Hierarchical and Ontogenic Positions Serve to Define the Molecular Basis of Human

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DevelopmentalCell,Vol.8,651–663,May,2005,Copyright©2005byElsevierInc. DOI10.1016/j.devcel.2005.03.004
Hierarchical and Ontogenic Positions Serve
to Define the Molecular Basis of Human
Hematopoietic Stem Cell Behavior
FarbodShojaei,1,2JenniferTrowbridge,1,2 define HSCs (Enver et al., 1998). Since transplantable
LisaGallacher,1LouYuefei,1DavidGoodale,1 HSCs can give rise to progenitors detected in vitro,
FrancisKaranu,1KrystaLevac,1 whereas in vitro progenitors lack multilineage repopu-
andMickieBhatia1,2,* lating function in vivo, a hierarchical arrangement of
1StemCellBiologyandRegenerativeMedicine hematopoieticcellscanbeassembledbasedonthese
RobartsResearchInstitute functionalassaysinwhichrepopulatingHSCsareposi-
100PerthDrive tionedattheapex,followedbymoreabundantprogeni-
London,OntarioN6A5K8 tors with more restricted proliferative and develop-
Canada mentalpotential(OrkinandZon,2002;Verfaillie,2002).
2DepartmentofMicrobiologyandImmunology Inadditiontothefunctionalrelationshipsthatdefine
TheUniversityofWesternOntario progenitorsandHSCs,theontogenicoriginofmamma-
London,OntarioN6A5K8 lianHSCsisalsorelatedtochangesinoverallHSCca-
Canada pacity (Geiger and Van Zant, 2002). Both proliferative
abilityanddifferentiativepotentialofHSCsdeclinewith
age, e.g., HSCs obtained from the fetus possess
Summary greater proliferative and differentiative capacity than
those isolated from adult mice (Geiger and Van Zant,
Themolecularbasisgoverningfunctionalbehaviorof 2002).Sincehematopoieticfunctionmustbesustained
humanhematopoieticstemcells(HSCs)islargelyun- forthelifetimeofanorganismandisthereforedepen-
known. Here, using in vitro and in vivo assays, we dentonHSCs,rigidcontrolofHSCsurvivalanddiffer-
isolate and define progenitors versus repopulating entiationdecisionsthroughouttheaginganddevelop-
HSCsfrommultiplestagesofhumandevelopmentfor ment process is critical (Geiger and Van Zant, 2002;
globalgeneexpressionprofiling.Accountingforboth Verfaillie, 2002). Despite these biological observations
the hierarchical relationship between repopulating and collective functionalunderstanding of mammalian
cells and their progenitors, and the enhanced HSC HSCs, the molecular mechanisms that tightly regulate
function unique to early stages of ontogeny, the hu- HSC physiology (Murdoch et al., 2001; Orschell-Tray-
man homologs of Hairy Enhancer of Split-1 (HES-1) coffetal.,2000;Domenetal.,2000)andcellfateremain
and Hepatocyte Leukemia Factor (HLF) were iden- poorly understood (Lemischka, 1999). In the current
tifiedascandidateregulatorsofHSCs.Transgenichu- study,weuseinvitroandinvivoassays,combinedwith
man hematopoietic cells expressing HES-1 or HLF prospectiveisolationofcellpopulationsdefinedtopos-
demonstratedenhancedinvivoreconstitutionability sess properties of either progenitors or repopulating
thatcorrelatedtoincreasedcyclingfrequencyandin- HSCsfrommultiplestagesofhumandevelopment,for
hibition of apoptosis, respectively. Our report identi- globalgeneexpressionprofiling.Werevealtworegula-
fies regulatory factors involved in HSC function that torytranscriptionfactorsinvolvedinhumanHSCfunc-
elicit their effect through independent systems, sug- tionthatelicittheireffectthroughindependentmecha-
gestingthatauniqueorchestrationofpathwaysfun- nismscapableofcontrollingstemcellbehaviorinvivo.
damentaltoallhumancellsiscapableofcontrolling
stemcellbehavior. Results
Introduction FunctionalCharacterizationandIsolationofHuman
HematopoieticProgenitorsandRepopulatingStem
Rare hematopoietic stem cells (HSCs) are responsible CellsduringHumanDevelopment
for production and maintenance of mature human Based onour currentknowledge of humanHSC char-
bloodcells(Morrisonetal.,1995).HSCsaredefinedby acteristics,molecularprofilingthatdiscriminatesgenes
twofunctionalproperties:vastproliferativeself-renewal expressedinHSCsfromthoseexpressedinmorema-
ability, and multilineage hematopoietic differentiation tureprogenitors(hierarchical)andthosegenesthatdis-
potential (Morrison et al., 1995).In the human, cellular tinguishfunctionallyuniqueHSCsatdifferentstagesof
characteristicsofprimitivehematopoieticcellsandpu- mammalian development (ontogenic) should be taken
tativeHSCscanonlybeexperimentallyinvestigatedby intoaccounttobestdefinethemolecularbasisofHSC
using surrogate approaches (Dick, 1996). In vitro pro- behavior. Using this premise, subsets of lineage-
genitor assays are widely used to measure primitive depleted (Lin−) human hematopoietic cells with multi-
colony-forming unit (CFU) (Sutherland et al., 1989) ac- lineage repopulating capacity (Lin−CD34+CD38−), and
tivity,butthisassayisnotindicativeofstemcellfunc- Lin−hematopoietic cellscapableofin vitroprogenitor
tionsinceitisshorttermandlimitedtomyeloidprogen- function but devoid of in vivo repopulating ability
itordetection.Onlyinvivoxeno-transplantationassays unique to HSCs (Lin−CD34+CD38+), were isolated.
