MicroRNA125b expands hematopoietic stem cells and enriches for the lymphoidbalanced and lymphoidb.docx

MicroRNA125b expands hematopoietic stem cells and enriches for the lymphoidbalanced and lymphoidb.docx

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MicroRNA125bexpandshematopoieticstemcellsandenrichesforthelymphoidbalancedandlymphoidb

MicroRNA-125bexpandshematopoieticstemcellsandenrichesforthelymphoid-balancedandlymphoid-biasedsubsets

1.A.G.LisaOoia,

2.DebashisSahooa,

3.MaddalenaAdornoa,

4.YuleiWangb,

5.IrvingL.Weissmana,1,and

6.ChristopherY.Parka,1,2

+AuthorAffiliations

1.aInstituteofStemCellBiologyandRegenerativeMedicineandDepartmentsofPathology,ChemicalandSystemsBiology,andDevelopmentalBiology,StanfordUniversitySchoolofMedicine,Stanford,CA94305;and

2.bAppliedBiosystems,Inc.,FosterCity,CA94404

∙↵2Presentaddress:

MemorialSloan-KetteringCancerCenter,1275YorkAvenue,NewYork,NY10065.

1.ContributedbyIrvingL.Weissman,November3,2010（sentforreviewSeptember25,2010）

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Abstract

MicroRNAsprofoundlyimpacthematopoieticcellsbyregulatingprogenitorcell-fatedecisions,aswellasmatureimmuneeffectorfunction.Howevertodate,microRNAsthatregulatehematopoieticstemcell（HSC）functionhavebeenlesswellcharacterized.HereweshowthatmicroRNA-125b（miR-125b）ishighlyexpressedinHSCsanditsexpressiondecreasesincommittedprogenitors.OverexpressionofmiR-125binmouseHSCenhancestheirfunction,demonstratedthroughserialtransplantationofhighlypurifiedHSC,andenrichesforthepreviouslydescribedSlamf1loCD34−lymphoid-balancedandtheSlamf1negCD34−lymphoid-biasedcellsubsetswithinthemultipotentHSC（CD34-KLS）fraction.MatureperipheralbloodcellsderivedfromthemiR-125b–overexpressingHSCareskewedtowardthelymphoidlineage.Consistentwiththisobservation,miR-125boverexpressionsignificantlyincreasesthenumberofearlyB-progenitorcellswithinthespleenandinducestheexpansionandenrichmentofthelymphoid-balancedandlymphoid-biasedHSCsubsetviaanantiapoptoticmechanism,reducingthemRNAexpressionlevelsoftwoproapoptotictargets,BmfandKLF13.TheantiapoptoticeffectofmiR-125bismorepronouncedinthelymphoid-biasedHSCsubsetbecauseoftheirintrinsichigherbaselinelevelsofapoptosis.TheseeffectsofmiR-125bareassociatedwiththedevelopmentoflymphoproliferativedisease,markedbyexpansionofCD8+Tlymphocytes.Takentogether,thesedatarevealthatmiR-125bregulatesHSCsurvivalandcanpromotelymphoid-fatedecisionsattheleveloftheHSCbypreferentiallyexpandinglymphoid-balancedandlymphoid-biasedHSC.

MicroRNAsareaclassofevolutionarilyconservedsmallRNAsthatinducecleavageortranslationalrepressionoftargetmRNAsbybindingtopartiallycomplementaryseedsequencesfoundonthe3′UTRregionsoftargetmRNAs

（1）.MicroRNAsarepredictedtoprofoundlyaffectgene-expressionprofilesandregulatetheexpressionofhundredsofmRNAs（1–3）.

