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STEM CELL INFORMATION

AppendixE:StemCellMarkers
AppendixE.i.HowDoResearchersUseMarkerstoIdentifyStemCells?

Inrecentyears,scientistshavediscoveredawidearrayofstemcellsthathaveuniquecapabilitiestoselfrenew,grow
indefinitely,anddifferentiateordevelopintomultipletypesofcellsandtissues.Researchersnowknowthatmanydifferent
typesofstemcellsexistbuttheyallarefoundinverysmallpopulationsinthehumanbody,insomecases1stemcellin
100,000cellsincirculatingblood.And,whenscientistsexaminethesecellsunderamicroscope,theylookjustlikeanyother
cellinthetissuewheretheyarefound.So,howdoscientistsidentifytheseraretypeofcellsfoundinmanydifferentcellsand
tissuesaprocessthatismuchakintofindinganeedleinahaystack?Theanswerisrathersimplethankstostemcell
"markers."Thisfeaturedescribesstemcellmarkertechnologyandhowitisusedintheresearchlaboratory.Followingthisisa
listingofsomeofthecommonlyusedstemcellmarkers(seeAppendixE.ii.MarkersCommonlyUsedtoIdentifyStemCellsand
toCharacterizeDifferentiatedCellTypes).
Whatarestemcellmarkers?Coatingthesurfaceofeverycellinthebodyarespecializedproteins,calledreceptors,thathave
thecapabilityofselectivelybindingoradheringtoother"signaling"molecules.Therearemanydifferenttypesofreceptorsthat
differintheirstructureandaffinityforthesignalingmolecules.Normally,cellsusethesereceptorsandthemoleculesthatbind
tothemasawayofcommunicatingwithothercellsandtocarryouttheirproperfunctionsinthebody.Thesesamecellsurface
receptorsarethestemcellmarkers.Eachcelltype,forexamplealivercell,hasacertaincombinationofreceptorsontheir
surfacethatmakesthemdistinguishablefromotherkindsofcells.Scientistshavetakenadvantageofthebiologicaluniqueness
ofstemcellreceptorsandchemicalpropertiesofcertaincompoundstotagor"mark"cells.Researchersowemuchofthepast
successinfindingandcharacterizingstemcellstotheuseofmarkers.
Stemcellmarkersaregivenshorthandnamesbasedonthemoleculesthatbindtothestemcellsurfacereceptors.For
example,acellthathasthereceptorstemcellantigen1,onitssurface,isidentifiedasSca1.Inmanycases,acombination
ofmultiplemarkersisusedtoidentifyaparticularstemcelltype.Sonow,researchersoftenidentifystemcellsinshorthandby
acombinationofmarkernamesreflectingthepresence(+)orabsence()ofthem.Forexample,aspecialtypeof
hematopoieticstemcellfrombloodandbonemarrowcalled"sidepopulation"or"SP"isdescribedas(CD34/low,cKit+,Sca1+)
[4].
Researchersusethesignalingmoleculesthatselectivelyadheretothereceptorsonthesurfaceofthecellasatoolthatallows
themtoidentifystemcells.Manyyearsago,atechniquewasdevelopedtoattachtothesignalingmoleculeanothermolecule
(orthetag)thathastheabilitytofluoresceoremitlightenergywhenactivatedbyanenergysourcesuchasanultravioletlight
orlaserbeam(seeFigureE.i.1.IdentifyingCellSurfaceMarkersUsingFluorescentTags).Attheresearchers'disposalare
multiplefluorescenttagswithemittedlightthatdifferincolorandintensity.

FigureE.i.1.IdentifyingCellSurfaceMarkersUsingFluorescentTags.

