Isolation of an adult blood-derived progenitor cell population capable

Cwithoutbrake.<br><br> Afterwashingwithphosphate-bufferedsaline(PBS),cellswere re-suspendedinasmallvolume(1 Æ 5 33 Æ 0ml)ofX-vivo15 serum-freemedium(Cambrex,EastRutherford,NJ,USA)and subjectedtoaseconddensity-basedcellenrichmentstepusing eitherOptiPrep(Axis-ShieldPoCAS)orPercoll(GEHealth- care,AmershamBiosciences,Uppsala,Sweden).Cellssubjected toOptiPrepgradientwerecentrifugedfor30minat700 g , 21 ° Cwithoutbrake;CellssubjectedtoPercollwerecentrifuged for30minat1260 g ,13 ° Cwithoutbrake.Layersofcells havingadensityoflessthan1 Æ 072g/mlwerecollectedtoa 50mltubepre-PlledwithPBS,washedtwicewithPBSand cultured invitro .BothOptiprepandPercollgradientswere equallyeffective. CellCulture Synergeticcellpopulationcells,seededataconcentrationof 1 Æ 5 33 · 10 6 cells/mlinX-vivo15mediumsupplementedwith 10%autologousserum,wereculturedon25 l g/mlPbronectin (Chemicon,Temecula,CA,USA)orautologousplasmacoated dishes(Corning,Corning,NY,USA).Furtherdifferentiation wasachievedbygrowingtheSCPundercultureconditions speciPcforeachlineage.Uponterminationoftheculture,non- adherentcellswerecollectedandcombinedwiththemechan- icallydetachedadherentcells.TogenerateACPs,SCPcells wereculturedataconcentrationof1 Æ 5 33 Æ 0 · 10 6 cells/mlas describedaboveandfurthersupplementedwith1 310ng/ml vascularendothelialgrowthfactor(VEGF,R&DSystems, Minneapolis,MN,USA)and5IU/mlheparin(Kamada,Beit- Kama,Israel).Togenerateneuralcellprecursors(NCPs),1 Æ 5 3 2 Æ 5 · 10 6 SCPcells/mlweresupplementedwith10ng/mlbasic Pbroblastgrowthfactor(bFGF,R&DSystems),25ng/ml brain-derivedneurotrophicfactor(BDNF,PeproTech,Rocky Hill,NJ,USA),50ng/mlnervegrowthfactor(NGF,Pepro- Tech),and5IU/mlheparin.After8d,cellswerewashedand incubatedinX-vivo15mediumcontaining33%F12,2%B27 (Sigma-Aldrich,StLouis,MO,USA),10ng/mlbFGF,25ng/ mlBDNF,50ng/mlNGF,20ng/mlepidermalgrowthfactor (EGF,PeproTech),and5IU/mlheparin.Togeneratemyo- cardialcellprecursors(MCPs),2 Æ 0 33 Æ 0 · 10 6 SCPcells/ml wereculturedasdescribedaboveandfurthersupplemented with10ng/mlbFGFand5IUheparin.Tendaysafter cultureonset,3 l M5-azacytidine(Sigma-Aldrich)wasadded for24h. Tubeformationassay Tubeformationwastestedusingan invitro angiogenesisassay kit(Chemicon).BrieQy,harvestedACPs(0 Æ 1 30 Æ 4 · 10 6 cells/ ml)oracetylatedlowdensitylipoprotein(Ac-LDL)-DiO(BTI, Stoughton,MA,USA)pre-loadedACPs,wereculturedover- nightina96-wellplateusingM199medium(Sigma-Aldrich) containing10%autologousserum,10ng/mlVEGF,10ng/ml bFGF,5IU/mlheparin,and25 l g/mlendothelialcellgrowth supplement(ECGS;BTI)onextracellularmatrix(ECM)gel.<br><br> Tubeformationwasassessedvisuallyusinganinvertedlight microscope(NikonECLIPSETS-100;Nikon,Melville,NY, USA).Angiogenicpatternandvasculartubeformation werescoredaspreviouslydescribed(Kayisli etal ,2004):grade 0 3scatteredindividualcells;grade1-cellsbeginningtoalign witheachother;grade2 3organizationintovisiblecapillary-like structures;grade3-sproutingofsecondarycapillarytubes; grade4-closedpolygonsofcapillariesbeginningtoformand grade5-complexmesh-likecapillarystructures. Immunocytochemistry CellsweregrownonPermanox(Nunc,Rochester,NY,USA) slidesorloadedonslidesafterharvestingandPxedin3% paraformaldehyde(PFA,Sigma-Aldrich)for15minatroom temperature.Followinga30minnon-speciPcstainblocking step(4%normalserum,1%bovineserumalbumin(BSA),and 