三种干细胞的比较Isolation and Characterization of Mesenchymal Stem 2.docx

三种干细胞的比较Isolation and Characterization of Mesenchymal Stem 2.docx

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三种干细胞的比较IsolationandCharacterizationofMesenchymalStem2

SupplementaryMethods

CryopreservationofCB-MSC

MNCisolatedfromCBbydensitycentrifugation,andexpandedCB-MSC,BM-MSCandAT-MSCweresuspendedinculturemediumcontainingdimethylsulfoxide（DMSO,finalconcentration10%v/v）andfrozenatacoolingrateofapproximately-2˚C/minuteinaBicell（NihonFreezer）placedina-80˚Cfreezer（Sanyo）andthentransferredintoaLN2tank.CB-MSCatdifferentpassageswerecryopreservedbythestandardslowfreezingmethod.

IsolationofMSCfromhumanadiposetissueandbonemarrow

Humanadiposetissuefromhealthydonorsagedfrom21to49years（n=21）wasobtainedfromelectiveliposuctionproceduresunderanesthesiaafterinformedconsentwasobtained.Thecellswerederivedfromthefattyportionofliposuctionaspiratedasdescribedpreviously[Yoshimuraetal.,2006].Adiposetissuesweredigestedwith0.075%collagenase（Wako）at37˚Cfor30minutes,andnucleatedcellswereculturedatadensityof1×105cells/cm2ingelatin-coatedculturedishesinDMEMplus10%FBSwith2ng/mlFGF-1（PeproTech）and5ng/mlofheparin（Sigma-Aldrich）.Non-adherentcellswereremovedafterthreedays,andtheprimarycellswereculturedfor10days.Cellswerethendetachedandexpandedatthesamemedium.

FreshwholebonemarrowaspirateswerepurchasedfromVeritasPharmaceutical.BM-MSCwereisolatedasreportedpreviously[Kitamuraetal.,2004].Tenmlofthemarrowaspiratewerecombinedwith10mlofPBS,plasmaandfatlayerswereremovedfromthesupernatantaftercentrifuging,andtheremainingnucleatedcellswitherythrocyteswerepouredinto150-cm2flaskswith30mlofDMEMcontaining20%FBS.About10daysafterinitiationofprimarycultures,cellsweredetachedfromtheplatesandexpandedinthesamemedium.

Cellswereharvestedusing0.25%trypsin/0.5mMEDTA（Invitrogen）.Forexpansion,cellswereseededatadensityof1×104cells/cm2.Themediumwaschangedtwiceweekly.Allcellcultureinthisstudyweremaintainedundera5%CO2atmosphereat37˚Cinmediumcontaining1%antibiotics（100units/mlpenicillin,0.1mg/mlstreptomycin,Invitrogen）.Proliferationcurveswereobtainedbycountingdetachedcellseveryweek.

Cumulativepopulationdoublinglevel

Cumulativepopulationdoublinglevel（PDL）ateachsub-cultivationwascalculatedfromthecellcountusingtheequation[log10（NH）-log10（NI）]/log10

（2）=X,whereNI=inoculumnumber,NH=cellharvestnumber,andX=populationdoublings[Cristofaloetal.,1998].Thecellsobtainedfromprimarycultureweredefinedaspassage0andPDL0.CB-MSCusedinthisstudywerelessthan15PDLifnototherwisespecificallymentioned.

Flowcytometry

CB-MSCfromprimarycultureformingcoloniesandpassagecellswerestainedwithcombinationsofmonoclonalantibodiesasfollows:

