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Interactions of neurons, astrocytes and microglia with HUCB cell populations in stroke models

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Interactions of neurons, astrocytes and microglia with HUCB cell populations in stroke models migration, neuroprotection and inflammation
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Jiang, Lixian
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University of South Florida
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MCP-1
Hypoxia
Ischemia
Stem cell
Dissertations, Academic -- Pathology and Laboratory Medicine -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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ABSTRACT: Previous studies demonstrated that intravenous administration of human umbilical cord blood (HUCB) cells could improve behavioral and neurological recovery of stroked animals following middle cerebral artery occlusion (MCAO). In addition, HUCB cell recipients had less of an inflammatory response with less leukocyte infiltration. In these studies we explored how HUCB cells change the inflammatory response of neurons, astrocytes, and microglia to hypoxia/ischemia. Initiation of the inflammatory response occurs with the expression of chemokines. We determined that monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1alph (MIP-1α), which are upregulated in the brain early after a stroke, induce migration of HUCB cells to the site of injury. Neutralizing these chemokines with antibodies prevented migration in an in vitro migration assay. We next explored the interaction of the whole HUCB mononuclear cell fraction, as well as subpopulations from within the mononuclear fraction (T cell alone, B cell alone, and monocytes/macrophage alone) with cultures of enriched neurons, astrocytes or microglia exposed to hypoxia in an oxygen, glucose deprivation paradigm. We showed that HUCB cells increased the cell viability of neurons and astrocytes, while decreasing cell viability of microglia. There was also a change in the cytokine secretion profile from the cells exposed to HUCB cells under hypoxic conditions. These results suggested that chemokines, MCP-1 and MIP-1α increased in stroked brain, and they played an important role in recruitment of HUCB into the CNS after intravenous administration. Once inside the brain, HUCB could suppress the immune response by promoting microglial death and modulating the function of astrocytes. In addition, HUCB cells provide neuron protection against the injury caused by stroke. However, it is unlikely to contribute the effect of HUCB to a single population of HUCB.
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Dissertation (Ph.D.)--University of South Florida, 2008.
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by Lixian Jiang.
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InteractionsofNeurons,AstrocytesandMicrogliawithHUCBCellPopulationsinStrokeModels:Migration,NeuroprotectionandInflammationbyLixianJiangAdissertationsubmittedinpartialfulfillmentoftherequirementsfortheDegreeofDoctorofPhilosophyDepartmentofPathologyandCellBiologyCollegeofMedicineUniversityofSouthFloridaCo-MajorProfessor:AlisonEWilling,Ph.D.Co-MajorProfessor:SamuelSaporta,Ph.D.DonCameron,Ph.D.ThomasW.Klein,Ph.D.JuanSanchez-Ramos,PhD,M.D.DateofApproval:February19,2008Keywords:MIP-1,MCP-1,hypoxia,ischemia,stemcellCopyright2008,LixianJiang

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ACKNOWLEDGEMENTSSpecialappreciationisgiventomycoreadvisor,Dr.AlisonEWilling.IwanttosaythankyouforhersupportandencouragementinmyPh.Dstudies.Ialsoappreciatethehelpofmyco-coreadvisor,Dr.SameulSaporta.Healwayssupportedme,andgavemesuggestionsinmyresearch.IoffermyspecialthankstoDr.ChristopherPhelps.WithouthishelpandDrAlisonEWillingseffort,IwouldnothavebeenabletoreturntotheUnitedStatestofinishmyPh.D.whenIwasstuckinChinain2003.Manythanksaregiventothemembersofmydissertationcommittee:Dr.DonCameron,Dr.ThomasW.Klein,andDr.JuanSanchez-Ramos.SpecialthanksaregiventoDr.AlisonEWilling,Dr.SameulSaporta,Dr.JuanSanchez-Ramos,andDr.PaulSanbergfortheirsupportinmygreencardapplication.IacknowledgeIgotalotofhelpfromJeniferNewcomb,Dr.NingChen,Dr.SvitlanaGarbuzova-Davis,Dr.MaryNewman,Dr.KuzmenokandDonnaMorisson.Ireallyappreciatetheirhelp.

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iTABLEOFCONTENTSLISTOFTABLESiLISTOFFIGURESiABSTRACTiCHAPTERONE:INTRODUCTION11.1CharacterizingCordBlood11.2CordBloodasaTreatmentforCNSInjury41.3MechanismsofFunctionalRecoveryinStroke91.3.1CellReplacement91.3.1.1CharacterizationofNeuralPropertiesofHUCB101.3.1.2HUCBCellsReplaceNeuronsafterMCAO141.3.1.3HUCBInductionofNeurogenesis151.3.2NeurotrophicSupport171.3.3ImmuneSuppressionandDecreasedInflammation201.4DoHUCBCellsGetintotheBrain?281.5Hypothesis321.6SpecificAims32CHAPTERTWO:GENERALMATERIALSANDMETHODS342.1TheMiddleCerebralArteryOcclusionRatModel34

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ii2.1.1AnimalHousingandMaintenance342.1.2SurgicalProcedure342.2AnimalSacrifice352.3NeuralCellIsolationandCulture352.3.1PrimaryBrainCellIsolation352.3.2NeuronEnrichedCultures362.3.3AstrocytesEnrichedCultures372.3.4MicrogliaEnrichedCultures372.4HypoxiaCulture382.5MigrationAssay392.5.1MigrationChemotacticChamber392.5.2PreparationofBrainExtracts392.5.3PreparationofHUCBCells402.5.4BasicMigrationAssay402.6WesternBlotting412.6.1PreparationofTissueSample412.6.2GelPreparation412.6.3ProteinTransferfromGeltoNitrocelluloseMembrane412.6.4AntibodyIncubation422.7ELISA432.7.1PreparationofTissueSample432.7.2AssayProtocol43

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iii2.8Immunohistochemistry442.9MagneticCellSorting452.10ConfocalMicroscopy46CHAPTERTHREE:MIP-1ANDMCP-1INDUCEMIGRATIONOFHUMANUMBILICALCORDBLOODCELLSINMODELSOFSTROKE483.1Abstract483.2Introduction503.3MaterialsandMethods523.3.1MiddleCerebralArteryOcclusion(MCAO)523.3.2NeuralCellHypoxiaCulture523.3.3Immunohistochemistry533.3.3.1MIP-1(orMCP-1)DoubleLabeling533.3.3.2ChemokineReceptorImmunolabeling533.3.4WesternBlotting543.3.5MCP-1ELISA543.3.6CellMigrationAssay543.4Results553.4.1PresenceofMIP-1andMCP-1intheStrokedBrain553.4.2ChemotacticEffectofMCP-1andMIP-1onHUCBCellMigration613.4.3EffectofBrainTissueExtractonHUCBCellMigration61

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iv3.5Discussion65CHAPTERFOUR:HUMANUMBILICALCORDBLOODCELLSDEPRESSTHERATMICROGLIAINFLAMMATORYRESPONSESINVITRO694.1Abstract694.2Introduction714.3MaterialandMethods734.3.1IsolationofMicrogliaCells734.3.2CellCultureandHypoxiaTreatment744.3.3FDA/PIStaining754.3.4CytokineELISA764.4Results774.4.1.MicroglialCellViability774.4.2.HUCBCellViability784.4.3.MicrogliaMedia794.4.4.IL-1inMicrogliaMedia804.4.5.IL-10inMicrogliaMedia824.4-1,andIL-10inCordBloodMedia834.5Discussion84CHAPTERFIVE:HUMANUMBILICALCORDBLOODCELLSENHANCENEURONANDASTROCYTESURVIVALINHYPOXIACULTURE90

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v5.1Abstract905.2Introduction925.3MaterialandMethods945.3.1CellPreparation945.3.2CellCultureandHypoxiaTreatment955.3.3FDA/PIStaining965.3.4CytokineELISA975.4Results985.4.1NeuronCellViability985.4.2Neuronal995.4.3NeuronIL-1betaandIL-6expression1005.4.4NeuronIL-10expression1005.4.5Astrocytecellviability1011025.4.7AstrocyteIL-1betaexpression1035.4.8IL-6expression1035.4.9AstrocyteIL-10Expression104-1,IL-6andIL-10incordbloodmedia1055.5Discussion106CHAPTERSIX:THEINTERACTIONOFNEURONS,ASTROCYTES,ANDMICROGLIAWITHCD8,CD11b,CD19POSITIVECELLSDERIVEDFROMHUMANUMBILICALCORDBLOOD111

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vi6.1Abstract1116.2Introduction1136.3MaterialandMethods1156.3.1CellPreparation1156.3.2IsolationofMicroglia1166.3.3HUCBSubpopulationIsolation1176.3.4CellCultureandHypoxiaTreatment1186.3.5FDA/PIStaining1196.3.6CytokineELISAAssay1196.4Results120-10Expression1206.4.2.TheinteractionofCD11b+HUCBcellswithneurons,astrocytesandmicroglia1206.4.2.1EffectofCD11b+HUCBcellsonneuralcellviability1206.4.2.2IL-1andIL-6expressioninCD11b+HUCBcultureofneuralcells.1206.4.3.TheinteractionofCD8+HUCBcellswithneurons,astrocytesandmicroglia1266.4.3.1EffectofCD8+HUCBcellsonneuralcellviability1266.4.3.2IL-1andIL-6expressioninCD8+HUCB

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viicultureofneuralcells1286.4.4.TheinteractionofCD19+HUCBcellswithneurons,astrocytesandmicroglia1336.4.4.1.EffectofCD19+HUCBcellsonneuralcellviability1336.4.4.2IL-1andIL-6expressioninCD19+cellsHUCBcellsco-culturewithneuralcells.1356.5Discussion1406.5.1TheEffectofIL-1andIL-6onNeurons1416.5.2TheEffectofIL-1andIL-6onMicrogliaandAstrocytes1426.5.3EffectofCD11b+HUCBcellsonneuralcells1456.5.4EffectofCD8+HUCBcellsonneuralcells1466.5.5EffectofCD19+HUCBcellsonneuralcells146CHAPTERSEVENCONCLUDINGREMARKS1487.1Chemokines,MCP-1andMIP-1,AttractedHUCBtoTheInjurySiteFollowingStroke1507.2HUCBCellsPromotedMicrogliaDeathunderHypoxicConditions1527.3HUCBPreventedNeuronandAstrocyteHypoxiaInducedCellDeath1537.4TheInteractionbetweentheHUCBSubpopulationandNeuralCells156

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viiiREFERENCE155ABOUTTHEAUTHORENDPAGE

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ixLISTOFTABLESTable2.1ParametersofELISAassay44Table2.2Antibodiesusedforimmunochemicallabelingofcells45Table4.1ParametersofELISAassay77Table5.1ParametersofELISAassay95

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xLISTOFFIGURESFigure3.1.MCP-1andMIP-1expressioninneuronsafterstroke56Figure3.2.MCP-1andMIP-1expressioninastrocytesafterstroke57Figure3.3.MCP-1andMIP-1expressioninmicrogliaafterstroke58Figure3.4.MIP-1westernblotting59Figure3.5.ELISAassayofMCP-1inbraintissueandculturedneuralcells.60Figure3.6.MIP-1receptorCCR1,CCR5andMCP-1receptorCCR2expressioninHUCB62Figure3.7.ChemokinesdosedependentlyinducedHUCBcellmigration63Figure3.8.EffectofMCP-1andMIP-1onHUCBmigrationtobraintissueextract64Figure4.1.Microglialcellviability78Figure4.2HUCBcellviability79Figure4.3MicroglialIFNgammaexpression80Figure4.4MicroglialIL-1betaexpression81Figure4.5MicroglialIL-10expression82Figure5.1Neuronviability99Figure5.2100

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xiFigure5.4IL-10expressioninneuronalcultures.101Figure5.5AstrocyteViability102Figure5.6103Figure5.8IL-6expressioninastrocytes104Figure5.9AstrocyteIL-10expression105Figure6.1Neuralcellviabilitywith/withoutCD11b122Figure6.2IL-1betaexpressionwith/withoutCD11b124Figure6.3IL-6expressionwith/withoutCD11b126Figure6.4Neuralcellviabilitywith/withoutCD8128Figure6.5IL-1betaexpressionwith/withoutCD8130Figure6.6IL-6expressionwith/withoutCD8132Figure6.7Neuralcellviabilitywith/withoutCD19134Figure6.8IL-1betaexpressionwith/withoutCD19137Figure6.9IL-6expressionwith/withoutCD19139

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xiiINTERACTIONSOFNEURONS,ASTROCYTESANDMICROGLIAWITHHUCBCELLPOPULATIONSINSTROKEMODELS:MIGRATION,NEUROPROTECTIONANDINFLAMMATIONLixianJiangABSTRACTPreviousstudiesdemonstratedthatintravenousadministrationofhumanumbilicalcordblood(HUCB)cellscouldimprovebehavioralandneurologicalrecoveryofstrokedanimalsfollowingmiddlecerebralarteryocclusion(MCAO).Inaddition,HUCBcellrecipientshadlessofaninflammatoryresponsewithlessleukocyteinfiltration.InthesestudiesweexploredhowHUCBcellschangetheinflammatoryresponseofneurons,astrocytes,andmicrogliatohypoxia/ischemia.Initiationoftheinflammatoryresponseoccurswiththeexpressionofchemokines.Wedeterminedthatmonocytechemoattractantprotein-1(MCP-1)andmacrophageinflammatoryprotein1alpha(MIP-1),whichareupregulatedinthebrainearlyafterastroke,inducemigrationofHUCBcellstothesiteofinjury.Neutralizingthesechemokineswithantibodiespreventedmigrationinaninvitromigrationassay.WenextexploredtheinteractionofthewholeHUCBmononuclearcellfraction,aswellassubpopulationsfromwithinthemononuclear

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xiiifraction(Tcellalone,Bcellalone,andmonocytes/macrophagealone)withculturesofenrichedneurons,astrocytesormicrogliaexposedtohypoxiainanoxygen,glucosedeprivationparadigm.WeshowedthatHUCBcellsincreasedthecellviabilityofneuronsandastrocytes,whiledecreasingcellviabilityofmicroglia.TherewasalsoachangeinthecytokinesecretionprofilefromthecellsexposedtoHUCBcellsunderhypoxicconditions.Theseresultssuggestedthatchemokines,MCP-1andMIP-1increasedinstrokedbrain,andtheyplayedanimportantroleinrecruitmentofHUCBintotheCNSafterintravenousadministration.Onceinsidethebrain,HUCBcouldsuppresstheimmuneresponsebypromotingmicroglialdeathandmodulatingthefunctionofastrocytes.Inaddition,HUCBcellsprovideneuronprotectionagainsttheinjurycausedbystroke.However,itisunlikelytocontributetheeffectofHUCBtoasinglepopulationofHUCB.

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1CHAPTERONEINTRODUCTION1.1CharacterizingCordBloodHumanumbilicalcordblood(HUCB)isarichsourceofstemcellsthathavebeenusedtoreconstituteimmunecellsandbloodlineagesforthetreatmentofhematologicaldiseases(1).ThefirstclinicaluseofHUCBcellswasin1988onapatientwithFanconianemia(2).Sincethattime,morethan1000transplantshavebeenperformedaroundtheworldintreatmentofhematopoieticandgeneticdisordersincludinglymphoidandmyeloidleukemia,Fanconianemia,aplasticanemia,Huntersyndrome,Wiskott-Aldrichsyndrome,beta-thalassemia,andneuroblastoma(3).Cordbloodisthebloodcontainedintheumbilicalveinwithintheplacentalstumpthatisnormallydiscardedafterdeliveryoftheneonate.Cordbloodcanbecollectedafterclampingandcuttingtheumbilicalcord,immediatelyafterthebirthofthebaby.Comparedtobonemarrow,HUCBtransplantationhaslessmorbidityandmortality(4).CordbloodbanksarebeingestablishedinmanycountriesincludingtheUnitedStatesandstandardprotocolsforsampleprocessingandstoragedeveloped(5).Theactualpartofthecordbloodthatisusedforcelltransplantationisthemononuclearfraction,afractionofthebloodcontainingmainlymononuclear

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2cellsthatisobtainedbycellgradientseparationtechniques.Includedinthisfractionarethestemcells(HSC),whicharemulti-potentialandcanproliferateanddifferentiateintoalllineagesofhaematopoieticcells.HSCcellsareusuallyCD34positive(+)cells,althoughCD34+cellsconstituteaveryheterogeneouscellpopulation.ThemajorityofCD34+cellsexpressbothHLA-DRandCD38antigens,whilethemostprimitiveHSClacktheexpressionofHLA-DRandCD38(6,7).CD34+cellscanfurtherdifferentiateintothreedifferentprogenitorpopulations.Thefirstisthemultipotentprogenitors,orthecolonyformingunit-multipotential(CFU-Mix)progenitors.Thesecondoneisthemyeloidprogenitors,orcolonyformingunitgranulocyte-macrophage(CFU-GM)progenitors.Thethirdoneiserythroidprogenitors,anditsculturesarecharacterizedascolonyformingunitgranulocyte-macrophage-erythroid(CFU-E)andburstformingunit-erythroid(BFU-E)colonies.CFU-MIXprogenitorsexpresslow/undetectablelevelsofbothCD45RAandCD71,andCFU-GMprogenitorsareCD34+CD45RA+CD71locells,whileerythroidprogenitorsaremarkedwithCD34+CD45RAloCD71+(8,9).TheCD34+cellcontentinHUCBhasbeenshowntobearound1percentofthemononuclearfraction(10).OnemilliliterHUCBthereareabout8,000primitiveerythroidprogenitors(BFU-E),between13,000and24,000myeloidprogenitors(CFU-GM),andbetween1,000and10,000multipotentprogenitors(CFU-Mix)(11,12).Anothercomponentofcordbloodcellsisthelymphocyte,whichiscomprised

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3ofTandBcells,andaccountsforalmost41percentofthemononuclearfractionofHUCB(13).TcellsaredefinedbytheexpressionoftheCD3molecules,whichroughlyaredividedintoCD4orCD8positivecells.CD4positiveTcellsarearound55%oftheTcellpopulation,andCD8positivecellsaccountfortheremaining45%(14).TheproportionofCD4andCD8reflectsthematurityofTcells.Indevelopment,theratioofCD4toCD8Tcellsprogressivelyincreasesovertimeatleasttillsubjectsareadult(15).Actually,intheTcellspopulationtherearealsoasmallnumberofcellsthatexpressCD16and/orCD56withoutco-expressionofCD3.ThesecellsareNaturalKiller(NK)cells,andtheyareimportantagainstcancerforkillingcells(16).Incontrast,BcellsaredefinedbytheexpressionofCD19,aspecificmarkerofBcelllineage(17),whichstartstobeexpressedfromthedifferentiationofBcellprogenitors,continuesonpre-Bcell,andallthewaytomatureBcells(17).Bcellsareabout20%ofHUCB,anddeclineinadulthood(18).Monocytesarealsoacomponentofthemononuclearfaction,andtheymakeuptheremainingportionoftheHUCBmononuclearfractions(19).Monocytesarederivedfromcommittedmyeloidprogenitorcells,circulateinthebloodandentertissuestobecomeresidenttissuemacrophage.MonocytescanbeisolatedbyflowcytometryaccordingtotheircellsurfaceantigensCD11b,CD18,CD14,andCD16.CD11b/CD18arereceptorsonthesurfaceofmonocytes.TheyinteractwithintercellularadhesionmoleculeIontheendotheliumandlocalizemonocytes

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4tothesitesofinfection(20).Oncemonocytesenterdifferenttissues,theyexpresscertainenzymes,andcannon-specificallytakeupparticlessuchascolloidalcarbon,andspecificendocyticreceptors(21).Dendriticcells(DC)areantigenpresentingcells,andcanactivatenaveTcells.TheyaccountforaverysmallportionofHUCB.TheycanbegeneratedinvitronotonlyfromCD34+haemotopoieticstemcellsinbonemarroworcordblood(22),butalsofromperipheralbloodCD14+monocyteswithIL-4andGM-CSFstimulation(23).ImmatureDCcellsareCD1apositivecells,andwhentheygetmoremature,theyexpressCD83,CD80andCD86(24).Thesemarkersarealsoexpressedbymonocytes.Insummary,HUCBmononuclearfractionisaveryheterogeneouspopulationthatisroughlydividedintoTcells,Bcellsandmonocytes,andeachsubpopulationtakesonethirdofthewholemononuclearfractionofHUCB.StemcellsaswellasDCcellsonlyaccountfor1-3%ofthisHUCBfraction.1.2CordBloodasaTreatmentforCNSinjurySincehumanumbilicalcordblood(HUCB)cellsisrichinhematopoieticstemcells,itispossibleforthesecellstoproliferate,differentiateintoneurallikecellsandreplacethecelllosscausedbystroke.WhenHUCBcellsweredirectlydeliveredintorodentssubjectedtoMCAO,recipientanimalsshowedanimprovedneurologicalandbehavioralrecoverycomparedwithnon-transplanted

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5animals.AnimalswithpermanentMCAOgiven1,000,000HUCBcellsintrastriatallyweresignificantlylessactivethannontransplantedanimalsduringbothdarkandlightphasesofthelightcycle;activityoftransplantedanimalswassimilartotheirnovelbaselinebehaviorcomparedtoshamcontrols.Furthertransplantedanimalslearnedtostayontheplatforminthepassiveavoidancetasksignificantlymorequicklythantheanimalswithpermanentocclusionandnotransplant.Inaddition,transplantedanimalshadbetterbehavioralrecoveryintheelevatedbodyswingtestandsteptestcomparedtonontransplantedanimals(25).Weandotherlaboratoriesalsofoundthisfunctionalimprovementcouldbeachievedwithintravenous(i.v.)HUCBadministration(26).Forexample,CheninfusedHUCBviatailvein24hoursor7daysaftertransientMCAO.TheanimalswithHUCBadministeredat24hourspoststrokehadimprovedperformanceontherotarodtestandmodifiedneurologicalseverityscore(mNSS)comparedtocontrolanimals,whileanimalstreatedwithHUCB7daysafterMCAOonlyimprovedontherotarodtestbutnotthemNSS,suggestingtheimportanceofearlyintervention(26).Whenintraparenchymaladministrationwascomparedtoi.v.administration,bothtreatmentroutespromotedsignificantfunctionalimprovementcomparedwithstrokeonlyanimals(25).TheanimalsthatreceivedHUCBlearnedfasterthannontransplantedanimals(p<0.05).Interestingly,after2monthsonlyi.v.injectedanimalsmaintainedfunctionalimprovements,

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6particularlyintheSteptest.Fromapracticalstandpoint,eventhoughintravenousadministrationhadthesametherapeuticeffectasdirectadministration,ithasthegreatadvantageofbeingmoreeasilydelivered.ThedoseeffectofHUCBadministrationafterMCAOwasalsostudied(27).Significantreductionsinspontaneousactivitywereobservedinanimalstreatedwith106orhigherdosesofcordbloodcellsincomparisontomediatreatedcontrols.Reductionsinelevatedbody-swingbiasandimprovementsintheSteptestwereoptimalat107cellsanddidnotimprovefurtherathigherdoses(27).However,thehigherthedoseofcells,thesmallertheinfarctsizeinthestrokedanimals.ImprovedoutcomewasalsoobservedwhenHUCBcellswereadministeredtoanimalsthatunderwenthemorrhagicstroke(28).HUCBwasdeliveredbyintravenousinfusionintoanimalswithhemorrhagicinjuryinducedbyintrastriatalinjectionsofcollagenase.Theanimalsweresubjectedtoabatteryofneurologicalandbehavioraltestsat1dayafterinjury,whichwasfollowedbytheadministrationofumbilicalcordblood.Thefunctionalrecoverywastestedat6,and13daypostHUCBinfusion.HUCBsignificantlyimprovedbehavioralrecoveryintheSteptestatday6,andelevatedbody-swingtestatday13afterintravenousadministration,whileimprovedneurologicalscoreswereobservedday6today13(28).

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7ThefunctionofHUCBcellswasalsoevaluatedinratswithspinalcordinjuryinducedbyhemicompressionatT8/9withacalibratedaneurysmclip(29).OnemillionHUCBcellswereinfusedintoanimalsthroughthetailveinatday1orday5followingspinalcordinjury.Spontaneousactivitiesofanimalsweremonitoredwithadigitalcamerafor3weeks,andthebehaviorswerescoredbythestandarddevelopedbyBasso,BeattieandBresnehan(30).Itwasfoundthatfunctionalimprovementwasprogressivewithtimeinallspinalcordinjurygroups,whileanimalstreatedwithHUCBatday5afterspinalcordinjuryhadabetterfunctionalimprovementthananimalsthatreceivedHUCBatday1afterinjuryorthecontrolgroup.NosignificantdifferenceswereobservedbetweenanimalsthatreceivedHUCBatday1afterinjuryandthecontrolgroupatanytimepoint.HistologicalexaminationrevealedthatHUCBdidmigrateintotheinjurysite,andthereweremorecellsinanimalsthatreceivedHUCBatday5afterinjurythananimalsthatreceivedHUCBatday1afterinjury.ThedatasuggestedthatthefunctionalimprovementatleastpartiallydependsonhowmanyHUCBmigratedintotheinjurysite(29).Amyotrophiclateralsclerosis(ALS)isasevereCNSdiseasewithdiffusemotorneurondegeneration.Therearenoeffectivetreatmentsforthisdiseaseatthemoment.Recently,HUCBwasdeliveredintotransgenicALSmicethroughintravenousadministration,andthefunctionalrecoverywasexamined(31).

