationwasquan-tifiedbycountingOilredO-positivecellspercm2.AsshowninFigure6(a),theamountofOilredO-positivecellsintheimmunodepletedgroupwassignificantlyhigherthanthatofthecontrolgroup.Whenosteogenicdifferentiationwasexaminedusingabonenoduleformationassay,vonKossastainingrevealedthatcellsculturedafterimmunodepletionproducedsignificantlymorecalciumnodulesthancellsculturedwithoutimmunodepletion(Figure6(b)).Inaccor-dance,chondrogenicdifferentiationofID-cellswasmorepronouncedcomparedtocontrolcells(Figure6(c)).ThesedataindicatethatahigherpurityofmultipotentmouseMSCscanbeachievedafterimmunodepletionofCD11b,CD34,andCD45positivecells.Moreover,weobservedthatlatepassageID-mMSCs(atpassage8)retainedtheirrobustcapacitytodifferentiateintoadipocytes,osteoblasts,andchondrocytes(Figure7).
4.Discussion
Mesenchymalstemcells(MSCs)havegeneratedagreatdealofinterestandpromiseasapotentialsourceofcells
forcell-basedtherapeuticstrategiesfortissuerepairandregenerativediseases,primarilyowingtotheirintrinsicabilitytoselfrenewanddifferentiateintofunctionalcelltypesthatconstitutethetissueinwhichtheyexist.Atpresent,thereisalsoagreatinterestforgeneticallymodifiedMSCsincell-basedtherapyforcancersbasedontherationaleoftumor-homingpropertiesofMSCs.However,mostofMSCtherapeuticapplicationsarestillinpreclinicalstudies.
FriedensteinandhiscolleaguesfirstlyestablishedMSCsculturefromguineapigbyvirtueofMSCspreferentialattachmenttoplasticflasks[38],andtheoriginalmethodhasbeenfurtherusedforthecultureBMMSCsfromhumanandotherexperimentalanimals[17–22].Thismethodhas,however,proveninefficientformMSCsduetolowmMSCsnumberandcontaminationofhematopoieticcellsinthecultures.Tosolvethisproblem,manygroupshavedevelopedvariousmodifiedmMSCscultureconditionstoobtainsufficientnumbersofcellsintheshortesttimeandwiththehighesthomogeneity.Kopenetal.firstlydevelopedamethodtoeliminatemyelopoieticcellsusingananti-CD11bantibody,butthisprotocolcouldnotdepleteallthehematopoieticcells[33].Baddooetal.presentedamodifiedpurificationapproachbasedonimmunodepletion
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Figure6:InvitrodifferentiationofmMSCs.ThesameamountofnonimmunodepletedmMSCsandID-mMSCswereplatedandadipogenic,osteogenic,andchondrogenicdifferentiationwasevaluatedatpassage3.OilRedOstaining((a),×200),vonKossastaining((b),×40or×100)andcollagentypeIIstaining((c),×100)showthatID-mMSCsexhibitmuchmoredifferentiatedcells,indicatingahigherpurityofmultipotentMSCswithmodifiedharvestmethodandimmunodepletion.Representativepicturesareshownfromthreeindependentexperiments.∗P<.05,scalebar=100μm.
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Figure7:LatepassageID-mMSCsretaintri-lineagedifferentiationpotential.Whenexposedininductionmedium,passage8ID-mMSCscanstillexhibitdifferentiationpotentialintoadipocytes,osteoblasts,andchondrocytes,asshownbyOilRedOstaining((a)×200),vonKossastaining((b)×100)andcollagentypeIIstaining((c)×100).Representativepicturesareshownfromthreeindependentexperiments,scalebar=100μm.
ofCD11b/CD34/CD45positivecellsfromculturedBMcells[34].Inonestudy,positiveselectionwasperformedinanattempttoobtainamorehomogeneousmMSCspopulation[32].ButsincethereisnospecificmarkerforMSCs,othernon-MSCswereunavoidableintroducedintothecultures.Moreover,severalothertechniques,includ-ingoptimizationofculturedensityandfrequentmediumchange,havealsobeenproposedbuttheseprotocolsarenotstandardized[30,31].Therefore,itremainsnecessarytodevelopareliableandeasymethodtoisolateandexpandahomogeneouspopulationofmMSCsfrommouseBM.