0.1%TritonX-100;Sigma-Aldrich),cellswereincubated overnightat4 ° CinthedarkwithspeciPcanti-human antibodiesormatchednon-speciPcisotypecontrols.The variouscelllineageswerestainedusingthefollowing:ACPs- CD31-phycoerythrin(PE)orCD31-Quoresceinisothiocyanate (FITC)(eBioscience,SanDiego,CA,USA)andFITC-labelled Lectinfrom Ulexeuropaeus (Ulex-Lectin,Sigma-Aldrich); NCPs-Neu-N-Alexa488(Chemicon),GlialPbrillaryacidic protein(GFAP,DakoCytomation,Glostrup,Denmark),Nes- tin, b III-Tubulin,andOligodendrocyte(O4,R&DSystems); MCPs-cardiacTroponinT,Desmin,andConnexin43 (Chemicon).Goatanti-mouse(GaM)IgG-FITC,GaMIgGPE (Chemicon)wereusedasisotypecontrols.Theprimary antibodieswerevisualisedbyGaMIgG-FITC,GaMIgG-PE (Chemicon)orRabbitanti-mouseIgG-Cy3(JacksonImmu- noresearch,WestGrove,PA,USA).ForAc-LDLuptake,cells wereincubatedinthepresenceof0 Æ 8 l g/mlAc-LDL(Alexa Y.Porat etal ª 2006TheraVitaeLtd 2 JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology Fluor488AcLDL-Invitrogen,Carlsbad,CA,USAorAc-LDL- DiI 3BiomedicalTechnologies,Inc.,Stoughton,MA,USA)for 15minat37 ° C,afterwhichtheywerewashed,Pxedin3%PFA andstainedwithCD31-FITC,CD31-PE(eBioscience)or FITC-labelledUlex-Lectin(Sigma-Aldrich).Slidesweremoun- tedwithaQuorescentmountingsolutioncontainingthe nuclearstain4 ¢ ,6-diamidino-2-phenylindole(DAPI)(Vector, Burlingame,CA,USA)andexaminedoneitheranOlympus BX-50oraNikonE400microscopeequippedwithappropriate excitationandbarrierPlters.Slidesstainedwithhematoxylin andeosin(H&E)wereexaminedonaNikonE200light microscope. Flowcytometry HarvestedcellswerewashedinPBSandcellpelletswere re-suspendedin100 l lPBS,stainedwithspeciPcQuoro- chrome-conjugatedornon-conjugatedprimaryanti-human antibodiesorisotype-matchednon-speciPccontrols,incuba- tedinthedarkfor30minonice;incaseofnon-conjugated primaryantibodyitwasfollowedbyQuorochrome-labelled secondaryantibody.SCPswerestainedusingthefollowing antibodies:CD31-FITC,CD45-PE(eBioscience)andCD34- APC.ACPswerestainedusingCD14-FITC,CD31-PEor CD31-FITC,CD34-APC,CD117-APC(DakoCytomation), CD133-PE,CD144-FITC,KDR-PE,Tie-2-PE(R&DSys- tems),VWF 3FITC(Chemicon)andUlex-Lectin-FITC.NCPs werestainedusingNestinand b III-Tubulin.MCPswere stainedusingDesminandcardiacTroponinT.GaMIgG- FITCandGaMIgG-PEwereusedassecondaryantibodies.<br><br> InthecaseofCD31andCD34theresultsrepresentthe percentageofcellswithbrightintensity(CD31 Bright and CD34 Bright respectively);cellstainingwasconsideredbrightif stainingintensitywasatleast50timeshigherthanthe intensityofthecorrespondingisotypecontrolstaining.For Ac-LDLuptake,cellswereincubatedinthepresenceof 0 Æ 8 l g/mlAc-LDL(AlexaFluor488AcLDLorAc-LDL-DiI) for15minat37 ° C,afterwhichtheywerewashedand stainedwithFITC-orPE-conjugatedCD31.Exclusionof deadcellswasperformedusing7-aminoactinomycinD(7- AAD;eBioscience)staining.Intracellularstainingwascarried outoncellsPxedin3%PFAandpermeabilizedby0 Æ 1% TritonX-100.Fivehundredthousandcellspersamplewere stained;atleast10000cellulareventspersamplewere assessedbyQowcytometry(FACScalibur,BectonDickinson, Rockville,MD,USA)andanalysedby cellquestpro software(BectonDickinson).Theresultsareexpressedas mean±standarderror(SE)ofthepercentageofstained cells. Analysisofcytokinesecretion HarvestedcellswerewashedinPBS,cellpelletswere re-suspendedto1 · 10 6 cellsin1mlX-vivo15andgrown for24hin24-wellplates.Cytokinesecretiontothesuper- natantwastestedusingQowcytometry,applyingtheBD TM CBAHumanAngiogenesisKit(BectonDickinson). Calciumuptakeassay Ca 2+ inQuxthroughvoltage-gatedcalciumchannelsin responsetoneurotransmitterstimulationwith100 l mol/l glutamateand100 l mol/lGABA(Sigma-Aldrich),wasper- formedaspreviouslydescribed(HershPnkel etal ,2001).