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8AnimalsreceivingHUCBhadalongerlifespancomparedtocontrolanimals.HUCBcellswerefoundinthedegeneratingregionofthebrainandspinalcord,andsomeexhibitedneuralphenotypes.There,someHUCBcellsarepositivelylabeledwiththeantibodiesagainstneuralmarkersincludingNestin,IIIBeta-Tubulin(TuJ1),andglialfibrillaryacidicprotein(GFAP)(31).AnimalsreceivingahighdoseofHUCBcellshadasignificantlylongerlifespanthananimalswithlowdoseHUCBadministration(32).BasedonthesepromisingeffectsofHUCBtreatmentofALSmice,theclinicaltrialswithhematopoieticstemcellsareunderway.Jansonetal(33)isolatedCD34+stem/progenitorcellsfromperipheralblood,andCD34+cellsarebelievedtobethekeystemcellcomponentinHUCB.ThepurifiedCD34+cellswereintrathecallyinjectedinthreeALSpatients.After612months,noneofthepatientsreportedside-effects,butnoclinicalefficacywasseen(33).Althoughtheexpectedsuccesswasnotachieved,therewereatleastnoharmfuleffectstothepatients.Further,sincethetotalcasesaretoofewforstatisticalanalysis,itistooearlytomakeaconclusion.Sanfilipposyndromeisageneticdiseasewhichproducesadeficitinthealpha-N-acetylglucosaminidase(Naglu)enzyme,whichiscriticaltothebreakdownoftheglycosaminoglycan(GAG)heparinsulfateinthelysosome.Thediseaseischaracterizedbydiffuseneuralandotherorgandegeneration,andthepatientusuallydiesat11-12yearsofage.RecentstudiesshowedthatHUCBmaycorrectthisenzymedeficit,andprovideatreatmentforthisdisease.Inthe

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9laboratory,animalswithamutationintheNaglugenereceivedi.v.injectionsofmononuclearcellsfromHUCBthathadthenormalNagluenzymeonthe5thdayofpregnancyduringblastocystimplantation.TheHUCBcellswereincorporatedintotheembryoduringitsdevelopment,andthesecellsfunctionallycorrectedthedeficitinNagluenzymeexpression(34).Inaddition,HUCBtransplantationisapromisingapproachinthetreatmentofParkinsondisease(35).Intheexperiment,HUCBwasdeliveredintoParkinsondiseasemice(B6CBACa-AW-J/A-KCNJ6)throughintravenousadministration.Theotheranimalsweretransplantedwithbonemarroworuntreatedascontrol.Thelifespanofthesemicewasexamined.When50%ofthecontrolanimalsdied,only1outof10animalsfrombonemarrowtransplantedanimals,and2outof12HUCBinjectedanimalshaddied.After200days,thecontrolgrouponlyhad10%oftheoriginalanimalsalive,andbonemarrowtransplantedgrouphad20%.Interestingly,thesurvivalrateintheHUCBtreatedanimalswas33%.ThereweresignificantdifferencesbetweentheHUCBtreatedgroupandcontrolgroup.Inaddition,HUCBtreatedmicehadlessParkinsonsdiseasesymptoms.ItispossibleforthegraftedHUCBtopartiallycorrectthedeficitsduetoalossofdopamineneurons(35).1.3MechanismsofFunctionalRecoveryinStroke1.3.1Cellreplacement

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101.3.1.1CharacterizationofneuralpropertiesofHUCBInourresearchgroup,weroutinelyculturedthemononuclearfractionofHUCBcellsinDMEMwith10%FBS(36).TheculturedHUCBcellsapparentlyseparatedintotwodifferentfractions:anadherentsubpopulationandafloatingsubpopulation.TheadherentsubpopulationmainlyexpressedCD45,andafewcellsexpressedstemcellmarkerssuchasCD133andCD117.Thesecellscanhaveavarietyofmorphologies;somecellsarelargeandegg-shaped,othersarebipolar,orhavemultiplehair-likespines,aremultipolar,oraresmallandroundwithprocesses.Whilestillfloating,thecellsofthefloatingfractionremainsmallandround,exhibitinglessdifferentiatedproperties.However,oncethesecellswerereplated,theyadheredtotheculturedishandtheydifferentiatedanddevelopedlongprocess.TheexpressionofCD45intheculturedecreased,butCD133andCD117increased,suggestingthesecellsarestillprimitive,sinceCD133andCD117arestem/precursorantigens.Interestingly,evenwithoutanycytokinestimulation,someofthesecellsfromboththeadherentandnonadherentfractionsstillexpressedneuralmarkers.TheadherentcellsexpressedNestin,A2B5andGFAP(Glialfibrillaryacidicprotein),andeventheneurotrophinreceptorstrkA,trkB,andtrkC.Thepresenceofneurotrophinreceptorssuggeststhatthesecellshaveapotentialtobecomeneural-likecellsafterinduction.Inthefloatingcellfraction,allthemarkersincludingnestin,GFAP,TuJ1,andA2B5werefoundinculturedHUCB.S100,

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11GalC,trkB,trkCwerefoundtobeenrichedintheadherentfraction,whilelongtermcultureoffloatingcellsenrichednestinandTuJ1expression(36).Sanchez-RamosexaminedHUCBdifferentiationinvitro(37).HUCBcellsweretreatedwithretinoicacid(RA)+nervegrowthfactor(NGF),ortheywereculturedinfreshDMEM+fetalcalfserum(FCS).RA+NGFtreatedHUCBhadheterogeneousshapesfromflatepithelioidcellstosmallspindle-shapedcellswithbranchingneuriticprocesses,andtherewerefewercellsthaninuntreatedculture.RA+NGFincreasedtheproportionoftheMusashi-1positivecellsupto6.2%ofthetotalcells,and-tubulinIIIpositivecellsupto18.7%,whilearound1.5%cellstreatedwithDMEMexpressedMusashi-1,and8%ofcellstreatedexpressed-tubulinIII.Musashi-1isaRNAbindingproteinintheCNSofvariousspeciesandhumans(38).-tubulinIIIisaspecificmarkerofneurons,andisbelievedtobeinvolvedinneuronaldifferentiation(39).InDMEMculture,around34%ofthetotalcellswereimmunoreactiveforGFAP,amarkerofastrocytes,andthisnumberwas66.2%inRA+NGFtreatedcultures(37).DNAmicroarrayrevealedthatsomeneuralmarkerssuchasmusashi-1increasedintheRA+NGFtreatedgroup.RT-PCRfoundthatevenwithouttreatment,HUCBhadsomeneuralmRNAexpression,whichincludednestin(anearlyneuraldevelopmentmarker)andnecdin(amatureneuronmarker).RA+NGFpromotedHUCBneuraldifferentiation,whereasmusashi-1andpleiotrophin(neuriteoutgrowthpromotingprotein)wereonlyfoundintheRA+NGFgroup.Thisphenomenawas

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12moreapparentinwesternblottingwhenagroupofneuralmarkersweredetectedinbothuntreatedandRA+NGFtreatedHUCB,anddensitometricanalysisrevealedmoreproteinexpressedinRA+NGFtreatedHUCB.Theseneuralmarkersweremusashi-1,-tubulinIII,pleiotrophinandNeuN(37).ThesedatastronglysuggestedthatHUCBcellscandifferentiateintoneuralcells.ItwaslongthoughtthatthesedifferentiatedcellswerefromstemcellswithinHUCB,butwhetherthesestemcellswerehematopoieticornon-hematopoietichasbeendebated(40).StemcellswereeliminatedfromHUCBmononuclearfractionbyimmunomagneticsortingwithananti-CD34+antibody.TheremainingcellswereculturedinDMEM,andthecellswerereplatedatthedensityof10cells/cm2inthepresenceofepidermalgrowthfactor(EGF)oncethecellshadformedamonolayer.Itwasfoundthatthereplatedcellsproducedanumberofclonesattherateofabout10timesthecellnumberperweek.Thenewcellshadthemostprimitiveprofile,anddidnothaveanyhematopoieticphenotype.WhenstemcelldepletedHUCBcellsweretreatedwithRAaloneorincombinationwithbrain-derivedneutrophicfactor(BDNF),HUCBdifferentiatedintoneurallikecells,whichwererecognizedbyTuJ1,GFAPandGalCantibodies.TuJ1antibodyrecognizesneuronspecificclassIII-tubulin,GFAPantibodyisdirectedagainstastrocytes,andGalCisaspecificmarkerofoligodendrocytes.ThisphenomenonwasalsoobservedinHUCBco-culturewithprimarycorticalcells,wherethemarkersofallthreeneuralcelltypesweredetectedwith

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13immunostaining.Approximately40%ofHUCBcellsdifferentiatedintoneurons,30%differentiatedintoastrocytesand11%intooligodendrocytes,suggestingtheneuralenvironmentmaybecriticalforpromotingHUCBdifferentiationintoneuralcells(40).ThedifferentiationofHUCBcellswasalsostudiedinvivo(41).FreshlythawedorculturedHUCBcellsweredirectlyadministeredintothestriatumofrodents,andtheanimalswereinjectedwiththeimmunosuppressiveagentcyclosporinA(CSA)orsaline.BothfreshlythawedandculturedHUCBwerelocalizedwithina500-mradiusfromtheinjectionspot,andsomecellsevenmigratedalongthecorpuscallosum.ThemajorityofcellsdisplayedhematopoieticmarkersincludingCD33,CD44,CD45,CD51/61andCD90/Thy-1onthesurfaceoftheinjectedcells.Interestingly,someofthegraftedcellsexhibitedneuralphenotypesandwerepositivelylabeledwithNeuN(amarkerofneurons),GFAPandCD11b,eventhoughthepopulationisnotlarge,suggestingthesecellscoulddifferentiateintoneuralcellsintheanimalbrain.Interestinglytherewerenosignificantdifferencesinmajorhistocompatibilitycomplex(MHC)classIantigenbetweenratswithCSAandratswithoutit(41).ItseemstheHUCBgraftcausedlessofanimmuneresponse,andtheCSAinjectionwasnotcriticaltocellsurvival.Thepossiblemechanismwillbediscussedlaterinthischapter.

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14Inanotherstudy,theRA+NGFpretreatedHUCBwastransplantedintotheanteriorpartofthesubventricularzoneofneonatalpups(1-day-old),aplacewellknownforneurogenesisinadultbrain,andtheDMEMtreatedHUCBwasadministeredasthecontrol(42).Onemonthlater,animalswereperfused,thebraincryosectionedandimmunocytochemistrywascarriedouttoinvestigatetheneuralphenotypes.Around2%oftheumbilicalcordbloodcellsexpressedGFAPaftertransplantation,regardlessofthetreatment,andonlylessthan0.2%cellsofthetotalpopulationexpressedtheneuronalmarker-tubulinIII.TheseHUCB-derivedpositivecellsdidnotlookorbehavelikeatypicalneuralcells.Theydidnotmigratetotheolfactorybulbthroughtherostralmigratorystream,thedestinationofnewbornneuronsinthesubventricularzone.ThismaybebecausetheseHUCBderivedcellswerehighlydifferentiated,anddidnotbehavelikeadevelopingcell.1.3.1.2HUCBcellsreplaceneuronsafterMCAOStrokeisacerebralvasculardiseasecausedbyavarietyofinsults,andcharacterizedbycentralnervoussystem(CNS)symptomssuchasdizziness,vomitingandevenlosingconsciousness.Duetotheneuralcelllossafterinsult,evenwithfirstaidandthebestavailablecare,notallofthepatientscanfullyrecover.Currently,therearenoeffectivetreatmentsforthiskindofdisease.SinceHUCBisrichinstemcells,whichcouldproliferateanddifferentiateintoneurallikecells,itwasthoughtthatHUCBcellscouldreplacethecelllossafter

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15transplantation.Thisproposalhasbeenexamined.Inoneexperiment,animalsweresubjectedtomiddlecerebralarteryocclusion(MCAO)for2hours,andHUCBwastransplantedintravenously1or7daysafterstroke(26).ThecontrolgroupwasadministeredPBS.HUCBcellswerefoundscatteredinbonemarrow,spleen,muscle,heart,lung,andliver.Interestingly,HUCBwasalsofoundinsidetheipsilateralstrokebrain,andexpressedsomeneuralmarkersincludingNeuN,MAP-2,GFAP,andFVIIIproteinsasdeterminedbyimmunochemistry.ThepercentageofHUCBcellsthatexpressedNeuN,MAP-2,GFAP,andFVIIIproteinswas 2%, 3%, 6%,and 8%,respectively(26).Inothersimilarstudies,HUCBrecipientshadmuchbetterfunctionalrecoverythanthecontrolanimals,butfewHUCBcellsexistedinthestrokedanimalafterintravenousadministration.HistologicalexaminationalsodemonstratedtheHUCBgraftbyitselfdidnotreducetheinfarctvolume,anditonlyhadthiseffectwhencombinedwithmannitol,areagentforhyperosmolartherapythatincreasespermeabilityofthebloodbrainbarrier(43).SincetherearefewstemcellsintheHUCB,andonlyaverysmallportionofHUCBislikelytoinfiltratethestrokebrain,itishardtoimaginethatthesecellscouldfullyreplacethelostneurons.1.3.1.3HUCBinductionofneurogenesisHUCBmaypromoteneurogenesisinthestrokedanimal.Ithasbeenlong

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16thoughtthatneuronsinthebrainarehighlydifferentiated,andcannotregenerate.Thisdogmahasbeenchallengedbyevidencethatnewneuronsareborninthegerminalzoneofthehippocampusandsubventricularzoneinrodentsandhumansthroughoutlife(44).RecentresearchdemonstratedthatCD34+cellsfromHUCBcouldenhanceneurogenesisinthestrokedbrainafterintravenousadministration(45).AnimalsweresubjectedtoMCAO(middlecerebralarteryocclusion).Fortyeighthoursfollowingthesurgery,animalsweretransplantedwithCD34+orCD34cellsfromcordbloodthroughi.v.injection,andthecellswerepre-labeledwithafluorescentdye.Interestingly,itwasfoundthatthetransplantedCD34+cellswerenotco-stainedwithantibodytoaneuronal(neuron-specificnuclearprotein;NeuN)orastrocyticantigen(GFAP),suggestingthetransplantedcellsdidnotdifferentiateintoneurallikecells,anditwasunlikelyforthesecellstoreplacethelostfunction.Inaddition,theenhancedendogenousneurogenesiswasobservedinCD34+recipients,butnotinCD34-orPBStransplantedanimals,wherenewborncellsfromneurogenesiswerefoundinthesubventricularzone(SVZ)oftheinjuredhemisphere,andtheymigratedtotheischemiazone.Incontrast,nonewborncellswerefoundinthecontralateralhemisphere.Theseresultsrevealedanewmechanismofactionforstemcelltransplantationinstrokeanimals,whereCD34+cellsfromHUCBinitiallystimulatedangiogenesisandwhereangiogenesisfurtherpromotedneurogenesis.Thenewborncellsfromneurogenesismigratedintothenewlyrestoredcortexwherethesecellsmaturedandwereresponsibleforfunctionalrecovery(45).

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17Themaintenanceofcorticalwidthintheseanimalsisconsistentwithourobservationsintheintraluminalfilamentmiddlecerebralartery(MCA)modelwhendoseofcellsandtimingofHUCBadministrationareoptimized(27,46).OnecaveattotheseresultsisthatthemigrationofnewdaughtercellsfromtheanteriorSVZwaslimited,andthenewborncellsonlymovedafewcelldiametersafterHUCBstimulation(47).ItishardforthecellstomigratelongdistancesfromtheSVZtothedestination,theinfarctsite,andtoreplacethelosttissue.WhilethecorticalwidthisgreaterintheHUCBtreatedanimals,analternateexplanationisangiogenesisandastrogliosis.HUCBcontainsalargenumberofgrowthfactors,includingvascularendothelialgrowthfactor(VEGF),angiopoietin-1,angiopoietin-2,insulin-likegrowthfactors(IGFs).ItwaswellknownthatVEGF,angiopoietin-1andangiopoietin-2arepotentangiogenicfactors,andCD34cellshavethecapabilityofexpressingangiopoietin-1,angiopoietin-2.IntheCNS,althoughneuronsandmicroglialcellsarehighlydifferentiated,andhavelimitedcapabilitytoproliferate,astrocytesalsoproliferate.IGFexpressionincreasedshortlyafterthebraininjury,andthisexpressionwascorrelatedtoastrogliosis(48).AsimilarphenomenonwasobservedinMSplaques(49),whileIGFalsocanpromoteangiogenesisinstrokebrain(50).1.3.2NeurotrophicSupportHUCBmayalsoprovidetrophicsupporttotheinjuredbrain.Itis

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18richinneurotrophins,whichareimportantforbraindevelopmentandneuroprotection.FanetalcomparedneurotrophinmRNAexpressioninHUCBandadultperipheralblood.Theyexaminedbrain-derivedneurotrophicfactor(BDNF),glialderivedneurotrophicfactor(GDNF),nervegrowthfactor(NGF)andneurotrophin(NT).ExpressionofmRNAoftheseneurotrophinswashigherinHUCBcomparedtoadultperipheralblood.Inaddition,ELISAassayrevealedthattheproteinexpressionofBDNFandNT3inthesupernatantofHUCBwassignificantlyhigherthaninadultperipheralblood(51).BDNFisawellknownneuralgrowthfactorthatwasfirstdescribedbyBardeetal(52).BDNFisinvolvedinthedevelopmentofperipheralsensorygangliasubservingvariousdifferentmodalities(53),andcandecreasetissuelossinbrainwhenadministeredafterhypoxic-ischemicinjuryinneonatalanimals(54,55).Neurotrophin3(NT3)hassimilareffectsasBDNF,andcomplementsthetrophiceffectofBDNFintheCNS(56).NGFistheprototypicalneurotrophin,andNGFprotectscholinergicneuronsagainstaxotomy-inducedneurodegenerationandaged-relatedatrophy(57).Itcanpreventglutamateinducedinjurytoneuronsinthehippocampus(58),hypoglycemia,excitotoxicityandiron-induceddegeneration(59,60,61)inculture.Inaddition,thegrowthfactorsinHUCBcellsmaybeeffectiveagainstthehypoxicinjurycausedbystroke,andimproveneuralcellviability.Thehematopoieticcytokinecolonystimulatingfactor-1isagrowthfactorintheCNS.

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19Itplaysanimportantroleinmodulationofcellsurvivaland/ordifferentiationofneuronprogenitorcells,andmayplayanimportantroleinproliferationordifferentiationofneuraltissue(62).IGFisanothergrowthfactorexpressedbyHUCB,anditisawellknownangiogenicfactor.IGFisrequiredforvesselremodelinginCNS,anditisinvolvedinvesselformationduringbraindevelopment(50).Recently,ithasbeenshownthatbothbrain-derivedandcirculatingIGF-Iactsasaneuroprotectivesignalsintheadultbrain(63,64).IGF-Ialsoisimportantformediatingtheprotectiveeffectsofphysicalexerciseonthebrain(65).HUCBalsoexpressesFGF-2,apotentneurotrophicfactorintheCNS.FGF-2isconservativelyexpressedinsomeneuronsandmostastrocytesinintactbrain,andplaysanimportantroleinmaintenanceofneuralcellfunctionasalocaltrophicfactor(66).FGF-2expressionisupregulatedafterbraininjury(67),andstimulatestissuerepair,neuronsurvivalandaxonregenerationinCNS(68).TodetermineifHUCBcellshaveatrophiceffectwhenintravenouslyadministeredafterMCAO,thesecellswereinjected1hourpoststroke.AnimalsthatreceivedHUCBandmannitolhadimprovedfunctionalrecoverycomparedtocontrolanimals,andareducedinfarctvolume.Inaddition,theexpressionofGDNF,NGF,andBDNFwassignificantlyhigherinanimalsthatreceivedHUCBandmannitolcomparedtothecontrolgroup.WhenHUCBcellswerepretreatedwithgrowthfactorantibodiestoGDNF,NGF,andBDNF,thehistologicaland

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20functionalrecoverywasdiminishedintheHUCBandmannitoltreatedanimals(43).TheauthorconcludedthatthegrowthfactorsfromHUCBcontributedtofunctionalrecoveryachievedbyHUCB.However,peripheralbloodalsocontainstheneurotrophinsmentionedabove.Whyperipheralblooddoesnothaveasignificanteffectonstrokedbrainisnotclear.1.3.3ImmuneSuppressionandDecreasedInflammationHUCBgraftshavealowincidenceofmorbidityandmortalityinthetreatmentofmanyhaematologicalandnon-haematologicaldiseasesfollowinghigh-dosemyeloablativechemo-radiotherapycomparedtobonemarrowtransplantation(69).Clinicalresearchdemonstratedthatthislowermorbidityandmortalityisaresultofthelowincidenceofgraftversushostdisease(GVHD)inHUCBtransplantedpatients,andwhenGVHDdoesoccur,itusuallyislesssevere(70).TheGVHDisasevereinflammatoryresponse,whereTcellsandotherimmuneeffectorsinHUCBattackhosttissueandcausetissuedamage.Underphysiologicalimmuneconditions,thenaveTcellsexistinthethymusandre-circulatethroughcentralimmuneorgansincludinglymphnodes,untiltheyencounterantigen-presentingcells(APC),whichincludedendriticcells,macrophagesandBcells.ForTcellimmunity,dendriticcellsarecritical(71).ProfessionalAPCcellsabsorbforeignmaterialsandundergoantigenprocessing.TheprocessedantigenpeptidesarepresentedatthesurfaceofAPCwithMHC

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21(majorhistocompatibilitycomplex),(72).MHCbindsTCR(Tcellreceptor)onthesurfaceofTcells,andtriggersTcellfunction.BasicallyCD8+Tcells,andmaturesCD8+Tcellsintocytotoxiccells;whileMHCIIinteractswithCD4+Tcells,andCD4+TcellsbecomeThelpercells.TheactivatedThelpercellsproliferate,andbecomeeitherTh1cellsorTh2cells.Th1cellscanproduceinterferon-factor-beta(TNF-),whileTh2cellsproduceIL-4,IL-5andIL-13.Th1cytokineprofilefavorsmacrophageandCD8+cellactivity,suggestingitactivatesthecellularimmunesystem.Incontrast,Th2cytokineprofilestimulatesBcellactivity,andpromotesBcellantibodyproduction,suggestingTh2cellsfavorthehumoralimmuneresponse.Interestingly,eachprofilemayenhancethispolarizationbyautocrinemechanisms,andinhibittheotherpathway(73).Forexample,Th2cellssecretIL-4andIL-5,whichenhancetheTh2pathway,andatthesametime,Th2cellsalsoproducetheanti-inflammatorycytokinesIL-10,whichinhibitstheTh1pathway(73).Theimmaturityofcordbloodisexhibitedonalltheaspectsoftheimmunecascade.Forantigenpresentation,cordbloodhasimpairedAPCcells,especiallydendriticcells(DC),whichresultsinlessofanimmuneresponse.DCcellsdevelopnotonlyfromCD34+haemotopoieticstemcellsinbonemarroworcord

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22blood,butalsofrommonocytesunderthestimulationofinterleukin(IL)-4andgranulocytemacrophagecolony-stimulatingfactor(GM-CSF)invitro(74).Interestingly,itwasfoundthatcordbloodyieldedfewerDCcellscomparedtoadultperipheralbloodafter7daysincultureinthepresenceofIL-4andGM-CSF.Inaddition,thecordbloodderivedDCexhibitedlessendocytoticactivitythanadultperipheralblood,characterizedbylessuptakeofFITC-labeledparticles,suggestingDChadimpairedantigenpresentingability.WhentheseDCcellswereco-culturedwithCD3+Tcells,DCcellsfromcordbloodwerenotabletoactivatenaveTcellsaswellasDCsfromadultperipheralblood(75).Further,cordbloodDCcellsfavortheimmunetolerancepathway.Forexample,DCcellsareclassifiedintoDC1(myeloidDC,CD11c+,CD123-),DC2(lymphoidDC,CD11cCD123+),andiDC(lessdifferentiatedDC,CD11cCD123-)(76,77).ThemajorpopulationofcirculatingDCincordbloodisofthelymphoidlineage(DC2)andpossessesanimmaturephenotypethatfavorstheTh2pathway(78,79),whileDC1playsaroleinTH1effectorcelldevelopment(80,81).Th2pathwaymodulatesimmunetolerance(80).Inthecordblood,therearemorelymphoidDC2cellsthanthereareinadultblood,promotingthedevelopmentofTcellsintoTh2cells,anddownregulatingtheimmuneresponse(82).Further,theimmatureDCproducelessIL-12thanadultperipheralblood,theimportanteffectorforTh1typeactivation(83),whichalsofavorstheTh2pathway.WithrespecttoTcellimmunefunction,CD4+Tcellsareimmatureincord

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23blood.Forexample,CD31isadifferentiationmarkeronthesurfaceofTcells,anditisprogressivelyandirreversiblylostduringrepetitivestimulationofCD4+Tcells.Therearearound10-20%CD4+CD31-incordblood,whilethissubsetofcellsisundetectedinadultperipheralblood,suggestingCD4+cellaremoreimmatureincordblood(84).CD45isanextracellularantigenassistingThelpercellactivation,anditslength(CD45RA+)shortensfollowingThelpercellsactivation(CD45RO+).Therefore,CD45RA+isanindicatorofnaveTcells.Inoneexperiment,itwasfoundthatthereweretwiceasmanyCD45RA+/CD45RO-cellsincordbloodthaninadultperipheralblood.AnothermaturecellmarkerforCD4+cellsisL-selectin(CD62L)whichhasbeenshowntoindicatethatthecellisarecentthymicemigrant,andthereweresignificantlyfewerincordbloodthanadultperipheralblood(85).Thesephenomenawereconfirmedinanotherstudy.CD4+CD45RA+werecollectedfromcordbloodandadultperipheralblood,andculturedinRPMI-1640for72hours.FACSexaminationrevealedthattherewasalowermeanfluorescenceintensityofCD45onthesurfaceofcordbloodthanadultperipheralblood,suggestinganimpairedThelperactivationpathway.Inaddition,cordbloodhadfeweractivatedCD4+CD45RA+TcellsasdeterminedwithCD25expressionthanperipheralblood.CD25isareceptorofIL-2locatedonthesurfaceofCD4+cellsregulatingcellproliferation(86).CD8+cellsarealsoimmatureincordblood.Inoneexperiment(85),effectortypecytotoxicTcells(CTL,CD8+/CD45RA+/CD27-)andsuppressorT

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24cellsubsets(CD8+/CD57+/CD28-)wereabsentinHUCB,andtherewerefewerCD8+/CD11b+effectorTcellsinHUCBthanperipheralblood.Generally,theratioofCD4+/CD8+ishigherinHUCBthanperipheralblood,anditwasconsideredaHUCBindicatorofbloodimmaturity.ThedeficitofCD8+TcellsignificantlyimpairedthefunctionofTcells(87).Inoneexperiment,HUCBandperipheralbloodcellswerefreshlyprepared,orgrown5to7daysincultureaftercollection.Cytotoxiceffectorcellresponsesweredeterminedinastandard4h51Cr-releaseassay.NolyticactivitywasdetectedinfreshisolatedHUCBcells,withneitherNK-likelysisnorTcelllyticactivity.Interestingly,after5to7daysinculture,HUCBcellsdevelopedanonspecificcelllysis,suggestingHUCBslowlybuilduptheimmunesystem.Cutaneouslymphoidantigen(CLA)isaligandonthesurfaceofmaturedTcell,whichcanbindwithE-selectinexpressedbyskinvasculaturefacilitatingTcelladhesionandtransendothelialmigration(87).Comparedtoperipheralblood,CLAwasabsentincordbloodasdemonstratedintheabovestudy(87).SinceCD8+Tcellfunctionisdeficientincordblood,andthecytotoxicpathwayisretardedinresponsetopathogenstimulation,TcellimmuneresponsefavorstheCD4+pathway,whereimmunesignalstriggertheTh1orTh2cascade.Comparedtoperipheralblood,HUCBexhibitsanimmuneimmatureprofile,wheretheTh2pathway,ananti-inflammatoryresponse,isfavored.ScientistscomparedcytokineprofilesinbloodfromHUCB,fromtheneonatesonthe1st

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25and5thdayafterbirth,frommothersandfromcontrols(89).TheTh1cytokine,inHUCBcomparedtoadultblood,anditsconcentrationincreasedsignificantlyfromumbilicalcordbloodtoneonatesonthe5thdayafterdelivery.Inanotherstudy(90),cordbloodandadultperipheralbloodwerestimulatedwithT-cellreceptor(TCR)complex,andtheexpressionofcytokinesIL-10,IL-4,IL--4andIL-5.whileIL-4,IL-5andIL-10areindicesofTh2cells.Interestingly,IL-10wasproducedfasterinHUCBthanadultperipheralblood(90,91),whereonlyprimarystimulationtriggeredHUCBIL-10production,whileadultperipheralbloodrequiredrepeatedstimulations.Inaddition,IL-4andIL-12enhancedIL-10productioninthisstudy.TheresultsrevealedthatHUCBskewedTh1/Th2toTh2dominance,whichhadanti-inflammatoryeffectpartlythroughIL-10(90).BcellsinHUCBarealsoimmuneimmature.CD5isindicativeofprimitiveBcells(92),andCD5+BcellsarealsoknownasB1cells,whichareassociatedwithself-reactivity,autoimmunity,andleukemia.B1cellsproducepolyreactiveIgMandnaturalautoantibodiesbutnotIgG,andIgMantibodyisnotsufficientinhumoralimmunedefense.ThisisthereasonwhyimmunerestorationpatientshavemoresevereimmunodeficiencyafterHUCBtransplantation(93).ThepercentageofBcellsinHUCBiscontroversial,butmoststudiesdemonstratedthatHUCBhadmoreCD5+Bcellsthanadultperipheralblood(87).