(Dick,1996)areabletoassessthemultilineagedifferen- Lin−CD34+CD38−andLin−CD34+CD38+werepurified
tiation and proliferative self-renewing properties that from Lin− fractions of human fetal blood (FB), cord
blood(CB),andmobilizedperipheralblood(MPB)with
*Correspondence:[email protected] more than 99% purity upon reanalysis (Figure 1A). Al-
DevelopmentalCell
652
Figure1.FunctionalCharacterizationofPurifiedHumanFB,CB,andMPBHematopoieticSubsetswithInVitroandInVivoAssays
(A)IsolationofLin−CD34+CD38−andLin−CD34+CD38+populationsfromFB,CB,BM(notshown),andMPB.Lin−fromtheabove-mentioned
populations(biologicalandexperimentalreplicatesfromeachpreparationare detailedinTableS1)wereenrichedandstainedwithanti-
CD34-APCandanti-CD38-PEandweresortedaccordingtothesortinggatesassignedforCD34+CD38−(I)andCD34+CD38+(III)populations.
Reanalysisofsortedfractions(IIandIV)verifiedpurityofthepopulationsofinterestfromeachstageofontogeny.
(B)CFUcapacity:1200cellsfromeachpopulationwereplatedinmethylcellulosesupplementedwithSCF,GM-CSF,IL-3,andEPO,followed
bytotalcolonycountsafter14daysofincubationat37°C.
(C) Repopulating capacity: cells from each population were intravenously transplanted into NOD/SCID mice at a range of 5,000–10,000
Lin−CD34+CD38−cells,andfrom10,000to200,000Lin−CD34+CD38+cellsfromallhumanontogenicsourcesindicated.Humanengraftment
wasevaluated6weeksposttransplantbystainingcellsharvestedfromBMoftransplantedmicewithmonoclonalantibodyagainsthuman
CD45.ThelevelofengraftmentrepresentsthepercentageofhumanhematopoieticchimerismintheBMforrecipientmice.(“**”and“*,”
statisticallydifferent,p<0.05).
(D)BMcellsoftransplantedanimalswerestainedwithseveralcellsurfacemarkerstoexaminethepercentageofmyeloid(CD33,CD15),
lymphoid(CD19,CD20),andprimitive(CD34,CD38)cellsindicativeofmultilineagehumanhematopoieticengraftment.AveragesandSEM
foreachsubsetareprovidedinquadrantsbasedon4–8independentexperimentsforFB,CB,andMPBrepopulatingHSCsrespectively,
n=18.
(E)ThesubsetsindicatedwerestainedwithKi67-FITCantibodyaspertheprotocol(seeExperimentalProcedures)andanalyzedbyflow
cytometry.“*,”statisticallydifferent,p<0.01.
(F)ProposedmodeltoaccountforhierarchicalandontogenicpositionofhumanHSCsandnonrepopulatingprogenitors.
DefiningtheMolecularNatureofHumanHSCs
653
though MPB cells do not represent a normal stage of HSCs possess superior proliferative and self-renewal
human hematopoietic ontogeny (since primitive cells capacity compared to their aged counterparts. Based
are induced to the circulation via cytokine administra- onourfunctionalobservations,wesuggestamodelto
tion), MPB represents an established and accessible positionhumanHSCsthataccountsforbothhierarchi-
source of adult HSCs for fundamental investigation. cal and ontogenic characteristics of the primitive
Within each stage of ontogeny (fetal, neonatal, and hematopoietic compartment in the human (Figure 1F).
adult),clonogenicprogenitorcapacitymeasuredbythe ThismodelillustratesthathumanHSCsversusprogeni-
invitroCFUassay(Sutherlandetal.,1990)wassimilar tors are restricted to a specific population of hemato-
between Lin−CD34+CD38− and Lin−CD34+CD38+ cells, poieticcellsthroughoutlife,suggestingthatthehierar-
withadultMPBsourcespossessingfarlessclonogenic chical arrangement of the hematopoietic system is
progenitorcapacity,independentofthepopulationiso- conserved during development, but also accounts for
lated (Figure 1B). However, in vivo functional analysis higher proliferative potential of fetal HSCs, and the
by intravenous (IV) transplantation into immune-defi- gradualdeclineinthispropertywithinhumanHSCsto-
cientrecipientNOD/SCIDmicedemonstratedthatonly ward adult life. We suggest that the gene expression
Lin−CD34+CD38− from human FB, CB, and MPB pos- profiles that regulate functional capacity of human
sessed repopulating ability (Figure 1C). Therefore, these HSCsaremoreabundantlyexpressedinFBHSCsver-
purifiedpopulationsrepresentahierarchicaldistinction susneonatalandadultsources,andthattheseprofiles
within the human hematopoietic system in which arerestrictedtoHSCsversusprogenitorsatallstages
Lin−CD34+CD38−repopulatingcellsincludefunctional ofhumandevelopment.
human HSCs (termed “repopulating cells” herein) and
Lin−CD34+CD38+arerestrictedtotheprogenitorcom-
MolecularProfilingofPurifiedCellsComprising
partment (termed “nonrepopulating progenitors” herein).
theHumanHematopoieticHierarchy
This hierarchical relationship was conserved at all
throughoutHumanOntogeny
stages of human ontogeny, indicating that the pheno-
Utilizing the conserved hierarchical relationship be-
type used to define putative human HSCs and re-
tweenrepopulatingHSCsandnonrepopulatingprogen-
stricted progenitors is reliable and provides a useful
itors, and the ontogenic relationships among human
meanstodistinguishthesesubsetsthroughouthuman
HSCsfunctionallydefinedandmodeledinFigures1A–
development.
