Flow Cytometric Isolation and Clonal Identification of Self-Renewing

FlowCytometricIsolationandClonalIdenti cationofSelf-RenewingBipotentHepaticProgenitorCellsin

AdultMouseLiver

AtsushiSuzuki,1,2SayakaSekiya,1MakikoOnishi,1NaokoOshima,3HiroshiKiyonari,3

HiromitsuNakauchi,4andHidekiTaniguchi2,5

Theadultliverprogenitorcellsappearinresponsetoseveraltypesofpathologicalliverinjury,especiallywhenhepatocytereplicationisblocked.Thesecellsarehistologicallyidenti edascellsthatexpresscholangiocytemarkersandproliferateintheportalareaofthehepaticlobule.Althoughthesecellsplayanimportantroleinliverregeneration,theprecisecharacterizationthatdeterminesthesecellsasself-renewingbipotentprimitivehepaticcellsremainstobeshown.Hereweattemptedtoisolatecellsthatexpressacholangiocytemarkerfromtheadultmouseliverandperformsinglecell-basedanalysistoexaminepreciselybilineagedifferentiationpotentialandself-renewingcapabilityofthesecells.Basedontheresultsofmicroarrayanalysisandimmunohistochemistry,weusedanantibodyagainstCD133andisolateCD133 cellsvia owcytometry.Wethenculturedandpropagatedisolatedcellsinasinglecellcultureconditionandexaminedtheirpotentialforproliferationanddifferentiationinvitroandinvivo.Isolatedcellsthatcouldformlargecolonies(LCs)inculturegaverisetobothhepatocytesandcholangiocytesasdescendants,whilemaintainingundifferentiatedcellsbyself-renewingcelldivisions.TheclonogenicprogenyofanLC-formingcelliscapableofreconstitutinghepatictissuesinvivobydifferentiatingintofullyfunctionalhepatocytes.Moreover,thedeletionofp53inisolatedLC-formingcellsresultedintheformationoftumorswithsomecharacteristicsofhepatocellularcarcinomaandchol-angiocarcinomauponsubcutaneousinjectionintoimmunode cientmutantmice.Thesedataprovideevidenceforthestemcell-likecapacityofisolatedandclonallyculturedCD133 LC-formingcells.Conclusion:Ourmethodforprospectivelyisolatinghepaticprogenitorcellsfromtheadultmouseliverwillfacilitatestudyoftheirrolesinliverregen-erationandcarcinogenesis.(HEPATOLOGY2008;48:1964-1978.)

Abbreviations:Alb,albumin;DAPI,4’,6-diamidino-2-phenylindole;DDC,3,5-diethoxycarbonyl-1,4-dihydrocollidine;ES,embryonicstem;FAH,fumarylacetoacetatehydrolase;LC,largecolony;NOD/SCID,nonobesediabetic/severecombinedimmunode cient;NTBC,2-(2-nitro-4-tri uoromethylbenzoyl)-1,3-cyclohexanedione;qPCR,quantitativepolymerasechainreaction;RT-PCR,reverse-transcriptionpolymerasechainreation;SC,smallcolony.

Fromthe1DivisionofOrganogenesisandRegeneration,Post-GenomeScienceCenter,MedicalInstituteofBioregulation,KyushuUniversity,Fukuoka,Japan;the2ResearchUnitforOrganRegenerationandthe3LaboratoryforAnimalResourcesandGeneticEngineering,CenterforDevelopmentalBiology,RIKEN,Hyogo,Japan;the4LaboratoryofStemCellTherapy,CenterforExperimentalMedicine,InstituteofMedicalScience,UniversityofTokyo,Tokyo,Japan;andthe5DepartmentofRegenerativeMedicine,GraduateSchoolofMedicine,YokohamaCityUniversity,Kanagawa,JapanReceivedMarch31,2008;acceptedJuly28,2008.

SupportedinpartbytheProgramforImprovementofResearchEnvironmentforYoungResearchersfromSpecialCoordinationFundsforPromotingScienceandTechnologycommissionedbytheMinistryofEducation,Culture,Sports,Science,andTechnology(MEXT)ofJapan;Grant-in-AidsforScienti cResearchfromtheMEXTofJapan;andagrantfromtheLeadingProjectinJapan.

*Addressreprintrequeststo:AtsushiSuzuki,DivisionofOrganogenesisandRegeneration,Post-GenomeScienceCenter,MedicalInstituteofBioregulation,KyushuUniversity,3-1-1Maidashi,Higashi-ku,Fukuoka,Fukuoka812-8582,Japan.E-mail:suzukicks@bioreg.kyushu-u.ac.jp;fax:(81)-92-642-6793;orHidekiTaniguchi,DepartmentofRegenerativeMedicine,GraduateSchoolofMedicine,YokohamaCityUniversity,3-9Fuku-ura,Kanazawa-ku,Yokohama,Kanagawa236-0004,Japan.E-mail:rtanigu@med.yokohama-cu.ac.jp;fax:(81)-45-787-8963.

Copyright©2008bytheAmericanAssociationfortheStudyofLiverDiseases.PublishedonlineinWileyInterScience(doc.docsou.com).DOI10.1002/hep.22558

Potentialcon ictofinterest:Nothingtoreport.

AdditionalSupportingInformationmaybefoundintheonlineversionofthisarticle.1964

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