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26Incontrast,adultperipheralbloodhadmorematureBcellsexpressingCD19+/CD23+thanHUCB,andHUCBexhibitedimmuneimmaturitywithmoreCD19+/CD23-Bcells(84).Thisimmaturitywasalsoobservedatthelevelofthegene(94).Bcellsderivedarefromhematopoieticprogenitorcells,orpro-Bcells,aredesignatedasCD10+CD19-CD22-CD34+.Whenpro-BcellsdevelopintoBcells,theimmunoglobulinheavychainisrearrangedwhichensurescorrectIgHsecretion.HirosecomparedtheIgHgeneofBcellsubsetsfrompro-Bcell,pre-BcelltoBcellsinHUCBandinadultperipheralbloodthroughRT-PCR(94).Itwasfoundthatpre-BcellsderivedfromHUCBhadashorterNregionthanthosederivedfromadultperipheralblood.Inaddition,immunochemistrystainingrevealedthataBcelldifferentiationrelatedgene,theterminaldeoxynucleotidyltransferase(TdT)gene,wasnotdetectedincellsderivedfromHUCB.TheseresultsindicatedthattheHUCBBcelllineageisimmature.Anotherimportantmononucleatedpopulationincordblood,monocytes,isalsoimmature.Asprofessionalantigenpresentingcells,monocytescanactivateTandBcellimmunitywiththestimulationofpathogen.Inoneexperiment(95),monocytesderivedfromadultperipheralbloodinducedburst-promotingactivity(BPA)ofTcellsderivedfromadultperipheralbloodinvitrointhepresenceofphytohemagglutinin(PHA).Whenthemonocytesfromadultperipheralbloodwerereplacedwithmonocytesderivedfromcordblood,theBPAwassignificantlydecreased(95).Monocyteswerealsounresponsivetohepatocytegrowthfactor

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27(HGF)stimulation,apotenteffectorformonocyteactivationinadultblood.Inoneexperiment,JiangshowedthatHGFsignificantlyincreasedthemonocyteantigenpresentingcapacityofadultperipheralblood,andup-regulatedHLA-classIexpressioninadultperipheral,butdidnotinHUCB(96).ThecytokineprofilesofHUCBmonocytesweredifferentfromadultperipheralblood(97).MonocytesderivedfromHUCBproducedlessIL-1andTNF-thanthosederivedfromadultperipheralblood,andthecellswerelimitedinCD64+CD14+cells.Incontrast,monocytesderivedfromadultperipheralbloodhadmorecellstosecreteIL-1andTNF-,whichincludeCD64+CD14+cells,CD64+CD33+cellsandCD64+CD45RO+cells.Inaddition,cordbloodmonocyteslackCD11b,totheoflossofCD58andCD102expressionincordbloodcells,andalowintensitylabelingofCD29,CD54andCD102comparedtoadultblood(98).CD11b,CD11c,andCD49dbelongtotheintegrinfamilyandareveryimportantinplateletaggregation,inflammation,immunefunction,tumormetastasis,andtissuemigration(99).CD54,CD58andCD102areinvolvedincontrollingreceptormediatedsignal-transduction,asintercellularadhesionmolecules(100).ThedecreaseinexpressionofthesemarkersreflectsthelowintensityofimmuneresponseofHUCB.Thisinhibitedimmuneresponsewasalsoconfirmedinvivoinourlab(101).HUCBwasintravenouslyadministratedintorodentanimalsfollowing

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28MCAO.Twentyfourhoursafterdelivery,theinflammatorycellpopulationinstrokedbrainwasanalyzedwithflowcytometricanalysis.HUCBrecipientshadfewerCD45+/CD11b+andCD45+/B220cellsinbraincomparedtoMCAOonlyanimals,whiletherewasnosignificantdifferencebetweentreatedanduntreatedratsinthenumberofCD45+/CD3+andCD45+/NTcellsinthebrain.Inaddition,thepro-/anti-inflammatorycytokinesweremeasuredwithbothRNaseprotectionandELISA.Thepro-inflammatorycytokinesTNF,IL-1andIL-6significantlyincreasedshortlyafterstrokestimulation.WhenHUCBwasadministrated,thehighexpressionofpro-inflammatorycytokineswasdiminishedtoasimilarlevelasshamcontrol.Interestingly,HUCBadministrationalsodecreasedtheactivityofNF-thatinducesTNFexpression(102).1.4DoHUCBcellsgetintothebrain?TheargumentcanbemadethattheHUCBcellsdonotneedtogetintothebraintohavetheirtherapeuticeffects.Whilethebesteffectsoccurwithi.vasopposedtointrastriataldelivery,someofthecellscanbeobservedinthebrainafteri.v.administration(43).TransplantedHUCBcellswereobservedinthecortex,subcortex,andstriatumofrecipientrats(26).Sincebrainishighlyinsulatedbytheblood-brain-barrier(BBB),itisdifficultforlargemoleculestocrossthisbarrier.However,afterthestroke,the

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29BBBisdamagedandbloodcellsinthecirculatorysystemcouldleakthroughthedamagedbloodbrainbarrier(BBB)intotheregionoftheinfarct.ConsistentwiththepassiveleakageofcellsintothebrainistheobservationthatwhenHUCBcellsweredelivered7daysafterMCAO,animalshadlessmotorandneurologicalrecoverycomparedwiththeHUCBgrouptreated24hourafterMCAO(103).TheBBBisalmostrecoveredatthistime(104),andtherefore,fewerHUCBcellsmaybeabletocrosstheBBB.Onceinthebrain,Tcellsmayrecognizetheirantigen,triggeringaninflammatoryprocesswiththesubsequentproductionofcytokinesthatcouldactivateand/orfurtherdamagetheendotheliumoftheBBB,allowingthepassageoftheothercells.TheseTcellsalsoexpresschemokines(MIP-1/CCL3,MIP-1/CCL4)(105)andincreasetheexpressionofthechemokinesinastrocytesandperivascularmacrophages(106).Inaddition,strokeincreasessomechemokineexpressionintheCNS(107).Thisthenisasecondpossiblemechanism.TheelevatedchemokinesfacilitatethepassageofHUCBcellsintothebrain.Chemokinesaresmall,solublemoleculesknowntorecruitgranulocytes,macrophages,andotherinflammatorycellsinischemicbrainandcauseaninflammatoryresponse.Thechemokinesareclassifiedintofourdistinctsubfamiliesaccordingtothepositionandspacingoftheconservedcysteins,giventhepreferrednamesCXC,CC,C,andCXC3,alsoreferredtoasandneurotactin,respectively(108,109).Sincethefirstchemokine,IL-8,was

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30discovered(110),morethan50chemokineshavebeenreported,andtheyinteractwithatleast18receptors.Macrophageinflammatoryprotein(MIP)-1,andmonocytechemoattractantprotein-1(MCP-1)belongtothe-chemokinefamily,andhavebeenimplicatedaspotentialmodulatorsoflymphoidandmononuclearcellinfiltrationintotheCNS(111).MCP-1isa76aminoacidmoleculewith8-15kDweight,andoriginallywasidentifiedasanimmediateearlygeneproductinducedbyaplatelet-derivedgrowthfactorinmouse3T3fibroblastcells(112).MCP-1hashighlyspecificchemotacticpropertiesformonoccyteswithlittleeffectonleukocytesorlymphocytes(112,113).MCP-1expressionisprominentwithintheatheroscleroticlesion(114),inflamedsynovium,andglomerulus,andextensivelyexistsinperipheralorganssuchasischemickidney,liverandlung,andinrheumatoidarthritis(115).ThelevelofMCP-1isverylowinintactCNS,whilestroke,HIV,andmultiplesclerosissignificantlyincreaseitsproduction(116).MCP-1mRNAwasincreasedasearlyas4hoursintheipsilateralhemispherefollowingischemia,anduntil5dayspoststrokedependingontheanimalmodelandexaminationmethods(117,118),whereasthelevelofMCP-1inthecontralateralhemispherewassimilartocontrollevels.Doublestainingwithneuronspecificantigenorglialfibrillaryacidicprotein(GFAP)showedtheseMCP-1positivecellswerepredominantlyastrocytes,althoughsomeneuronswerealsolabeled(119).Otherstudiesfoundthathypoxia-ischemiaresultsinMCP-1expressioninmultiplecelltypesaroundthesiteofinjuryinneonatalrodentbrain(120).FollowingMCP-1expression,leukocytes

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31arefoundinthelesionedhippocampus.Inaddition,mutantmicewithaCCR2(MCPreceptor)deficithadneitherTcellsnormacrophageinfiltrationinthedenervatedhippocampus,suggestingacriticalroleforMCP-1anditsreceptorCCR2inleukocytemigration(121).MIP-1isapotentchemokine,andwasfirstisolatedfromsupernatantofendotoxin-stimulatedmurinemacrophages(122).Theprimaryisolationwasfurtherseparatedandyieldedtwodistinctbuthighlyrelatedproteins,MIP-1andMIP-1,whichshare68%identicalaminoacids(123).MIP-1isproducedbymanykindsofcells,suchasTcells,Bcells,neutrophilsandmacrophages,andalsohasbeendetectedinanimmortalizedmousemicroglialcellline(124),corticalastrocytesandmonocytes(125).StrokecanincreaseMIP-1expressionatthelesionsiteasearlyas1hrpostinsult,whilenochangeswereobservedinthecontralateralhemisphere(126,127).AninvivostudydemonstratedthatthisMIP-1expressionwascorrelatedwithmononuclearcellinfiltration(128).Thischemoattractiveeffectwasfurtherverifiedinvitro.Extractfromischemicbraincouldinducehumanbonemarrowstromalcellmigrationininterfacemigrationcultures,andthiscellmigrationcouldbeblockedbyMIP-1antibody(129).Similarresultswereachievedinexperimentswithlymphocytes(130),andmonocytes(131)aswellasothersubpopulationofmononuclearcells.Inaddition,accumulatingevidenceindicatesthatMIP-1alsohasvariouseffectsonthegrowthofhematopoieticprogenitorcells.MIP-1isanactivecomponentof

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32normalbonemarrowextractthatcaninhibittheproliferationofmultipotentialhematopoieticprogenitorcellsasdeterminedwiththespleencolony-formingunitassay(132,133).InvitroMIP-1enhancesgrowthofmaturemyeloidprogenitors,butsuppressesthegrowthofimmatureones(134,135).ItwasalsodocumentedthatMIP-1couldmodulatehematopoieticprogenitorcelladhesiveproperties(136).Therefore,alongwiththeleakagethroughBBB,strokewouldalsoenhancesomechemokineexpressionaroundtheischemiasite,whichinturncouldinduceHUCBcellstomigrateacrosstheBBBafterintravenousadministration.1.5HypothesisInthisstudy,wehypothesizedthatMCP-1andMIP-1expressionincreasesinthedamagedhemisphereafterstrokeandfacilitateHUCBcellmigrationintotheischemicbrain.Onceinsidethebrain,HUCBcellscouldsuppresstheinflammatoryresponse,andsupportneuronandastrocytesurvivalinthehypoxiccondition.Thishypothesishasbeenexaminedusinginvivoandinvitroexperimentalmodels.1.6SpecificAimsAim1.VerifythechemotacticeffectofMCP-1andMIP-1inischemic

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33brainextractsonHUCBmigrationinvitro.Aim2.DeterminetheeffectsofHUCBcellsoncellsurvivalofneurons,astrocytesandmicrogliainvitro.Aim3.DetermineiftheeffectofHUCBcellsonneurons,astrocytesandmicrogliaisdependentonaspecificsubpopulationofHUCBcells.

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34CHAPTERTWOGENERALMATERIALSANDMETHODS2.1TheMiddleCerebralArteryOcclusionRatModel2.1.1AnimalhousingandmaintenanceAdultSpragueDawleyrats(averagebodyweightrange:17525g)wereusedinthisstudy.TheanimalswerehousedintheVivariumintheUniversityofSouthFlorida,CollegeofMedicine.Animalshadfreetoaccesstowaterandfood,andweremaintainedina12hourdark/lightcycleinatemperaturecontrolledroomfor7dayspriortosurgery.2.1.2SurgicalProcedureThesurgerywasperformedundertheguidelinesoftheNationalInstituteofHeathasapprovedbytheUniversityofSouthFloridaInstitutionalAnimalCareandUsecommittee.AnimalswereanesthetizedwithIsoflurane(2-5%inO2deliveredat2L/min)first,andoncetheanimalswereinadeepsleepasindicatedbynoresponsetotoepinch,thegasdeliverywasreducedto1liter/min,andtheneckskinshavedanddisinfectedwithiodineand70%alcohol.Usingbluntdissection,therightcommoncarotid,externalcarotid,internalcarotidandpterygopalatinearterieswereexposed.Thecommoncarotidarterywascarefullyseparatedfromthevagusnerveandsurroundingtissues.Atemporarytiewas

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35placedonthepterygopalatineinordertostopbloodflowandprevententryoftheembolusintothisartery.Theexternalcarotidarterywastiedoffwithtwo5-0silksuturesandsacrificedbycuttingjustabovethebifurcationoftheinternalandexternalcarotidarteries.Theemboluswasinsertedthroughtheexternalcarotidandguidedintotheinternalcarotidartery,pastthebaseoftheskulltotheoriginofMCA,adistanceofapproximately25mm.Theexternalemboluswaspermanentlytiedin,andtheskinwassutured.Eachanimalwasputonawarmingblanketuntiltheyrecoveredconsciousness.Theanimalswerereturnedtotheirhomecagesuntiltheywereeuthanatized24hourslater.2.2AnimalSacrificeAnimalswerecarriedtothesurgeryroom,andthecagecoverwasreplacedwithanair-sealedcoverhavingaconnectiontoaCO2tank.AnimalswereeuthanizedbyCO2.Whentherewasnoresponsetostimulation,orthetoepinch,animalswereguillotined.Theskullswereopened,andbrainswerecarefullyremovedwithaspatula.Thebrainswereimmediatelysnapfrozeninliquidnitrogenandstoredforfutureuse.2.3NeuralCellIsolationandCulture2.3.1PrimaryBrainCellIsolationPregnantSpragueDawleyrats(HarlanSpragueDawley)werehousedwithfreeaccesstofoodandwaterfor2days.Whenthepupshaddevelopedtoembryonicday17(E17),themotherwassacrificedasdescribedabove,the

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36abdomenwasopened,andthepupswereremovedfromthemothersbelly.Theheadsofpupswerecutwithscissors,andwereplacedincoldDMEMonice.Allneuralcellsisolationprocedureswereperformedonice.Theskullswereopenedwithfineforcepsunderadissectingmicroscope,andthebloodvesselandmeningesofthebrainwerecarefullyremoved.ThebrainwasmincedwithascalpelinEarle'sBalancedSaltSolution(EBSS)containing20g/mlDNaseand0.3%BovineSerumAlbumin(solutionA)onice.Followingthis,thehomogenatewascentrifugedat500xgfor2min,andthepelletwascollectedandincubatedinEBSScontaining0.025%trypsin,60g/mlDNaseand0.3%BovineSerumAlbumin(solutionB)at37Cfor15mintodissolvetheconnectivetissuebetweencells.Trypsinizationwasstoppedwith10%fetalcalfserum(FCS),andthesolutionwascentrifugedagainat500gfor2min.ThepelletwasresuspendedinsolutionAandtrituratedthroughafire-polishedPasteurpipette.Thesolutionwasallowedtosettlefor4minutesandthesupernatantwascollected.ThenmoresolutionAwasaddedtothepelletbeforerepeatingthisprocesstwice.Thecollectedsupernatantwascombinedtogetherandcentrifugedat500gfor5min.Thepelletwithcellswasresuspendedinculturemedium(DMEMsupplementedwith10%(v/v)FCS).2.3.2NeuronEnrichedCulturesFollowingtissuecellisolation,theisolatedcellswereplatedatadensityof2.5105cells/cm2inaflaskcoatedwithpoly-L-lysine.After3daysinculture,mediumwaschangedtoNeurobasal(500ml)andsupplementedwith10ml50X

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37B-27,5mlAntibiotic/Antimycotic,2.5mlglutamine,0.5ml1:1000glutamate.TenM5-fluro-2-deoxyuridineand10Muridinewereaddedtoinhibitgrowthofallothercelltypesexceptneurons.Usingthisprocedure,8590%ofthecellswereneuronsasdeterminedbycellularmorphology.2.3.3AstrocytesEnrichedCulturesTheisolatedbraintissuecellswereplatedin50mlflaskcoatedwithpoly-L-lysineatthedensityof2.5105cells/cm2.DMEM(Gibco)with10%FCSwasaddedintoflask,andcellswereculturedat37oCin5%CO2inanincubator.Themediumwaschangedeverytwodays.Sinceastrocytesgrowfasterthanotherneuralcells,theytakeoverthecellcultureafter5daysinvitro(DIV)withminorneuronalandmicroglialcontaminationasdeterminedwithcellularmorphology.2.3.4MicrogliaEnrichedCulturesTheisolatedbraintissueswereculturedwithDMEM(supplementedwith10%FCS)at37Cin5%CO2withoutmediumchangeuntilconfluency.Theconfluentculturewasplacedonarotaryshaker,andwasagitatedfor1houratroomtemperature.Theadherentmicrogliadonotfirmlysticktothewalloftheflaskasastrocytesdo,thereforetocollectthem,themicrogliawerefloatedinthemedia,andthemediawascollectedandspundown.ThecellpelletwasresuspendedinserumfreeDMEMandwasplatedat100,000cells/mlintoapolylysinatedcultureflaskandmaintainedat37Cwith5%CO2for1hour.Under

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38theseconditions,microgliaattachedtothewalloftheflaskquicklyandfirmly,whileoligodendrocytesremainedlooselyattached.Theoligodendrocyteswereeasilyfloatedfromthewalloftheculturedishesbygentlyshakingwiththehand.ThemediumwasremovedgentlyandfreshserumfreeDMEMwasaddedtothedishes,andtheharvestprocedurewasrepeatedtwice.Theremainingcellsonthewallofdisheswerepurifiedmicroglia.Theculturemedium(DMEMwith10%FBSand0.1%gentamicin)wasadded,andcellsculturedtilluse.2.4HypoxiaCultureThegastightchamberwaspurchasedfromBillups-RothenbergInc(CA,USA).Thechamberwasputinsidetheincubatorandmaintainat37oC.AYshapeplasticpipeconnectedboththenitrogentankandCO2tankwiththechamber.Thegastankswereturnedon,andflowadjustedsothat5%CO2and95%N2enteredtheincubator.Beforehypoxia,thegas-tightchamberandthelowglucosecolorfreeDMEMmedium(Gibco)wereflushedwiththemixedgasfor10minutestoexcludeanyoxygen.Thentheplateswerewashedwiththeflushedmedium,andreplenishedwiththismedium,andputintothegas-tightchamber.Theplateswereexposedtohypoxia(5%CO2and95%N2)at37oCfor2hour(62).Thecontrolgroupwastreatedwiththesamevolumeofthehypoxia-treatedlowglucosecolorfreeDMEMmedium(Gibco)inanincubatorat37oCundernormoxicconditionsfor2hour.Afterthat,themediawereharvested.Somewellsinplatesweretreatedwith10%paraformaldehydefor5minutes.The

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39paraformaldehydewaswashedawaybyPBS3timeswith10minutesperwash,andthecellsstoredforfutureuse.2.5MigrationAssay2.5.1MigrationChemotacticChamberA96-ChemotxChamber(NeuroProbe)wasusedfortheseassays.Thechamberisa96-wellplateconsistingofbottomwellsthatholdthesamplesorchemicalattractantsandatopplate,whichisapolycarbonatemembranewith5mporesizeandiswherethecellsareplaced.Thetopplateissecurelyattachedtothebottomwellplatecreatingadifferentialgradient.2.5.2PreparationofBrainExtractsThenitrogenpreservedtissuesamplewasweighed.Thebloodvesselsandmeningeswereremovedunderadissectingmicroscope.TissuesampleswereplacedintoclearDMEMmediacontaining1%FBS(Fetalbovineserum),1%Sodiumpyruvateand1%Glutmax,0.1%gentamicinand4tabletsproteaseinhibitorcocktail(in50mlvolume)at150mg/mltissueweight,thenhomogenizedandcentrifugedat2000gfor20minutes.Thesupernatantswerecollectedandfilteredthroughsyringefilters.Alltheseoperationswereperformedonice,andthetissueextractwaskeptat4oCfornextdayuse.

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402.5.3PreparationofHUCBcellsFrozensamplesofHUCB(SaneronCCEL,Inc)werethawedinto10mLofclearDMEM(Gibco)supplementedwith5%fetalbovineserum(Gibco),gentamicin(50g/mL,Sigma),5%sodiumpyruvateand5%Glutamax,andcentrifugedfor7minutesat1500rpm.Thesupernatantwasremovedandcellswereresuspendedin1mLofthesamemedia.Theviabilityofallsampleswasdeterminedbythetrypanbluedyeexclusionmethod.Onlycellswith70%oraboveviabilitywereused.Cellswerethenpipetteddirectlyintothetopwellofthemigrationchambers.2.5.4BasicMigrationAssayTissueextracts(300L)werepipettedintothebottomwellsofa96-wellplate.Cellsweredirectlypipettedintothetopwellataconcentrationof100,000cellsper60Landthetopwellplatewasplaceddirectlyontopofthebottomwellplate.Themigrationchamberthenwasplacedinawater-jacketincubatorat37oCwith5%CO2.Eachsample,controlandstandardwasperformedintriplicate.Attheendofmigrationthetopwellplatewasremoved.Thebottomplatewascentrifuged,and200mlofmediaremoved.Themigratedcellnumberwascountedwithacellviabilityassay.Briefly,onevialofCellTiter-GloSubstratefromthiskitwasmeltedinto10mlCellTiter-GloBuffer,and100mlofthesolutionwasaddedintotheplate.After10minutes,theplatewasreadinaplatereadertodeterminethenumberofcellsthathadmigratedintothelowerchamber.