1F, we established a strategy to define the molecular
InadditiontotheconsistencyofHSCandprogenitor
basisofhumanHSCbehaviorthroughglobalgeneex-
functionwithinthesetwosubsets,detailedanalysisof
pressionanalysisofpurifiedsubsetsbyusingmicroar-
engraftedanimalsindicatedagradualdeclineinhuman
ray.Toprovideanindepthassessment,severalexperi-
HSCrepopulatingabilityfromFBtoCBtoMPB,where
mental and biological replicates were used (Table S1,
both the level and frequencyof human engraftment in
see the Supplemental Data available with this article
FBwasgreaterthanCBand,inturn,greaterthanMPB
online). To simultaneously account for hierarchy and
(Figure1C).Furthermore,thelevelandfrequencyofre-
ontogenicpositioningofHSCs,ourstrategyformolec-
populating capacity are higher in FB HSCs versus CB
ular analysis used several filters (Figure 2A). First, we
andMPBHSCs,and,inturn,CBHSCcapacityishigher
applied hierarchical discrimination that identified all
than that of MPB HSCs (Figure 1C). Other than a pro-
genes expressed in repopulating Lin−CD34+CD38−
pensity for more myeloid versus lymphoid differentia-
fractionsofFB,CB,bonemarrow(BM),andMPBthat
tion arising from reconstituting FB HSCs, all sources
weredifferentiallyexpressedfromnonrepopulatingpro-
of human HSCs possessed multilineage differentiation
genitors at each stage of ontogeny. Adult BM was
capacity in vivo (Figure 1D). The gradual reduction of
added in these analyses to provide an additional
reconstitution capacity from FB to CB to adult MPB
source of adult HSCs. This filter provided genes
HSCspromptedustoexaminecellcyclestatus(Glimm
uniquely enriched in repopulating human HSCs at all
etal.,2000)ofLin−CD34+CD38−cellsandLin−CD34+
stages of human development and included 366 and
CD38+progenitorsfromallstagesofhumanontogeny
329genesup-anddownregulated,respectively(Tables
byusingKi67 staining(Glimmetal., 2000)(Figure1E).
S3andS4).Self-organizingmap(SOM)(Tamayoetal.,
Higher cycling activity in all nonrepopulating progeni-
tors compared to repopulating cells confirmed the 1999) clustering provided correlative nodes of dif-
unique quiescent state associated with de novo iso- ferentiallyexpressedgenesidentified(FigureS1).Inthe
lated HSCs (Srour et al., 2001; Murdoch et al., 2001). second filter, ontogenic discrimination was applied to
However, consistent with reconstituting capacity (Fig- identify HSC-enriched transcripts that were preferen-
ure1C),FBLin−CD34+CD38−cellsdemonstratedgreater tially expressed in early versus later stages of human
cyclingactivitycomparedtosimilarcellsfromCBand development (Figure 2A). Third, we further selected
MPB (p < 0.05, Figure 1E). Collectively, these data re- genes based on the hypothesis that transcription
vealthatdifferencesbetweenrepopulatingcapacityof factorsarelikelytoplayasubstantiveroleinHSCregu-
HSCsisolatedfromdifferentstagesofhumanontogeny lation (Ramalho-Santos et al., 2002). This bioinformat-
correlate with differences in cell cycle activity that are ics strategy, unique to human hematopoietic cell mo-
likelytoaffecttheinvivoproliferativecapacityofHSCs. lecular profiling, resulted in the identification of 16
DespitethefactthattheHSCshavesimilarcellsur- candidate transcription factors (Figure 2B), of which
face phenotypes and functional properties at various STAT-1(Jenkinsetal.,2002),Id3(Cooperetal.,1997),
stagesofhumandevelopment,ourresultsdemonstrate Ets1(Dieterlen-Lievreetal.,1993),HLF(CrableandAn-
anontogenicdistinctionofhumanrepopulatingHSCs, derson, 2003), and HES-1 (Kunisato et al., 2003) have
butnotofmorematureprogenitors:ontogenicallyearly beenpreviouslyimplicatedinprimitivehematopoiesis.