ThestandardmethodthatisusedforisolationofmurineBM-derivedMSCsistoflushBMcellsfromhindlegsofmice.However,withthismethod,thefrequencyofcollectedMSCsisratherlow[42].OurharvestmethodinvolvesnotonlyflushingoutBMcells,butalsocrushingthebonesofhindlegsandvertebraeaswellasenzymaticaltreatmentoftheboneswithCollagenase(Figure1(a)).StromalcellsarepresentintheendosteumwhereasmaturehematopoieticcellsexistinthecenteroftheBM[43,44].Becauseofthisdeeplocation,itisdifficulttoobtainenoughMSCsevenafterstrongflushingduringmarrowcellharvest.Inthisstudy,usingthemodifiedisolationmethod,wecouldisolate19.4%moreBM-nucleatedcellsthanthestandardmethod.CFU-Fassay,whichwasusedtodetectandevaluatethefrequencyoftheMSCsinfreshBM,hasfurthermorebeenusedtovalidatetheeffectivenessofthismodifiedmethod(Figures2(b)and2(c)).
AfterharvestingBMwithourmodifiedprotocol,weperformedmagneticcellsortingafterthefirstpassageusingnegativeselectionwithCD11b/CD34/CD45antibodies-coupledmicrobeadsasproposedbyBaddooetal.[34](Figure1(b)).Twoconsecutiveroundsofimmunodepletion,removingrespectivelyCD11b-positivemonocyticcellsandCD34-positivehematopoieticstemcellscandepletethemajorpartofcontaminatedcells,andthethirdroundofCD45-positivecellsdepletioncouldfurthereliminateallresidualcontaminatedhematopoieticcells.WefoundthatafterimmunodepletiontheMSCspopulationappearedmuchmorehomogeneousshowingmostlyspindle-shaped,cells(Figure4),whileunderthestandardexpansioncondi-tions,culturesusuallyremainedmorphologicallyhighlyhet-erogeneousuntilpassage5-6,presentinground,polygonal,spindle-shapedandflattenedcells.Somedegreeofmorpho-logicalheterogeneitycouldstillbeobserveduptopassage6–8ormore.AlthoughtheCD11b/CD34/CD45immunode-pletionprotocoldidremovealmostallthecontaminatedhematopoieticcellsfromtheBMcultures,wehavetonoticethatthenucleatedcellyieldinourmousestrainwaslowaftertheimmunodepletionstepincombinationwiththestandardharvestmethod(3.7%onaverage).However,weobservedthatafterCD11b/CD34/CD45immunodepletionincombi-nationwithourmodifiedBMharvestmethod,theyieldofnucleatedcellswasnearlytwicehigher(Figure5(a)).Theimmunodepletedcellscultureduptopassage3werepositiveforCD90,CD73,andSca-1buttotallynegativeforCD14,CD34,andCD45(Figure5(b)),whilecontrolcellswerestillsignificantlycontaminatedwithCD45+cellsandslightlycontaminatedwithCD14+andCD34+cells(Figure5(c)).Inadditiontomorphologyandphenotype,thebiologicalpropertythatuniquelyidentifiesMSCsistheircapacityfortri-lineagemesenchymaldifferentiation.So,wefurthertestedthetri-lineagedifferentiationabilityofID-mMSCsandcontrolcellsatpassage3.ID-mMSCsandcontrolcellswerebothabletodifferentiateintoadipocytes,osteoblasts,andchondrocytesdeterminedbyOilRedO,vonKossaandtypeIIcollagenstainingwhenexposedtorespectivelyadipogenic,osteogenic,andchondrogenicmedia.However,withthesameinitialcellnumbersandinductionconditions,ID-mMSCsexhibitedamorevigoroustri-lineagedifferentiationpotentialascomparedtothecontrolcells(Figure6)andcanretaintheirrobustdifferentiationpotentialatleastuntilpassage8(Figure7).Basedonthemorphology,phenotype,anddifferentiationpotentialinvitro,wecanconcludethatimmunodepletedmMSCsaremorepurifiedintheearlypassages.
JournalofBiomedicineandBiotechnology5.Conclusion
Takentogether,thecurrentstudypresentsaneffective,quickandeasy-to-performmethodforenrichmentandpurificationofmurineBMderivedmesenchymalstemcellsinvitrobycombiningmechanicalcrushingandCollage-nasetreatmentatthemomentofharvestandanegativeimmunodepletionstepduringtheinvitroexpansionculture.ThisprotocolcanfacilitatetheinvitroandinvivostudyofmMSCs,bothforexaminingtheirbiologicalpropertiesaswellastheirtherapeuticpotentialinvariousmurinediseasemodels.
Acknowlegments
TheauthorswouldliketothankNicoleArras,AngeloWillems,VeerleDeGreef,andWimRenmansfortheirexperttechnicalassistance.TheirresearchworkissupportedbygrantsfromtheFoundationforScientificResearch(FWO),theVrijeUniversiteitBrussel(HOA),the“VlaamseLigategenKanker”Belgium.S.XuissupportedbyaCSC-VUBscholarship.
ConflictsofInterests
Noconflictingfinancialinterestsexist.
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