<br><br> BrieQy,harvestedcellswereculturedovernighton33mm glassslidescoatedwithpoly- l -lysine.Cellswereincubatedfor 30minwith5 l mol/lFura-2acetoxymethylester(AM;TEF- Lab,Austin,TX,USA)in0 Æ 1%BSAinNaClRinger 9ssolution. Afterdyeloading,thecellswerewashedinRinger 9ssolution, andthecoverslidesweremountedinachamberthatallowed thesuperfusionofcells.FreecellularCa 2+ levelmeasuredby Fura-2thatwasexcitedat340nmand380nmandimaged witha510nmlong-passPlter.Theimagingsystemconsisted ofanAxiovert100invertedmicroscope(Zeiss,Go ¨ ttingen, Germany),PolychromeIImonochromator(TILLPhotonics, Planegg,Germany),andaSensiCamcooledcharge-coupled device(PCO).Fluorescentimagingmeasurementswere acquiredwithImagingWorkbench2(AxonInstruments, FosterCity,CA,USA). Statisticalmethods Theresultsarepresentedasmean±SEofindependent experiments.Statisticalanalyseswasperformedusingtwo- tailedStudent 9s t -test; P £ 0 Æ 05wasconsideredasigniPcant difference.Thecorrelationgraphwasanalysedbyanonpar- ametrictwo-tailedanalysis( graphpadprism software;Graph- PadSoftware,SanDiego,CA,USA).<br><br> P £ 0 Æ 05wasconsidereda signiPcantdifference. Results CharacterisationofSCP Peripheralbloodmononuclearcellsobtainedfromindividual normalblooddonationswereusedinindependentexperi- mentstoisolatetheSCP.CD45cellscomprisedmorethan 85%,inbothPBMCandenrichedSCP(datanotshown).As canbeseeninFig1A1andA2,thepercentageofCD34 Bright cellsintheSCPwas3 Æ 5-foldhigherthaninthePBMC population.Furthermore,thepercentagesofCD31 Bright , CD34 + CD45 ) /Dim andCD34 Bright cellsintheSCPandthe PBMCpopulationswere67 Æ 2±3 Æ 5%,3 Æ 12±0 Æ 58%and 0 Æ 36±0 Æ 07%vs.18 Æ 2±1 Æ 5%,1 Æ 04±0 Æ 18%and0 Æ 09± 0 Æ 02%respectively(Fig1B).CulturedSCPcellsadheredto theculturedishsurfaceand,after4dofculture,twomain typesofcellmorphologies,mitoticandmultinucleated,were observed(Fig1C).Themorphologyofthemultinucleatedcells andtheexpressionofCD31onbothSCPandACPssuggested thatsomeofthemmaybeosteoclastsormegakaryocytes,both Blood-derivedMultipotentProgenitorCellPopulation ª 2006TheraVitaeLtd JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology 3 characterisedbyCD51/CD61,receptoractivatorofnuclear factorkappaBligand(RANKL)anditsdownstreamindicator tartrate-resistantacidphosphatase(TRAP).However,their speciPcnatureandbiologicalactivitywerenotdetermined duringthecultureperiodandwillbeaddressedinfuture studies. Theseresultsdemonstratedthepreferentialexpressionof CD31 Bright ,CD34 + CD45 ) /Dim andCD34 Bright intheSCPthat can,subsequenttoculturingonPbronectinorplasma,giverise tocellsthatexhibitapronounceddifferentiationpotential.