DevelopmentalCell
654
DefiningtheMolecularNatureofHumanHSCs
655
IdentificationofHES-1andHLFasCandidate profilingstudies(Georgantasetal.,2004;Ivanovaetal.,
RegulatorsofHumanHematopoietic 2002). To functionally examine a potential role of HES-1
RepopulatingStemCells and HLF in human HSCs, full-length cDNA coding se-
Ofthe16candidatesidentified(Figure2B),HES-1and quencesforeachgeneweresubclonedintoaretroviral
HLFexpressionbestmirroredthefunctionalmodelwe vector underthe controlof a constitutivelyactive pro-
predictedtobethemostrelevanttooverallHSCbeha- moter and upstream of the reporter gene-enhanced
vior(Figure1F).Quantitativereal-timePCR(Q-RT-PCR) greenfluorescentprotein(GFP)(Figure2F).HumanCB
using freshly isolated FB, CB, and adult BM and MPB Lin− cells containing both CD34+CD38− and CD34+
Lin−CD34+CD38− cells demonstrated a declining pat- CD38+subsetswereusedtoevaluatetheroleofHES-1
ternofexpressionfromFBtoneonataltoadultsources and HLF based on subsequent functional readout of
(Figure2C).Inaddition,theratioofHES-1andHLFex- repopulating HSCs and nonrepopulating progenitors
pressionbetweenLin−CD34+CD38−repopulatingcells overexpressing the transduced gene. Following trans-
and Lin−CD34+CD38+ progenitors was consistently duction of primary human Lin− CB cells, the in vitro
greater than 1 (i.e., HSC expression > progenitor ex- proliferative capacity, phenotype, progenitor potential,
pression)atallstagesofontogeny,andthisratiodeclined and in vivo repopulating function of HES-1 and HLF
fromearlytolatestagesofHSC/progenitordevelopment transgenichuman hematopoieticcells werecompared
(Figures 2D and 2E). This pattern of expression was to vector-transduced controls. Our experimental strat-
unique to HES-1 and HLF-1 in comparison to other egyis illustratedin Figure2G. HES-1,HLF, andvector
genesexaminedinasimilarmanner(datanotshown). alone retrovirally transduced hematopoietic cells were
Accordingly, HES-1 and HLF were selected for further analyzed by flow cytometry for GFP and cell surface
functionalanalysis. CD34expression(Figure2H).Nodifferenceinretroviral
HES-1 is a basic-helix-loop-helix transcriptional re- transduction(number ofGFP+cells, Figure2I) ortotal
pressor that inhibits differentiation of many cell types numberofprimitiveCD34+cellswasdetectable(Figure
(Ishibashietal.,1994;Kageyamaetal.,2000;Nakamura 2J).ThesetransgenichumancellsoverexpressingHES-1
et al., 2000) and is a downstream target of the Notch and HLF were used in functional in vitro and in vivo
signaling pathway (Karanu et al., 2000b, 2001, 2003; assays to evaluate the affects of these genes on pro-
Kumanoetal.,2003;Varnum-Finneyetal.,2000).Are- genitorandHSCbehavior,respectively.
cent study has shown that HES-1 is able to maintain
the reconstitution ability of mouse CD34−Lin−c-Kit+ HES-1andHLFAugmentHumanHematopoietic
Sca-1+HSCs(Kunisatoetal.,2003).HLFiscloselyre- StemCellActivityInVivo
lated to zipper-containing transcription factors that TransductionofHES-1orHLFhadnoeffectoninvitro-
have a role in developmental stage-specific gene ex- detected progenitors, suggesting that overexpression
pression (Inaba et al., 1992), and it has recently been of these genes does not impact functional properties
showntoactivatetheLMO2promoter(CrableandAn- of nonrepopulating cells. Transduced (GFP+) hemato-
derson, 2003) necessary for initiation of mammalian poietic progenitor frequency or lineage composition
embryonichematopoiesis(Warrenetal.,1994).Inaddi- were not affected by overexpression of the HES-1 or
tion,HLFwasfoundtobeinvolvedinacutehumanleu- HLF transgene (Figures 3A and 3B) since erythroid,
kemias when fused to the E2A basic-helix-loop-helix macrophage, and granulocytic CFU subtypes were
transcription factor (Honda et al., 1999; Inaba et al., equallyidentified.
1992)andwasrecentlyidentifiedintwoothermolecular Similar numbers of HES-1-, HLF-, and vector-trans-
Figure2.IdentificationofCandidateTranscriptionFactorsRegulatingHumanHSCswithaUniqueStrategyBasedonHierarchicalandOnto-
genicPositioning
(A) Using global gene expression analysis of human Lin−CD34+CD38− from FB, CB, BM, and MPB and Lin−CD34+CD38+ progenitors,
transcriptswereinterrogatedbymicroarrayanalysis.Bioinformatics-basedpriorityfilterswereappliedasschematicallyillustratedandin-
cludedhierarchicalandontogenicdiscrimination,followedbytheselectionofcandidateDNAbindingtranscriptionfactors.
(B)Atotalof16transcriptswereidentifiedbyusingthestrategyshownin(A).Acompletelistofdifferentiallyregulatedgenesisavailableas
TableS3.Abbreviations:POLD4,polymerasedelta4;TEF4,transcriptionalenhancerfactor4;HES-1,hairyenhancerofsplit;LIM,zincbinding
domain present in Lin-11, Isl-1, Mec-3; HLF, hepatic leukemia factor; PAX8, paired box gene8; Id3, inhibitor of DNA binding3; HOXA6,
homeoboxA6;ETS1,erythroblastosisvirusE26oncogenehomolog1;STAT1,signaltransducerandactivatoroftranscription;ZNFP1&2,zinc
fingerprotein;EST,expressedsequencedtag;MBLL,melanogastermuscleBlindB;PHRET1,PHdomaincontainingproteininretina1.