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412.6WesternBlotting2.6.1PreparationofTissueSampleThebrainsinliquidnitrogenweresagitallycutintostrokedsideandnon-strokedside.Theconnectivetissuesincludingvesselsandmeningeswereremoved,thebrainwasmincedandputinto3volumesoflysisbuffer(100.0mllysisbuffercontains1.0MTris-HCl(pH8.0),0.5MEDTA,1.0MDTT,1.3MSpermine,3.1MSpermidine,50mlGlycerol,4tabletsproteaseinhibitorcocktail,2.5MKCLand47.7mlH2O).Thetissuemixturewassonicatedfor1minute,thetissuelysatescentrifugedat10,000gfor10min,andthesupernatantscollected.2.6.2GelPreparationTissuelysateproteinconcentrationswereassayed(BCAProteinAssaykit,PierceBiotechnology,IL),andmixedwithlaemmlibuffer(BioRad,Inc)containing5%mercaptoethanol,anddenaturedat100oCfor10minutes.Whilethesampleswerebeingdenatured,12%SDSpolyacrylamidegel(Invitrogen,Inc)wasloadedontherunningapparatus,anditwasfilledwith1XNuPagerunningbuffer(Invitrogen,Inc).Thesampleswerethenspundown,andthesameamountofproteinwasloadedoneachlaneofthegel;themolecularweightmarker(seeblue,InvitrogenInc)wasloadedtoindicatetherunningfront.Theelectrophoresisapparatuswasplacedinacoldroom,andrunat100Vfor15minutes,thenthevoltagewasswitchedto60Vandrunovernight.2.6.3ProteintransferfromGeltoNitrocelluloseMembrane

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42Beforetransfer,thenitrocellulosemembrane(Invitrogen,Inc),blottingpaper,andfiberpadsweresoakedin1XNuPagetransferbuffer(Invitrogen,Inc)for15minutes.Thegelwasunloadedandthetransfersandwichassembledinthesequenceoffiberpad,onesheetofblottingpaper,gel,nitrocellulosemembrane,onesheetofblottingpaperandfiber.Thesandwichwasplacedinthetransferbuffer,andtheairbubbleswererolledoutwitharod.Thesandwichcassettewasloadedintothetransferringapparatuswiththenitrocellulosemembranefacingthepositiveanode.Thentheapparatuswasfilledwith1XNuPagetransferbuffer,andtheblottransferredtonitrocelluloseat100VforIhourinacoldroom.2.6.4AntibodyIncubationAftertransfer,thenitrocellulosemembranewaswashedthreetimesfor15minutesinTTBSbuffercontaining2.42gTris-Base,8.0gNaCl,and0.1%Tweeninoneliterofdistilledwater.Thenthemembranewasblockedwith5%non-fatmilkpreparedin1XTTBSwithout0.1%Tweenfor1hour.Themembranewasfurtherwashedwith1XTTBS(3X5minutes),andplacedintheprimaryantibodysolutionforincubationovernight.Theantibodysolutionwaspreparedwith25mlTTBSand0.1gnonfatmilk,andtheconcentrationofantibodywasadjustedaccordingly.Themembranewasthenwashedthreetimesasdescribedabovebeforeincubationinhorseradishperoxidase-conjugatedsecondaryantibodysolutionin25mlTTBSand0.1gnonfatmilkfor1hour.Theconcentrationoftheantibodywasadjustedaccordingly.Themembranewas

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43washedthefinalthreetimeswith1XTTBS.Themembranewasprobedusingenhancedchemiluminescencedye(ECL,AmershamPharmaciaBiotech,Piscataway,NJ)andautoradiographed.2.7ELISA2.7.1PreparationofTissueSampleThebraintissueinliquidnitrogenwasweighed,andtheconnectivetissuesincludingbloodvesselsandmeningesremoved.Thetissuewasputin3volumesoftissuelysisbuffer(0.1MTrisbuffer,0.9%,NaCL,0.5%NP-40,1mMEDTA,1%Aprotinin,0.5mg/LLeupeptin,0.7mg/L,2mMPMSF),andwassonicatedfor1minute.Thetissuelysateswerecentrifugedat10,000gfor10min,andthesupernatantswerecollected.Alltheseprocedureswereperformedonice.ThentissuelysateproteinconcentrationsweredeterminedwithaBCAProteinAssaykit(PierceBiotechnology,IL),andtheconcentrationwasadjustedtothesameconcentrationineachsamplewithlysisbuffer.2.7.2AssayProtocolELISAwasperformedaccordingtothemanufacturersprotocol.Briefly,thestandarddilutionbufferwasaddedtothezerowell,andthewellsreservedforchromogenblankwereleftempty.Thestandardsolutionwasaddedtothewellswith100lvolume,and50lsamplesfromtissueextractormediumwith50lstandarddilutionbufferwereaddedintotheplatestomakeallthewells

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44havethesamevolume.Then,50lofbiotinyltatedanti-IFN(oranyothercytokinetobetested,seeTable2.1)solutionwereaddedintoeachwellexceptthechromogenblanks.Theplateswerecoveredandincubatedfor30minutesatroomtemperature.Theplateswerewashed4times,and100lstreptavidin-HRPworkingsolutionwasaddedtoeachwellexceptthechromogenblankwell.After45minutesofincubation,theplateswerewashed4times,and100lstabilizedchromogenfromthekitwasaddedineachwell.Theplatethenturnedblueanditwasfurtherincubatedfor30minutes.Thereactionwasstoppedwiththe100lstopsolution.Thentheconcentrationofcytokineswasdeterminedonaplatereaderwithlightabsorbanceat450nm.Table2.1ParametersofELISAassayCytokinesStandardCurveSensitivity IL-10-2000pg/ml>3pg/ml IFN-0-1400pg/ml>13pg/ml IL-602000pg/ml>8pg/ml IL-100-1000pg/ml>5pg/ml 2.8ImmunohistochemistryThebrainsstoredin4%paraformaldehydeweretransferredinto20%sucrose(20gsucrose,80mlH2O)overnight,frozen,sectionedonacryostatata30mthickness,andstoredinWalthersAntifreeze(NaH2PO4x1H2O1.389g,Na2HPO45.45g,H2O400ml,EthyleneGlycol300ml,Glycerol300ml)until

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45furtheruse.Forimmunohistochemistry,sectionswerewashedthreetimes(10minuteseachtime)with0.1MPBS(pH.7.3).Afterwashing,sectionswereincubatedwithblockingsolution(10%normalserum,3%TritonX100in0.1MPBS)for1hour,thenincubatedwithprimaryantibodyat4oCovernight(seeTable2.2).Thesectionswerewashedthreetimes(10minuteseachtime)with0.1MPBS(Ph7.3),thenincubatedwithsecondaryantibodyinblockingsolution(10%normalserum,3%TritonX100in0.1MPBS)atroomtemperaturefor1hour.Thesecondaryantibodieswereeitherrhodamine-conjugatedIgGorFITC-conjugatedIgG.Thesectionsweremountedandexaminedunderepifluorescence.Table2.2.AntibodiesusedforimmunohistochemicallabelingofcellsAntigenHostSpeciesCrossreactivityDilutionSource MCP-1RabbitRatMouse1:100Novus MIP-1GoatRatMouse1:100SANTACRUZ OX-42MouseRatNotconfirmed1:100Abcam TubulinMouseMouseHuman,Mouse,Rat1:100Chemicon GFAPMouseRatHuman,Mouse,Rat1:100Chemicon CCR5GoatHumanNottested1:200Capralogics CCR1RabbitHumanMouse1:200Abcam CCR2MouseHumanNottested1:200Abcam 2.9MagneticCellSorting

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46Cryo-preservedHUCBcells(SaneronCCELTherapeutics,Inc,TampaFL)werefreshlythawedinto10mlPBS(PH.7.3),andcentrifugedat1000rpmfor10minutes.Thecellpelletwasresuspendedin1mlPBS,andthecellnumberwascountedwithtrypanblueexclusionmethods.PBSwasrefilledto10ml,andthecellswerecentrifugedagain.Thecellnumberwasadjustedto107per90lin1XBDImage,andHUCBcellswereincubatedwith10lanti-humanCD8magneticnanoparticles(BDBioscience,cat.No.557765),anti-humanCD11bmagneticbeads(MACS,No.130049601),oranti-humanCD19magneticnanoparticles(BDBioscience,cat.No.551521)atroomtemperaturefor15minutesrespectively.Thecellsolutionwaswashedwith1XBDImagebuffer20times,andsupernatantwasremovedcompletely.Thecellpulletwassuspended,andcellconcentrationadjustedto2X107cells/mlincold1XBDImagebuffer.Thelabeledcellsuspensionwastransferredtothepositive-fractioncollectiontubes(12X75mmtubes),andthepositive-fractiontubeswereplacedintothemagneticholderfor6minutes.Thepositivelabeledcellswereattachedtothewallofthetube,whilethenegativecellssanktothebottomofthetubeorfloatedinthemedium.Thesupernatantwasremoved,andcellsortingrepeatedtwiceasdescribedabove,exceptthecellswereincubatedfor2minutesinsteadof6minutes.Thepositivecellswerewashedandcollectedwith1XBDImagebuffer.2.10ConfocalmicroscopyThespecimenisimmunolabeledasdescribedinsection2.8.Beforeconfocalmicroscopy,theslideswereexaminedunderconventionalfluorescent

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47microscopy,confirmthestaining,andidentifycandidatesforconfocalmicroscopy.ForfluorescencereportingwithAlexa488(FITC,green),thelaser458/476/488/514nmlineisneeded.Forfluorescence633,thelaser633lineisneeded.Iftheslidesarestainedwithfluorescence568,thelaser568lineisneeded.Mountslides,andturnontheconfocalmicroscopeandcomputer.Choosethelaserlinesofchoiceandfilterparameters.TheseparametersincludeI3,N2.1andY5.I3isforblueexcitationandgreenemission(FITC,Alexa488,etc.).N2.1isforgreenexcitationandredemission(Rhodamine,etc.)andY5isforredexcitationandlongredemission(CY5,Alexa633,etc.).Oncetheseareset,settheotherscan-relatedparametersthatincludeMode,ScanSpeed,ScanFormat,Pinholesize,ZoomfactorandImageAverage.Theslidesarethenscannedandimagessaved.

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48CHAPTERTHREEMIP-NDMCP-1INDUCEMIGRATIONOFHUMANUMBILICALCORDBLOODCELLSINMODELSOFSTROKE3.1AbstractBACKGROUNDANDPURPOSE:Monocytechemoattractantprotein-1(MCP-1)andmacrophageinflammatoryprotein(MIP-1)are-chemokines,implicatedinmonocyteinfiltrationintothecentralnervoussystem(CNS)underpathologicalconditions.Wepreviouslyshowedthatbothinvivoandinvitrohumanumbilicalcordbloodcells(HUCB)migratetowardbraininjuryaftermiddlecerebralarteryocclusion(MCAO).WehypothesizedthatMCP-1andMIP-1mayparticipateintherecruitmentofHUCBtowardstheinjury,andinvestigatedthepossiblemechanisminculture.METHODS:Sprague-DawleyratsweresubjectedtoMCAOand24hourslatertheproductionofMCP-1andMIP-1inthebrainwasexaminedwithimmunohistochemistry,ELISA,orwesternblotting.ThechemotacticeffectofMCP-1andMIP-1forHUCBwereevaluatedwithinterfacecultureassays,andtheexpressionofMCP-1receptorCCR2andMIP-1receptorCCR1,CCR5onthesurfaceofHUCBwasalsoexamined.RESULTS:MCP-1andMIP-1expressionwasincreasedinastrocytes,microgliaandneuronsofischemicbrain.Interfacecultureassayrevealedthatthebrain

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49extractfromischemichemispheresignificantlypromotedHUCBcellmigrationcomparedwiththeextractfromthecontralateralside,andthiscellmigrationwasneutralizedwithpolyclonalantibodiesagainstMCP-1orMIP-1.Also,chemokinereceptorswereconstitutivelyexpressedonthesurfaceofHUCBcells.CONCLUSIONS:IschemiasignificantlyinducesMCP-1andMIP-1expressionaroundtheinfarct.Theup-regulatedchemokinescanbindcellsurfacereceptorsonHUCB,andinducecellmigration,demonstratingamechanismforinfiltrationofsystemicallydeliveredHUCBcellsintotheCNSinvivo.KeyWords:MCP-1,MIP-1,MCAO,HUCB

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503.2IntroductionIntravenousdeliveryofhumanumbilicalcordbloodcells(HUCB)decreasesneuraldamageinrodentssubjectedtomiddlecerebralarteryocclusion(MCAO),andprovidesbetterbehavioralandneurologicalrecoveryinarodentmodelofstrokecomparedtodirectintracranialdelivery(25).Whileanadditionaladvantageofthesystemicrouteistheeaseofdelivery,howthesecellsmigrateintothebraintoproducetheireffectsisnotclear,sincethebrainisinsulatedbythebloodbrainbarrier(BBB)andhasimmuneprivilege.OnepossibilityisthatBBBisdisruptedaftertheischemicinsultandgranulocytes,macrophages,andotherinflammatorycellsleakintotheischemicbrainpassively.Asecondpossibilityisthatstrokeup-regulatedchemoattractants,whichpromoteHUCBcellmigrationintothebrain.Oneofthepotentialchemoattractantsismonocytechemoattractantprotein-1(MCP-1).MCP-1hasbeendetectedininjuredbrainafterstroke(121),predominantlyinastrocytes(137).Otherstudiesfoundthathypoxia-ischemiaincreasesMCP-1expressioninmultiplecelltypesaroundthesiteofinjuryinneonatalrodentbrain(119).FollowingMCP-1expression,leukocytescouldbefoundinthelesionedhippocampus.Inaddition,mutantmicewithaCCR2(MCP-1receptor)deficithadneitherTcellsnormacrophageinfiltrationinthedenervatedhippocampus,suggestingacriticalroleforMCP-1anditsreceptorCCR2inleukocytemigration(121).AnotherpotentialchemoattractantimplicatedinthetraffickingoflymphoidandmononuclearcellintotheCNSismacrophageinflammatoryprotein(MIP-1).

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51Itisupregulatedasearlyas3to6hrpoststrokeintheipsilateralhemispheretothestroke,whereasthelevelofMIP-1inthecontralateralhemispherewassimilartocontrollevels(120,127).MIP-1mRNAwasalsofoundpresentinanimmortalizedmicrogliacellline,corticalastrocytesandmonocytesinculture(125).Invivo,MIP-1expressionwascorrelatedwithmononuclearcellinfiltration(128).Further,extractsfromischemicbraininducedhumanbonemarrowstromalcellmigrationinculture,andthiscellmigrationcouldbeblockedbyanMIP-1antibody,suggestingachemoattractanteffectofMIP-1(129).InthisstudyweexaminedwhetherMIP-1andMCP-1expressionincreasedinthedamagedhemisphereafterstrokeandifthepresenceofthesechemokinesinextractsofstrokebrainwouldinduceHUCBmigrationinanexvivoassaysystem.

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523.3MaterialsandMethods3.3.1MiddleCerebralArteryOcclusion(MCAO)TwentySpragueDawleyratswererandomlyassignedtotheMCAOornormalgroupsaspreviouslydescribed(25).Briefly,animalswereanesthetizedwithisoflurane(2-5%in2L/minO2).Therightcommon,externalandinternalcarotidarterieswereisolatedandanembolusinsertedretrogradelythroughtheexternalcarotid,intotheinternalcarotid,pastthebaseoftheskulltotheoriginoftheMCA(approximately25mmfrominsertion).Thefilamentwaspermanentlyanchoredinplaceandtheincisionclosed.Allanimalswereeuthanized24hpostsurgery,andthebrainswereharvested.Someofthebrainswerefixedwith4%paraformaldehydeinPBS,andtheotherswereimmediatelyfrozeninliquidnitrogen.3.3.2NeuralCellHypoxiaCultureNeurons,astrocytesandmicrogliawereisolatedfromfetalratbrainatembryonicday(E)17asdescribed(138,139),andgrownseparatelyinculturetoconfluency.Beforehypoxia,thegas-tightchamber(129,140)wasflushedwith5%CO2and95%N2andtheplateswashedwithlowglucosecolorfreeDMEMmedium.Forthehypoxiagroup,mediumwaschangedto1mlhypoxiapre-treatedlowglucosecolorfreeDMEMandculturesplacedinthegas-tightchamber.Thecultureswereexposedtohypoxia(5%CO2and95%N2)at37oCfor2hours(141).Thecontrolgroupwastreatedwithlowglucosecolorfree

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53DMEMundernormoxicconditionsfor2hour.Themediawereharvested,andcellspreparedforwesternblotting.3.3.3Immunohistochemistry3.3.3.1MIP-1(orMCP-1)doublelabeling .Thefixedbrainsweresectionedonacryostatat30mthick.Thesectionswereblockedwith10%goatserum,0.3%tritoninPBSfor1hour,andthenincubatedwithprimaryantibodycocktailat4oCovernight.ThecocktailconsistedofgoatantiMIP-1(SantaCruz;1:100)orrabbitantiMCP-1(Novus;1:100)witheithermouseantiOX-42(CD11b/c)(Abcam;1:100),mouseantiTuJ1,(Chemicon;1:400)orchickenantiGFAP(Chemicon;1:100).Afterwashing,thesectionswereincubatedwithasecondaryantibodycocktailofrhodamine-conjugatedgoatanti-rabbitIgG(MolecularProbes;1:500)andFITC-conjugatedgoatanti-mouseIgG(MolecularProbes;1:500).Thesectionsweremountedandexaminedwithaconfocalmicroscope.3.3.3.2ChemokineReceptorImmunolabeling. HUCBsmearswerefixedwith4%paraformaldehyde,washedandthenairdried.Theslideswerestainedwithprimaryandsecondaryantibodyasdescribedabove.Theprimaryantibodyusedwasgoatanti-humanCCR5(CapralogicsInc,1:200),rabbitanti-humanCCR1,andmouseanti-humanCCR2(AbcamInc,1:200).SecondaryantibodieswereFITC-conjugatedgoatanti-mouseIgGorrhodamine-conjugatedgoatanti-rabbitIgG(MolecularProbes;1:500).

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543.3.4WesternBlottingThebrainswerecutsagitallyintostrokeside(ipsilateraltoMCAO)andnon-strokeside(contralateraltoMCAO)andthetissuelysed.AnequivalentamountofproteinwasloadedonSDS-12%polyacrylamidegelandtransferredtonitrocellulosepaper.Themembraneswereimmunoblottedwithanti-ratMIP-1(Chemicon)followedbyhorseradishperoxidase-conjugatedsecondaryantibody.Afterthefinalwash,membraneswereprobedusingenhancedchemiluminescencedye(ECL,AmershamPharmaciaBiotech,Piscataway,NJ)andautoradiographed.Neuralcellsfromcultureweretreatedsimilarly.3.3.5MCP-1ELISAAssayThebrainswerecutsagitallyintostrokedsideandnon-strokedside.Tissueswereweighed,placedinclearDMEM(Gibco,150mg/ml),homogenizedandcentrifugedat2000gfor20minutes.Thesupernatantswerecollected,filteredandadjustedtothesameproteinconcentration.TheELISAassaywasperformedaccordingtomanufacturersprotocol(AmershamBioscience),andtheconcentrationofchemokinewasdeterminedonaplatereaderatabsorbanceof450nmand550nm.3.3.6CellMigrationAssayThestandard(chemokineprotein)ortissueextractsamples(300L)withorwithoutMCP-1orMIP-1antibodies(1:100)werepipettedintothebottomwellsofa96-wellplate.FreshlythawedHUCBcellsweredirectlypipettedinto

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55thetopwellataconcentrationof100,000cellsper60L.Themigrationchamberwasincubatedat37Cwith5%CO2from4hoursto24hours.Thetopwellplatewasthenremovedandthebottomplatewascentrifuged,and200lmediaremoved.ThenumberofmigratedcellswasdeterminedwithCellTiter-GloLuminescentCellViabilityAssay,(Promega)accordingtothemanufacturersprotocol.Theplatewasreadinaplatereader.Themigrationassayswereperformedtwice.Eachsample,controlandstandardwasperformedintriplicate.3.4Results3.4.1PresenceofMIP-1andMCP-1inthestrokedbrainBothMCP-1andMIP-1werefoundinthebrain24hourafterMCAOusingimmunolabeling.Nopositivestainingwasfoundinthecontralateralhemisphere.DoubleimmunochemistrystainingrevealedthatbothMCP-1andMIP-1werefoundinneurons(Fig3.1),astrocytes(Fig3.2),andsomemicroglia(Fig3.3).MIP-1expressionwasverifiedwithwesternblotting(Fig3.4A)fromextractsofstrokedbrain.Further,inenrichedcultures,MIP-1wasexpressedinculturedastrocytesandneurons.Afterhypoxia,MIP-1increasedinneuronscomparedtoneuronsonlyexposedtonormoxiaconditions(Fig3.4B).MCP-1expressionwasverifiedwithELISA.Itwassignificantlyincreasedonthestrokedsideofthebraincomparedtothecontralateralsideafterstroke(p<0.05)(Fig3.5A).Inenrichedcultures,hypoxiainducedMCP-1expressionparticularlyinmicrogliaandastrocytes(p<0.05)(Fig3.5B).

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56 Fig.3.1.MCP-1andMIP-1expressioninneuronsafterstroke.StrokedratbrainwasimmunolabelledwithMCP-1orMIP-1(red;A,D,respectively),andthenlabeledwithTuJ1(neurons;green).BothMCP-1andMIP-1couldbefoundinneuronsoftheinfarctedhemisphere(C,F,mergedimages).(G,HarenegativecontrolsforMCP-1andMIP-1,respectively).

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57 Fig.3.2.MCP-1andMIP-1expressioninastrocytesafterstroke.StrokedratbrainwasimmunolabelledwithantibodiestoMCP-1orMIP-1(red,A,Drespectively)andGFAP(astrocytes,B,E,green).BothMCP-1andMIP-1couldbefoundinneuronswithintheinfarctedhemisphere(mergedimagesC,F)(G,HarenegativecontrolsforMCP-1andMIP-1,respectively)..

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58 Fig.3.3.MCP-1andMIP-1expressioninmicrogliaafterstroke.StrokedratbrainwasimmunolabelledwithMCP-1orMIP-1(red,A,Drespectively),andthenlabeledforCD11b(microglia,B,E;green).BothMCP-1andMIP-1couldbefoundinmicrogliaoftheinfarctedhemisphere(mergedimageC,F)(G,HarenegativecontrolsforMCP-1andMIP-1respectively)..

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59A B Figure3.4.MIP-1westernblotting.A)MIP-1waspresentinthestrokedbrain(R),andthepositivecontrol(C).Listhenon-strokedside.B)MIP-1expressioninhypoxiaandnormoxiaculturedastrocytes,neuronsandmicroglialcells(HA:hypoxiaastrocytes;NA:normoxiaastrocytes;HN:hypoxianeurons;NN:normoxianeurons;HM:hypoxiamicroglia;NM:normoxiamicroglia).

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60AB MCP-1ExpressioninBrain020004000600080001000012000StrokeSideNon-strokeSide C o n c e n t r a t i o n ( p g / m L ) * MCP-1Expressionincondition culturedneuralcells020406080100120140160180MicrogliaAstrocytesNeurons C o n c e n t r a t i o n ( p g / m L ) Hypoxia Normoxia*Figure3.5.MCP-1inbraintissueandculturedneuralcells.A)MCP-1expressionincreasedonthestrokedsideofthebrainasshownwithELISA.B)Afterenrichedcultureswereexposedtohypoxia,MCP-1concentrationinthemediaofmicrogliaandastrocytecultureswassignificantlyhigherthaninneuronalmedium(*,p<0.05).

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613.4.2ChemotacticeffectofMCP-1andMIP-1onHUCBcellmigrationMIP-1receptorsCCR1andCCR5aswellasMCP-1receptorCCR2wereallexpressedonthecellsurfaceofHUCBcells(Fig3.6)suggestingthatHUCBcellscouldrespondtoexpressionofthesechemokines.Indeed,MIP-1inducedmigrationinvitro,especiallyatthelowestdose,30ng/ml(p<0.05;Fig3.7A).HUCBcellmigrationtoMCP-1reacheditsmaximumataconcentrationof600ng/mlalthoughthiswasnotsignificantlydifferentfromthelowerdoses.Incontrast,the400ng/mlconcentrationwassignificantlydifferentfromthe30ng/ml,50ng/mland100ng/mldoses,respectively(*p<0.05;Fig3.7B).3.4.3EffectofbraintissueextractonHUCBcellmigrationTissueextractfromthestrokedbraininducedmoreHUCBcellstomigrateacrossamembranethanextractofthenon-strokedsidedid(p<0.05;Fig3.8A).AntibodiestobothMCP-1andMIP1-antibodydepressedmigrationtowardnon-strokedvalues(p<0.05).

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62 Figure3.6.MIP-1receptorsCCR1(A),CCR5(B)andMCP-1receptorCCR2(C)werepresentonthesurfaceofHUCBcells(x100).

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63AB MIP-1alpaDoseResponseForHUCB Migration010020030040050030ng50ng100ng200ng400ng600ngMIP-1alpaDose M L u m / E MCP-1DoseResponseForHUCB Migration0200400600800100030ng50ng100ng200ng400ng600ngMCP-1Dose M L u m / E*Figure3.7.ChemokinesdosedependentlyinducedHUCBcellmigration.A)MIP-1atadoseof30ng/mlproducedoptimalHUCBmigrationafter12hourincubation,comparedtoallotherMIP-1concentrations(p<0.05).B)MCP-1doseresponseshowsthatHUCBcellmigrationreacheditsmaximumataconcentration600ng/ml,butowingtothevariance,therewasnosignificantdifferencebetweenthisdoseandtheothers.Incontrast,the400ng/mlconcentrationwassignificantlydifferentfromthe30ng/ml,50ng/mland100ng/mldoses,respectively(*p<0.05).

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64AB EffectofMCP-1onHUCBMigrationto BrainTissueExtract0500010000150002000025000300003500040000strokenon-strokeMCP-1Antibody C e l l N u m b e r* EffectofMIP-1alphaonHUCBMigrationtoBrainTissueExtract020004000600080001000012000140001600018000strokeno-strokeMIP-1antibody C e l l N u m b e r#Figure3.8.EffectofMCP-1andMIP-1onHUCBmigrationtobraintissueextract.A)ExtractsfromthestrokedsideofthebrainattractedmoreHUCBcellscomparedtothenonstrokedside(*p<0.05),andthischemotacticeffectwassignificantlydepressedbyMCP-1antibody;B)MIP-1antibodyalsodepressedthemigrationofHUCBtoischemicbrainextract(#,p<0.05).

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653.5DiscussionMIP-1wasobservedontheipsilateralinjuredsideofthebrain24hoursafterMCAOasdeterminedwithimmunohistochemistryandwesternblotting.OurresultsareconsistentwithpreviousstudiesthatreportedMIP-1expressionasearlyas1hourafteronsetoftheMCAOandwhichpeakedat4-6hourpostsurgeryintheinjuredhemispheres(120,127).MIP-1hasalsobeenfoundtoincreaseafterotherbraininjuriessuchasischemia(142),stabwound(143),hypoxia,olfactorytargetablation(144).ThemajorityofMIP-1inthebodyisproducedbycirculatingmonocytes.IntheCNS,theinducibleMIP-1couldexistinastrocytes,microglia,endotheliacellsorneurons.Controversyexists,withsomegroupsfindingMIP-1intheastrocytes(127,145)whileothersreportthatitisonlyproducedbyMac-1mRNA-positivecellsincludingmicroglia/macrophages(142).Babcocketal(121)observedthattheMIP-1wasexpressedbyglialcellsandcoulddirectleukocytestotheCNSafteraxonalinjury(137).OtherinvestigatorsindicatedMIP-1wasexpressedbyneuronalcellsinculture(145).ImmunostainingshowsthatMIP-1canbeinallthreeneuralcells,butwesternblottingshowsMIP-1wasmainlyproducedbyastrocytesandneurons.Theinconsistencybetweenthesestudiesmostlikelyreflectsdifferencesbetweenanimalmodelsandcellculturetechniques.

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66SimilartoMIP-1,MCP-1wasalsofoundintheischemicbrainaroundtheinfarct.ELISAassayrevealedthattheamountofMCP-1onthestrokedsidewas7foldgreaterthanthecontralateralside.MCP-1wasproducedbyneurons,astrocytesandmicrogliacells.ThisisconsistentwithpreviousworkshowingthatMCP-1wasdetectedasearlyas3to6hrpoststrokeintheipsilateralhemisphere(121)andpeakedat2-3dayspostMCAO(137).Whenweexaminedculturesenrichedforneurons,astrocytesormicroglia,MCP-1wasmainlyexpressedinmicrogliaandastrocytesafterhypoxia.Usingimmunochemistrydoublestaining,ChefoundthemajorityMCP-1positivecellswereastrocytes.Thisisconsistentwithworkinbothcortico-striatalslice(146)andamousemodelofischemia(137).However,whatleadstoMCP-1andMIP-1upregulationafterstrokeisnotknown.RecentstudiesrevealedthattheproductionofMCP-1andMIP-1wasregulatedbypro-andanti-inflammatorycytokinesinducedbytheischemicinsult.Interleukin(IL)-1productionincreasedshortlyaftertheonsetofthestroke(147);followingthat,MCP-1andMIP-1productionincreased.Further,dexamethasoneandIL-10(bothanti-inflammatory)significantlyreducedMCP-1expression.However,inknockoutmicewhereinterleukin-1convertingenzyme(ICE),acysteineproteasethatcleavesinactivepro-IL-1togeneratematureIL-1,wasremoved,MCP-1expressionwassimilartowildtypeanimalswhenanimalsunderwentseverehypoxia.Withalessseverehypoxicincident,ICE-/-didattenuateMCP-1expressioncomparedtowildtypeanimals(148).The

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67inductiveMCP-1expressionisregulatedbyligandsthattriggernuclearfactorkappaB(NF-B)DNAbinding.HighdosesofIL-6treatmentremainedwithouteffect(149).TheearlyexpressionofIL-1couldactivatetheNF-rtoinduceexpression.ThefunctionalNF--1,andinducesMIP-1production.WhenIL-1expressionisblocked,MIP-1expressionisinhibited(150).TheproductionofMCP-1andMIP-1couldinturnelicitbloodcellmigrationintothebrain.Inastabwoundmodel,MIP-1inducesTcellandneutrophilinfiltrationintothebrain3daysaftersurgery;monocytes/macrophageswerepresentintheinjuredarea12dayspostinjury(143).Inahumanblood-brain-barriermodel,MIP-1expressionincreasedshortlyafteramyloid-betastimulation,andwasfollowedbymonocytemigrationfromthebloodsidetothebrainside(151).Further,inMIP-1knockoutanimals,thereisadecreaseinCD8dendriticcellmigrationintotheCNSafterinfectionwithmousehepatitisvirus(MHV)(152).Inaddition,bothMCP-1andMIP-1areinvolvedininflammatorycellrecruitmentinothertissues(153,154)afterinfection.MCP-1andMIP-1increaseinhippocampusafterentorhinodentatelesionspriortoTcellandmacrophagemigrationintothedenervatedhippocampus.WhenCCR2(MCP-1receptor)knockedoutanimalswereused,thecellmigrationwasquenched,demonstratingacriticalrolefortheCCR2ligandMCP-1/CCL2inleukocytemigration.CellularinfiltrationwasnotalteredbyamutationtotheCCR5receptortowhichMIP-1andRANTES/CCL5bind(137).Thedeletionof

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68MCP-1mayalsoresultinup-regulationofothercytokines(155),suggestingakeyroleofMCP-1inacytokinenetwork.ThesefindingssuggestedthatMCP-1andMIP-1mayberesponsibleforHUCBcellinfiltrationintotheCNSafterintravenousadministrationinstrokedanimals.However,itcanbearguedthatalargeenoughdifferenceinspecieshomologycouldresultinnoHUCBresponsetorodentchemokinesignals.ThisisunlikelysinceourinterfacemigrationsystemdidinduceHUCBmigration,andexhibitedadoseeffect.ThiscouldbeduetotheconservationofbothMCP-1andMIP-1ligandsandtheirreceptorsacrossspecies(156,157).Inaddition,wefoundconstitutiveexpressionofMCP-1andMIP-1receptorsonHUCB.Inconclusion,wehypothesizethatHUCBmigrationafterischemiabeginswithIL-1secretionattheinjurysite,whichinitiatesMCP-1andMIP-1secretioninactivatedastrocytes,microgliaandsomeneurons.TheaccumulatedMCP-1andMIP-1formaconcentrationgradientandwhencombinedwiththereceptoratthesurfaceofHUCBcells,inducemigrationofsystemicallyadministratedHUCBcells.