<br><br> CharacterisationofACPs TheSCPseedingefPciencywas38 Æ 4%±2 Æ 4%( n ¼ 14). Whengrownfor5dinamediumcontainingautologous serum,heparinandVEGF,SCPdifferentiatedintoACPs exhibitingthecharacteristicelongated,spindle-shapedmor- phology(Fig2A).Despitelosingthismorphologyfollowing harvesting,ACPsretainedtheabilitytorenewfullydifferen- tiatedculturesofelongatedandspindle-shapedcellswhenre- platedonaPbronectinsurfacefor24h(Fig2B).The functionofdifferentiatedACPswastested invitro :angiogenic potencywasassessedbymicroscopicexaminationofvascular tubeformationpattern18 348haftercellseedingonECM. Semi-closedandclosedpolygonsofcapillariesandcomplex mesh-likecapillarystructureswereobservedandscoredas grade4 35(Fig2C).Thesetube-formingcellsoriginated mainlyfromACPscapableofAc-LDLuptake(datanot shown).Supportivecytokinesecretionby10 6 ACPcells culturedfor24hinserum-freemedium(X-vivo15)was assessedusingtheQowcytometry-basedCBAkit(BD Biosciences).Resultsshowthat,whencomparedtoX-vivo 15control,ACPssecretedinterleukin(IL)-8 (10107±1108pg/ml),VEGF(165±6pg/ml)andangioge- nin(615±62pg/ml(Fig2D)butnottumournecrosisfactor (TNF)andb-FGF(datanotshown).Immunostainingofcells harvestedandPxedonslidesshowedtypicalangiogenic characteristicsofconcomitantbindingofUlex-Lectinand uptakeofAc-LDL(Fig3A1,A2andB).Flowcytometry assessmentofACPsshowedexpressionofthestemcell markersCD34(23 Æ 6±3 Æ 6%ofthecells),CD133 (10 Æ 1±2 Æ 1%)andCD117(7 Æ 0±1 Æ 9%)andendothelial/angi- ogenicmarkersKDR(10 Æ 2±4 Æ 8%),Tie-2(31 Æ 8±4 Æ 2%), CD144(24 Æ 4±5 Æ 3%),vonWillebrandfactor(VWF; 30 Æ 1±8 Æ 1%)andCD31 Bright (67 Æ 9±4 Æ 5%).Additionally, 68 Æ 2±7 Æ 6%ofACPsshowedconcomitantbindingofUlex- (A1) (B)(C) (A2) Fig1.<br><br> Characterisationofthesynergeticcellpopulation(SCP).(A)Flowcytometryanalysisofexpressionofmultipotenthaematopoieticcellular markerCD34,detectedusinganti-CD34-APConfreshlypreparedperipheralbloodmononuclearcells(PBMC)(A1)andonSCP(A2).(B)Flow cytometryanalysisofPBMCandSCPstainedwithanti-CD31-Quoresceinisothiocyanate(FITC)(left y -axis;greyhistogramsrepresentPBMC;black histogramsrepresentSCP),anti-CD45-PEandanti-CD34-APC(right y -axis;greystripedhistogramrepresentsPBMC;blackstripedhistogram representsSCP).Thepercentageofcellsexpressingthemarkersispresentedasmean±SE(CD31 Bright , n ¼ 16;CD34 + CD45 ) /Dim , n ¼ 40and CD34 Bright , n ¼ 18),statisticallysigniPcant( P <0 Æ 01)differencesaremarkedbyasterisks.Matchedisotypecontrolantibodystainingresultsare deductedfromspeciPcantibodyresults.(C)RepresentativemorphologicaloverviewofSCPcellsafter4-dculture.Cellnucleiarestainedwith haematoxylin;mitoticcellsindicatedbywhitearrows;multinuclearcellsindicatedbyblackarrows. Y.Porat etal ª 2006TheraVitaeLtd 4 JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology LectinanduptakeofAc-LDLand58 Æ 8±4 Æ 3%oftheACPs bothexpressedCD31 Bright anddisplayeduptakeofAc-LDL (Fig3CandD).Moreover,themajorityofCD31 Bright cells showedbothbindingofUlex-Lectin(92 Æ 9±5 Æ 2%;Fig4A1 andA2)anduptakeofAc-LDL(86 Æ 9±2 Æ 9%;Fig4B1,B2and D).ConcurrentexpressionofCD31 Bright anduptakeofAc- LDLwereconsequentlyusedtodePnethedifferentiatedACPs. ThisspeciPcattribute,clearlyobservedondifferentiatedACP cells,waslimitedtoSCPcells(Fig4CandD).Similar characterisationresultswereobtainedwhencellswerecultured inplatescoatedwitheitherPbronectinorautologousplasma thatcanbesafelyusedforthedevelopmentoftherapeutic cellularproducts.Anaverageof25 Æ 1±3 Æ 7 · 10 6 CD31 Bright- xAc-LDLcellswasgeneratedfrom450mlblood( n ¼ 14).