(C) Expression profile of HES-1 and HLF in freshly isolated Lin−CD34+CD38− cells derived from all stages of human ontogeny using
Q-RT-PCR.
(DandE)Q-RT-PCRwasusedtoanalyzetheratioof(D)HES-1and(E)HLFexpressioninLin−CD34+CD38−toLin−CD34+CD38+fromFB,
CB,andMPBsources.AllQ-RT-PCRanalysiswasnormalizedtoGAPDHtranscript,anddifferentialexpressionwascalculatedbyusingthe
(cid:1)(cid:1)Ctequation.“*,”statisticallydifferentthancorrespondingexpressioninLin−CD34+CD38+cells,p<0.05.
(F)Full-lengthcDNAofHES-1wasclonedintheEcoRIandXhoIsites,andHLFfull-lengthcDNAwasclonedintheEcoRIandSalIsitesof
theMIEVretrovirusvectorasillustrated.
(G) Schematic illustration of experimental design to functionally analyze the effect of HES1 and HLF overexpression by using retroviral
genereconstitution.
(H) Lineage-depleted population from human CB was transduced with viral supernatant containing HES-1, HLF, and vector alone. Gene
transferefficiencywasdeterminedbythepercentageofGFP+cells6daysposttransductionbyusingflowcytometryandprimitivehematopoi-
eticphenotypeusingCD34expression.
(IandJ)Summaryofthetotalnumberof(I)GFP+and(J)CD34+GFP+transgeniccells6daysposttransduction.
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656
Figure3.FunctionalAnalysisofHLFandHES-1OverexpressioninPrimitivePopulationsofHumanHematopoieticCells
(A)NumberofclonogenicprogenitorstransducedwithHES-1(bluebars),HLF(yellowbars),andvector(blackbars)byusingtheCFUassay.
Atotalof1000transgenic(GFP+sorted)cellswereplatedintomethylcellulosesupplementedwithSCF,GM-CSF,IL-3,andEPO,anddif-
ferentialcolonycountsofCFUsubtypes(erythroid,macrophage,andgranulocyte)weredeterminedandcomparedafter14days.
DefiningtheMolecularNatureofHumanHSCs
657
ducedcellswereintravenouslytransplantedintorecipi- HES-1andHLFActIndependentlytoEnhanceCell
entNOD/SCIDmicetoinvestigate theinvivoeffectof CycleandInhibitApoptosisofHuman
thesegenesonrepopulatinghumanHSCs.Multilineage HematopoieticStemCells,Respectively
humanreconstitutionofHES-1andHLFtransgenichu- Based on the robust effect of HES-1 and HLF in en-
manHSCsdidnotdifferfromvector-transducedrepop- hancing human HSC function independent of HSC-
ulatingcells,andbothlymphoidandmyeloidlineages, specifichomingpropertiestothebonemarrowintrans-
along with primitive subpopulations of CD34+ cells, planted recipients, we sought to understand the
weredetectableatsimilarfrequenciesinengraftedre- underlying mechanism involved through the consider-
cipients(Figure3C).ThissuggeststhatHES-1andHLF ation of fundamental pathways that impact all human
do not affect lineage differentiation of human HSCs. celltypes,suchascellcycleandapoptoticstatus.Brd-U
However, mice transplanted with HES-1 or HLF trans- incorporation and 7-AAD staining of transgenic cells
genicHSCsrevealedadramaticincreaseinthelevelof wereusedtodeterminethecellcyclestatusbyanalyz-
BM reconstitution and number of HSCs, as indicated ing the frequency of primitive CD34+ dormant (G –G )
0 1
byincreasedfrequencyofengraftedrecipients,n=35 and cycling (S and G +M) cells (Figure 4A). While HLF
2
(Figure 3D). Based on the levels of human chimerism, transgenicandvectorcontrolcellsweresimilartoeach
human HSC capacity was enhanced by w6-fold upon other with respect to distribution among the different
overexpression of HES-1 and w4-fold upon overex- stagesofthecellcycle,agreaterproportionofHES-1
pressionofHLFcomparedtovector-transducedHSCs transgenic cells was actively cycling, and a corre-
(Figure3G).Therefore,despitethelackofeffectonpro- spondingly smaller proportion was found to be quies-
genitor capacity, HSC repopulation potential was dra- cent (Figure 4B). Based on these observations, we
matically enhanced in transgenic cells overexpressing sought to evaluate the affect of HES-1 on cell cycle
HES-1 or HLF. These functional analyses validate our activityontransgenichumanHSCscomparedtoHLF-
originalstrategytoidentifykeymolecularregulatorsof and vector-transduced cells in vivo by Brd-U labeling.