(2001TereseWinslow)
Describedherearetwoapproachesofhowresearchersusethecombinationofthechemicalpropertiesoffluorescenceand
uniquereceptorpatternsoncellsurfacestoidentifyspecificpopulationsofstemcells.Oneapproachforusingmarkersasa
researchtooliswithatechniqueknownasfluorescenceactivatedcellsorting(FACS)(seeFigureE.i.2.LookingforaNeedlein
aHaystack:HowResearchersFindStemCells)[1,3,5].ResearchersoftenuseaFACSinstrumenttosortouttherarestem
cellsfromthemillionsofothercells.Withthistechnique,asuspensionoftaggedcells(i.e.,boundtothecellsurfacemarkers
arefluorescenttags)issentunderpressurethroughaverynarrownozzlesonarrowthatcellsmustpassthroughoneata

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time.Uponexitingthenozzle,cellsthenpass,onebyone,throughalightsource,usuallyalaser,andthenthroughanelectric
field.Thefluorescentcellsbecomenegativelycharged,whilenonfluorescentcellsbecomepositivelycharged.Thecharge
differenceallowsstemcellstobeseparatedfromothercells.Theresearchersnowhaveapopulationofcellsthathaveallofthe
samemarkercharacteristics,andwiththesecellstheycanconducttheirresearch.

FigureE.i.2.LookingforaNeedleinaHaystack:HowResearchersFindStemCells.

(2001TereseWinslow,LydiaKibiuk,CaitlinDuckwall)
Asecondmethodusesstemcellmarkersandtheirfluorescenttagstovisuallyassesscellsastheyexistintissues.Often
researcherswanttoassesshowstemcellsappearintissuesandindoingsotheyuseamicroscopetoevaluatethemrather
thantheFACSinstrument.Inthiscase,athinsliceoftissueisprepared,andthestemcellmarkersaretaggedbythesignaling
moleculethathasthefluorescenttagattached.Thefluorescenttagsarethenactivatedeitherbyspeciallightenergyora
chemicalreaction.Thestemcellswillemitafluorescentlightthatcaneasilybeseenunderthemicroscope.
Geneticandmolecularbiologytechniquesareextensivelyusedtostudyhowcellsbecomespecializedintheorganism's
development.Indoingso,researchershaveidentifiedgenesandtranscriptionfactors(proteinsfoundwithincellsthatregulate
agene'sactivity)thatareuniqueinstemcells.Scientistsusetechniquessuchaspolymerasechainreaction(PCR)todetectthe
presenceofgenesthatare"active"andplayaroleguidingthespecializationofacell.Thistechniquehasishelpfulto
researcherstoidentify"geneticmarkers"thatarecharacteristicofstemcells.Forexample,agenemarkercalledPDX1is
specificforatranscriptionfactorproteinthatinitiatesactivationoftheinsulingene.Researchersusethismarkertoidentify
cellsthatareabletodevelopisletcellsinthepancreas.
Recently,researchershaveappliedageneticengineeringapproachthatusesfluorescence,butisn'tdependentoncellsurface
markers.Theimportanceofthisnewtechniqueisthatitallowsthetrackingofstemcellsastheydifferentiateorbecome
specialized.Scientistshaveinsertedintoastemcella"reportergene"calledgreenfluorescentproteinorGFP[2].Thegeneis
onlyactivatedor"reports"whencellsareundifferentiatedandisturnedoffoncetheybecomespecialized.Onceactivated,the
genedirectsthestemcellstoproduceaproteinthatfluorescesinabrilliantgreencolor(seeFigureE.i.3.MicroscopicImageof
FluorescentLabeledStemCell).ResearchersarenowcouplingthisreportingmethodwiththeFACSandmicroscopicmethods
describedearliertosortcells,identifythemintissues,andnow,trackthemastheydifferentiateorbecomespecialized.

FigureE.i.3.MicroscopicImageofFluorescentLabeledStemCell.