<br><br> Interestingly,anon-parametrictwo-tailedanalysisof11 individualblooddonationsconPrmedasigniPcantnegative correlation( r ¼ ) 0 Æ 74, P <0 Æ 01)betweenpercentagesofcells expressingthemultipotenthaematopoieticstemcellmarker CD34andthepercentageofcellsexhibitingtheangiogenic differentiationphenotypeofCD31 Bright xAc-LDL(Fig4E). CharacterisationofNCPs Synergeticcellpopulationcultureswereinducedtodifferen- tiateintoNCPsandanaverageof13 Æ 5 · 10 6 ( n ¼ 5)NCPs weregeneratedfrom450mlblood.Thesecellsdeveloped irregularperikarya,fromwhichPlamentousextensionsspread andcontactedneighboringcells,forminganet-likeorganisa- tion(Fig5A).NCPsexpressedtheneuralprogenitormarkers Nestinand b III-Tubulin,typicalofnewlydifferentiated neurons(Fig5BandC),andNeu-N,anuclearproteinpresent inneurons(Fig5D).Othercellsfromtheseculturesexpressed O4andGFAP,oligodendrocyteandastrocytemarkers,(Fig5E andF).Flowcytometryanalysisshowedthat49 Æ 4±6 Æ 3%and 34 Æ 0±5 Æ 9%ofNCPsexpressedNestinand b III-Tubulin respectively(Fig5G).Inadditiontodemonstratingneural lineage,thedifferentiatedNCPsrespondedtotheneurotrans- mittersglutamateandGABA,asdetectedbycalciuminQux throughvoltage-gatedcalciumchannels(Fig5H). CharacterisationofMCPs Inpreliminaryexperiments( n ¼ 3)SCPcultureswere inducedtodifferentiateintoMCPs.Morphologically,MCPs appearedelongatedwithdarkcytoplasm,possiblyindicating highproteincontent(Fig6A).Furthermore,thecells expressedthemyocardialmarkerscardiacTroponinT(Fig6B) andthegapjunctionmarkerConnexin43(Fig6C).Flow cytometryanalysisshowedtheexpressionofDesminand cardiacTroponinT(on19 Æ 7%and52 Æ 3%ofcellsrespectively) (Fig6DandE).<br><br> Lineage-speci;cdifferentiation ThespeciPcityofthedifferentiationprocessesissummarised inTableI.Incontrasttodifferentiated,lineage-speciPc (A) (C) (D) (B) Fig2. Characterisationofangiogeniccellprecursors(ACPs).Morphology,immunostainingandfunctionalexamination.Microscopicmorphology illustrating:(A)Typicalelongated,spindle-shapedcells,(B)RenewalofACPculturemorphology.HarvestedACPswerereplatedfor24hon Pbronectin-coated24-wellplates.(C)Tubeformationassay:arrowsindicatecellorganisationintotube-likestructures.(D)Cytokinesecretion by10 6 ACPcellsculturedfor24hinserum-freemediumwasassessedusingtheQowcytometry-basedCBAkit.Mediumwithnoculturedcellsservedas control.Secretionofinterleukin-8(left y -axis;blackhistogramrepresentssecretionbyACPs;greyhistogramrepresentsmediumcontrol);VEGFand Angiogenin(right y -axis;blackstripedhistogramsrepresentACPsecretion;greystripedhistogramrepresentsmediumcontrol). Blood-derivedMultipotentProgenitorCellPopulation ª 2006TheraVitaeLtd JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology 5 precursors,freshlyisolatedSCPcellsfailedtoexpressACP, NCPorMCP-speciPcmarkers.Theydidnotgeneratetube-like structuresandlessthan1%ofSCPcellsshowedconcomitant expressionofCD31 Bright andAc-LDLuptake(Fig4C),char- acteristicstypicalofACPs.Furthermore,theyexpressedneither theNCPmarkers b III-TubulinandGFAPnortheMCP markersConnexin43andcardiacTroponinT.Differentiated cells,ontheotherhand,expressedonlytheirlineage-speciPc markersbutnotthosetypicaloftheotherlineages:ACPsthat expressedspeciPclineagecharacteristics,suchasCD31,KDR andTie-2,signiPcantAc-LDLuptakeandbindingofUlex- LectindidnotexpresstheNCP-speciPcmarkers b III-Tubulin andGFAPortheMCP-speciPcmarkersConnexin43and cardiacTroponinT;differentiatedNCPsthatexpressedNeu- N, b III-Tubulin,andGFAPdidnotexpresstheMCPmarkers cardiacTroponinTandActin;andMCPsthatexpressed cardiacTroponinT,DesminandConnexin43didnotexpress b III-TubulinandGFAP,markersofNCPs.