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69CHAPTERFOURHUMANUMBILICALCORDBLOODCELLSDEPRESSTHERATMICROGLIALINFLAMMATORYRESPONSESINVITRO4.1Abstract:BACKGROUNDANDPURPOSE:SystemicadministrationofHumanUmbilicalCordBlood(HUCB)cellsimprovesbehavioralandneurologicalrecoveryinthemiddlecerebralarteryocclusion(MCAO)modelofstroke.Further,animalsreceivingHUCBcellshavealesssevereinflammatoryresponsecomparedtoMCAOonlyanimals.However,theunderlyingmechanismisnotclear.WeproposedthatthefunctionalrecoveryisduetothedecreasedmicroglialinflammatoryresponseinHUCBtransplantedanimals.METHODS:Inthisstudy,primaryculturesenrichedformicrogliawereexposedtohypoxicornormoxicconditionseitherwithorwithoutHUCBcellsandfluoresceindiacetate/propidiumiodide(FDA/PI)labelingwasusedtodeterminecellviability.ThecytokineexpressionwasexaminedbyELISA.RESULTS:FDA/PIstainingrevealedanincreaseincelldeathinthemicrogliaHUCBcellco-cultureafterhypoxiaexposurecomparedtoallothergroups.Accompanyingtheincreasedcelldeath,theconcentrationofIL-1inthehypoxiaexposedco-culturemediumsignificantlyincreasedincomparisontothehypoxiaexposedmono-culturedgroup.Hypoxiaalsoincreasedtheexpressionofthepro-

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70-inflammatoryIL-10wasinhibited.CONCLUSIONS:OurdatasuggestthatHUCBcellsdepressedtheinflammatoryresponseafterMCAObydirectlyaffectingmicroglialactivity.Thiswasconsistentwithourinvivoobservationsofdecreasedinfiltrationoractivationofmicroglial/macrophagelikepopulationsinthebrainsofHUCBtreatedstrokedrats,whichmayhavecontributedtotheirrecovery.Keyword:Humanumbilicalcordblood,microglia,inflammatoryinhibition

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714.2IntroductionTheintactbrainisanimmuneprivilegedislandsecuredbytheblood-brain-barrier(BBB).Withinthecentralnervoussystem(CNS),therearenoprofessionalantigenpresentingcells,andtherearefewcellsexpressingmajorhistocompatibilitycomplex(MHC)classIandIImolecules(158).However,thisimmuneprivilegecouldbedisruptedbystroke.Microgliabecomeactivated,andexpressesMHCantigensandco-stimulatorymolecules(159).Theactivatedmicrogliacanacquireaphagocyticphenotypetoclearneuralnecrosisanddebris(160).Microgliaalsosecretecytotoxiccytokines,freeradicalsandnitricoxide,anddisruptstheBBB(161).Inaddition,microgliacansecretechemokinestorecruitleukocytesfromthecirculatorysystem,whichinturnparticipateintheneuroinflammatoryresponse.-1,andIL-10areofparticularinterest.IFNwasincreasedinstrokedbrainfollowingMCAOinrodents(162),andmicroinjectionofIFN/LPSintotherathippocampuscausedadelayedneuronalapoptosisinthehippocampuswhichpeaked7daysafteradministration(163).WhenIFNwasknockedoutintheDownssyndromemouse,neuronshadimprovedgrowthandviability(164).Inaddition,IFNcanalsoaffectmicroglialvulnerabilitybypromotingtheirdeathunderstressfulconditions(165).SimilartoIFNIL-1isalsoexpressedshortlyafterfocalcerebralischemia.Itexacerbatestheinfarctvolumeafterischemiainrodents(166),whileinhibitionof

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72IL-1expressioncanpreventneuronalnecrosisorapoptosiscausedbyinjury(166).IL-1alsocanmediatemicroglialactivationandproliferation(167).In-1,IL-10isananti-inflammatorycytokine(168).Itsuppressesmostofthepro-inflammatorycytokinesproducedinglialcells(169,170).Changesinthebalancebetweenpro-andanti-inflammatorycytokinesmayaffectmicrogliaproliferationandactivity.Ourpreviousresearchhasshownthatsystemicadministrationofhumanumbilicalcordblood(HUCB)producedbetterneuralbehavioralrecoverythancontrolanimalsaftermiddlecerebralarteryocclusion(MCAO)inrodents(25).SinceHUCBisrichinprogenitorcells(1),itwasthoughtthatthisfunctionalrecoverycouldbeattributedtodifferentiationofthetransplantedcellsandreplacementoflostcells.However,immunohistologicalexaminationfoundthatfewinfusedHUCBcellssurvived,andamongthemonlyfewcellsexpressedneuralmarkers.Itisunlikelythatthesecellsproliferateanddifferentiateenoughtoreplacethecelllosscausedbystroke.Interestingly,HUCBinfusedanimalsexhibitedanti-inflammatorypropertieswithadecreasedinfiltrationofCD45/CD11b-positive(+)andCD45/B220+cellsintothebrain.Thechangeswereaccompaniedbydecreasedexpressionofsomepro-inflammatorycytokines,decreasednuclearfactorkappaB(NF-kappaB)DNAbindingactivity,andincreasedexpressionofanti-inflammatorycytokines(171).Therefore,thefunctionalrecoveryoftheHUCBtreatedratscouldbetheresultofthesuppressedinflammatoryresponse.

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73Therefore,weproposedthattheHUCBcellsinducedneuroinflammatorysuppressionbymodulatingmicroglialactivityandrelatedcytokineexpression.Inthisstudy,asimulatedischemia/hypoxiamodelwasused.Primarymicrogliawereisolatedfromembryonicday(E)17ratpupsandco-culturedwithHUCBcellsinculture.Thecellviabilityaswellasthecytokineexpressionwereexaminedanddiscussed.4.3MaterialandMethods4.3.1IsolationofMicrogliacellsMicrogliawereisolatedfromfetalratbrainatE17accordingtotheproceduredescribedelsewhere(138).ThebrainwasmincedinEarle'sBalancedSaltSolution(EBSS)containing20g/mlDNaseand0.3%BovineSerumAlbumin(solutionA)onice.Followingthis,thehomogenatewascentrifugedat500gfor2min,andthepelletwascollectedandincubatedinEBSScontaining0.025%trypsin,60g/mlDNaseand0.3%BovineSerumAlbumin(solutionB)at37Cfor15mintodissolvetheconnectivetissuebetweencells.Trypsinizationwasstoppedwith10%fetalcalfserum(FCS),andthesolutionwascentrifugedagainat500gfor2min.ThepelletwasresuspendedinsolutionAandtrituratedthroughafire-polishedPasteurpipette.Thesolutionwasallowedtosettlefor4minutesandthesupernatantwascollected.ThenmoresolutionAwasaddedtothepelletandtheprocessrepeatedtwice.Thecollectedsupernatantwas

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74combinedtogetherandcentrifugedat500gfor5min.Thepelletwithcellswasresuspendedinculturemedium(DMEMsupplementedwith10%(v/v)FCS)andplatedatadensityof2.5105cells/cm2inaflaskcoatedwithpoly-L-lysine.Isolatedcellswereculturedat37Cwith5%CO2withoutmediumchangefor5days.Aftermediumchange,cellsweremaintainedincultureuntilthyehadgrowntoconfluency.Theconfluentculturewasplacedonarotaryshakerandagitatedfor1hourat37C.Themediawascollectedandspundown.ThecellswereresuspendedinserumfreeDMEMandplatedat100,000cells/mlintocultureflasksandmaintainedat37Cwith5%CO2for1hour.Looselyadherentoligodendrocyteswerefloatedfromthewalloftheculturedishesbygentlyshakingwiththehand.ThemediumwassuckedoffgentlyandmoreserumfreeDMEMwasaddedtothedishes.Thisprocedurewasrepeatedtwice.Theremainingadherentcellswerepurifiedmicroglia.Theresultingcellswereculturedwithmedium(DMEMwith10%FBSand0.1%gentamicin)untiluse.4.3.2CellCultureandHypoxiaTreatmentMicrogliacellsweregroupedintohypoxiamono-culture,hypoxiaco-culture,normoxiamono-cultureandnormoxiaco-culture.Beforehypoxia,thegas-tightchamber(129,140)aswellasthelowglucosecolorfreeDMEMmediumwasflushedwith5%CO2and95%N2for10minutes.Thenallplateswerewashed,andrefilledto1mlwiththepre-treatedmedium.Forco-culture,aninsertwassetintothemicroglialplate,andfreshlythawedHUCBcellsweredirectlypipettedintotheinsertsataconcentrationof500,000cells/well.The0.4

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75mmillipore(MilliporeInc)membraneallowsmediumtoflowfreelyin/out,whilethecellsareheldintheinsert.Hypoxiagroupswereexposedtohypoxia(5%CO2and95%N2)at37oCfor2hour,whilethenormoxiagroupsweretreatedsimilarlyundernormoxicconditionsfor2hour.Afterthat,themediawereharvestedforfurtheruse.Ascontrols,thesameamountoffreshlythawedHUCBcellsweredirectlypipettedintothesametreatedwellsexceptnopre-culturedmicroglia,andtheHUCBcellswereexposedtohypoxiaornormoxiaasdescribedabove.After2hoursculture,themediawerecollectedandstoredat-80Cuntiluse.4.3.3FDA/PIStainingCellviabilitywasdeterminedbyFluoresceinDiacetate/PropidiumIoidide(FDA/PI)staining.Briefly,FDAstocksolution(5mg/mlinacetone)andPIstocksolution(1mgin50mlPBS)werepreparedandstoredat4C.FDAworkingsolution(5lFDAstocksolutionin1mlPBS)waspreparedfreshlybeforeuse,and100lFDAworkingsolutionwasaddedtothe30lPIstocksolutionasFDA/PIworkingsolution.Then30lFDA/PIwasaddedintoeachwellcontaining600lPBS,andcellswereincubatedatroomtemperaturefor5minutes.ForHUCBcellsonthemembranesoftheinserts,theinsertwastransferredintoanotherwellcontaining600lPBS,and30lFDA/PIwasaddedintoeachwell.Photomicrographsweretakenunderepifluorescencefromfivefieldsofeachsample,whichincludesthecenter,left,right,upanddown,andthenumberoflive(FDAlabeled)ordead(PIlabeled)cellswerecountedusing

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76ImageProPlussoftware.Theexperimentwasrepeatedintriplicate.Theviabilitywasdeterminedasmeansempercentageoflivecells.ThesedatawerethenanalyzedusingStudentsttests.4.3.4CytokineELISAAssayTheELISAwasperformedaccordingtothemanufacturersprotocol(AmershamBioscience).Briefly,thestandarddilutionbufferwasaddedtothezerowell,andthewellsreservedforchromogenblankwereleftempty.Thestandardsolutionwasaddedtothewellswith100lvolume,and50lofmediasamplewith50lstandarddilutionbufferwereaddedintotheplatestomakeallthewellshavethesamevolume.Then,50lofbiotinylatedanti-IFN(oranyothercytokinetobetested,seeTable4.1)solutionwereaddedintoeachwellexceptthechromogenblanks.Theplateswerecoveredandincubatedfor30minutesatroomtemperature.Theplateswerewashed4times,and100lstreptavidin-HRPworkingsolutionwasaddedtoeachwellexceptthechromogenblankwell.After45minutesofincubation,theplateswerewashed4times,and100lstabilizedchromogenfromthekitwasaddedineachwell.Theplatethenturnedblueanditwasfurtherincubatedfor30minutes.Thereactionwasstoppedwith100loftheStopsolution.Thentheconcentrationofcytokineswasdeterminedonaplatereaderwithlightabsorbanceat450nm.Foreachcondition,therewere6samples,andeachsamplewasexaminedintriplicate.Thedataareexpressedasmean+ semconcentrationinpg/mlandwereanalyzedusingStudentsttests.

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77Table4.1.ParametersofELISAassayCytokinesStandardCurveSensitivity IL-10-2000pg/ml>3pg/ml IFN0-1400pg/ml>13pg/ml IL-100-1000pg/ml>5pg/ml 4.4Results:4.4.1.MicroglialcellviabilityCellviabilitywasdeterminedwithFDA/PI.Living(FDAlabeled,green)anddead(PIlabeled,red)cellswereobservedinallcultureconditions(Fig4.1A).Cellcountingrevealedthatunderhypoxicconditions,microgliaculturedwithHUCBhadalowercellviabilitycomparedtoothergroups(Fig4.1B,p<0.05),whereastherewasnosignificantdifferenceincellviabilitybetweenco-culturedandmonoculturedgroupsinnormoxicconditions.

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78AB MicrogliaViabilityafterHypoxiaExposure020406080100120Hypoxia-HUCBHypoxia+HUCBNormoxia-HUCBNormoxia+HUCB V i a b i l i t y ( % ) *Fig4.1.Microglialcellviability.A.FDA/PIstaininginmicroglia(x20).Live(FDAlabeled)cellswereingreen,whilePIlabeleddeadcellswereinred.B.Moremicrogliacellsdiedintheco-cultureconditionafterhypoxiaexposurecomparedtoallothergroups(*p<0.05)4.4.2.HUCBcellviabilityHUCBcellviabilitywasdeterminedbyFDA/PI.Living(FDAlabeled,green)anddead(PIlabeled,red)cellswereobservedinallcultureconditions(Fig4.2A).CellcountingshowstherewasnosignificantdifferenceinHUCBcellviabilitybetweenallgroups(Fig4.2B).

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79A B HUCBCellViability717273747576777879NormoxiaMonocultureHypoxiaMonocultureNormoxiaMicrogliaCocultureHypoxiaMicrogliaCoculture V i a b i l i t y ( % ) Fig4.2.HUCBcellviability.A.FDA/PIstaininginHUCB(x20).Live(FDAlabeled)cellsweregreen,whilePIlabeleddeadcellswereinred.B.Therewasnosignificantdifferenceofcellviabilitybetweengroups.4.4.3theconditionedmicrogliamediawasexaminedwithELISA.hadatendencytobehigherthanmonoculturedmicrogliaunderhypoxicconditions(Fig4.3).ThelevelofIFNlowerthanallnormoxicgroups(Fig4.3,*.#p<0.05).Therewasnosignificantdifferencebetweenthenormoxicgroups.

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80 MicrogliaIFNgammaexpression020406080100120140Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m l )*#Fig4.3.IFNinmicroglialcultures.TherewassignificantlylessIFNinthehypoxicmono-culturescomparedtoboththenormoxicmono-culturesandnormoxicco-cultures,respectively(*,#p<0.05).Interestingly,eventhoughtherewasatendencyforthehypoxicco-culturemediumtohavemoreIFN-culturemedium,therewasnosignificantdifferencebetweenhypoxiaHUCBco-cultureandothergroups.4.4.4.IL-1inmicrogliamediaHypoxiaexposuresignificantlyincreasedIL-1concentrationinmediafrommono-culturedmicrogliacomparedtoallgroups(*p<0.05).Hypoxiaalso

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81increasedIL-1intheco-culturedmicrogliacomparedtonormoxicco-culturedmicroglia,buttheconcentrationwasatthesimilarlevelasthenormoxicmono-culturedmicroglia.Interestingly,HUCBcellsdecreasedIL-1concentrationcomparedtothecorrespondingmono-culturedmicrogliainbothhypoxiaornormoxiaconditions.Therewasasignificantdifferencebetweenco-culturedandmono-culturedmicrogliaundernormoxicconditions(Fig4.4,#,p<0.05). MicrogliaIL-1betaexpression0100200300400500600700800Hypoxia+HUCBHypoixa-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m l ) ***###Fig4.4.MicroglialIL-1betaexpression.IL-1concentrationsignificantlyincreasedinhypoxiaexposedmono-culturescomparedtoitsnormoxicmono-culturegroup(##,p<0.05),andasimilarincreaseofIL-1wasobservedinhypoxiaexposedco-culturescomparedtonormoxiaexposedco-culture(**,p<0.05).Inaddition,therewasasignificantdifferenceofIL-1concentrationbetweenmono-culturedandco-culturedgroupsunderhypoxia(*p<0.05),andthisdifferenceexistedbetweennormoxicgroups(#p<0.05).

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824.4.5.IL-10inmicrogliamediaIL-10concentrationsweresimilarinthenormoxicmono-culturesandco-cultures,anddidnotchangeinthehypoxicexposedmono-culture.However,intheHUCBmicrogliaco-cultureexposedtohypoxia,therewasasignificantdecreaseintheIL-10concentration(Fig4.5,*,p<0.05). MicrogliaIL-10Expression0102030405060708090100Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m L )*Fig4.5.MicroglialIL-10expression.Therewasnosignificantdifferencebetweenmono-culturedandco-culturedmicrogliaundernormoxiacondition,andtheconcentrationofIL-10inthehypoxiaexposedmono-culturewasatthesamelevelofthesetwonormoxicgroups.TherewasasignificantdifferenceinIL-10concentrationbetweenthehypoxiaexposedmono-culturegroupandtheHUCBmicrogliaco-culturegroupexposedtohypoxia(p<0.05).

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83-1,andIL-10incordbloodmedia-1andIL-10wereundetectedinallHUCBalonemedia,includingnormoxicculturedHUCBmediaandhypoxicculturedHUCBmedia

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844.5DiscussionThisisthefirststudytoexplorethecrosstalkbetweenmicrogliaandcordbloodinvitrounderhypoxicconditions.ItwasfoundtheHUCBthemselvesweretoleranttohypoxicconditions.WhenweexaminedtheeffectofHUCBonmicroglia,undernormoxicconditionstheHUCBhadnoeffectoncellviabilityofthemicroglia.Incontrast,HUCBcellssignificantlypromotedmicroglialcelldeathunderhypoxicconditions,eventhoughhypoxiaalonehadnoeffectonmicroglialviability.Thismayexplainthedepressedimmuneresponseobservedinourpreviousstudies(171).However,themechanismunderlyingtheseobservationsisnotasclear.comparedtonormoxicgroups,althoughithadlesseffectonco-culturedmicrogliathanmono-culturedmicroglia.Asapro-predominantlyproducedbyTlymphocytesandnaturalkillercells.Itisalsoproducedbyastrocytesandmicroglialcellsinthecentralnervoussystem(172,173).Theconcentrationisusuallylowunderphysiologicalconditions.However,sionintheischemichemisphere(162),andexogenousadministrationexacerbatestheischemia-inducedbraindamage(17amajordiseasepromotingcytokine,andsthediseaseinMSpatients(175).Thisadverseeffectisextensive,andnotjustlimitedtoneurons.Itcouldinduceoligodendrocytedeathbyapoptosisasshowninvitro(176).Inanotherstudy,

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85lvulnerabilitytoFasLmediatedcytotoxicity(165),whileantioxidantscouldneutralizetheadverseeffectofFasL.Itsuggestedthatfreeradicalswereinvolvedinthisprocess.CordbloodisrichintheHUCBaloneculturesoriginatedfromthemicroglia,butitdoesnotexcludethepossibilitythatthavebeenmediatedbycordblood.Cordbloodcellsarespeciallyadaptedtolowoxygentensionsandmaythereforeberesistanttooxygendeprivation.Incontrast,normoxiaandespeciallyhyperoxiaactivateleukocytesandincreasecytokinesexpression(177).Thechangeincytokineproductionreflectsthesignalexchangeamongcordbloodcellsthroughthemedium,whichmayfurtheraffectmicroglialthelowIconcentrationinallhypoxicgroups,andrelativelyhigherconcentrationinnormoxia.Underhypoxicconditions,thetendencyforhigherconcentrationsof-culturemediumcomparedtothemono-culturesmayintoxicatethemicroglia,leadingtomicroglialcelldeath.Inthemicrogliamono-culutres,hypoxiastimulatedsecretionofthepro-inflammatorycytokine,IL-1whileIL-1productioninthehypoxicco-culturegroupwasnotsignificantlydifferentfromnormoxicmono-culturedmicroglia.IL-1isapro-inflammatorycytokinebelongingtotheTh1immuneresponse,andis

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86cytotoxictoneuralcells.IL-1isamatureproductofcytokineIL-1interactionwithIL-1convertingenzyme(ICE),andissecretedfromcells(178).IL-1combineswithitsreceptortoplayapivotalroleinregulationoftheinflammatoryresponseintheCNS.ItcanrecruitleukocytesintotheCNSfromperipheralbloodvessels,mediatethereleaseoffreeradicals,protease,glutamateandinflammatorycytokinesfromglialcells(179),andincreasesinstantlyafterstressorstimulation.TheearlyincreaseinIL-1expressioninresponsetoischemia/reperfusionsuggeststhatIL-1isthecauseofbraindamageratherthanaconsequence(180).Further,intracranialventricularadministrationofIL-1increasestheinfarctsizecausedbyischemia,whileapplyingIL-1receptorantagonist(IL-1Ra)orblockingconversionofIL-1toIL-1inmicewithmutantICEhavebeenshowntoreduceneuronaldamageafterfocalcerebralischemiainrodents(166).Interestingly,thisIL-1toxiceffectonlyaffectsneuronsintheCNS,andisnotassociatedwithmicroglialcelldeath.Forinstance,inIL-1knock-outmice,microgliahadasimilarcelldeathprofileaswild-typemicrogliainresponsetoLPSandATP(181).Inaddition,IL-1evenactedasapluritoptentgrowthfactorforglialcells(182).Wheninfusedintothebrain,IL-1stimulatesastrogliosisandneovascularizationatthesiteofinjury(183),andpromotesoligodendrocyteproliferationandremyelinationafterCNSinjury(184).Invitro,IL-1canpromotemicroglialproliferationafterN-methyl-d-asparate(NMDA)exposure.WhenIL-1productionwasinhibited,microglialrecoverywasretardedandtherewasadecreaseinDNAreplication(167).OurstudydemonstratedthathypoxiadoesactivateandincreaseIL-1expressioninmono-culturedmicroglia,

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87whichwasconsistentwithpreviouswork(185).ThisinducedIL-1mayworkasaneurotrophicfactortopreventhypoxia-inducedmicroglialcelldeath.WhenHUCBcellswereco-culturedwithmicroglia,therewasadecreaseinthehypoxiainducedIL-1releasecomparedwithmonoculture,whichcouldleadtoadecreaseinhypoxicresistence,leadingtocelldeathcomparedtohypoxicco-culture.Inthisstudy,noIL-1wasobservedinmediumfromHUCBalonecultures,thereforetheIL-1wasfrommicroglialcells.Inaddition,asuppressedIL-1productionwasobservedintheco-culturednormoxiaconditioninthisstudy.Sincenotoxicfactorswerepresentduringnormoxia,thelowlevelofIL-1hadnoeffectoncellviability.Incontrasttothepro-inflammatorycytokines,IL-10isananti-inflammatorycytokineproducedbyavarietyofcelltypesincludingtype2helperTcells,Bcells,macrophages,andmicroglialcells(168).IL-10hasneuroprotectiveproperitesagainstglutamateinducedneuronandoligodentrocytedeath(186),hypoxia-ischemia(187),LPS,andinterferon(188).IL-10cancounteractthetoxiceffectofendotoxinduringhypoxia-ischemiaintheperinatalbrain(189).IL-10caninhibitnitricoxideproductionandnitricoxidesynthaseactivityinmurinemacrophages,andblockstheabilityofmacrophagestoactasantigen-presentingcells(168),whichresultsinenhancedanti-inflammatoryaction.Inaddtion,IL-10candirectlysuppressbothproductionofpro-inflammatorycytokinesandexpressionofcytokinereceptorsinmicroglia(169,170).Forinstance,IL-10caninhibitexpressionoftheinflammatorycytokineIL-6invitro(190).IL-6caninduce

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88anorexia(191),fever(192)andhyperalgesia(193),anditssuppressionreducesthebraininflammatoryresponsecausedbykainicacid(194).Therefore,ourobserveddecreaseinproductionofIL-10inco-culturesofmicrogliawithHUCB,mayhavereducedthemicroglialresistancetothedetrimentaleffectofhypoxiainourstudy.However,howHUCBcellsmodulatecytokineexpressionisstillunclear.Humanumbilicalcordbloodcellsmaybeimmunetolerantcellsandproducelesscytokineswhenstimulated.HanetalcomparedtheinflammatorycytokineproductionincordbloodwithadultbloodfollowingstimulationwithPhorbolmyristateacetatePMA,IonomycinandPhytohaemagglutinin(PHA)/IL12.Theproductionofacytotoxiccytokineprofiles(IL-1,IL-1,IL-12,TNF)wassignificantlylessincordbloodcomparedwithadultblood(195).Inanotherexperiment,cordbloodcellswereexposedtoLPS,anditwasfoundthattheyhadasimilarcytokineexpressionprofileasreportedinHanswork(196).CordbloodcellshadlessTNF-andmoreIL-8expressioncomparedtoadultcells(53).FurtherstudiesrevealedcordbloodcellscontainalargenumberofimmatureTcells,andhasfewantigenpresentingcells(197).However,thecurrentknowledgeisstillnotenoughtoelucidatewhycytokineproductionincordbloodcellsafterstimulationislessthanintheadult.Thegeneprofileofcordbloodcellsandtheactivationofsignalingpathwaysinresponsetoinflammatorystimulirequirefurtherstudy.

