人胎盘组织来源造血干／祖细胞及其特性

人胎盘组织来源造血干／祖细胞及其特性 人胎盘组织来源造血干,祖细胞及其特性 ;;:7;l:‰cMedicne中国组织工程研究与临床康复第J2誊第2J期2008一o5—2O 出版 JournalofClinicalRehabilitativeTissueEngineeringResearchMay20,2008Vo1.12No.21 Propertiesofhematopoieticstem/progenitorcellsderived fr0mhUmanplacentatissues冰?? ZhangTao,FangNing,ChenDai-xiong,LiuZu?lin,LiuJin?wei,WanWei-hong,QiYing,X iaoJian?hu XiaoYu KeyLaboratoryof CellEngineeringof GuizhouProvince, 山cAffiliated HospitalofZunyi Medica1College, Zunyi563003. GuizhouProvince, China ZhangTao?.Doctor, Associatedprofessor, KeyLaboratoryof CellEngineeringof GuizhouProvince, 山cAffiliated HospitalofZunyi MedicalCollege, Zunyi563【x】3. GuizhouProvince, China oceanzt@163com Correspondenceto: FangNing,Senior Experimentalist.Key LaboratoryofCell Englneenngot GuizhouProvince, 山cAmliated HospitalofZunyi Medica1College, Zunyi563【x】3. GuizhouProvince, China Supportedby: GuizhouScienceand TechnologyProgram, No2051; 2o03JGY005 7一lO—l9 Received:2【x】 Accepted:2007—1t-03 (07—50一t0—5663/1-1) ZhangFangN, ChenDX,LiuZL, LiuJW,wanWH. QiY?XiaoJH,Xiao Y.Propertiesof hematopoietic stem/progenitor cellsderivedfrom humanplacenta tissues.Zhongguo ZuzhiGongcheng YanjiuyuLinchuang Kangfu2008;12(21): 4172-4176(China) 【wwwzglckf.com/ zglckffejournal/ upfiles/08—21/ 21k-4172(ps)paq 4172 Abstract BACKGRoUND:Asiswellknownthathematopoieticstemandprogenitorcells(HSPCs)c ontaininbonemarrow,peripheral blood.andcordblood.Recentstudiesfoundthathumanplacentatissue(alsoexistsinHSPC s.Butsofarthepropertyand differentiationcapacityofhumanPT—HSPCsisnotyetknown.Furthermorethecomposit ionoflymphocytesubpopulationsand immunogenicityregardedtohumanPT-HSPCsarcalsounclear. oBJECTIVE:ToverifywhethertherearemoreHSPCsinhumanPTthanthoseinhumanum bilicalcordblood(UCB).to investigatetheircapacitiesofproliferationanddifferentiation,andtoanalyzethephenotyp esoflymphocytesubpopulationsin human DESIGN.TIM[EANDSETrING:OpeneXperimentswereperformedattheKeyLaborato ryofCellEngineeringofGuizhou ProvincefromJanuary20o4t0Deeember2()o6. SETTING:KeyLaboratoryofCellEngineeringofGuizhouProvince.theA伍 liatedHospitalofZunyiMedicalCollege. MATE砒 ALS:TwelvehumanplacentaandUCBsamplesthroughcesareandeliveryWel?ecollectedasepticallywitl1theinforrned consentsofparturientsderivedfromMaternityDepartmentoftheAffihatedHospitalofZunyiMedicalCollege.Themalnreagents weredetailedasfollows:lymphocytesubpopulationsanalysisreagentsSimultestIMK-lymphocyteKit.CD34absolutecounting reagentsKit(BectonDickinson);CD34MultisortKit,FITCconjugatedCD38monoclonalantibody,anti—FITCmicrobeadsand MS,LSminiMACSsegregatingcolumns(MiltenyiBiotec). M[ETHoDS:UCBsampleswerel:ldilutedwithRPMI—l640containing0.1volumefracti onoffetalbovineserumandthe mononuclearcells(MNCs)wereisolatedonFicoil—Histopaquebycentrifugationfor30 minutes.TheMNCsattheinterfacewere collectedandwashedwitl1PBS.SinglecellssuspensionliquidofhumanPTwaspreparedbymechanicalmethodcombinedwitl1 O.25g,Lcollagenasedigestion.Atierthat,theplacentasamplesunderwentthesameprotocolasusedinUCBtoisolateMNCs.The percentageofCD34CD38?.CD34CD38+HSPCsandthephenotypeoflymphocytesubpo pulationsderivedfromhumanr_MNCs wereanalyzedbyflowcytometry(FCM).CD34CD38?,CD34CD38一 