HSC behavior in vivo that are distinct from regulators Brd-Uispassivelytakenupbycellsandstablyincorpo-
ofnonrepopulatinghematopoieticprogenitors. ratedintotheDNAofcellsundergoingmitoticdivision,
Since HSC capacity was evaluated in vivo by IV thereby providing a retrospective method to evaluate
transplantation, increased repopulating capacity of actively dividing parent cells that give rise to Brd-U+
transgenicHES-1andHLFhumanHSCsmaybedueto progeny.Recipientmiceweretransplantedwithhuman
enhancedabilitytohometotherecipientbonemarrow HSCsoverexpressingHES-1orHLFandwereadminis-
compartment.Cellsurfaceexpressionofproteinsasso- tered Brd-U for 3 days prior to sacrifice between 5–6
ciated with increased homing capacity, CD44, CD49, weeksposttransplantation.Ourexperimentaldesignis
andCXCR4,wereequallyexpressedonHES-1-,HLF-, illustrated in Figure 4C. Mice transplanted with trans-
andvector-transducedcells(Figure3E).Tofunctionally genic human HES-1 cells demonstrated a larger num-
evaluatetheeffectofHES-1andHLFonhomingability ber of primitive CD34+ cells incorporating Brd-U as
of HSCs, human cells overexpressing HES-1 and HLF comparedtoeitherHLF-orvector-transducedrepopu-
were seeded directly into the femoral bone marrow latingHSCs(Figure4D).ThesedataindicatethatHES-1
space of recipient mice by using intrafemoral bone is able to augment proliferative capacity of transgenic
marrowtransplantation(IBMT)(Mazurieretal.,2003a). humanHSCsinvivo,whereasHLFoverexpressionhad
HSC capacity of transgenic cells delivered by IBMT noinfluenceoncellcyclestatusofhumanHSCs.
wascomparedtoHSCstransducedwithvectoralone. While HES-1 overexpression increases cycling of
SimilartoresultsobservedafterIVtransplantation(Fig- primitive cells, HLF transgenic cells had a similar cell
ure 3D), IBMT of transgenic HSCs demonstrated that cycling status profile to vector-transduced cells, sug-
human HSC activity was equally enhanced by overex- gesting that HLF confers enhanced repopulation ca-
pression of HES-1 and HLF, n = 9 (Figure 3F). These pacity on human HSCs via an alternative mechanism
resultsindicatethatHES-1andHLFdonotlikelyaffect (Figures 4A–4E). Given previous observations of the
homingcapacityofhumanHSCs,andpromptedeluci- ability of HLF to inhibit apoptosis (Hitzler et al., 1999),
dationofalternativemechanisms. we investigated the apoptotic status of transgenic
(B)RepresentativeexamplesHES-1,HLF,andvectoralonetransgenicprogenitorsdetectedintheCFUassayconfirmingtransgeneexpression
inmultiplehematopoieticlineages.
(C)ComparisonofmultilineagehumanhematopoieticreconstitutionfromHES-1,HLF,andvectortransgenicrepopulatingHSCsintheBMof
transplantedmice.Approximatelyequalnumbersoftransgenic(GFP+)cells(averageof75,000)weretransplantedineachmousebyusinga
totalofsevenindependenthumanCBsamples,n=35.MouseBMwasstainedwithanti-CD45-APC(toidentifyhumancells);anti-CD10and
anti-CD19-PE(toidentifyBlymphoidlineage),anti-CD13,-CD14,-CD15,and-CD33-PE(toidentifymyeloidlineage);andanti-CD34(toidentify
primitivehumanhematopoieticcomponent)antibodiesandanalyzedbyflowcytometry6weeksposttransplantationofhumancells.
(D) The level of human hematopoietic repopulating capacity was compared as a percentage of human GFP+ transgenic cells, and the
frequency of engrafted mice intravenously transplanted with transgenic HES-1-, HLF-, and vector alone-transduced cells 6 weeks after
transplantationisshown.“*,”theaveragelevelofengraftmentisstatisticallydifferentfromthelevelofrepopulationinmicetransplantedwith
vector-transducedHSCs,p<0.05.
(E) Frequency of transgenic cells expressing cell surface molecules CD44, CD49, and CXCR4 associated with HSC homing to the bone
marrowcompartment.
(F)Theaveragelevelofhumanhematopoieticrepopulation(inset)fromHES-1,HLF,andvectoralonetransgenicHSCsdelivereddirectlyto
therecipientbonemarrowsitebyintrafemoralbonemarrowtransplantation(IBMT),n=9.“*,”theaveragelevelofengraftmentisstatistically
differentfromthelevelofrepopulationinmicetransplantedwithvector-transducedHSCs,p<0.05.
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658
Figure4.CharacterizationofCellCycleandApoptosisRegulationinHES-1-,HLF-,andVector-TransducedPrimaryHumanHematopoietic
Cells
(A)HumanCBCD34+cellstransducedwithHES-1,HLF,andvectoralonewerepulsedwithBrd-Ufor2hr,followedbyfixationandwashing,
andwerestainedwithanti-Brd-U-APCand7-AADtoanalyzethefractionofcellsatdifferentstagesofthecellcycle,includingG0+G1,S,
andG2+Mphases.
(B)QuantitativesummaryofcyclingandnoncyclingHES-1(bluebars),HLF(yellowbars),andvectoralone(blackbars)transgeniccells.“*,”
statisticallydifferentfromHLF-andvector-transducedcells,p<0.05.
(C)Schematicillustrationoftheexperimentaldesignusedtoevaluatemitoticallyactivecellsrepopulatingrecipientmice.