<br><br> (A1) (B) (D) (A2) (C) Fig3. CharacterisationofACPs.ArepresentativePeldofharvested,slide-Pxed,speciPcallylabelledACPswasimagedusingtwowavelengthPlters: (A1)acetylatedlowdensitylipoprotein(Ac-LDL)-Dilimagedat565nm.(A2)Ulex-Lectin-Quoresceinisothiocyanate(FITC)imagedat505nm.Cells thatshowedonlyAc-LDLuptakeareindicatedbyredarrows;cellsstainedsolelybyUlex-Lectin-FITCareindicatedbygreenarrows;andcellsthat showconcomitantexpressionofbothAc-LDLuptakeandbindingofUlex-Lectinareindicatedbywhitearrows.FlowcytometryofharvestedACPs stainedwith:(B)Ulex-Lectin-FITCandAc-LDL-Dil;(C)anti-CD34-APC,CD133-PE,CD117-APC,KDR-PE,Tie-2-PE,CD144-FITC,VWF-FITC, CD31-FITCandAc-LDL-Dil.(D)Thepercentageofcellsexpressingthemarkersispresentedasmean±SE(CD34, n ¼ 33;CD117, n ¼ 29;CD133, n ¼ 5;KDR, n ¼ 10;Tie-2, n ¼ 21;CD144, n ¼ 11;vonWillebrandfactor(VWF), n ¼ 9;CD31 Bright , n ¼ 24;andCD31 Bright xAcLDL, n ¼ 14). MatchedisotypecontrolantibodystainingresultsarepresentedinFigS1anddeductedfromspeciPcantibodyresults.<br><br> Y.Porat etal ª 2006TheraVitaeLtd 6 JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology Discussion Mostattemptstodevelopstemcelltherapyhavefocusedon thedirectisolationormobilisationofBMcells(Gussoni etal ,1999;Fuchs&Segre,2000;Kalka etal ,2000;Lagasse etal ,2000,2001;Bianco&Robey,2001;Forbes etal ,2002; Badorff etal ,2003;Grove etal ,2004;Guo etal ,2004, Morrison etal ,1997;Petite etal ,2000;Ramiya etal ,2000; Stock&Vacanti,2001;Rehman etal ,2003;Losordo& Dimmeler,2004;Matsubara,2004).Blood-derivedadult stemcellsarecurrentlybeingevaluatedasapotentialsource ofdifferentcelllineages.Recentreportshavedescribed (A1) (B1)(B2) (A2) (C)(D) (E) Fig4. CharacterisationofCD31 Bright ACPs.AsinglePeldofharvested,slide-Pxed,speciPcallylabelledACPswasimagedusingtwowavelengthPlters: (A1)anti-CD31-PEimagedat565nm.(A2)Ulex-Lectin-FITCimagedat505nm.Cellsstainedsolelybyanti-CD31areindicatedbyredarrows;cells stainedsolelybyUlex-Lectinareindicatedbygreenarrows;andcellsthatshowconcomitantexpressionofbothanti-CD31andUlex-Lectinare indicatedbywhitearrows.(B1)Anti-CD31-PEimagedat565nm.(B2)UptakeofAc-LDL-Alexa488imagedat505nm.Cellsstainedsolelybyanti- CD31areindicatedbyredarrows;cellsthatshowedonlyAc-LDLuptakeareindicatedbygreenarrows;andcellsthatshowconcomitantexpressionof bothanti-CD31andAc-LDLuptakeareindicatedbywhitearrows.Flowcytometryanalysisofconcomitantexpressionofanti-CD31-FITCand uptakeofAc-LDL-DiI.(C)SCP(day0ofculture)and(D)ACP(day5ofculture).(E)NegativecorrelationbetweentheexpressionofCD34andthe concomitantexpressionCD31anduptakeofAc-LDL-DiIonACPs.Thecorrelation,generatedfrom11individualblooddonations,resultedfroma nonparametrictwo-tailedanalysisusing graphpadprism software. Blood-derivedMultipotentProgenitorCellPopulation ª 2006TheraVitaeLtd JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology 7 proceduresforthegenerationofprogenitorcellsfrom peripheralblood.Assmus etal (2002)usedpuriPed,cultured cellsfromperipheralbloodinaclinicalstudy(TOPCARE- AMI)butdidnotdemonstratemultiplelineagepotentialfor thesecells,whileotherreportsdemonstrateddifferentiation intomultiplelineages,butonasmallscale(Abuljadayel, 2003;Rehman etal ,2003;Zhao etal ,2003;Dobert etal , 2004;Romagnani etal ,2005).<br><br> (A) (D) (E) (F) (G) (H) (B) (C) Fig5. Characterisationoftheneuralcellprecursors(NCPs).Morphology,immunostainingandfunctionalexaminationofneuralprogenitors.