CD14（M5E2;allophycocyanin[APC];BDBioscience）,CD29（K20;fluoresceinisothiocyanate[FITC];BeckmanCoulter）,CD31（WM59;FITC;BDBioscience）,CD34（581;APC;BeckmanCoulter）,CD44（G44-26;FITC;BDBioscience）,CD45（H130;APC;BDBioscience）,CD73（AD2;phycoerythrin[PE];BDBioscience）,CD90（F15-42-1-5;PE;BeckmanCoulter）,CD105（SN6;APC;eBioscience）,CD146（P1H12;PE;BDBioscience）,CD166（3A6;PE;BDBioscience）,CD271（C40-1457;PE;BeckmanCoulter）,HLA-ABC（B9.12.1;FITC;BeckmanCoulter）,HLA-DR（L243（G46-6）;PE;BDBioscience）,andNG2（7.1;PE;BeckmanCoulter）.IsotypecontrolsincludedmouseIgG1-APC,IgG1-FITCandIgG1-PEfromBeckmanCoulter.SK-N-MC,neuroepithelioma,andSK-MEL-28,malignantmelanoma,celllineswereusedasCD271andNG2positivecontrolcells,respectively,obtainedfromATCC.

Foranalysis,cellswereresuspendedinPBSand7-AAD（BDBioscience）wasaddedfordiscriminationofliveanddeadcells.TheanalysiswascarriedoutoneitheraFACSCaliburorFACSCanto（BDBioscience）.CellQuestandBDFACSDivaSoftware（BDBioscience）wereusedtoacquiredata,respectively.DataanalysisinvolvedpostacquisitiongatingusingFlowJosoftwareversion7.6.1（TreeStar）.

Telomeraseactivityandtelomerelengthassay

Telomeraseactivitywasassessedusingthetelomererepeatamplificationprotocol（TRAP）kit（Roche）accordingtothemanufacturer’sinstructions.DNAwasextractedbyusingEasy-DNA™Kit（Invitrogen）and1µgofDNAwasused.TelomerelengthwasdetectedbyusingaTeloTAGGGTelomerelengthAssay（RocheDiagnosticsCorp.）followingthemanufacture’sinstructions.

SizedistributionofTRF（telomererestrictionfragment）wascomparedwithaDNAlengthstandardandevaluatedutilizingthemultianalysersoftware（Bio-Rad,Hercules,CA,USA）.

Fluorescenceinsituhybridization（FISH）analysis

CBunitsofmaleinfantswereusedtoassesscontaminationofmaternalcellsintheCB-MSC（n=8）.FISHwasperformedusingaCEPX-αsat/Y-satIIIKit（Abbott）includingspectrumorange-labeledX-centromericandspectrumgreen-labeledY-centromericDNAprobesasdescribepreviously[Iguraetal.,2004].

Cytogeneticanalysis

MetaphasespreadswereobtainedfromeachCB-MSCcellline（n=5）atpassages8-16.Inbrief,culturedcellsweretreatedwithcolcemid（0.02μg/ml）for2htoaccumulatemetaphasesandcellsweresuspendedinahypotonicsolutionof0.075MKClat37˚Cfor15minutes.ThenthecellswerefixedwithCarnoy’sfixative（3:

1methanolaceticacid）andair-driedonglassslides.Toevaluatecytogeneticstabilityofeachcellline,theslideswerestainedwithbothHoechst33258andquinacrinemustard（Q-banding）[Satohetal.,1993].Over30well-bandedmetaphaseswerekaryotypedandanalyzedaccordingtotheinternationalsystemforhumancytogeneticnomenclature[Shafferetal.,2004].

Differentiationtochondrocytes,osteocytesandadipocytesinvitro

Chondrogenicinductionwasperformedasdescribedpreviously[Iguraetal.,2004;Zhangetal.,2006a;Zhangetal.,2006b].Briefly,2×105cellswereplacedina15-mlpolypropylenetubeandcentrifugedtoapellet.Thepelletwasculturedat37˚Cina5%CO2incubatorin500μlofchondrogenicmediumcontaining10ng/mltransforminggrowthfactor-β3（TGF-β3:

R&DSystems）and500ng/mlbonemorphogeneticprotein-2（BMP-2:

AstellasPharma）inbasicmediumwhichincludedhighglucoseDMEMsupplementwith10-7Mofdexamethasone,50μg/mlascorbate-2-phosphate,40μg/mlproline,100μg/mlpyruvate（Sigma-Aldrich）and50mg/mlITS+Premix（6.25μg/mlinsulin,6.25μg/mltransferrin,6.25ng/mlseleniousacid,1.25mg/mlbovineserumalbuminand5.35mg/mllinoleicacid;BDBiosciences）.TheculturewascontinuedthreeweeksandpelletsculturedwithoutTGF-β3andBMP-2servedasacontrol.Fortheidentificationofoptimalconditionsforchondrogenicdifferentiation,500ng/mlBMP-2,10ng/mlTGF-β3,10μg/mlinsulin,300ng/mlinsulin-likegrowthfactor-1（IGF-1）（PeproTech）,and10ng/mlFGF-2weretestedindependentlyasadditivestothebasicmedium.

Osteogenicinductionwasperformedasdescribedpreviously[Takahashietal.,2004].Inbrief,CB-MSCwereseededatadensityof1.5×104cells/cm2in12-wellplasticdishesandculturedfor3weeksinDMEM（lowglucose）plus10%FBSsupplementedwithorwithout0.1μMdexamethasone,50μMascorbicacid-2-phosphateand10mMβ-glycerophosphate.

Adipogenicinductionwasperformedasdescribedpreviously[Iguraetal.,2004;Zhangetal.,2006b].Briefly,CB-MSCwereseededatadensityof2×104cells/cm2inDMEM（lowglucose）plus10%FBSandcultureduntilreachingconfluence.Thenculturesweretreatedfor3dayswithadipogenic-inductionmediumcomposedofDMEM（highglucose,Sigma-Aldrich）,10%FBS,1μMdexamethasone,10μg/mlinsulin,0.2mMindomethacin,and0.5mM3-isobutyl-l-methy-xanthin（IBMX;Sigma-Aldrich）.ThiswasfollowedbymaintenanceusingadipogenesismaintenancemediumcomposedofDMEM（highglucose）plus10%FBScontainingfor4days.Thiscyclewasrepeatedthreetimes.

Detectionofdifferentiationinvitro

Chondrogenicdifferentiationwasevaluatedbymeasuringthesizeandweightofpellets,bystainingsectionsofpelletsbytoluidinebluetodetectproteoglycan（extracellularmatrix）,byandimmunohistochemicalanalysiswithanti-typeIandtypeIIcollagenantibodiesasdescribedpreviously.Alkalinephosphatase（ALP）andvonKossastainingswereusedtodetectosteogenesis.Oil-Red-Ostainingwasusedtodetectadipogenesisasdescribedpreviously[Iguraetal.,2004].

ForanalysisofosteogenicdifferentiationofCB-MSC,ALPactivitywasmeasuredbyp-nitrophenolphosphatesubstratebuffer[Kitamuraetal.,2004].Activitywasrepresentedbyp-nitrophenolwhichwasreleasedafterincubationfor30minutesat37˚CandwasnormalizedforDNAcontents.DNAcontentwasmeasuredbyQuant-iTTMPicoGreen®dsDNAReagentandkits（Invitrogen）followingthemanufacturer’sinstructions.

Calciumcontentandosteocalcincontentweredetectedincelllayersin12-welldishesafter2weeksofosteogenicinductionasdescribedpreviously[Kitamuraetal.,2004].CalciumcontentwasmeasuredusingcalciumCkit（Wako）,andosteocalcincontentwasmeasuredusinghumanosteocalcinEIAkit（TakaraBio）.

InvivochondrogenesisandosteogenesisofCB-MSC

AnimalstudieswereapprovedbytheInstitutionalAnimalCommitteeoftheIMSUT.ChondrogenesisofCB-MSCinvivowasexaminedbytransplantationofCB-MSCsubcutaneouslyinto6-week-oldnudemice（n=4）（CLEAJapan）for3weeksasdescribedpreviously[Zhangetal.,2006a].Briefly,1×106cellsin100µlmediumwereseededintoacollagensponge（5mmindiameterby3mminthickness,BDBiosciences）andthecompositewasculturedfor2weeksinchondrogenicmediumandthentransplantedintosubcutaneouspocketsofnudemice.After3weeks,thecompositewastakenout,embeddedinparaffi