DefiningtheMolecularNatureofHumanHSCs
659
cells. Annexin-V staining for preapoptotic cells (Figure Severalcandidateregulatorsofhumanstemcellfate
4F)revealedasignificantlysmallerAnnexin-V+popula- have also been implicated or originally identified from
tioninHLFtransgenicCD34+cellscomparedtoeither human cancers. As such, transformation events and
HES-1-orvectorcontrol-transducedCD34+cells(Fig- pathwaysinfluencedbytransformationmayberelated
ure 4G), suggesting that HLF transgenic cells are less to the regulators of human HSCs (Pardal et al., 2003).
prone to apoptotic cell death. Since mechanisms and Genetic rearrangement of HLF with E2A has been ob-
targets of apoptotic control are species and cell served in human leukemias and associated with re-
context specific (Danial and Korsmeyer, 2004), we ducedapoptosisinleukemicprogenitors(Kurosawaet
aimedtofurthercharacterizethepotentialtargetgenes al.,1999).UsingPCRamplification,wewereunableto
associatedwithapoptoticcontrolviaHLFthatspecifi- detectHLF-E2AtranscriptsinprimaryHLF-transduced
cally affect human hematopoietic cells. Within the cells(Figure5B),indicatingthatourobservationofHLF
context of hematopoietic cells, SODD (Takada et al., apoptotic inhibition is independent of primary human
2003), TOSO(Song andJacob, 2005), andBcl-2 (Cory cellsharboringHLF-E2Amutations.Similarly,theNotch
etal.,2003)havebeenidentifiedasfactorscontrolling pathwayhasbeenimplicatedinhumanleukemias,and
apoptosis.Accordingly,primitivehumanhematopoietic ithasbeenshowninnonhumansystemstoaffectHES-1
cellstransducedwithHES-1orHLFwereisolatedand expression. To evaluate whether HES-1 could be
examined quantitatively for changes in SODD, TOSO, targetedforregulation,humanHSCswerepurifiedand
and Bcl-2 expression compared to vector-transduced treated with Jagged-1 (Karanu et al., 2000b). Short-
cells (representative Q-RT-PCR analysis, Figure 4H). termstimulationofpurifiedcellswithJagged-1demon-
OurresultsdemonstratethatoverexpressionofHLFin strated an increase in HES-1 expression within 24 hr,
primitivehumanhematopoieitccellsinducesBcl-2ex- compared to protein control (IgG)-treated cells (Figure
pression,buthasnoeffectonthelevelsofeitherSODD 5C). Upregulation of HES-1 could be sustained by 72
or TOSO (Figure 4I). In contrast, cells’ overexpression hr,butitrequiredtheadditionofJagged-1(Figure5C),
of HES-1 did not alter Bcl-2, SODD, or TOSO levels, suggesting that HES-1 could be upregulated but re-
providingfurtherevidenceoftheinabilityofHES-1,as quirescontinualstimulationsimilartothatachievedby
comparedtoHLF,toinfluenceapoptosisoftransgenic overexpressionofHES-1intransgenicHSCsregulated
cells(Figure4I).WesuggestthatHLFtargetsantiapo- by retroviral vectors. We suggest that HES-1 and HLF
ptoticpathwaysassociatedwithcomplexBcl-2regula- enhance the reconstitution capacity of human HSCs
tion in human HSCs, similar to effects observed by throughindependentsystems,andthatpathwaysgov-
using Bcl-2 mutations in mouse HSCs (Domen et al., erning fundamental cell physiology, which include cell
2000).Takentogether,ourresultsrevealthatHES-1and cycle regulation and apoptotic control, are capable of
HLF promote cell cycling and reduce apoptosis,
modulatinghumanHSCfunction.
respectively, thereby enhancing repopulating capacity
ofhumanHSCsviaindependentpathways.
TocharacterizeHES-1andHLFgenetargets,andto Discussion
further investigate potential downstream signaling of
these two transcriptional regulators, transgenic cells The molecular basis that defines human HSCs has
wereisolatedandanalyzedbyQ-RT-PCR.Aswouldbe been under intensive investigation (Georgantas et al.,
predicted, HES-1- and HLF-transduced cells demon- 2004;Ivanovaetal.,2002;Ramalho-Santosetal.,2002;
strated a 20- and 27-fold greater level of HES-1 and Steidlet al.,2002),but fewinsightsinto complexHSC
HLF expression, respectively, compared to vector- behaviorhaveemerged.Thefrequencyofrepopulating
transduced cells (Figure 5A). Consistent with their ob- HSCs or progenitors enriched in all sources of human
served independent mechanisms of action, neither Lin−CD34+CD38− and Lin−CD34+CD38+ cells sug-
HES-1 nor HLF affected the expression of the other geststhatthevastmajorityofcellswithinthesepopula-
(Figure 5A), suggesting limited overlap between their tionsareinfactnotHSCsorprogenitors,andtherefore
respectivepathways.Similartootherstudies(Ronchini these purified subsets represent extremely hetero-
andCapobianco,2001),HES-1upregulatedexpression geneous populations not likely suitable for molecular
ofCDK-2buthadnoeffectonarecentlyidentifiedpu- profiling (Iscove et al., 2002). Despite these concerns,
tativetargetgeneofHLF,LMO2(CrableandAnderson, our unique strategy allowed for identification of two
2003). Conversely, HLF upregulation increased LMO2 candidates’ genes capable of regulating repopulating
expression(Figure5A)buthadnoeffectonCDK-2ex- HSCs and not progenitors, demonstrating the impor-
pression. The distinct roles ofHES-1 and HLF are fur- tanceofthesegenesinfunctionalfeaturesusedtode-
ther supported by the distinct clustering location of fine HSCs enriched in the Lin−CD34+CD38− popula-
thesetwogenesinSOManalysis(FigureS1). tion. Arrival at this result suggests that functional
(DandE)(D)Representativeanalysisand(E)summaryofhumanhematopoieticcellsrepopulatingthebonemarrowofrecipientNOD/SCID
thathaveincorporatedBrd-Uafterinvivoadministration.“*,”statisticallydifferentfromHLF-andvector-transducedcells,p<0.05.