(2001TereseWinslow)

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Thesediscoverytoolsarecommonlyusedinresearchlaboratoriesandclinicstoday,andwilllikelyplayimportantrolesin
advancingstemcellresearch.Therearelimitations,however.Oneofthemisthatasinglemarkeridentifyingpluripotentstem
cells,thosestemcellsthatcanmakeanyothercell,hasyettobefound.Asnewtypesofstemcellsareidentifiedandresearch
applicationsofthembecomeincreasinglycomplex,moresophisticatedtoolswillbedevelopedtomeetinvestigators'needs.For
theforeseeablefuture,markerswillcontinuetoplayamajorroleintherapidlyevolvingworldofstemcellbiology.

References
1. Bonner,W.A.,Hulett,H.R.,Sweet,R.G.,andHerzenberg,L.A.(1972).Fluorescenceactivatedcellsorting.Rev.Sci.
Instrum.43,404409.
2. Eiges,R.,Schuldiner,M.,Drukker,M.,Yanuka,O.,ItskovitzEldor,J.,andBenvenisty,N.(2001).Establishmentof
humanembryonicstencelltransducedclonescarryingamarkerofundifferentiatedcells.Curr.Biol.11,514518.
3. Herzenberg,L.A.andDeRosa,S.C.(2000).MonoclonalantibodiesandtheFACS:complementarytoolsfor
immunobiologyandmedicine.Immunol.Today.21,383390.
4. Jackson,K.,Majka,S.M.,Wang,H.,Pocius,J.,Hartley,C.J.,Majesky,M.W.,Entman,M.L.,Michael,L.H.,Hirschi,K.K.,
andGoodell,M.A.(2001).Regenerationofischemiccardiacmuscleandvascularendotheliumbyadultstemcells.J.Clin.
Invest.107(11),13951402.
5. Julius,M.H.,Masuda,T.,andHerzenberg,L.A.(1972).Demonstrationthatantigenbindingcellsareprecursorsof
antibodyproducingcellsafterpurificationwithafluorescenceactivatedcellsorter.Proc.Natl.Acad.Sci.U.S.A.69,
19341938.
AppendixE.ii.MarkersCommonlyUsedtoIdentifyStemCellsandtoCharacterizeDifferentiatedCellTypes
MarkerName

CellType

Significance

Fetalliver
kinase1(Flk1)

Endothelial

Cellsurfacereceptorproteinthatidentifiesendothelialcellprogenitormarkerofcellcellcontacts

Smoothmuscle
cellspecific
myosinheavy
chain

Smoothmuscle

Identifiessmoothmusclecellsinthewallofbloodvessels

Vascular
endothelialcell
cadherin

Smoothmuscle

Identifiessmoothmusclecellsinthewallofbloodvessels

Bonespecific
alkaline
phosphatase
(BAP)

Osteoblast

Enzymeexpressedinosteoblastactivityindicatesboneformation

Hydroxyapatite

Osteoblast

Minerlizedbonematrixthatprovidesstructuralintegritymarkerofboneformation

Osteocalcin(OC)

Osteoblast

Mineralbindingproteinuniquelysynthesizedbyosteoblastmarkerofboneformation

BloodVessel

Bone

BoneMarrowandBlood
Bone
morphogenetic
proteinreceptor
(BMPR)

Mesenchymalstem
andprogenitorcells

Importantforthedifferentiationofcommittedmesenchymalcelltypesfrommesenchymalstemand
progenitorcellsBMPRidentifiesearlymesenchymallineages(stemandprogenitorcells)

CD4andCD8

Whitebloodcell(WBC)

CellsurfaceproteinmarkersspecificformatureTlymphocyte(WBCsubtype)

CD34

Hematopoieticstem
cell(HSC),satellite,
endothelialprogenitor

Cellsurfaceproteinonbonemarrowcell,indicativeofaHSCandendothelialprogenitorCD34alsoidentifies
musclesatellite,amusclestemcell

CD34+Sca1+Lin
profile

Mesencyhmalstemcell
(MSC)