cellsubsetsisolatedbymagnetic—activatedcellsorting (MACS)fromhumanPTwereusedtocarryoutcolony--formingcultureincludinggranulocyte/macrophagecolony??formingunit (CFU—GM).burstformingunit—erythroid(BFU-E)andmixedcolony—formingunit( CFU—Mix)inordertoassesstheircapacitiesof hematopoieticprogenitorcells?proliferationanddifierentiation.Inparallel.UCBsamplesunderwentthesameprotocolsfor comparison. MAINOUTCoMEMEASURES:PercentcompositionsofCD34HSPCs.hematopoieticprogenitors?lineagecolony—forming capacitiesofCD34HSPCs.phenotypesandcompositionsoflymphocytesubpopulationsbothinPTandUCB. RESULTS:ThepercentageofCD34cellscontainedinhumanPTwas8.8timeshigherthanthatofinUCB(P<0.01).Thetotal numberoflymphocytes,Tcells(CD3?D2),Bcells(CD19),Th(CD3? D4)andTh,rSratiowereapparentlylowerinhuman placenta.whilethenumberofCD8CD28一 TsuPPressorcellswerehighercomparedtoUCBsamples(P<0.00.Among CFU—GM.BFU—EandCFU—MixfrequenciesofCD34+CD38?cellssubsetweremuc hhigherthanthatofCD34CD38一(P<0.00. withinthesamephenotypeofcellsubsets.however,thenumberofeachcolony—forminguhitwassimilarbetweenPTandUCB(P> O.O51. CoNCLUSION:HumanPTisricherinCD34CD38一.CD34+CD38+HSPCsandbothofthemhavetheabilitiesofproliferatingand GM,BFU.EandCFU—Mix.ConsideringthathumanPThavealo direndatin2intoCFU— werlymphocytesubpopulationsand higherTscells.humanPTmightbeaalternativeandsuitablesourceofHSPCsforclinicaltra nsplantation. 1NTR0DUCTl0N SinceKnudtzonelalfirstdiscoveredthat umbilicalcordblood(UCB)wasricherin hematopoieticstemandprogenitorcells(HSPCs), transDlantationusingcordbloodhasbecomean importantmeanoftreatmentforhematopoietic functionfailure,hematopoieticmalignancyand somegeneticdiseases【引.However.thenumber ofHSPCscontainedineachsampleofUCB cannotaffordthehematoDoieticreconstitutionof anadultpatient.Thus,searchinganewsource whichcontainsricherHSPCsisvervinevitable. RecentstudiesfoundthatDlacentatissuealso containHSPCs.Butsofarthepropertyand difflerentiationcapacitvofhumanplacenta derivedHSPCsarenotvetknown.Thiswork usednowcVtometrVtoinvestigatethe DhenotVpesofnucleatedcel1sinhuman placentatissue(PT)andisolatedhumanPT- CD34CD38,CD34CD38?HSPCsbV magnetic—activatedcel1sorting(MACS).We f0undthathumanPTcontainedmoreHSPCs comparedwithUCB,andbothCD34CD38, CD34CD38?HSPCsderivedfromhumanPT canDroliferateanddiff.erentiateinto colony—f0rmingunit(CFU—GM)?burstf0rming unit.ervthroid(BFU—E),mixedcolony—f0rming unit(CFU—Mix).Furthermore,our蟊taalso shownthathumanPTcontainedlowerT lymphocytesandhigherTsuppressorceUs.Al1 thesedatasuggestedthathumanplacenta whichwasusual1vdiscardedcouldbea valuablealtemativesourceoftransDlantable HSPCs. Po.B12o0.s}le?ng】】o0o413385o83@sim.com Zhangzela1.Propertiestffhematopoieticstem~progenitorcellsderived,mhumanplacent atissueandtheirproperties… MATERIALSANDMETH0DS Materlals TheexperimentwasperformedattheKeyLaboratoryof CellEngineeringofGuizhouProvincefromJanuary2004 tODecember2006.TwelvehumanplacentaandUCB samplesthrOughcesareandeliverywerecollected asepticallywiththeinformedconsentsofparturients derivedfrommaternitydepartmentofourhospitalandthe experimentwasapprovedbytheEthicsCommitteeofthe