SeveralstudieshavedemonstratedthatmiRNAsmayregulatelineage-fatedecisionsinhematopoieticdevelopment.Forexample,ectopicexpressionofmiR-181inlineage-negativemousebone-marrowcellsleadstoexpansionofBcellsbutadiminutionofTcells（4）.Myeloid-specificmiR-223hasbeenimplicatedingranulocyticdevelopment,withmiR-223knockoutmiceexhibitingincreasednumbersofgranulocyteprogenitorsandmaturecells（5）.DeficiencyofmiR-150leadstoexpansionoftheB1B-cellcompartment,whereasectopicexpressionofmiR-150impairsBcelldevelopment（6）.Inaddition,miR-150,whichishighlyexpressedinthemegakaryocyticlineage,canbiasdifferentiationofmegakaryocyte-erythroidprogenitors（MEP）towardthemegakaryocyticfateattheexpenseoferythrocytes（7）.Morerecently,studiessuggestthatmiRNAs,suchasmiR-29a,mayregulatehematopoieticstemcell（HSC）self-renewal,asevidencedbytheaberrantinductionofself-renewalinprogenitorpopulationsbymiRNAshighlyexpressedinHSCandhumanacutemyeloidleukemia（AML）（8）.

WeareinterestedinidentifyinggenesthatregulateHSCfunctionandundertookanefforttoidentifymiRNAsthataredifferentiallyexpressedinHSC.WefoundthatmiR-125bisexpressedathighestlevelsinmouseHSC,andthatmiR-125bexpressiondecreasesprogressivelyascellsdifferentiatetomyeloidandlymphoidcommittedprogenitors,withmiR-125bexpressedatsignificantlyhigherlevelsincommonlymphoidprogenitors（CLP）comparedwiththecommonmyeloidprogenitors（CMP）.TotestthebiologicalfunctionofmiR-125binHSC,weoverexpressedmiR-125binhighlypurifiedHSCusinglentiviralvectors.OverexpressionofmiR-125bincreasedHSCengraftmentincompetitivetransplants,andweconfirmedthatthiseffectwasaresultofcell-autonomouseffectsonHSCandnotcommittedprogenitorsbyrecapitulatingthephenotypethroughserialtransplantationofhighlypurifiedHSC.Inaddition,miR-125binducedanexpansionoftheHSCcompartmentinpartbyinhibitingexpressionofatleasttwoantiapoptotictargetgenes,Bmf（Bcl2modifyingfactor）andKLF13（Krueppel-likefactor13）.BothtargetswereidentifiedaspotentialmiR-125btargetsinvivobyevaluatingpurifiedstemandprogenitorpopulations.TheHSCexpansionwasassociatedwithalymphoiddifferentiationbiasintheperipheralblood.InasmallfractionofthemiR-125btransplantedmice,weobservedalymphoproliferativedisease.

Concurrently,O'Connelletal.（9）foundthatmiR-125bishighlyexpressedinthestemandprogenitor-cellcompartmentofthemousebonemarrowandthat1,000-foldoverexpressionofmiR-125binthehematopoieticstemandprogenitorpopulationsgaverisetoamyeloproliferativediseasethatprogressedtoAML.Guoetal.（10）alsofoundthatmiR-125aishighlyexpressedinthestem-andprogenitor-cellcompartmentofthebonemarrow.Overexpressionofmir-125aalsoexpandedtheHSCcompartmentviaanantiapoptoticmechanism,possiblybytargetingtheproapoptoticprotein,Bak1.OurobservationsconfirmthesefindingsandextendthembydemonstratingthatmiR-125bcaninducepreferentialexpansionofthepreviouslydescribedlymphoid-balancedandlymphoid-biasedHSC（phenotypicSlamloCD34−andSlamnegCD34−KLS）populations.

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Results

ExpressionProfilingofHematopoieticPopulations.

ToidentifymiRNAsthatmayregulateHSCfunction,wefirstanalyzedthemiRNAexpressionprofilesofmultiplehematopoieticpopulations.Wedouble-sortedHSCandcommittedprogenitorpopulationsfromthebonemarrowofC57BL/6-Thy1.1（BA）micebasedoncell-surfacemarkersdefinedbyourlaboratoryandothers（11）:

HSC（c-kit+Sca-1+Lin−Flk2−CD34−）,multipotentprogenitor（MPP）flk−cells（c-kit+Sca-1+Lin−Flk2−CD34+）,MPPflk+cells（c-kit+Sca-1+Lin−Flk2+CD34+）,CLPcells（Ly6c−Flk2+IL7R+CD27+CD4−B220−CD19−CD11c−）,CMPcells（c-kit+Sca-1−Lin−FcGloCD34+）,granulocyte-monocyteprogenitor（GMP）cells（c-kit+Sca-1−Lin−FcGhiCD34+）,andMEPcells（c-kit+Sca1−Lin−FcGloCD34−）.MicroRNAsweremeasuredfromtotalRNAisolatedfromeachcellpopulationusingapreviouslydescribedTaqManreal-timePCRstrategy（8）.Fiveindependentlysortedbiologicreplicates,eachrepresentingfivepooledmice,wereusedforthesestudies.OuranalysisrevealedthatmiR-125bexpressionisconsistentlyandsignificantlyhigherinHSC（>twofold,P<0.05）comparedwithallotherprogenitorpopulationsassayed（Fig.1）.Inparticular,theexpressionlevelofmiR-125bissignificantlyhigherintheCLPpopulationcomparedwiththeCMPpopulation.AsimilartrendwasobservedinhumanHSCandcommittedprogenitorcellpopulations;however,thedifferenceswerenotstatisticallysignificant（P>0.05comparingHSCwithallprogenitorpopulations）,likelyreflectingthegeneticheterogeneityofthehumancellpopulations.

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Fig.1.

MicroRNA-125bishighlyexpressedinHSCsandMPPcellsinmiceandhumans.（A）NormalizedexpressionlevelofmiR-125bwasdeterminedbyquantitativePCRusingmiRNATaqmanprobesindouble-sortedmousehematopoieticcellpopulations:

HSC（c-kit+Sca1+Lin−CD34−Flk2−）,MPPFlk−（c-kit+Sca1+Lin−CD34+Flk2−）,MPPFlk+（c-kit+Sca1+Lin−CD34+Flk2+）,CLP（Ly6c−Flk2+IL7R+CD27+CD4−B220−CD19−CD11c−）,CMP（c-kit+Sca-1−Lin−FcGloCD34+）,GMP（c-kit+Sca-1−Lin−FcGhiCD34+）,andMEP（c-kit+Sca1−Lin−FcGloCD34−BM）.Expressionwasnormalizedagainstmmu-mir-16（n=5）.（B）NormalizedexpressionlevelsofmiR-125bweredeterminedbyquantitativePCRusingmiRNATaqmanprobesindouble-sortedbonemarrowhumanhematopoieticcellpopulations:

HSC（Lin−CD34+CD38−CD90+CD45RA−）,MPP（Lin−CD34+CD38−CD90−CD45RA−）,CMP（Lin−CD34+CD38+CD123+CD45RA−）,GMP（Lin−CD34+CD38+CD123+CD45RA+）,andMEP（Lin−CD34+CD38+CD123loCD45RA−）.Expressionwasnormalizedagainstsno-R2.（n=5）.Thedifferenceswerenotstatisticallysignificant,possiblybecauseofheterogeneityofthehumanhematopoieticpopulations.ErrorbarsdenoteSEM.

Mir-125bexistsintwolocationsinthemouseandhumangenomes.Mmu-miR-125b1,foundonmousechromosome9,andhsa-miR-125b1,foundonhumanchromosome11,existinaputativepolycistronicclusterwithmiR-let7a2andmiR-100（Fig.S1）.Mmu-miR-125b2（mousechromosome16）andhsa-miR-125b2（humanchromosome21）existinaclusterwithmiR-let7c1andmiR-99a.Interestingly,notallmiRNA-125bclustermembershaveexpressionlevelsthatcovarywithmiR-125binearlyhematopoieticprogenitors.Specifically,onlymiR-99aandmiR-100appeartodemonstratesimilarprofilestomiR-125b,withhighestexpressionlevelsinHSCandgraduallydecreasinglevelsastheprogenitorsdifferentiate.BothmiR-99aandmiR-100alsodemonstratehigherexpressionlevelsintheearlylymphoidprogenitorscomparedwiththeearlymyeloidprogenitors.TheseresultssuggestthatthesemiRNApolycistronsmayberegulatedbyposttranscriptionalmechanismsinearlyhematopoiesis.

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