IdentifiesMSCs,whichcandifferentiateintoadipocyte,osteocyte,chondrocyte,andmyocyte

CD38

AbsentonHSC
PresentonWBC
lineages

CellsurfacemoleculethatidentifiesWBClineages.SelectionofCD34+/CD38cellsallowsforpurificationof
HSCpopulations

CD44

Mesenchymal

Atypeofcelladhesionmoleculeusedtoidentifyspecifictypesofmesenchymalcells

cKit

HSC,MSC

CellsurfacereceptoronBMcelltypesthatidentifiesHSCandMSCbindingbyfetalcalfserum(FCS)
enhancesproliferationofEScells,HSCs,MSCs,andhematopoieticprogenitorcells

Colonyforming
unit(CFU)

HSC,MSCprogenitor

CFUassaydetectstheabilityofasinglestemcellorprogenitorcelltogiverisetooneormorecelllineages,
suchasredbloodcell(RBC)and/orwhitebloodcell(WBC)lineages

Fibroblast
colonyforming
unit(CFUF)

Bonemarrow
fibroblast

Anindividualbonemarrowcellthathasgivenrisetoacolonyofmultipotentfibroblasticcellssuchidentified
cellsareprecursorsofdifferentiatedmesenchymallineages

Hoechstdye

AbsentonHSC

FluorescentdyethatbindsDNAHSCextrudesthedyeandstainslightlycomparedwithothercelltypes

Leukocyte

WBC

CellsurfaceproteinonWBCprogenitor

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Leukocyte
commonantigen
(CD45)

WBC

CellsurfaceproteinonWBCprogenitor

Lineagesurface
antigen(Lin)

HSC,MSC
DifferentiatedRBCand
WBClineages

Thirteento14differentcellsurfaceproteinsthataremarkersofmaturebloodcelllineagesdetectionofLin
negativecellsassistsinthepurificationofHSCandhematopoieticprogenitorpopulations

Mac1

WBC

Cellsurfaceproteinspecificformaturegranulocyteandmacrophage(WBCsubtypes)

Muc18(CD146)

Bonemarrow
fibroblasts,endothelial

Cellsurfaceprotein(immunoglobulinsuperfamily)foundonbonemarrowfibroblasts,whichmaybe
importantinhematopoiesisasubpopulationofMuc18+cellsaremesenchymalprecursors

Stemcell
antigen(Sca1)

HSC,MSC

Cellsurfaceproteinonbonemarrow(BM)cell,indicativeofHSCandMSCBoneMarrowandBloodcont.

Stro1antigen

Stromal
(mesenchymal)
precursorcells,
hematopoieticcells

Cellsurfaceglycoproteinonsubsetsofbonemarrowstromal(mesenchymal)cellsselectionofStro1+cells
assistsinisolatingmesenchymalprecursorcells,whicharemultipotentcellsthatgiverisetoadipocytes,
osteocytes,smoothmyocytes,fibroblasts,chondrocytes,andbloodcells

Thy1

HSC,MSC

CellsurfaceproteinnegativeorlowdetectionissuggestiveofHSC

CollagentypesII
andIV

Chondrocyte

Structuralproteinsproducedspecificallybychondrocyte

Keratin

Keratinocyte

Principalproteinofskinidentifiesdifferentiatedkeratinocyte

Sulfated
proteoglycan

Chondrocyte

Moleculefoundinconnectivetissuessynthesizedbychondrocyte

Adipocytelipid
bindingprotein
(ALBP)

Adipocyte

Lipidbindingproteinlocatedspecificallyinadipocyte

Fattyacid
transporter
(FAT)

Adipocyte

Transportmoleculelocatedspecificallyinadipocyte

Adipocytelipid
bindingprotein
(ALBP)

Adipocyte

Lipidbindingproteinlocatedspecificallyinadipocyte

Ychromosome

Malecells

Malespecificchromosomeusedinlabelinganddetectingdonorcellsinfemaletransplantrecipients