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89Therefore,HUCBcellsmaypromotemicroglialcelldeathbymodulatingthepro-/anti-inflammatorycytokineexpressionafterhypoxiaischemiaexposure.TheincreasedmicrogliallossafterculturewithHUCBcellsduringhypoxiaischemiaexposurefurtherdiminishedthepro-inflammatorycytokine,IL-1,production,leadingtolessenedinflammation.HUCBcellsmayhaveasimilarroleinvivo;systemicallydeliveredHUCBcellscoulddepresstheinflammatoryresponseafterMCAObydirectlyinteractingwithanddestroyingactivatedmicroglia,whichinturnmaypromotebetterrecoveryafterstroke.

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90CHAPTERFIVEHUMANUMBILICALCORDBLOODCELLSENHANCENEURONANDASTROCYTESURVIVALINHYPOXIACULTURE5.1Abstract:BACKGROUNDANDPURPOSE:Humanumbilicalcordblood(HUCB)cellsattenuatedtheneurologicalandfunctionaldeficitsinstrokedanimalsafterintravenoustransplantation,andthisfunctionalrecoverydidnotrequireHUCBcellsurvivalintheinjuredarea.Thisphenomenondecreasedthelikelihoodthata-*/directinteractionbetweenHUCBandneuralcellsisnecessary.Incontrast,somesolublemoleculescouldinfuseintotheinjuredbrainfromtheperipheralbloodandplayprotectivefunctions.Therefore,somesmallmolecules,suchasinflammatorycytokines,maybeinvolvedinthefunctionalrecovery.METHODS:Inthisstudy,thepro-inflammatory/anti-inflammatorycytokineswereexamined.AneuronalcelllineandprimaryastrocytesfromE17ratswereculturedaloneorwithHUCBcellsinnormoxicandhypoxicconditions.TheconditionedmediumwascollectedandcytokineexpressionincludingIFN-1,IL-6andIL-10wasexaminedwithELISA,andcellviabilitywasinvestigatedwithFDA/PIstaining.RESULTS:ThedatarevealedthatHUCBcellssignificantlyimprovedneuronandastrocyteviabilityunderhypoxicconditions.NoIL-1weredetectedin

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91astrocyteorneuronalmedium.WhileIL-6wasnotobservedinculturedneuronmedium,IL-6wasonlydetectedinhypoxiaco-culturedastrocyteandnormoxiaculturedastrocyte,andtheconcentrationinhypoxiaco-culturedastrocytemediumwassignificantlylowerthanitwasinthenormoxiaculturedastrocytemedium.Interestingly,theanti-inflammatorycytokineIL-10washigherinhypoxiaexposedHUCBco-culturedconditionscomparedtoneuronandastrocytesmono-cultures.CONCLUSIONS:ThesedatasuggestedHUCBmaymodulatethepro-/anti-inflammatorycytokineexpression,whichinturnmayprotectneuronsandastrocytesfromhypoxiainducedcelldeath.KeyWord:Humanumbilicalcordblood,Neuron,Astrocytes,IFNgamma,IL-10

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925.2IntroductionThebrainisoneofthemostcomplicatednetworksinnature.Yet,neurons,whichcomposethesenetworks,arethemostvulnerablecellsinthebrain.Fiveminutesofhypoxiacanresultinirreversibledamage.Unlikeneurons,astrocytesaremoreresistanttohypoxia,andtheirfatesaredeterminedbythedegreeanddurationofischemia(198).Astrocytesaredispersedthroughoutthebrainbutarepositionedbetweenthebloodvesselsandneuronsandfunctionascommunicators.Ischemia/strokecancauseastrocyteactivationandgliosis,whichactstoprotecttheneurons.Forexample,activatedastrocytescanbalancethefluidmovementsbetweentheintracellularandextracellularspace,andhavetheabilitytotakeupglutamatetoreduceexcitotoxicity,andbufferionicmovements(198).Inaddition,astrocytescanreleasetrophicfactors,whichinturnpromoteblood-brainbarrierregeneration(199).Whenastrocytesupportivegrowthfactor--glialfibrillaryacidicprotein(GFAP)ismutant,astrogliosisisblocked,andblood-brainbarrierreconstructionisretardedinanimal(200).Therefore,topromotefunctionalrecoveryandenhanceneuronsurvival,astrocyteshouldbethefundamentaltherapeuticstrategyafterstrokeinjury.Previousstudieshavedemonstratedthathumanumbilicalcordblood(HUCB)cellshaveneuralprotectiveeffects,andHUCBtransplantationcouldamelioratethefunctionalandcognitivedeficitinstrokeanimalsfollowingmiddlecerebralarteryocclusion(MCAO)(25).However,themechanismwasnotclear.Furtherstudiesindicatedthattheneuralprotectiveeffectcouldbeachievedeven

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93withouttheHUCBcellstakingupresidenceinthecentralnervesystem(CNS),suggestingsomesmallmoleculesmaycrosstheblood-brainbarrierandinduceneuroprotection.Moleculesofinterestaresomepro-/anti-inflammatorycytokines.IL-1expressionatthemRNAleveliselevatedwithinminutesandattheproteinlevelwithinhoursinresponsetostroke(201).Itsdeleteriouseffectonbrainishighlyrelevanttoischemia/reperfusion.InhibitionofIL-1withtheexogenousantagonistIL-ra,whichisbelievedtoblockallactionsofIL-1andexertnootheractions,markedlyandreproduciblyreducesneurallosscausedbycerebralischemia,braintrauma,orinfusionofneurotoxinsinrodent(202).WhileexogenousIL-1couldexacerbatetheseverityoftheinjury(203),anotherpro-inflammatorycytokineinterferongammamayhaveasimilarfunction.IFNisnotnormallypresentinthebrainparenchyma,butatstressconditions,itcanbeproducedbyinfiltratingTcellsandNKcells(204directeffectonneuralinjuryfollowingischemia,butitcouldelevateneuralsensitivitytodeleteriousfactors(17expressionofinduciblenitricoxidesynthase,cyclooxygenase-2,andoxygenfreeradicals,whicharepotentialcytotoxinstoischemicbrain(205).Incontrast,IL-10playsaneuralprotectiveroleinischemicbrain.IL-10cansuppressproductionofpro-inflammatorycytokineincludingIL-1,IL-andTNF,whileitalsoenhancesIL-1ra,thenaturalantagonistofIL-1(206).

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94Exogenouspre-treatmentwithIL-10decreasedtheinfarctvolumeinstrokeanimals(207),whilenullificationofIL-10expressioninIL-10knockoutmiceexacerbatedthedamagefollowingmiddlecerebralarteryocclusion(208).Thus,wehypothesizedthatthebetterbehavioralscoreafterHUCBsystemicadministrationcouldbeafunctionofHUCBcellinducedneuralandastrocyticprotection,whichwasachievedbychangesinpro-/anti-inflammatorycytokinesexpression.5.3MaterialandMethods5.3.1CellPreparationRatembryoniccerebrocorticalneuronalcellswerepurchasedfromCelprogenInc(Cat#12030-01),andculturedinthemediumprovidebythesamecompanyaccordingtotheirprotocols.AstrocyteswereisolatedfromfetalratbrainatE17(138).ThebrainwasmincedinEarle'sBalancedSaltSolution(EBSS)containing20g/mlDNaseand0.3%BovineSerumAlbumin(solutionA)onice.Followingthis,thehomogenatewascentrifugedat500gfor2min,andthepelletcollectedandincubatedinEBSScontaining0.025%trypsin,60g/mlDNaseand0.3%BovineSerumAlbumin(solutionB)at37Cfor15mintodissolvetheconnectivetissuebetween

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95cells.Trypsinizationwasstoppedwith10%fetalcalfserum(FCS),andthesolutionwascentrifugedagainat500gfor2min.ThepelletwasresuspendedinsolutionAandtrituratedthroughafire-polishedPasteurpipette.Thesolutionwasallowedtosettlefor4minutesandthesupernatantwascollected.ThenmoresolutionAwasaddedtopelletandtheprocessrepeatedtwice.Thecollectedsupernatantwascombinedandcentrifugedat500gfor5min.Thepelletwithcellswasresuspendedinculturemedium(DMEMsupplementedwith10%(v/v)FCS)andplatedatadensityof2.5105cells/cm2inaflaskcoatedwithpoly-L-lysine.Isolatedcellswereculturedat37Cwith5%CO2andthemediumchangedonday2andday4invitro(DIV),andthecellsweresubculturedon7DIVgrowntoconfluency.Sinceneuronsandmicrogliadonotflourishunderthesecultureconditions,themajorityofthecellswereastrocytes,whichwasconfirmedbycellularmorphology.5.3.2CellCultureandHypoxiaTreatmentNeurons(astrocytes)weregroupedintohypoxiamono-culture,hypoxiaco-culture,normoxiamono-cultureandnormoxiaco-cultureconditions.Beforehypoxia,thegas-tightchamber(129,140)aswellasthelowglucosecolorfreeDMEMmediumwasflushedwith5%CO2and95%N2for10minutes.Thenallplateswerewashed,andrefilledto1mlwiththepre-treatedmedium.Forco-culture,aninsertwassetintheplate,andfreshlythawedHUCBcellsweredirectlypipettedintotheinsertsataconcentrationof500,000/well.The0.4mmillipore(MilliporeInc)insertsallowmediumtofreelycirculatethroughtheentire

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96culturewhilenotallowingthecellstopass.Hypoxiagroupswereexposedtohypoxia(5%CO2and95%N2)at37oCfor2hour,whilethenormoxiagroupshadthesamemediaasthehypoxiagroupbutweremaintainedundernormoxicconditionsfor2hour.Asanadditionalcontrol,HUCBcellsculturedaloneweretreatedinhypoxiaandnormoxiaconditionsasdescribedabove.Afterthat,themediawereharvestedforfutureuse.5.3.3FDA/PIStainingCellviabilitywasdeterminedbyFluoresceinDiacetate/PropidiumIoidide(FDA/PI)staining.Briefly,FDAstocksolution(5mg/mlinacetone)andPIstocksolution(1mgin50mlPBS)werepreparedandstoredat4C.FDAworkingsolution(5lFDAstocksolutionin1mlPBS)waspreparedfreshlybeforeuse,and100lworkingsolutionwasaddedtothe30lPIstocksolutionasFDA/PIworkingsolution.Then30lFDA/PIwasaddedintoeachwellcontaining600lPBS,andcellswereincubatedatroomtemperaturefor5minutes.Photomicrographsweretakenunderepifluorescencefromfivefieldsforeachsample,whichincludesthecenter,left,right,upanddown,andthenumberoflive(FDAlabeled)ordead(PIlabeled)cellswerecountedusingImageProPlussoftware.Theviabilityofeachsamplewasdeterminedasthepercentageoflivecellsandwasexpressedasmeansem.ThesedatawerethenanalyzedusingStudentsttests.

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975.3.4CytokineELISATheELISAwasperformedaccordingtothemanufacturersprotocol(AmershamBioscience).Briefly,thestandarddilutionbufferwasaddedtothezerowell,andthewellsreservedforchromogenblankwereleftempty.Thestandardsolutionwasaddedtothewellswith100lvolume,and50lsamplesfrommediumand50lstandarddilutionbufferwereaddedintotheplatestomakeallthewellshavethesamevolume.Then,50lofbiotinyltatedanti-(oranyothercytokinetobetested,seeTable5.1)solutionwereaddedintoeachwellexceptthechromogenblanks.Theplateswascoveredandincubatedfor30minutesatroomtemperature.Theplateswerewashed4times,and100lstreptavidin-HRPworkingsolutionwasaddedtoeachwellexceptthechromogenblankwell.After45minutesofincubation,theplateswerewashed4times,and100lstabilizedchromogenfromthekitwasaddedineachwell.Theplatethenturnedblueanditwasfurtherincubatedfor30minutes.Thereactionwasstoppedwiththe100lstopsolution.Thentheconcentrationofcytokineswasdeterminedonaplatereaderwithlightabsorbanceat450nm.Foreachcondition,therewere6samples,andeachsamplewasexaminedintriplicate.Thedataareexpressedasmean+ semconcentrationinpg/mlandwereanalyzedusingStudentsttests.

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98Table5.3.ParametersofELISAassayCytokinesStandardCurveSensitivity IL-10-2000pg/ml>3pg/ml 0-1400pg/ml>13pg/ml IL-602000pg/ml>8pg/ml IL-100-1000pg/ml>5pg/ml 5.4Results:5.4.1NeuronCellViabilityHypoxiadecreasedneuronalviabilityinhypoxicneuronmono-culture,whileHUCBcellssignificantlyprotectedneuronsfromthehypoxia-inducedcelldeath(Fig5.1*p<0.05).

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99AB NeuronViability95.59696.59797.59898.59999.5Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C e l l V i a b i l i t y ( % ) *Fig.5.1Neuronviability.A.FDA/PIstainingofneuron(x20).Live(FDAlabeled)cellswereobservedingreen,whilePIlabeleddeadcellswerered.B.Neuronviability.5.4.2NeuronalIFNExpressionconcentrationwasdetectedinthemediumfromnormoxicneuron-HUCBcellco-culturebutwasundetectableorbelowthesensitivityoftheELISAintheotherconditions(Fig5.2).However,therewasatendencyforconcentrationtobehigherinhypoxicco-culturethaninhypoxicmono-culture.

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100 IFN-gammaExpressioninNeuron0510152025303540Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m L )inneuronalcultures.inmediumfromneuron-HUCBco-cultureundernormoxicconditions,whereasitwasbelowthelevelofsensitivityintheotherconditions.5.4.3NeuronIL-1betaandIL-6expressionIL-1andIL-6wereundetectableintheneuronalcultures.5.4.4NeuronIL-10expressionIL-10wasobservedinhypoxicco-culturemedium,whileitwasundetectableinothergroups.

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101 IL-10ExpressioninNeuron020406080100120Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m l )Fig5.4.IL-10expressioninneuronalcultures.IL-10wasdetectedinthehypoxicco-culturegroup,butitwasbelowthesensitivitylevelintheotherconditions.5.4.5AstrocytecellviabilityViabilityofastrocyteswasgreaterintheHUCBco-cultureunderhypoxicconditionscomparedtohypoxicmonocultures(Fig5.5B*,p<0.05).Whiletherewerenosignificantdifferencesbetweenothergroups.ThisisclearlyvisibleinthephotomicrographsofFDA/PIlabelingofthecellsinFig5.5A.

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102AB AstrocytesViability020406080100120Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C e l l V i a b i l i t y ( % )*Fig.5.5.AstrocyteViability.A.FDA/PIlabelingofastrocytes(x20).Live(FDAlabeled)cellswereobservedingreen,whilePIlabeleddeadcellswerered.B.Astrocyteviability5.4.6Astrocyteconcentrationwasbelowthesensitivitylimitsoftheassayinhypoxicco-culturesofHUCBcellsandastrocyte,andnormoxicmono-culture.Itwasonlydetectedinhypoxicmono-culturesandnormoxicco-cultures(Fig.5.6).

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103 AstrocytesIFN-gammaexpression024681012141618Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m L )Fig5.6inthemediumfromhypoxicmono-cultureandnormoxicco-culture,whiletheconcentrationsintheothergroupswerebelowthedetectionleveloftheassay.5.4.7AstrocyteIL-1betaexpressionNoIL-1levelwasobservedintheastrocyteculture.5.4.8IL-6expressionInastrocytes,IL-6wasobservedinthenormoxiaastrocyteco-culturecondition.Afterhypoxia,therewasasignificantdecreaseinIL-6concentrationintheco-culture(Fig5.4*,p<0.05).IL-6wasnotexpressedintheotherconditions.

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104 IL-6ExpressioninAstrocytes0100200300400500600700Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m l )*Fig5.8IL-6expressioninastrocytes.NoIL-6wasdetectedinhypoxicandnormoxicmono-culturemedium,andtherewasasignificantdifferencebetweenhypoxicco-cultureandnormoxicco-culture(*,p<0.05).5.4.9AstrocyteIL-10ExpressionHUCBsignificantlyelevatedIL-10concentrationinhypoxiaculturedastrocytescomparedtohypoxicmonoculture(*p<0.05,Fig5.9).Inaddition,IL-10fromhypoxicco-culturewassignificantlyhigherthanitwasinnormoxicco-culture(#,p<0.05)andnormoxicmono-culture(##,p<0.05).

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105 IL-10ExpressioninAstrocytes020406080100120Hypoxia+HUCBHypoxia-HUCBNormoxia+HUCBNormoxia-HUCB C o n c e n t r a t i o n ( p g / m l )*###Fig5.9AstrocyteIL-10expression.IL-10wassignificantlyhigherinhypoxicco-culturemediumcomparedtothemediuminhypoxicmono-culture(*,p<0.05).Itwasalsosignificantlyhigherthannormoxicco-culture(#,p<0.05)andmono-culture(##,p<0.05).Nootherdifferenceswerefoundamongothergroups.5.4.10IFN-1,IL-6andIL-10incordbloodmedia-1,IL-6andIL-10wereundetectableinallHUCBalonemedia,includingnormoxicculturedHUCBmediaandhypoxicculturedHUCBmedia.

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1065.5Discussion:Inthisstudy,theFDA/PIassaywasusedtoexaminethecellviabilityofneuronsandastrocytes.ItwasfoundthatHUCBcellscouldsignificantlyimproveneuronandastrocytesurvivalduringhypoxia.SincetherewasaMilliporemembraneatthebottomoftheinserts,whichseparatedtheHUCBcellsfromtheculturedneuralcells,andonlyallowedmediaacrossthemembrane,itisunlikelythatincreasedsurvivalwasafunctionofdirectinteractionbetweenHUCBwiththeneuronsorastrocytes.Therefore,solublemoleculesmayplayacriticalroleinthisprocess.Theexpressionofpro-/anti-inflammatorycytokineswasalsoexaminedinnormoxicco-cultureconditions,andthswasbelowthedetectionleveloftheassay.mostlyproducedbyTcells(209),andneuronsarenotthemajorcellularsource-culturesunderhypoxicornormoxicconditions.WhenHUCBcellswereadded,therewasnditioncomparedwiththemonoculturegroup,suggestingthemajorsourcewasfromHUCBcells.However,-culturedconditions,thereforeitismostcells.Butthemechanismisnotclear.Inthisexperiment,eventhoughthedetectiblelevelunderhypoxicconditions,therewasatendencyfor

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107fromtheco-culturemediumtobehigherthanitwasfrommono-culture.However,thislowlevelobservedinhypoxicmono-cultureandnormoxicco-thedetectablelevel.torforastrocytes,promotingastrocyteproliferationandevenastrogliosis(210,211).However,extensiveastrocyteactivationhasseroussideeffects.ReactiveastrogliosisafterCNSinjuryhasbeenconsideredamajorimpedimenttoaxonalregeneration.IFalsomodulatesMHCclassIandclassIIantigenexpressiononastrocytes.MHCclassIcanelevateastrocytesusceptibilitytolysisbyclassIrestrictedTlymphocytes,andMHCclassIImakesastrocytesfunctionasantigenpresentingcells,whichenhancaninducecytotoxinsincludingnitricoxide(212),freeradicals,TNF,IL-1production,andallofthemmayexacerbatethebraindamage.OurresultssuggestedthatHUCBcellscouldsuppressastrocyteactivityunderhypoxicconditions,whichinturnmayinhibittheinflammatoryresponse.IL-1isapro-inflammatorycytokineandplaysakeyroleinmodulationoftheinflammatoryresponseintheCNS.ThecellularsourceofIL-1intheCNSincludesallcelltypesofthebrain,althoughitisundetectableinnormalhealthybrain.TheIL-1productionprobablyistheresultofcellinteractionsintheCNS,especiallywithastrocytes.Forexample,theproductionofIL-1inastrocytes

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108wasdemonstratedinvivowithvariousanimalmodels(213).Incontrast,astrocyteenrichedculturesfailedtoproduceanyIL-1underexposuretoawiderangeofstimuliincludingLPS,IL-1,TNF(214),suggestingsomemoleculessecretedbyothercellsareimportantforIL-1productioninastrocytes.ThesecretedIL-1canbindtheIL-1receptoronthesurfaceofastrocytes,andregulatethefunctionoftheastrocyte.AstrocytesarerichinIL-1receptors(215).WhenthesereceptorsareboundwithIL-1,itcouldgreatlydecreaseIL-1concentrationinmedium.ThismaybethereasonIL-1wasundetectableinourstudy.AsforIL-1inneuronalculture,evidencesuggestedthatwhileneuronsmayproduceIL-1,itisnotnormallydetected(216).Ourdatawereconsistentwiththis,sincenoIL-1wasobservedinthenormoxicneuronalmonoculture.IntheCNS,theneurons,astrocytesandmicrogliaarethecellularsourceofIL-6(217).IL-6functionsasaneurotrophininresponsetoneuralinjury.Itcanincreasethesurvivalrateofprimaryneuronsinculture(218),andprotectprimaryneuronsagainstglutamatetoxicity,andratcholinergicneuronsagainsttheneurotoxiceffectofN-methyl-D-aspartate(219).Inglialcells,IL-6promotesastrocyteproliferation,andisbelievedtobeinvolvedinastrogliosis(220).However,over-expressionofIL-6isdeleterioustotheCNS.ThehighlevelofIL-6isassociatedwithneurodegeneration,breakdownoftheBBB,angiogenesis,increasedexpressionofcomplementproteins,andimpairedlearning(221,222).ChronicIL-6expressionmaycauseprofoundcerebellarpathology,andsignificantlychangetheelectricalandsynapticactivityofPurkinjeneuronsin

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109mice(223).Incontrast,IL-6knockoutmicehadacorrelativedecreaseinbraindamagecomparedtocontrolanimalsfollowingafocalcryoinjurytothefronto-parietalcortex(194).However,wherethedosageofIL-6turnsfrombeneficialtodeleteriousisnotclear,anditgreatlydependsondifferentanimalspecies,animalmodelsandmicroenvironment.Definitely,lowconcentrationsofIL-6arenotharmfulforneuralcells.Inthisstudy,IL-6wasnotdetectedinanyoftheneuronalgroups,suggestingIL-6wasnotinvolvedintheHUCBneuroprotection.Asforastrocytes,IL-6wasonlyobservedintheco-culturecondition,andIL-6levelwassignificantlyhigherinnormoxiccomparedtohypoxiccondition.Atthishighlevel,thepotentialharmofIL-6maybelessenedsincenostressfulstimuliexisted.Underhypoxicconditions,IL-6levelwassignificantlyhigherintheco-culturemediumthaninmonoculture,whichmayhavedecreasedastrocytesdeathinHUCBco-culturedgroup.Unlikethepro-inflammatorycytokines,IL-10isananti-inflammatorycytokine,whichfunctionsasaneurotrophicfactorandinflammatoryinhibitor.Themajorcellularsourceismacrophage.IL-10hasthecapacitytoinhibittheproductionofsolubleinflammatorymediators,includingoxygenornitrogenderivedfreeradicals,andavarietyofcytokines.TreatmentwithIL-10couldsignificantlyattenuatethebraindamageinstroke(224),andhasbeenshowtoimprovetheneurologicalrecoveryintraumaticbraininjury(206).ThefunctionalrecoveryobservedwithHUCBdeliveryafterstrokemaybeattributedtotheanti-inflammatoryeffectsofIL-10,aswellastotheneurotrophiceffectofthiscytokine.

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110Inourstudy,therewasmoreIL-10inhypoxiaexposedco-culturedgroupscomparedtothemonoculturegroupsforbothneuronsandastrocytes.OurresultssuggestedthatHUCBcouldselectivelydecreasetheproductionofpro-inflammatoryIFNgamma,andincreasethelevelofanti-inflammatorycytokineIL-10inhypoxicmedia.Thisinhibitedinflammatoryresponsecouldprotectneuronsandastrocytesfromhypoxiainducedcelldeath.