AffiliatedHospitalofZunyiMedicalCollege. Fluorescein—labeledmonoclonalantibodiesofCD moleculesarelistedinTablel;lymphocytesubpopulations analysisreagentsSimultestIMK—lymphocyteKit.CD34 absolutecountingreagentskitwerepurchasedfromBecton Dickinson;CD34MultisortKit,FITCconjugatedCD38 monoclonalantibody,anti—FITCmicrobeadsandmini MACSsegregatingcolumns(MS,LS)werepurchasedfrom Histopaque,agarareproductsof MiltenyiBiotec;Ficoll— Sigma;RPMI一1640,IMDM,FBS,andDESwereproducts ofGibco;IL一3,GM—CSF?methvlcellulosewerepurchased fromIntergenCompany. Methods Samplescollection:UCBsamplewerecollectedinsterile 250mLglassbottleswithheparin(20U/mL).Beforeseparation, thesamplewere1:ldilutedinRPMI—l640confining0.1 vohmefractionoffetalbovineserumandthemononuclearcells (MNCs)wereisolatedbycentrifugationonFicoll—Histopaque (1.077g/mL,Sigma;USA)at400×gfor30minutes. ?CsattheinterfacewerecollectedandwashedwithPBS. Singlecellssuspensionliquidof mechanicalmethodcombined humanPTwaspreparedby with0.025%collagenase digestionfor20minutes.Afterthat,theplacentasamples underwentthesameprotocolasusedinUCBtoisolateMNCs asdescribedabove. Immunophenotypicanalysisoflymphocytesubpopulations byflowcytometry:MNCsofPTandUCBwereincubated withmouseIgGtoblocknonspecificbindingtoFcreceptor andthenstainedwithapanelofmonoclonalantibodieslisted above(Table1).Flowcytometryanalysiswasperformed usingaFACScaliburcytometer(BectonDickinson).Atleast 10000eventswererequiredforeachanalysis. IsolationofCD34HSPCssubsetsbyMACS:After 1ssN|673.8225CN2ll539RcoDEN:ZLKHAH MNCswerewashedandcounted.theCD34cellsfraction bothfromPTandUCBwereisolatedbyMiniMACS columnsfMS/LS1andCD34IsolationKitaccordingtothe instructionofmanufacturer(MiltenyiBiotec;Germany). Briefly,MNCswereincubatedwithaFcRblockingreagent f0rl0minutesandthereafterlabeledwithCD34microbeads antibodyfQBEND/10)for30minutesat4?to8?.C:ells werethenwashedonceinacoldsolutionofPBScontaining 0.5%BSAand2mmol,LEDTAbeforepassingtheentireeell suspensionthroughaMiniMACScolumninamagneticfield wheretheCD34microbead—labeledcellswereretainedonthe column.TheCD34fractionwasrecoveredbyreleasingthe magneticfieldandelutingthepositivecellsfromthecolumn. Usually,thepurityofcellsisolatedwasgreaterthan80%,as determinedbyFCManalysisandtheviabilityofthecells isolatedwasover95%bvl%trypanbluestain.Inorderto isolatetheCD34CD38andCD34CD38一subsetsofCD34 cells,theFITCconjugatedCD38monoclonalantibody combinedwithanti—FITCmicrobeadswereusedaccording totherecommendationofmanufacturer. Colony—formingassays:Thecolony—formingassaysof CD34CD38andCD34CD38cellsisolatedfrombothPT andUCBwereperformedasfollows.TheassaysforBFU—E. CFU—MixwasperformedinalmLmixtureofIscove?s modifiedDulbecco?smediun(GIBIC0)containing5×l0 cells,0.9%methvlcellulOse,0.3volumefractionoffetal bovineserum(GIBICO),50umol/L2一mercaptoethanol, l%bovineserumalbumin(BSA),2mmoI/LL—glutamine. ForCFU—GMassaV_lmLmixtureofRPMI—l640 containing5×l0cells,0.3%agar,0.2volumefractionof donorequineserum(DES),2mmoI/LL—glutaminewere used.Severalgrowthfactorswereaddedtosustainthe formationofCFU.GM(50ng/mLGM—CSF),BFU—E f2ng/mLIL一3,l0U/mlEPO),andCFU—Mix(10ng/mL IL一3,l0ng/mLGM—CSF.3U/mLEP0).AUtheassayswere