ImmunophenotypeofLineage：Lineage：Lin-Sca-1+c-kit+,LKS+(SudaetSPcells(Goodelletal)Sidepopulation(Ho33342blue/redlow)

(Nakauchietal) (Jacobsenetal) (Slamfamily)(Morrisonet (MorrisonetLKS+CD34-CD150+CD48-orLKS+CD34-Wang,L.D.andWagers,A.J.2011,NatRevMolCellJ?rgenAdolfsson,etal.2005,Lin-IL-7Rα+Sca-1loc-E.Passegue,etal.2005,

SidepopulationHoechstAllisonMayle,etal.2005,(Cellcycle)和細胞增殖活性氧簇(ROS,reactiveoxygenDNAPI,Hoechst(Ho),DAPI,7-

Ki67andDNA

RNAdyeandDNAPyroninY(PY)andHo

PyroninPyronin

Ho

BrdU(orEdU)andDNA

CFSE(CFDA-SInvitro:culture1-2hInvivo(HSC):Short-term:i.p.-----12-16Long-term:drinkwater---7AnnexinVandDNA

碎片活細胞碎片活細胞

TUNELandDNA

RichardXuFeng,etal.2009,胞內ROS分析(

KennethD.Gibbs,etal.2011,(PopulationCAFC(cobblestoneareaformingcell

(移植)(歸巢

ysiseveryMicroarrayRNA-seq

Gene 功能分析

orKnock-down(Singlecell 14

(Mac-1,Gr-1,Ter119,

SinglecellRyoYamamoto,etal.2013,BM ntation(BMT)andSerialBMTCompetitiveBM ntation(cBMT)coupledwithserialSinglecell HuiCheng,etal.2013,MethodsMol

Congenicstrainof2212122121DefineHSCsin nt1sttrans >6month2ndtrans ntationabilityST-1st ntation<3or1st ntation>3month,butnotmulti-No2nd ntation1st ntation<1Nomulti-RyoYamamoto,etal.2013,移植細胞數量(全骨髓細胞1、halfaPBS+2mMEDTA。

SS+SS+SMMS:stemM:matureWTCD45.1+cellsEngraftmentinBM(%)WT

I°III°II° 供體細胞：全骨髓細胞或HSCLKS+orLT-CD45.1+Lin-Sca-1+c-kit+CD45.1+Lin-Sca-1+c-kit+CD34-CD150+MethodMethod

MiyamotoK,etal.2007,CellStemBTMBTM

IreneM.Min,etal.2008,CellStemRUs(RepopulatingUnits):indicatetheamountofpopulatingactivity(functionalqualityofHSCs),measurethefunctionalpotentialoftheunknownsourceofHSCsagainstasetknownnumberofHSCs,providequalitativeoratbest tativeinformationabouttheHSCswithinagivenpopulation—itcannotdistinguishbetweenthenumberofHSCsortheirquality(progenyproducedperHSC)1x105WTBMcells=1Donor Competitorcell

=DonorCompetitor

1xCD45.1cell33.6x5=2.585x105BMModifiedfromRyoYamamoto,etal.2013,HideoEma,etal.2005,DevelopmentPositive:onelineagereconstitution>YoheiYoheiMorita,etal.2010,8micepercelldose每個劑量大于8只小鼠3celldoses(大于3個劑量dosesof

2x105BM

(16

1、WholeBMcells8x1031x106+2x105competitorsBM2、Purifiedcellse.g.LKS+cells?????Try…2x105competitorsBMPositive:Alllineages(T,B,M)reconstitution>全部positive沒有意義。所選的劑量中，必須要有negative< Poissonstatistics(泊松分布):當 CRUs(competitiverepopulatingunits):numberofstemcells tyofCRU計算：L-Calc( IfIfGFPnormalizeto+JianweiWang,etal.2012,HSCSeanMorrisonLinhengLiToshioSudaPaulFrenetteHSCSelf-renewalvs.IrvingWeissmanHiromitsuNakauchiStuartOrkinMargaretGoodellAndreasT