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111CHAPTERSIXTHEINTERACTIONOFNEURONS,ASTROCYTES,ANDMICROGLIAWITHCD8,CD11B,CD19POSITIVECELLSDERIVEDFROMHUMANUMBILICALCORDBLOOD6.1Abstract:BACKGROUNDANDPURPOSE:Whenintravenouslyadministeredintoratsthathaveundergonemiddlecerebralarteryocclusion(MCAO),HUCBcellsdecreasedleukocyteinfiltrationintothestrokedbrain,whichwasaccompaniedbyadecreaseinpro-inflammatorycytokineproductioninthecentralnervoussystem(CNS).However,themechanismofactionunderlyingthisresponseisnotclear.METHODS:Inthisstudy,threemainsubpopulationsofHUCBcells(CD8+Tcells,CD11b+monocytesandmacrophage,andCD19+Bcells)wereisolatedfromHUCB,andculturedwithorwithoutneurons,astrocytesandmicrogliaindividuallyinhypoxiafor2hours.Themediumwascollectedandthecytokines,includingIL-1,IL-6,IL-10,andI.RESULTS:ItwasfoundthatCD11b+andCD19+cellssignificantlydecreasedmicrogliaviabilityinCD11b+(orCD19+)HUCBandmicrogliaco-culturesincomparisontomicroglialmonocultureunderhypoxicandnormoxicconditions,whileCD8+cellssignificantlydecreasedastrocyteviabilitybutnotmicroglial

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112viability.Nosignificantchangesincytokineexpressionwereobservedinthisstudy.CONCLUSIONS:ThedatawerepartlyconsistentwiththepreviousstudythatHUCBcellspromotedmicroglialcelldeath.TheneuroprotectiveeffectofHUCBmaybeattributedtotheeffectcausedbyinteractionsbetweenthesubpopulationsofHUCB,whichcannotbeachievedinCD8+,CD11b+orCD19+alone.Keyword:humanumbilicalcordblood,CD8,CD11b,CD19,inflammatorycytokines

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1136.2IntroductionTheHUCBcellshavebeenappliedintheclinicasatransplantationtreatmentforseverehematopoieticdiseasesformanyyears.HUCBcontainssimilarnumbersofstemcellsasbonemarrow,anditisusedintransplantationtherapyforreconstitutionofthebloodlineagesafterseveremyeloiddepletion(225).Comparedtobonemarrow,HUCBhasalowerincidenceofmorbidityandmortality,andgraft-versus-hostdiseases(226).WhenHUCBwasintravenouslyadministeredintostrokedanimals,therewasadecreasedCD45/CD11bpositive(+)andCD45/B220+cellularinfiltrationobservedinthestrokedbrainofrecipients.ThisdecreasewasaccompaniedbyadecreaseinmRNAandproteinexpressionofpro-inflammatorycytokinesandadecreaseinnuclearfactorappaB(NF-B)DNAbindingactivityinthebrainofstrokedanimalstreatedwithHUCB(171).Inaddition,weobservedmicroglialcelldeathwhenthesecellswereculturedwithHUCBunderhypoxicconditions,whileHUCBpreventedhypoxia-inducedcelldeathofneuronsandastrocytes.Thesechangesincellviabilitymaybecoupledwithpro-/anti-inflammatorycytokineimbalances,andmayaccountforthedecreasedinflammatoryresponseafterHUCBtransplantation.However,whichsubpopulationofHUCBhasthisfunctionisnotclear.HUCBisaveryheterogeneouscellpopulation.Amongthecellsinthepreparationarethreemainsubpopulations,includingTcells,Bcellsandmonocytes.Thesecellshavespecificlinagemarkers,CD8(orCD4),CD19andCD11brespectively.CD8+cellsarecytotoxicTcellsactivatedbyantigen

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114presentingcellswithpathogenattackingeffects(227).Incontrast,CD4+cellsareactivatedbyclassIIMHCantigenpresentingcells,anddonotattackpathogensdirectly.Instead,theycanmodulatecytokineexpression,andregulatetheimmuneresponseashelpercells(228).CD8+cellscanalsoproducecytokines(229,230)similartoCD4+cells.Inaddition,CD8+cellsproducechemokines(231,232),whichmayworkwithcytokinesandindirectlypromotetargetdestructionand/orrecruitadditionaleffectorpopulationstothesiteofinjury.Therefore,CD8+cellswerestudiedinthisexperiment.CD11b+cellsaremonocytes.Underhealthyphysiologicalconditions,CD11b+existsinintracellulargranules.CD11bisamarkeronthesurfaceofmonocytes,andincreasesfollowingstimulation.IncreasedCD11bpromotesfirmattachmentofthemonocytestotheendothelium,whichthenallowstransendothelialmigrationintotheinflamedtissue(233).Thetranslocatedmonocyteshaveendocytoticfunction,andbecomemicrogliaormacrophagedependingontheirdifferentlocations.CD11b+cellsproducefreeradicalsandcytokinesincludingTNF-,IL-1,andIL-6inresponsetoantigenstimulation(234).CD19isaB-lineage-specificantigen,amoleculeexpressedonnormalandneoplasticBcells(235).Bcellsnotonlyproduceantibodyinresponsetoantigeninvasion,butalsoplayavarietyofimmunoregulatoryrolesthroughtheirantigenpresentingabilityandthroughcytokineandchemokineproduction(236,237).Inthisstudy,CD8+,CD11b+,orCD19+cellswereisolatedfromHUCBthroughmagneticcellsorting,andculturedwithneurons,astrocytesand

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115microgliarespectively.Thepro-/anti-inflammatorycytokineexpressionwasinvestigatedanddiscussed.6.3MaterialandMethods6.3.1NeuronandAstrocytePreparationRatembryoniccerebrocorticalneuronalcellswerepurchasedfromCelprogenInc(Cat#12030-01),andculturedinthemediumprovidedbythecompany.AstrocyteswereisolatedfromfetalratbrainatE17(238).ThebrainwasmincedinEarle'sBalancedSaltSolution(EBSS)containing20g/mlDNaseand0.3%BovineSerumAlbumin(solutionA)onice.Followingthis,thehomogenatewascentrifugedat500gfor2min,andthepelletcollectedandincubatedinEBSScontaining0.025%trypsin,60g/mlDNaseand0.3%BovineSerumAlbumin(solutionB)at37Cfor15mintodissolvetheconnectivetissuebetweencells.Trypsinizationwasstoppedwith10%fetalcalfserum(FCS),andthesolutionwascentrifugedagainat500gfor2min.ThepelletwasresuspendedinsolutionAandtrituratedthroughafire-polishedPasteurpipette.Thesolutionwasallowedtosettlefor4minutesandthesupernatantwascollected.ThenmoresolutionAwasaddedtothepelletandtheprocessrepeatedtwice.Thecollectedsupernatantwascombinedandcentrifugedat500gfor5min.Thepelletwithcellswasresuspendedinculturemedium(DMEMsupplementedwith10%(v/v)FCS)andplatedatadensityof2.5105cells/cm2inaflaskcoatedwithpoly-L-lysine.Isolatedcellswereculturedat37Cwith5%CO2andmediawere

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116changedonday2andday4afterculture,andcellsweresubculturedonday7andgrowntoconfluency.Sinceneuronsandmicrogliacannotproliferateanddieinlongtermculture,themajorityofcellsleftoverareastrocytes.6.3.2IsolationofMicrogliaMicrogliawereisolatedfromfetalratbrainatE17(239).ThebrainwasmincedinEarle'sBalancedSaltSolution(EBSS)containing20g/mlDNaseand0.3%BovineSerumAlbumin(solutionA)onice.Followingthis,thehomogenatewascentrifugedat500gfor2min,andthepelletcollectedandincubatedinEBSScontaining0.025%trypsin,60g/mlDNaseand0.3%BovineSerumAlbumin(solutionB)at37Cfor15mintodissolvetheconnectivetissuebetweencells.Trypsinizationwasstoppedwith10%fetalcalfserum(FCS),andthesolutionwascentrifugedagainat500gfor2min.ThepelletwasresuspendedinsolutionAandtrituratedthroughafire-polishedPasteurpipette.Thesolutionwasallowedtosettlefor4minutesandthesupernatantwascollected.ThenmoresolutionAwasaddedtothepelletandtheprocessrepeatedtwice.Thecollectedsupernatantwascombinedtogetherandcentrifugedat500gfor5min.Thepelletwithcellswasresuspendedinculturemedium(DMEMsupplementedwith10%(v/v)FCS)andplatedatadensityof2.5105cells/cm2inaflaskcoatedwithpoly-L-lysine.Isolatedcellswereculturedat37Cwith5%CO2withoutmediumchangefor5days.Thecellswerecultureduntilconfluency,andtheconfluentculturewasplacedonarotaryshakerandagitatedfor1hourat37C.Themediawascollectedandspundown.ThecellswereresuspendedinserumfreeDMEM

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117andplatedat100,000cells/mlintocultureflasksandmaintainedat37Cwith5%CO2for1hour.Looselyadherentoligodendrocyteswerefloatedfromthewallofculturedishesbygentlyshakingwiththehand.ThemediumwasremovedgentlyandmoreserumfreeDMEMwasaddedtothedishes.Thisprocedurewasrepeatedtwice.Theremainingadherentcellswerepurifiedmicroglia.Theresultingcellswereculturedwithmedium(DMEMwith10%FBSand0.1%gentamicin)untiluse.6.3.3HUCBsubpopulationisolationCD8+Tcells,CD11b+monocytesandCD19+BcellswereisolatedfromHUCBwithmagneticcellsortingmethods.Briefly,cryopreservedHUCB(SaneronCCELTherapeutics,Inc,TampaFL)wasfreshlythawedinto1XBDImagebuffer(BDbioscience,cat.No.552362),andthecellconcentrationwasadjustedto107cellsper90l.TheHUCBcellswereincubatedwith10lanti-CD8linkedmicrobeads(BDBioscience,cat.No.557765),anti-CD11blinkedmicrobeads(MACS,No.130049601),oranti-CD19microbeads(BDBioscience,cat.No.551521)atroomtemperaturefor15minutes.Thesolutionwascentrifugedat1000gfor10minutes,andwashedwith1XBDImagebuffer20times,andsupernatantwasremovedcompletely.Thepelletwasresuspended,andthecellconcentrationwasadjustedto2X107cells/mlincold1XBDImagebuffer.Thelabeledcellsuspensionwastransferredtothepositive-fraction

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118collectiontubes(12X75mmtubes),andplacedintothemagneticholderfor6minutes.Thepositivelylabeledcellswereattachedtothewallofthetube,andnegativecellssanktothebottomofthetubeorremainedsuspendedinthemedium.Thesupernatantwasremoved,andthecellsonthewallwerecollected.Thecollectedcellswereresuspendedandthecellsortingprocedurewasrepeatedtwicewith2minuteincubations.6.3.4CellCultureandHypoxiaTreatmentNeurons(astrocytes,ormicroglia)weregroupedintohypoxiamono-culture,hypoxiaco-culture,normoxiamono-cultureandnormoxiaco-culture.Beforehypoxia,thegas-tightchamber(129,140)aswellaslowglucosecolorfreeDMEMmediumwasflushedwith5%CO2and95%N2for10minutes.Thenallplateswerewashed,andrefilledto1mlwiththepre-treatedmedium.Forco-culture,aninsertwassetinplate,andCD8+,CD11b+orCD19+HUCBcellsweredirectlypipettedintotheinsertsattheconcentrationof100,000/well.The0.4mmillipore(MilliporeInc)insertsallowmediumtoflowfreelyinandoutoftheinsertswhilekeepingtheHUCBsubpopulationcellsandneuralcellsseparated.Hypoxiagroupswereexposedtohypoxia(5%CO2and95%N2)at37oCfor2hour,whilethenormoxiagroupswereculturedinlowglucosemedia,butundernormoxicconditionsfor2hour.Afterthat,themediawereharvestedandstoredat-80oCforfutureanalysis.

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1196.3.5FDA/PIStainingCellviabilitywasdeterminedbyFluoresceinDiacetate/PropidiumIodide(FDA/PI)staining.Briefly,FDAstocksolution(5mg/mlinacetone)andPIstocksolution(1mgin50mlPBS)werepreparedandstoredat4C.FDAworkingsolution(5lFDAstocksolutionin1mlPBS)wasfreshlypreparedbeforeuse,and100lFDAworkingsolutionwasaddedto30lPIstocksolutiontoformtheworkingsolution.Then30lFDA/PIwasaddedintoeachwellcontaining600lPBS,andthecellswereincubatedatroomtemperaturefor5minutes.Photomicrographsweretakenunderepifluorescence,andthenumberoflive(FDAlabeled)ordead(PIlabeled)cellswerecountedusingImageProPlussoftware.Thenumberofsurvivingcellsisexpressedasapercentageofthetotalpopulation/fieldofview.Thesedataareaveragedandexpressedasmean+ semandthenanalyzedusingStudentsttests.6.3.6CytokineELISAAssayTheELISA-1,IL-10andIL-6wereperformedbyPierceEndogenBiotechnologyusingtheSearchlighttechnology(PierceBiotechnology).ThecommercialSearchlightplateswerepre-coatedwithspecificantibodieswitha2X2patternineachwell.Thestandardandsampleswereaddedintotheplateaccordingtotheinstructions,andtheplateswereincubatedatroomtemperatureonehourorlongenoughfortheproteinpresentinsampleandstandardtocapturetheantibody.Theplateswerethenincubatedwithstreptavidin-horseradishperoxidase(HRP),andtheluminescentsignalwasmeasuredina

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120platereader-1,IL-6andIL-10analyzedusingArrayVisioncustomizedsoftware.Thedataareexpressedasmean+ semconcentrationinpg/mlandwereanalyzedusingStudentsttests.6.4Results-10expression.ndIL-10wereundetectableunderalltheconditions.6.4.2.TheinteractionofCD11b+HUCBcellswithneurons,astrocytesandmicroglia6.4.2.1EffectofCD11b+HUCBcellsonneuralcellviabilityThecellviabilitywasassayedwithFDA/PIstaining,photographedandquantifiedusingImageProPlussoftware.CD11b+cellssignificantlydecreasedmicroglialviabilitycomparedtothemono-culturedmicrogliaunderhypoxia(*,p<0.05)andnormoxia(#,p<0.0),whileithadnoeffectonastrocyticandneuronalviability(Fig6.1).6.4.2.2IL-1andIL-6expressioninCD11b+HUCBcultureofneuralcells.Nosignificantdifferenceswereobservedbetweengroups,buttherewasatendencyforIL-1concentrationtobehigherintheCD11b+/co-culturedmicrogliathanthemono-culturedmicrogliaunderhypoxicaswellasnormoxicconditions(Fig6.2).Incontrast,neuronsandastrocyteshadatendencytohave

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121alowerIL-1concentrationinmediumfromtheco-culturescomparedtomono-culturemediuminnormoxicconditions.Thesimilartrendwasobservedinthehypoxiaculture(Fig6.2).Interestingly,IL-6concentrationfollowedasimilarpatternasIL-1.IL-6tendedtobehigherinco-culturemediumthanmono-culturemediumunderhypoxicaswellasnormoxicconditions,whileitwasoppositeinneuronsandastrocytes(Fig6.3).Nosignificantdifferenceswereobservedinanygroups.A MicroglialViabilitywith/withoutCD11b 0102030405060708090Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b V i a b i l i t y ( % )*

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122B AstrocyteViabilitywith/withoutCD11b8486889092949698Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b V i a b i l i t y ( % )#C NeuronCellViabilitywith/withoutCD11b848586878889909192939495Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b V i a b i l i t y ( % )Fig6.1Neuralcellviabilitywith/withoutCD11b.CD11b+HUCBcellssignificantlydecreasedmicroglialviabilityinco-culturecomparedtomicrogliamonocultureunderhypoxiaexposure(*p<0.05).Similarphenomenonwereobservedinthenormoxicconditionwithco-culturedmicrogliahavinglowerviabilitythanmicrogliainmonoculture(#,p<0.05).Therewerenosignificantdifferencesamongneuronalgroupsandastrocyticgroups.

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123A IL-1betaExpressioninMicrogliaCulture with/withoutCD11b01020304050607080Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b C o n c e n t r a t i o n ( p g / m L )B IL-1betaExpressioninAstrocytesCulture with/withoutCD11b0246810121416Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b C o n c e n t r a t i o n ( p g / m L )

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124C IL-1betaExpressioninNeuronCulturewith/without CD11b00.511.522.533.544.55Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b C o n c e n t r a t i o n ( p g / m L )Fig6.2.IL-1betaexpressionwith/withoutCD11b.CD11b+HUCBcellshadatendencytohavedecreasedIL-1concentrationinhypoxiaexposedneuronandastrocytes,whileIL-1concentrationtendedtobehigherinco-culturedmicrogliathaninmono-cultureunderhypoxicaswellasnormoxicconditions.Nosignificantdifferenceswereobservedbetweengroups.

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125A IL-6ExpressioninMicrogliaCulturewith/without CD11b0100200300400500600Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b C o n c e n t r a t i o n ( p g / m L )B IL-6ExpressioninAstrocytesCulturewith/without CD11b050100150200250300350400450Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b C o n c e n t r a t i o n ( p g / m L )

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126C IL-6ExpressioninNeuronCulturewith/without CD11b020406080100120140160180Hypoxia+CD11bHypoxia-CD11bNormoxia+CD11bNormoxia-CD11b C o n c e n t r a t i o n ( p g / m L )Fig6.3.IL-6expressionwith/withoutCD11b.Nosignificantdifferenceswereobservedbetweenallgroups.However,inthehypoxicornormoxicconditions,IL-6tendedtohavealowerconcentrationinCD11b+HUCBco-culturedastrocytesandCD11b+co-culturedneuronscomparedtoastrocytemono-cultureandneuronmono-culturerespectively,whileinmicroglialcultures,theoppositewastrue.6.4.3.TheinteractionofCD8+HUCBcellswithneurons,astrocytesandmicroglia6.4.3.1EffectofCD8+HUCBcellsonneuralcellviabilityUnlikeCD11b+cells,CD8+cellshadnoeffectsonmicroglialandneuronalviability.InsteadCD8+cellssignificantlydecreasedtheastrocyteviability

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127comparedtomono-culturedastrocytesunderhypoxic(*,p<0.05)ornormoxic(#,p<0.05)conditions(Fig6.4)A MicrogliaViabilitywith/withoutCD8 707580859095100Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 V i a b i l i t y ( % )B AstrocyteViabilitywith/withoutCD8788082848688909294Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 V i a b i l i t y ( % )*#

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128C NeuronViabilitywith/withoutCD891929394959697Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 V i a b i l i t y ( % )Fig6.4Neuralcellviabilitywith/withoutCD8.CD8+cellssignificantlydecreasedco-culturedastrocyteviabilitycomparedtomono-culturedastrocytesunderhypoxicconditions(*p<0.05).Thiswastrueinnormoxicconditionsaswell(#p<0.05).CD8didnotsignificantlyaffecttheviabilityofneuronandmicrogliainculture.6.4.3.2IL-1andIL-6expressioninCD8+HUCBcultureofneuralcells.CD8+HUCBcellsincubatingwithastrocyteshadatendencytoincreaseIL-1concentrationcomparedtomono-culturesofastrocytesunderhypoxicornormoxicconditions;thegreatesteffectwasobservedinthenormoxiccondition.IL-1inneuronandmicroglialcultureswasundetectable(Fig6.5).

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129HypoxiatendedtoincreaseIL-6productioninthemediumcollectedfromneuronalandmicroglialcultures,buttherewerenosignificantdifferencesbetweentheco-cultureandmono-culturegroups(Fig6.6).Fortheastrocytecultures,CD8+HUCBcelltendedtoincreaseIL-6concentrationinthemediaincomparisonofthemono-culturesunderhypoxicandnormoxiccondition(Fig6.6).A IL-1betaExpreesioninMicrogliaCulture with/withoutCD800.511.522.533.544.5Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 C o n c e n t r a t i o n ( p g / m L )

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130B IL-1betaExpressioninAstrocytesCulture with/withoutCD80102030405060708090Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 C o n c e n t r a t i o n ( p g / m L )C IL-1betaExpressioninNeuronCulturewith/without CD800.511.522.533.54Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 C o n c e n t r a t i o n ( p g / m L )Fig6.5IL-1betaexpressionwith/withoutCD8.IL-1wasnegligibleinthemediafromneuronalandmicroglialcultures,andnosignificantdifferenceswereobservedbetweenallgroups.However,intheastrocyteculturetherewasatendencyforincreasedIL-1presenceinmediafromCD8+HUCB/astrocyteco-cultureinbothnormoxiaandhypoxiaconditions.

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131A IL-6ExpressioninMicrogliaCulturewith/without CD8020406080100120Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 C o n c e n t r a t i o n ( p g / m L )

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132B IL-6ExpressioninAstrocytesCulturewith/without CD8050100150200250300350400Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 C o n c e n t r a t i o n ( p g / m L )C IL-6ExpressioninNeuronCulturewith/withoutCD8020406080100120140160180Hypoxia+CD8Hypoxia-CD8Normoxia+CD8Normoxia-CD8 C o n c e n t r a t i o n ( p g / m L )Fig6.6IL-6expressionwith/withoutCD8.Nosignificantdifferenceswereobservedbetweenallgroups.ButCD8+HUCBcellstendedtoelevateIL-6concentrationwhenco-culturedwithastrocytesinhypoxicornormoxicconditionscomparedwithmono-culturedastrocytes.Forallconditions,therewasatendencyforhypoxiaexposedculturestohaveahigherIL-6concentrationinmediacomparedtothecorrespondingnormoxiagroups.

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1336.4.4.TheinteractionofCD19+HUCBcellswithneurons,astrocytesandmicroglia6.4.4.1.EffectofCD19+HUCBcellsonneuralcellviabilityCD19+HUCBcellssignificantlydecreasedmicroglialviabilitycomparedtomono-culturedmicrogliaexposedtohypoxia(*,p<0.05).Similarresultswereobservedinnormoxiacultures(#,p<0.05).Interestingly,lowerastrocyteviabilitywasobservedinnormoxicCD19astrocyteco-cultureincomparisontohypoxicastrocytemonoculture(##,p<0.05)(Fig6.7).A MicrogliaViabilitywith/withoutCD190102030405060708090100Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 V i a b i l i t y ( % )*#

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134B AstrocyteViabilitywith/withoutCD1986878889909192939495Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 V i a b i l i t y ( % )C NeuronViabilitywith/withoutCD1991929394959697Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 V i a b i l i t y ( % )Fig6.7Neuralcellviabilitywith/withoutCD19.Itshowsmicrogliainco-culturewithCD19+HUCBcellshadalowerviabilitythanthosemonocultureunderbothhypoxic(*,p<0.05),andnormoxiccondition(#,p<0.05).

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1356.4.4.2IL-1andIL-6expressioninCD19+cellsHUCBcellsco-culturewithneuralcells.NosignificantdifferencesofIL-1concentrationwereobservedinthemediabetweengroups(Fig6.8),butmediumfromCD19+HUCBmicrogliaorastrocytesco-culturestendedtohaveanincreaseIL-1concentrationincomparisontotherespectivemono-culturesinhypoxicornormoxicconditions.Thistendencywasmostpronouncedintheastrocyteco-cultureinnormoxia(Fig6.8).HypoxiatendedtodecreaseIL-6concentrationinneuronalculturecomparedtothenormoxiccultures,andthemediafromneuronalco-culturetendedtohaveahigherIL-1concentrationthanthemediacollectedfrommono-culturedneuronsunderhypoxicconditions.Thesamewastrueinnormoxia,althoughnosignificantdifferencesinIL-6wereobserved(Fig6.9).

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136A IL-1betaExpressioninMicrogliaCulture with/withoutCD19012345678Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 C o n c e n t r a t i o n ( p g / m L )B IL-1betaExpressioninAstrocytesCulture with/withoutCD190102030405060708090Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 C o n c e n t r a t i o n ( p g / m L )

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137C IL-1betaExpressioninNeuronCulture with/withoutCD190123456Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 C o n c e n t r a t i o n ( p g / m L )Fig6.8.IL-1betaexpressionwith/withoutCD19.Inbothhypoxicandnormoxicconditions,IL-1concentrationtendedtoincreaseinCD19+co-culture.

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138A IL-6ExpressioninMicrogliaCulturewith/without CD190102030405060708090Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 C o n c e n t r a t i o n ( p g / m L )B IL-6ExpressioninAstrocytesCulturewith/without CD19050100150200250300Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 C o n c e n t r a t i o n 9 p g / m L )

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139C IL-6ExpressioninNeuronCulturewith/without CD19020406080100120Hypoxia+CD19Hypoxia-CD19Normoxia+CD19Normoxia-CD19 C o n c e n t r a t i o n ( p g / m L )Fig6.9.IL-6expressionwith/withoutCD19.Co-culturingneurons,astrocytes,microgliawithCD19+HUCBcellshadnosignificanteffectonIL-6secretionintotheculturemedium.

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1406.5DiscussionPreviousstudiesfoundthathumanumbilicalcordblood(HUCB)cellscanpromotemicroglialdeath,andsimultaneouslypreventastrocyticandneuronalcelldeathfromhypoxia.However,themechanismsthatunderlietheseeffectswereunknown.Inthisstudy,CD8+,CD11b+andCD19+cellswereisolatedfromHUCB,andincubatedwithmicroglia,neurons,andastrocytesindividually.ItwasfoundthatCD11b+andCD19+HUCBcellssignificantlydecreasedmicroglialviabilitycomparedtomicrogliamonoculturesunderhypoxicandnormoxicconditions.Interestingly,CD8+HUCBcellsdecreasedastrocyteviabilityunderhypoxicandnormoxicconditions.Noothersignificantdifferencesincellviabilitywerefound.ThesechangesincellviabilitywerenotaccompaniedbysignificantchangesinIL-1orIL-6concentration.-10werenotdetectedinanyofthecultures,whichsuggestthatthesecytokinesatleastwerenotinvolvedinneuralcelldeathwhentheneuralcellswereco-culturedwithsubpopulationsofHUCB.ThiswasnottruewhenHUCBcellsinteractedwithmicroglia,astrocyte-culturepromotedmicroglialdeath,whileIL-10benefitedastrocyteandneuronsurvivalunderhypoxicconditionsaswediscussedintheprevioustwochapters.ThissuggeststhattheinteractionbetweeneachsubpopulationofHUCBiswhatmodulates-10expression.