performedintriplicate35mmPetridishes.Culturewere performedat37?inawhole—humidifiedatmosphereat 5%CO,.Colonycountingwascarriedoutondayl4 post—culture. Statisticalanalysis:Statisticalanalyseswereca~iedout withSPSSl3.0softwareandrepresentedasMean?SD. Intergroupcomparisonsweremadebyt-test.AvalueofP< 0.01wasregardedassignificantdifference. RESUU-s HulnanPTcontaInsricherInHSPCs Theresults(Table2)indicatedthatthepercentageof CD34cellsfromahumanPTwas8.8timeslargerthan thatofUCBwhileCD34CD38一andCD34CD38cells were4.6andl1.9timeslargerthanUCB.respectively. TheseresultssuggestedthathumanPTcontainsricher HSPCsthanUCB. tymphocytesubpOpIatIOnsInhumanPT ExceptforNKcells(CD16CD56/CD3?),thepercentageof lymphocytes,Tcells(CD3,CD2)andBcells(CDl9)in PTwereobviouslylowerthanthatofinUCB.Inaddition,T helpercells(CD3/CD4)andTh厂11sratioinPTwerealso apparentlylowerthanthatinUCB(Tlable3). 4173 , v.zglckfcomZhang17,etalPropertiesofhematopoieticstern/progem?torcellsderived(“nhu manplacentatissueandtheirproperties ComparisonofTcellssubpOpuIatiOnsbetweenPTand UCB InPT,ThelpercellssubpopulationsThl(CD4/CD45RA) andTh2fCD4/CD45R01were(3.65+_2.70)%and (5.07+_2.15)%,respectively.ComparedwithUCBwhose counterpartswere(24.31?8.66)%andr4.64+_3.98)%Th1 wasapparentlylowerinplacenta(P<0.01)whileTh2had notsignificantdifference(P>0.05,Figure1).Among CD8TcellssubDopulationsinPcytotoxicTcells (CD8/CD28)andTsuppressorcells(CD8/CD28?)were (12.34+_2.93)%and(13.05+_3.31)%,respectively.Cytotoxic TcellswereobviouslydecreasedwhileTsuppressorcells wereapparentlyincreasedcomparedwithcordbloodwhose counterpartswere(25.85+_4.36)%and(4.35+-0.44)%fP< 0.01,. CellpurificationandrecoveryofCD34+HSPCs AfierMNCswereisolatedbyMACS.thepurityofCD34 HSPCsderivedfromUCBandPTwere(8639?l1_271% and(73.32?l3.40)%.respectively,whiletherecoverywere (63.05?l0.14)%and(56.29+_8.16)%,respectivelY (Figure1). Colony-formingcapacitiesofCD34subsets ResultsareshowninTable4andFigure2.Withinthe samephenotypecellsubsets.thefrequenciesOfCFU—GM. BFU—E.andCFU—MixweresimilarbetweenPTandUCB. BothforPTandUCBhowever,thefrequenciesOfal】 three—typecoloniesgeneratedfromCD34CD38cells werehigherthanCD34CD38?cells.Theseresults confirmthatbothPTandUCB.andtheCD34CD38 subsetconsistedprimarilyofstemorprogenitorscells andtheCD34CD38subsetbelongedtomoremature progenitors. 4l74 0 42 CD38FJTC a:ProportionaldistributionofCD34 cellsi/1MNCsfromUCB 10”…7;4 CD38FffC c:ProportionaldistributionofCD34 cellsinMNCsfromPT Figure ,噜 CD38FrrC b:ThepurityofCD34cellsafter sortingofMNCsbvMACSInUCB 寸 CD38FITC d:ThepurityofCD34cellsafter sortingofMNCsbyMACSinPT Flowcytometer(FCM)analysisdemonstratingthepurityof CD34cellsafterplacentatissue(PT)andumbilicalcord blood(UCB)一mononuclearcells(MNCs)beingsortedby magnetic—activatedcellsorting(MACS) O.Box1200.Shenyang110004kf23385083@sina.com c0 ?102l0.. 7震ww礓k~comZhange.Prop?h?啪poie”c耵e嘶雠csved『romhmnnp州 Btissueand|rprop? criterionforevaluatingHSCs,sofurtherinvestigatingtheir biologicalpropertiessuchashoming,invivohematopoietic reconstitution,flexibility,etc.aremeaningfulforassessing humanPTasapotentialsourceoflransplantableHSPCs. 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