(A) MicroscopicexaminationofneuralprogenitorcellsmorphologyshowsirregularcellbodiesfromwhichPlamentousextensionsspreadandcreate connections,forminganet-likestructure.Slide-Pxedneuralprogenitorcellsstainedwith:(B)anti-Nestindetectedbygoatanti-mouse(GaM) immunoglobulinG(IgG)-Quoresceinisothiocyanate(FITC);(C)anti- b III-TubulindetectedbyGaMIgG-FITC(positivecellsmarkedbyarrows);(D) anti-Neu-N-Alexa488;(E)anti-O4detectedbyGaMIgG-Cy3;(F)anti-GFAPdetectedbyanti-mouseIgG-Cy3.(G)Flowcytometryanalysisresults, presentedasthepercentagemean±SEofharvestedPxedneuronalprogenitorcellsstainedwithanti- b III-Tubulinandanti-NestindetectedbyGaM IgG-FITC.MatchedisotypecontrolantibodystainingresultsarepresentedinFigS2anddeductedfromspeciPcantibodyresults.(H)ResultsofCa 2+ releasetestfollowingactivationofneuralprogenitorcellswithglutamateandGABA. Y.Porat etal ª 2006TheraVitaeLtd 8 JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology Thesynergeticcellpopulation,describedhereforthePrst time,containsincreasednumbersofCD34 + CD45 ) /Dim and CD31 Bright multipotentcellsbutnotcellsexpressingmature lineagemarkers.TheSCPcanbeinducedtolineage-speciPc differentiation.Ourapproachprovidesthemeanstosimply andreliablyobtainmorethan10 7 differentiatedprecursorcells from450mlofblood.Forexample,ameanof25 Æ 1 · 10 6 ACPsobtainedfrombloodsamplesiscomparablewith(or evenhigherthan)theamountofspeciPcprogenitorcells obtainedfrom10 9 BMcells(Dobert etal ,2004;Schachinger etal ,2004;Pompilio etal ,2005;Strauer etal ,2005).<br><br> TheSCP,amultipotentcellpopulation,ispuriPedbasedon celldensityandisthereforemoreafQuentthanPBMCsin progenitorcells,asdemonstratedbythelevelsofCD34 Bright , CD34 + CD45 ) /Dim andCD31 Bright cells.UnderspeciPcculture conditions,cellsoftheSCPcandifferentiateintoangiogenic, myocardialandneurallineages.Furtherstudiesareneededto explorecultureconditionsunderwhichtheSCPmaydiffer- entiatetoothercelllineages. AngiogeniccellprecursorsgeneratedfromtheSCP expressedCD34,CD117andCD133,typicalofmultipotent haematopoieticstemcellsaswellasKDR,Tie-2,CD144,VWF, CD31 Bright anddisplayedbothUlex-LectinandAc-LDL uptake,whichistypicalofangiogenic/endothelialcells. CD31/PECAM-1isexpressedonhematopoieticprogenitor cellsandisamajorconstituentoftheendothelialcell intercellularjunction,whereupto10 6 moleculesareconcen- trated(resultinginCD31 Bright cells)(Sheibani etal ,1999).<br><br> CD31isnotpresentonPbroblasts,epithelium,muscle,or othernonvascularcells(Newman,1997);thusevaluationof CD31 + cellinvolvementintheangiogenicprocessesisof particularinterest.PreviousstudiesreportedthatCD31 + cells demonstratetheabilitytodifferentiateintoendothelialcells andsigniPcantlyimprovesymptomsinmodelsofmyocardial infarction(Kanayasu-Toyoda etal ,2003;Kawamoto etal , 2003).OurdatasupporttheimportanceofCD31asamarker (A) (D) (E) (B) (C) Fig6. Characterisationofthemyocardialcellprecursors(MCP).MorphologyandimmunostainingofMCPs.(A)Microscopicexaminationof morphologyshowselongatedcellswithdarkcytoplasm(markedbyarrows).Harvestedslide-PxedMCPsstainedwith:(B)anti-cardiacTroponinT detectedbygoatanti-mouse(GaM)immunoglobinG(IgG)-Cy3and(C)anti-mouseConnexin43detectedbyGaMIgG-FITC.Flowcytometry analysisofcardiomyocyteprogenitorsstainedwith:(D)anti-cardiacTroponinTdetectedbyGaMIgG-phycoerythrin(PE)and(E)anti-Desmin detectedbyGaMIgG-PE.Matchedisotypecontrolantibodystainingresultsarepresentedinhistograms6Dand6EandinFigS3. TableI.