(FandG)(F)HES-1-,HLF-,andvector-transducedCD34+cellswerefixedandstainedwithAnnexin-V-APC,andthepercentageofpreapo-
ptoticcellsissummarizedin(G)andexpressedasthepercentageofapoptoticcellsrelativetovectorcontrol-transducedcells.“*,”statistically
differentfromHES-1-andvector-transducedcells,p<0.05.
(HandI)(H)RepresentativeQ-RT-PCRofBCL-2,SODD,andTOSOexpressioninhumanhematopoieticcellstransducedwithHES-1,HLF,or
vectoraloneasindicated,andthe(I)averagelevelofgeneexpressionsummarized.GAPDHwasusedasahousekeepingcontroltonormalize
cDNAinput.Theaveragelevelofgeneexpressionwasbasedontheresultsoffourindependentexperiments.“*,”theBCL-2geneexpression
levelisstatisticallydifferentfromthatinHES-1-andvector-transducedcells,p<0.05.
DevelopmentalCell
660
Figure5.TargetGeneRegulationinHES-1-,HLF-,andVector-TransducedPrimaryHumanHematopoieticCells
(A)Q-RT-PCRofHES-1,HLF,CDK-2,andLMO2geneswithcDNAfromprimaryhumanhematopoieticcellstransducedwithHES-1orHLFas
indicated.“*,”statisticallydifferent,p<0.05.
(B)ConventionalPCRofcDNA.Lanes:1,molecularmarker;lanes2,3,and4,primaryhumanCBcellstransducedwithHLF;lane5,positive
controlusingdiagnosedhumanacutelymphoblasticleukemiasamplecontainingHLF-E2Arearrangement;lane6,No-RTcontrol.
(C)RegulationofHES-1inpurifiedLin−CD34+CD38−cellsevaluatedbyQ-RT-PCRinresponsetoJagged-1treatmentat24and72hras
comparedtocontrol-treatedcells.“*,”statisticallydifferent,p<0.05.
assays used to determine the frequency of human re- capableofregulatinghumanHSCcapacityviamodula-
populatingcellsinpurifiedsubsetsunderestimateHSC tion of the cell cycle involving upregulation of CDK-2
content, and/or these purified populations are more geneexpression.Unexpectedly,ouranalysisalsoiden-
molecularly homogeneous than originally predicted. tified the DNA binding transcription factor HLF as a
Since our study identifies two candidate genes that uniquely associated factor involved in maintaining the
modulate the repopulation capacity of human HSCs, repopulating capacity of human HSCs by conferring
ouruniquestrategythataccountsforbothhierarchical antiapoptotic effects and preventing premature cell
andontogenicpositioningofhumanHSCsthemselves deaththataremediatedinpartbyBcl-2regulation.The
mayhaveenabled thesefindings.Byfirst defininghu- ideathatHES-1andHLFimpactHSCfunctionviatwo
manHSCsandprogenitorsviaprospectivepurification differentmechanisms, andthatthese mechanismsact
anduseofestablishedfunctionalassays,ourapproach throughfundamentalpathways(cellcycleandapopto-
to classify genes involved in HSC regulation could be sis)commontoallhumancells,suggeststhatspecific
tested via gene reconstitution of identified candidates HSC genes may not molecularly define the basis of
byusingthesamefunctionalcriteriaofHSCrepopula- HSC behavior. Alternatively, a unique combination of
tion onwhich the studywas founded. Webelieve that moregeneralsignalingpathwaysactiveinthemajority
ourreportrepresentsademonstrationofthepowerof ofspecializedcelltypesmaydefinethemolecularsig-
molecular profiling when based on functional criteria nature of human stem cells (Pyle et al., 2004) (e.g.,
andbiologicaloutcomes. genes requiredto establishHSCs duringdevelopment
Identification of HES-1 as one of the upregulated arenotrequiredtosustainormolecularlydefineHSCs).
transcriptsinbothhierarchicalandontogeniccompari- However,independentcategoriesthatdelineatesignal-
sonsunderscorestheroleoftheNotchpathwayinhu- ingpathwaysandtranscriptionfactorsthatare“essen-
manHSCregulation(Karanuetal.,2000b;2001,2003; tial”versusthosethat“augment”existingHSCfunction
Ohishi et al., 2002; Varnum-Finney et al., 2000). Al- are emerging. These categories may be related, but
thoughtheimportanceofNotchsignalingtoHSCswas they may allow access to unique components of HSC
previously appreciated, details of the cellular mecha- behavior.Thosethatfallinthecategoryofessentialare
nismwereunknown.Ourcurrentreportprovidesinde- likelytodisruptHSCfunctionuponinactivationand,as
pendentevidenceforHES-1asaputativeNotchtarget recentstudiesindicate,donotseeminglyincludeJag-