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1416.5.1TheeffectofIL-1andIL-6onneuronsAsaninflammatorycytokine,IL-1isexcitotoxictoneurons.CliniccasesdemonstratethatIL-1levelsignificantlyincreasesincerebrospinalfluidshortlyafterseveretraumaticinjuryinhuman,andthisincreaseisassociatedwithneuronaldamageintheipsilateralhemisphere(240).PatientswithhighIL-1inCNShaveissueswithmetabolicregulation,temperatureregulation,sleep,foodandwaterintake,andmemoryfunctions.Thesedeleteriouseffectshavebeenfoundinanumberofanimalmodelsofneurodegenerativediseases,includinghypoxic-ischemic(H/I)insult,braintrauma,multiplesclerosis,andAlzheimersdisease.Inabraininjuryanimalmodelinducedbymetabotropicglutamate(MGlu)injection,aprogressiveneuronallossexistedincortexandstriatum;atleastinstriatumthisneuronalinjuryoccurredwithinasimilartimeframeandregionasIL-1expression(241).Inaddition,anexacerbatedneuronalcelllosswasobservedinIL-1receptorantagonistknockoutanimalsafterinjectionofbeta-amyloid(A),whereIL-1signalisenhancedsincethereisalossoftheIL-1receptorsphysiologicalantagonist(242).Incontrast,inhibitionofIL-1activity,eitherthroughneutralizingantibodies,orthroughblockadeofthereceptor,amelioratedfunctionaloutcome(243).However,IL-1alonedoesnotinducedeathinotherwisehealthyneurons.Therefore,itishardtoevaluatetheroleofIL-1inneuronalculture.Recently,Vivianietal(2003)determinedthatIL-1hadnoeffectoncellviabilityofprimaryhippocampalneuronsinculture,butitsignificantlyincreasedN-methyl-D-asparticacid(NMDA)-inducedcelldeath.IL-1caused30%moreneuronaldeaththanNMDAtreatmentonly.Thiscelldeath

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142couldbeblockedbybothlavendustinAandPP2(4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine),thetyrosinekinaseinhibitors,suggestingthiscelldeathwasmediatedthroughthetyrosinekinasepathway(238).UnlikeIL-1,theliteraturestronglysuggeststhatIL-6isaneurotrophicfactorinvitroratherthananinflammatorycytokine.Forexample,IL-6stimulatedPC12cellstosproutneuralprocesses,andexpressanumberofneuronalmarkersafterstimulation.Further,IL-6wasshowntoprotectdopaminergicneuronsfromtheinjurycausedby1-methyl-4-phenylpyridinium(MPP+)invitro(244).Inaddition,IL-6notonlysupportsthesurvivalofcatecholaminergiccellsbutalsocholinergicneurons(245,246).6.5.2TheeffectofIL-1andIL-6onmicrogliaandastrocytesAsforastrocytesandmicroglia,IL-1servesasamediatorofcellactivation,andpromotesglialcellproliferation.Wheninfusedintoratbrain,IL-1inducesastrocytegliosis,andenhancesimmunoreactivityofGlialFibrillaryAcidicProtein(GFAP)(247).Inacorticectomyinjurymodel,astrocytesfailedtoup-regulateexpressionoftheneurotrophicfactorCiliaryneurotrophicfactor(CNTF)followingCNSinjuryinmicedeficientinIL-1,andthisabnormallackofCNTFelevationcouldberescuedbytheapplicationofexogenousIL-1(239).Inacuprizonemodelofdemyelinationandremyelination,theauthorsshowedIL-1deficientmicefailedtoremyelinateproperly,andthisfailurecorrelatedwithdelayeddifferentiationofoligodendrocyteprecursorsandwithalackofastrocytic

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143andmicroglialproductionofInsulin-likegrowthfactor1(IGF-1)(184).Inaddition,IL-1hasanindirecttrophiceffectonglialcells.Inatraumaticbraininjury(TBI)model,IL-1antagonistdiminishestheinflammatoryresponsecausedbyinjury,andatthesametime,antagonistalsoinhibitsNervegrowthfactor(NGF)productioninastrocytes,whichinturnhamperedglialandotherneuralcellsrecovery(214).ThegliosiseffectofIL-1isalsosupportedbyresultsobtainedinvitrousingprimaryastrocyteandmicrogliacultures.ApplicationofexogenousIL-1tosuchculturesinducesIL-6,CNTF,andNGFexpression(248).Mostofthesecytokinesandgrowthfactorsareknowntoinduceneuronalandglialgrowthandsurvival.Forexample,inprimaryastrocyteculturesfromneonatalrats,IL-1wasshowntoinduceproliferationofastrocytes(249),aswellasincreasingGFAPimmunostaining.Inanotherstudy,IL-1wasshowntopromotemicroglialproliferationafterNMDAinjury,whileIL-1supplementwasnotinuse,microglialrecoverywassloweddownwithablockageofcellproliferation(167).Inenrichedbraincellculture,theauthorevenarguedthatIL-1waspivotalfortheactivationofmicrogliaandgliosis(247).IL-6hasasimilareffectonastrocytesandmicroglia.FurtheritisbelievedthatthegliosisinducedbyIL-1ismediatedthroughtheIL-6pathway.Inoneexperiment,cytokinesIL-6,IL-1andTNFweredeliveredintotheleftcerebro-ventricularsystemofahealthyratthroughconvectionenhanceddelivery(CED)methods.GliosisoccurredintheleftcerebralcortexinIL-6andIL-1treatedgroups,whilegliosiswasnotseenintheTNFtreatedgroups.Whentheanimals

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144wereco-treatedwithpropranololorIL-10,areagentattenuatingthelevelofIL-6,thegliosisdecreaseddramaticallytonormallevels(250).InaParkinsonmousemodelinducedby1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),microgliosiswasobservedinthesubstantianigraparscompacta,wheremicrogliosisstartedfromday1,andpeakedatday7.WhenIL-6mutantanimalswereused,therewasnomicrogliosis,suggestingIL-6iscrucialformicrogliosis(251).Invitro,astrocytesculturedfrommaturebovinebrainwereshowntoproliferatemorerapidlyafterIL-6treatment(252).Inastrogliomas,IL-6appearstopromotegrowthinanautocrinemanner(220),andthisconstitutivelyexpressedIL-6sustainsastrogliomagrowth.NeutralizingIL-6withanantibodyorblockingIL-6receptorinhibitsastrogliomacellproliferation(220).Incontrast,overproductionofIL-6inatransgenicmousewasassociatedwithastrogliosisandincreasedmicroglialramification(253).Takentogether,thesedatastronglysuggestedthatthefunctionofIL-1andIL-6istopromotegliosis.Theactivatedmicrogliaandastrocytesproducechemokines(MCP-1,IL-8,IP-10)(255)-1,TNF)(256,257),andadhesionmolecules(ICAM-1,VCAM-1,E-selectin)(258,259),mostofwhichareknowntobestronglypro-inflammatory.Inaddition,activatedmicrogliaandastrocytesalsoproduceNOandotherfreeradicals(260).Thesesmallmoleculesservetosafeguardthebrainagainstpathogeninvasion,andclearawaydamagedordyingcells;ontheotherhand,theseelevatedpro-inflammatorycytokinesand

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145freeradicalscouldexacerbatethedamagecausedbyinjuryandcausecelldeath(261).6.5.3EffectofCD11b+HUCBcellsonneuralcellsInco-culturesofmicrogliaandCD11b+HUCBcells,therewasanapparenttendencyfortheconcentrationofIL-1tobehigherthaninthemonoculturegroupsunderhypoxicandnormoxicconditions.AsimilarpatternwasobservedforIL-6concentrationinmicrogliaculture.TogetherIL-6andIL-1shouldpromotemicrogliosis.However,withthelimitedtimeinculture(2hours)inthisstudy)itishardtoseehowmicrogliacouldbecomegliotic,sincetheDNAandproteinpreparationforcellproliferationtakestime.Incontrast,theseoverlappinggliosiseffectsofIL-1andIL-6mayturnouttobeafunctionoffreeradicalreleasewhichcouldcausemicroglialdeathaswediscussedabove.Asforneurons,thelevelofIL-1inallgroupswasverylowandthereforeitisunlikelythatthiscytokinecouldaffectneuronalviability.SinceIL-6worksasaneurotrophicfactor,itsslightchangeinconcentrationinthedifferentgroupsdidnotimpairneuronalviability.AsimilarIL-1andIL-6patternwasobservedinastrocyteculture,wherebothIL-1andIL-6workedasastrogliosiseffectors.ThismaybethereasonthatnosignificantdifferencesincellviabilitywereobservedinCD11b+treatedneuronsandastrocyteswithhypoxiaexposure.

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1466.5.4EffectofCD8+HUCBcellsonneuralcellsThepatternofglialcelldeathandIL-1andIL-6cytokineexpressionobservedinCD8+treatedastrocytecultureswassimilartothatoftheCD11b+treatedmicroglialcellcultures.ItispossiblethattheoverlappingeffectsofIL-1andIL-6triggeredthepotentialgliosisandfreeradicaloutburst,whichmayhaveinducedastrocytedeath.Incontrast,thechangesofIL-1andIL-6inmicrogliaandneuronculturewereslightandmayexplainwhytherewasnosignificantcelldeathinCD8+treatedmicroglialandneuronalcultures.6.5.5EffectofCD19+HUCBcellsonneuralcellsInCD19+celltreatedmicroglia,IL-1levelappearedhigherthaninthemicroglialmono-cultureconditionsunderbothhypoxiaandnormoxia.UnlikeCD11b+treatedcells,thedifferencesinconcentrationofIL-6werenotsignificantindifferentmicrogliaculturesasshowninFig6.9,perhapsbecauseoflimitedsamplesize.WecanonlyhypothesizethatthecombinedreleaseofIL-6andIL-1mayhaveresultedinexcessfreeradicalproductionthatinducedmicroglialdeath.Interestingly,highlevelsofIL-1wereobservedinthenormoxicastrocyteCD19co-culture,andtheeffectofthishighIL-1wasenhancedbyhighlevelofIL-6inthesameculture.ThismaybethereasonthatasignificantviabilitydifferencewasobservedbetweenhypoxicastrocytemonocultureandnormoxicastrocyteCD19+co-culture(Fig6.7).SincethevariancesofIL-1andIL-6wereslightinneurongroups,therewerenosignificantviabilitydifferencesamongneuroncultureswith/withoutCD19treatment.

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147Takentogether,theseresultsshowedthatCD11bandCD19derivedHUCBcellssignificantlycausedmicroglialdeathinmicrogliaco-culturecomparedtomicrogliamonoculture,whileCD8derivedHUCBcellssignificantlyimpairedastrocyteviabilitycomparedtoitsmonoculture.ThecellviabilitychangeswereassociatedwithincreasedIL-1andIL-6,whichmayhavetriggeredthefreeradicalcascade,andcausedcelldeath.Interestingly,noobviousneuralprotectionwasobservedinanyculturewithHUCBsubpopulations.However,whenHUCBwasco-culturedwithmicroglia,astrocyteandneuronsindividually,itpromotedmicrogliadeath,andpreventedneuronalandastrocyticdeathunderhypoxicconditions.ThisinconsistencymaybeattributabletotheinteractionsofthedifferentsubpopulationofHUCBwhentheyaremixedtogether.Forexample,theanti-inflammatorycytokineIL-10wasfoundinthemediafromHUCBtreatedneuralcellsinChapter4and5.IL-10canmodulategliosisbyregulationofIL-6production(250),preventneurondeath(262)andinhibitfreeradicalproductioninimmuneresponse(263).Therefore,thehighastrocytedeathrateinco-culturesofastrocytesandCD8+HUCBcellsispossiblycausedbytheoverproductionofIL-6andIL-1,whichmaynothappeninHUCBculturewhereIL-10attenuatestheproductionofIL-6,andindirectlyaffectsIL-1effects.ButhowthesubpopulationsofHUCBinteractwitheachotherisnotclear,andremainsforfurtherinvestigation.

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148CHAPTERSEVENCONCLUDINGREMARKSTheactualpartofthecordbloodthathasbeenusedforcelltransplantationisthemononuclearfraction,whichcontains1%orlessstemcells,41%Tcells,20%Bcells,and38%monocytes(13,14,19).HUCBtransplantationcansignificantlyimprovethefunctionalrecoveryinanimalmodelsofParkinsondisease,spinalcordinjury,amyotrophiclateralsclerosis(ALS),Sanfilipposyndrome,andstroke(3).TheHUCBhasbeendelivereddirectlyintothesiteofinjury,orthroughintravenousadministration.Whenthetwodeliverysystemswerecomparedinstrokedanimals,intravenousadministrationhadsimilareffectsonfunctionalrecoveryearlyafterstrokeasintracranialadministration.However,thefunctionalrecoveryinintravenouslytreatedanimalswasmaintainedlongerthaninanimalswithintracranialadministration(25).Interestingly,thisfunctionalrecoverywasshowntobedosedependentinstrokedanimals(27).Animalsthatreceived106ormorecellshadmuchbetterbehavioralrecoverythananimalsthatreceivedfewercordbloodcells,andthisfunctionalrecoverywasassociatedwithasmallerinfarctvolumeincomparisontocontrolanimals.ItwaslongthoughtthatstemcellsfromHUCBcouldproliferateanddifferentiateintoneurallikecellsthatcouldreplacethecellslostduringthestroke.

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149ThispossibilityhasbeenconfirmedinthatinfusedHUCBcellswerefoundintheparenchymaoftheinjuredbrain,andexpressedneuronalandastrocyticmarkers(26).Andtheextentoffunctionalrecoverywasdirectlyrelatedtotheamountofcellsinfiltratingintotheinjuredsite.However,howHUCBcellscouldgettotheinjurysiteafterintravenousadministrationwasnotclear.Onepossibilitywasthatthebloodbrainbarrierwasdisruptedafterinjury,andthecordbloodcellscouldpassivelyleakedintotheinjuredbrain.Anotherpossibilitywasthatsomechemokines,MCP-1andMIP-1,caninduceleukocytemigrationalongaconcentrationgradient.Itwasfoundthattheexpressionofchemokinesincreasedinstantlyafterstroke,andtheincreasewasassociatedwithmononuclearinfiltrationintotheinjurysite(118).Therefore,HUCBcouldactivelymigratetotheinjurysiteundertheinfluenceofchemokines.Interestingly,somestudiesindicatedthattherearefewstemcellsinHUCB,andafterintravenousadministration,onlyfewcellscouldinfiltratethroughthebloodbrainbarriertotheinfarctarea(47).Inaddition,amongtheinfiltratedcells,fewofthemdifferentiatedintoneurallikecellsastheyusuallydoinvitro,wheretheirneuralpropertiescouldbedeterminedwithimmunostainingforneuralmarkersincludingNeuN,TuJ1,GFAP,etc.(37).Therefore,thecellreplacementmechanismisunlikely.Recently,itwasfoundthatHUCBreducedthelevelofCD45/CD11b+andCD45/B220+cellsinstrokedbrainfollowingintravenousadministration(171).ThedecreasedcellinfiltrationwasaccompaniedbyadecreaseinmRNAandproteinexpressionofpro-inflammatorycytokinesanda

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150decreaseinnuclearfactorkappaB(NF-kappaB)DNAbindingactivityinthebrain.Thisworkwasconsistentwiththecomparativestudyofinflammatoryphenotypesbetweencordbloodandadultblood.Cordbloodcellshaveimmaturephenotypesofinflammationwithcompromisedexpressionofpro-inflammatorycytokinesandhighproductionofanti-inflammatorycytokinesincludingIL-10comparedtoadultbloodcells.Whentransplantedinclinic,thereisalowerincidenceofgraftversushostdisease(GVHD)inHUCBrecipients.IfGVHDdoeshappen,itsseverityisless(70).Therefore,thesuppressedinflammatoryresponsecouldbethemajormechanismcontributorytothefunctionalrecoveryinstrokedanimalsfollowingHUCBadministration.Inthisstudy,thesubpopulationsofneuralcellswereisolatedfromembryonicday17rats,andtheywereco-culturedwithHUCBoritssubpopulations.TheroleofchemokinesonHUCBmigrationwasalsointhefirstaimofthisproject.Followingthat,whetheritwasjustcytokinesorthecellularinteractionsresponsibleforinflammationwasinvestigated.7.1Chemokines,MCP-1andMIP-1,attractedHUCBtotheinjurysitefollowingstrokeThefirstquestionofthisprojectishowHUCBcellscangettothesiteofinjuryinthestrokedbrainafterintravenousadministration.Onepossibilityistheblood-brain-barrierwastemporarilyopenedafterinjury,whichallowedHUCBcellstopassivelyleakintotheCNS.AnotherpossibilityisthatHUCBcellscan

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151activelymigrateintotheinjurysiteundertheinfluenceofchemokines.ChemokineMCP-1andMIP-1wereofinterestsincetheyareamongthemostpotentchemokinesintheCNS,andtheyincreasedshortlyafterinjury.Forexample,inamodelofhippocampaldegenerationcausedbyaxonalinjury,CCR2(MCP-1receptor)mutationledtotheabsenceoflymphocyteinfiltrationintheCNS,whichwasnotreversiblebythepresenceofanyotherchemokines,suggestingthecriticalroleofMCP-1inlymphocytemigration(121).OurexperimentsshowedthattheexpressionofMCP-1andMIP-1increasedafterstrokeinrodentanimalsasdeterminedbywesternblottingandimmunohistochemicalstaining.TheincreasedMCP-1andMIP-1werelocatedintheipsilateralhemisphereofinjuryside,andwaspositivelyco-stainedwithastrocyte,neuron,andmicroglialmarkers.However,thisincreasedMCP-1andMIP-1wereofratorigin,andmaynothaveinducedhumancordbloodcellmigration.Therefore,theireffectonHUCBmigrationwasfurtherexaminedwithexvivomigrationassays.TheresultsindicatedthatratspecificMCP-1orMIP-1alonepromotedHUCBcellmigration.TissueextractsfromstrokedbrainsignificantlypromotedHUCBmigrationcomparedtotissueextractsfromthecontralateralhemisphere,andthismigrationwasneutralizedbyapplicationofMCP-1orMIP-1antibodiesinthemedium.TheseresultswerefurtherconfirmedbytheconstitutiveexpressionoftheMCP-1receptor,CCR2andMIP-1receptors,CCR1andCCR5onthesurfaceoftheHUCBcells.Therefore,

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152chemokines,MCP-1orMIP-1,mayparticipateinmodulationofHUCBinfiltrationintheinjurysiteafterintravenousadministration.7.2HUCBcellspromotedmicrogliadeathunderhypoxicconditions.TheabovestudydemonstratedthatHUCBcellswereabletomigrateacrosstheblood-brain-barrierundertheattractionofchemokinesafterintravenousadministration.Onceinsidethebrain,HUCBcellscanrescuethefunctionaldeficitcausedbystroke.However,themechanismisnotclear.Inamodelofstroke(26),ALS(29)andtraumaticbraininjury(264),theinfusedHUCBcellswerefoundintheparenchymaoftheinjuredbrainandexpressedtheneuronalandastrocyticmarkers.ThesefindingssuggestedthatHUCBcellscoulddifferentiateintoneurallikecellsandreplacethecelllosscausedbyinjury,whichleadtothefunctionalimprovement.However,recentstudiesindicatedthatthecellreplacementmechanismwasunlikelysincefewHUCBcellswerefoundintheinjuredbrainafterinfusion(45).Interestingly,adecreasedimmuneresponsewasobservedinHUCBinfusedanimalincomparisontothecontrolgroup(171),whichcouldberesponsibleforrescuingtheratfromfunctionaldeficitscausedbystroke.However,howHUCBcellsmodulateimmuneresponseisnotclear.Inthisstudy,theinteractionbetweenmicrogliaandHUCBcellswasexamined.TheFDA/PIassayshowedthatHUCBcellssignificantlypromotedmicroglialcelldeathduringhypoxiaexposurecomparedtothecellviabilityin

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153hypoxicmono-culturedmicroglia.Accompanyingtheincreasedcelldeath,theconcentrationofIL-1inthehypoxiaexposedco-culturemediumwassignificantlylowerincomparisonwithhypoxiaexposedmono-culturedgroup.Hypoxiaincreasedtheamountofthepro-anddecreasedtheamountofanti-inflammatoryIL-10intheHUCBandmicrogliaco-culturedgroup.PreviousstudiesdemonstratedthathighIFNcouldincreasemicroglialsensitivitytoinjury,andpromotecelldeath(165).Incontrast,IL-1isatrophicfactorformicroglia.IL-1couldpreventmicrogliafrominjurycausedbyN-methyl-D-aspatate(NMDA)exposure,andpromotecellrecovery,whileblockageofIL-1inhibitedcellproliferation.Further,IL-1injectionintobrainpromotesastrogliosisandneovascularizationatthesiteofinjury.IL-10isananti-inflammatorycytokine,andcanprotectneuronsandoligodentrocytesfromdeathcausedbyglutamate(42),hypoxic-ischemia(265),LPS(266),andinterferon(266).Theunbalancedstateofpro-/anti-inflammatorycytokinesmayresultinthecelldeathofmicrogliaafterhypoxicexposure.7.3HUCBpreventedneuronandastrocytehypoxiainducedcelldeathPreviousstudiesdemonstratedthatHUCBreducedinfarctvolumeinthebrainofstrokedanimalsincomparisontotheuntreatedanimal(27).ThisreducedinfarctvolumecouldbetheresultofthereductionininflammatoryresponsecausedbyHUCBcells.Inanotherexperiment,HUCBcellsweretransplanteddirectlyintoparenchyma,wheresproutingnervefibersfromthenondamagedhemispherewereobservedinthedamagedsideofbraininHUCB

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154celltreatedanimals(267).Incontrast,animalreceivingsalinedidnotshowobvioussproutingofnervefibers(267).AsimilareffectofHUCBcellswasalsoobservedinthemodelofspinalcordinjury,whereHUCBsignificantlyreducedthesizeofthecysticcavityatthesiteofinjury,andanapparentsproutingofneuralaxonsintotheinjurysite(268),suggestingHUCBcellshaveneurotrophiceffects.However,therewasnodirectevidencethatHUCBsupportsastrocyteandneuronsurvivalunderhypoxicconditions.Inthisstudy,theeffectofHUCBcellsonneuronsandastrocyteswasinvestigated.HUCBcellssignificantlyincreasedneuronandastrocyteviabilityafterhypoxia.IFNproductionwasobservedinhypoxicandnormoxicco-cultureofneurons-culturewasundetectable.IFNinhypoxiaco-culturewasverylow.Incontrast,IFNwasonlydetectedinthemediumofhypoxicmono-cultureandnormoxicco-cultureofastrocyte,andIFNwasundetectableinotherastrocytegroups.IFNhastwofacesinintoxicationandnutritionofastrocytes.Asaneurotrophicfactorforastrocytes,IFNpromotesastrocyteproliferationandevenastrogliosis(210,211).However,extensiveastrocyteactivationhasserioussideeffects.ReactiveastrogliosisafterCNSinjuryhasbeenconsideredamajorimpedimenttoaxonalregeneration.IFNalsomodulatesMHCclassIandclassIIantigenexpressiononastrocytes.MHCclassIcanelevateastrocytesusceptibilitytolysisbyclassIrestrictedTlymphocytes,andMHCclassIImakesastrocytesfunctionasantigenpresentingcells,whichenhancestheinflammatoryresponse

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155cytotoxinsincludingnitricoxide(212),freeradicals,TNF,IL-1production,andallofthemmayexacerbatethebraindamage.Asaneurotrophicfactorforastrocytes,thelowlevelofIFNastrocytehypoxicco-cultureincreasedthevulnerabilityofastrocytesexposedtohypoxia.OurresultssuggestedthatHUCBcellscouldsuppressastrocyteactivityunderhypoxicconditions,whichinturnmayinhibittheinflammatoryresponses.Whileinneuronalcultures,IFNwasonlyfoundintheco-cultureconditions,andthelevelofIFNinhypoxicco-culturegroupswasverylow,whichmaynotaffectneuronalviabilityduringhypoxiaOurdataalsoindicatedthatnoIL-1wasdetectedinastrocytemedium.Inaddition,thelevelofIL-1wasverylowinthemediumofneuronculture.Asapro-inflammatorycytokine,IL-1istoxictoneuronsandastrocytes.IthasbeendemonstratedthatIL-1couldincreasethecalciumentryintothecell,whichisstronglyimplicatedincelldeath(238).Inaddition,exogenousadministrationofIL-1intoanimalbraininjuredthroughtrauma,ischemia,orexcitotoxicstimulimarkedlyincreasedtheextentofinjury(203),whileneutralizationofIL-1withitsantibodyorIL-1receptorantagonistmarkedlydecreasedthebraininjuryresultingfromMCAOorintracerebralinjectionoftoxicchemicals(269).ThetoxiceffectofIL-1wasdependentonconcentration.Whenitsconcentrationwashigh,IL-1hadaneuralprotectiveprofile,suchasinhibitionofglutamaterelease,inhibitionofcalcium-aminobutyricacid(GABA)(201).Inourstudy,IL-1concentrationwaslowinall

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156groups,andwasnotlikelyinvolvedinneuronandastrocytecellsurvivalorcelldeath.IncontrasttoIL-1,IL-6wasnotdetectableintheneuronalculture,anditwasonlyobservedinthemediumofco-culturedastrocytes.TherewasagreatsignificantdifferenceofIL-6betweenhypoxicco-culturedastrocytesandnormoxicco-culturedastrocytes(*,p<0.05).Interestingly,theanti-inflammatorycytokineIL-10washigherinco-culturedneuronsunderhypoxicconditionsincomparisontoothergroups.Thesimilarresultswereobservedinastrocyteculture.ItiswellknownthatIL-6hasaneuralprotectiveeffectagainsttheinjurycausedbyglutamate,orN-methyl-D-aspartate(NADA)(219),andinducedastrogliosisinvitro(220).WhenIL-6wasblockedwithanIL-6competitiveinhibitor,theneuroprotectiveeffectdisappeared(270).SimilartoIL-6,IL-10hasaneuroprotectivefunctionagainstlipopolysaccharide(LPS)inducedneuronaldamage(271).Interestingly,IL-10canpromoteglialcellproliferationathighconcentration,whileitinhibitsDNAreplicationofglialcellsatlowconcentration(272).Inourstudy,IL-6andIL-10mayprovideprotectiveeffectstoastrocytesandneuronsexposedtohypoxia.7.4TheinteractionbetweentheHUCBsubpopulationandneuralcellsOurstudieshavedemonstratedthatHUCBcanpreventneuronsandastrocytesfromhypoxia-inducedcelldeath,andpromotemicrogliacelldeathunderhypoxicconditions.Theseeffectswereaccompaniedbymodulationinthe

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157pro-/anti--1,IL-6andIL-10betweenHUCBandneuralcells.However,whichsubpopulationofHUCBproducestheseeffectsisnotknown.Inthisstudy,Tcells(CD8+),Bcells(CD19+)andmonocytes(CD11b+)wereisolatedfromHUCBbymagneticcellsorting.Thesecellswereculturedwith/withoutneurons,astrocytesandmicrogliaundernormoxicandhypoxicconditionsrespectively.Cellviabilityandcytokineexpressionwereexamined.Interestingly,thereweredifferencesintheeffectofthesubpopulationonneurons,astrocytesandmicroglia.Bcells(CD19+)andmonocytes(CD11b+)HUCBcellsdecreasedmicrogliasurvivalafterhypoxiacomparedtosurvivalinmicroglialmonoculture.Incontrast,theCD8+Tcellssignificantlydecreasedastrocytecellviabilityafterhypoxiaexposurecomparedtoviabilityinastrocyte-10,IL-6andIL-1didnotsignificantlychangeinanyofthesecultures,suggestingtheymaynotbecriticalplayersintheobservedcelldeath.NoneuroprotectiveeffectwasobservedineitherBcells,Tcellsormonocyteco-culturedwithneurons.Thereasonisnotclear.ItispossiblethattheinteractionbetweenHUCBsubpopulationisneededtoinduceneurotrophiceffectsofHUCBforneuralsurvivalunderhypoxicexposure.

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ABOUTTHEAUTHORLixianJiangreceivedhisBachelorsDegreeinBiologyfromHunanNormalUniversityin1994andaM.S.DegreeinNeurosciencefromAcademyofMilitaryMedicalSciencein1997inChina.Then,heworkedinTraumaCenterof304thhospital,BeijingasaresearchscientistuntilheenteredthePh.D.programinUniversityofSouthFlorida.Besideshisachievementsinstemcellresearch,Mr.LixianJiangshowsgreatinterestinlegalscienceandintellectualpropertylaw.HeiscurrentlyanattorneyinPeoplesRepublicofChina,andapatentagentinUSA.Since2006,Mr.LixianJiangworkedinStemCellTherapyInternational,Inc,astemcellpubliccompanylocatedinTampaBayareaasChiefOperationOfficer,andpatentagent.Heisexcellentinfinancialanalysis,IPmanagement,patentdraftingandprosecution.


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Interactions of neurons, astrocytes and microglia with HUCB cell populations in stroke models :
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ABSTRACT: Previous studies demonstrated that intravenous administration of human umbilical cord blood (HUCB) cells could improve behavioral and neurological recovery of stroked animals following middle cerebral artery occlusion (MCAO). In addition, HUCB cell recipients had less of an inflammatory response with less leukocyte infiltration. In these studies we explored how HUCB cells change the inflammatory response of neurons, astrocytes, and microglia to hypoxia/ischemia. Initiation of the inflammatory response occurs with the expression of chemokines. We determined that monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1alph (MIP-1), which are upregulated in the brain early after a stroke, induce migration of HUCB cells to the site of injury. Neutralizing these chemokines with antibodies prevented migration in an in vitro migration assay. We next explored the interaction of the whole HUCB mononuclear cell fraction, as well as subpopulations from within the mononuclear fraction (T cell alone, B cell alone, and monocytes/macrophage alone) with cultures of enriched neurons, astrocytes or microglia exposed to hypoxia in an oxygen, glucose deprivation paradigm. We showed that HUCB cells increased the cell viability of neurons and astrocytes, while decreasing cell viability of microglia. There was also a change in the cytokine secretion profile from the cells exposed to HUCB cells under hypoxic conditions. These results suggested that chemokines, MCP-1 and MIP-1 increased in stroked brain, and they played an important role in recruitment of HUCB into the CNS after intravenous administration. Once inside the brain, HUCB could suppress the immune response by promoting microglial death and modulating the function of astrocytes. In addition, HUCB cells provide neuron protection against the injury caused by stroke. However, it is unlikely to contribute the effect of HUCB to a single population of HUCB.
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