<br><br> Summaryoflineagecharacteristicsexpressionbyfreshly isolatedSCPcellsandbyspeciPcangiogenic,neuralandmyocardial lineageprecursorcells. CelltypeSCPACPNCPMCP ACPcharacteristics: LDLxCD31 Bright (%)<1%58 Æ 8%±4 Æ 3%NTNT Tubeformation(0 35)05NTNT NCPcharacteristics: b III-Tubulin(+/ ) ) )) + ) GFAP(+/ ) ) )) + ) MCPcharacteristics: CardiactroponinT(+/ ) ) ))) + Connexin43(+/ ) ) ))) + SCP,synergeticcellpopulation;ACP,angiogeniccellprecursors;NCP, neuralcellprecursors;MCP,myocardialcellprecursors;LDL,low densitylipoprotein;GFAP,glialPbrillaryacidicprotein;NT,nottes- ted. Blood-derivedMultipotentProgenitorCellPopulation ª 2006TheraVitaeLtd JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology 9 formultipotentprogenitorcellsthatcandifferentiateintoa varietyoflineagesincludingtheACPlineage.Upondifferen- tiationintoACPs,CD31 Bright cellsacquireendothelialcell- speciPccharacteristics,suchastheuptakeofAc-LDL.We foundthatthemajorityofCD31 Bright cellsintheACP population,butnotinthesourceSCP,demonstratedAc-LDL uptakeandbindingofUlex-Lectin.TheseCD31 Bright xAc-LDL positivecellsshowedtypicalmorphologyofelongated,spindle- shapedcellsthatnotonlyexpressedACPmarkersbutalso showedspeciPcangiogenicbiologicalactivity:secretionof tissueregenerationfactors,suchasIL-8(alsoknownasthe chemokineCXCL8),VEGFandangiogenin(Han etal ,1997; Wiedlocha,1999;Rivera etal ,2001;Pruijt etal ,2002;Li etal , 2003)andformationoftube-likestructures(Kayisli etal , 2004).<br><br> Thus,wedescribehereamethodologyforcharacterisingthe angiogenic/endotheliallineagebasedonconcomitantexpres- sionofCD31 Bright anduptakeofAc-LDL.Usingthisapproach, weobservedanegativecorrelation( r ¼ ) 0 Æ 74, P <0 Æ 01) betweenthepercentagesofdifferentiatedangiogeniccells (CD31 Bright xAc-LDL)andundifferentiated,multipotent,hae- matopoieticCD34 + cellsthatcouldindicatethedifferenti- ation/multipotentialstatusoftheangiogenicpopulations. Synergeticcellpopulation-derivedNCPsexpressedthe neuronalandglialmarkersNestin, b III-Tubulin,Neu-N, GFAPandO4(Steindler&Pincus,2002;Goolsby etal ,2003) andrespondedtoneurotransmitterstimulation,whereasSCP- derivedMCPsexpressedDesmin,cardiacTroponinTandthe gapjunctionmarkerConnexin43(Grounds etal ,2002; Nygren etal ,2004). ThespeciPcityofdifferentiationprocessescanbedemon- stratedbythefactthattheSCPdoesnotexpresslineage- speciPcmarkersandthatthedifferentiatedcellsstrictlyexpress highlevelsofspeciPccharacteristics,butnotthoseofother lineages.<br><br> Theisolationofmultipotentcells,thenatureoftheinter- actionsbetweenthecellularelementsoftheSCP,andthe methodsrequiredtofacilitateproductionofadditionallineage- speciPcprogenitorswillbethesubjectoffuturestudies.The vitalityandplasticityshownbytheSCPandthecommitted precursorcellsgeneratedthereofcanpotentiallyformthebasis forsafeandeffectiveautologouscelltherapiesapplicabletoa widerangeofclinicaldisorders.However,thebiologicalactivity ofthelineagespeciPcprecursorsshouldbeaddressed invivo in ordertoevaluatetheirtherapeuticpotential. Acknowledgement ThisstudywasfundedbyTheraVitaeLtd. 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Thismaterialisavailableaspartoftheonlinearticlefrom http://www.blackwell-synergy.com Y.Porat etal ª 2006TheraVitaeLtd 12 JournalCompilation ª 2006BlackwellPublishingLtd, BritishJournalofHaematology<br><br>

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