Karyotype

Mostcelltypes

Analysisofchromosomestructureandnumberinacell

Albumin

Hepatocyte

Principalproteinproducedbytheliverindicatesfunctioningofmaturingandfullydifferentiatedhepatocytes

B1integrin

Hepatocyte

Celladhesionmoleculeimportantincellcellinteractionsmarkerexpressedduringdevelopmentofliver

CD133

Neuralstemcell,HSC

Cellsurfaceproteinthatidentifiesneuralstemcells,whichgiverisetoneuronsandglialcells

Glialfibrillary
acidicprotein
(GFAP)

Astrocyte

Proteinspecificallyproducedbyastrocyte

Microtubule
associated
protein2(MAP
2)

Neuron

DendritespecificMAPproteinfoundspecificallyindendriticbranchingofneuron

Myelinbasic
protein(MPB)

Oligodendrocyte

Proteinproducedbymatureoligodendrocyteslocatedinthemyelinsheathsurroundingneuronalstructures

Nestin

Neuralprogenitor

Intermediatefilamentstructuralproteinexpressedinprimitiveneuraltissue

Neuraltubulin

Neuron

Importantstructuralproteinforneuronidentifiesdifferentiatedneuron

Neurofilament
(NF)

Neuron

Importantstructuralproteinforneuronidentifiesdifferentiatedneuron

Neurosphere

Embryoidbody(EB),
ES

ClusterofprimitiveneuralcellsincultureofdifferentiatingEScellsindicatespresenceofearlyneuronsand
glia

Noggin

Neuron

Aneuronspecificgeneexpressedduringthedevelopmentofneurons

O4

Oligodendrocyte

Cellsurfacemarkeronimmature,developingoligodendrocyte

O1

Oligodendrocyte

Cellsurfacemarkerthatcharacterizesmatureoligodendrocyte

Cartilage

Fat

General

Liver

NervousSystem

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Synaptophysin

Neuron

Neuronalproteinlocatedinsynapsesindicatesconnectionsbetweenneurons

Tau

Neuron

TypeofMAPhelpsmaintainstructureoftheaxon

Cytokeratin19
(CK19)

Pancreaticepithelium

CK19identifiesspecificpancreaticepithelialcellsthatareprogenitorsforisletcellsandductalcells

Glucagon

Pancreaticislet

Expressedbyalphaisletcellofpancreas

Insulin

Pancreaticislet

ExpressedbybetaisletcellofpancreasPancreas

Insulin
promoting
factor1(PDX1)

Pancreaticislet

Transcriptionfactorexpressedbybetaisletcellofpancreas

Nestin

Pancreaticprogenitor

Structuralfilamentproteinindicativeofprogenitorcelllinesincludingpancreatic

Pancreatic
polypeptide

Pancreaticislet

Expressedbygammaisletcellofpancreas

Somatostatin

Pancreaticislet

Expressedbydeltaisletcellofpancreas

Pancreas

PluripotentStemCells
Alkaline
phosphatase

Embryonicstem(ES),
embryonalcarcinoma
(EC)

Elevatedexpressionofthisenzymeisassociatedwithundifferentiatedpluripotentstemcell(PSC)

Alpha
fetoprotein(AFP)

Endoderm

ProteinexpressedduringdevelopmentofprimitiveendodermreflectsendodermaldifferentiationPluripotent
StemCells

Bone
morphogenetic
protein4

Mesoderm

Growthanddifferentiationfactorexpressedduringearlymesodermformationanddifferentiation

Brachyury

Mesoderm

Transcriptionfactorimportantintheearliestphasesofmesodermformationanddifferentiationusedasthe
earliestindicatorofmesodermformation

Cluster
designation30
(CD30)

ES,EC

SurfacereceptormoleculefoundspecificallyonPSC

Cripto(TDGF1)

ES,cardiomyocyte

GeneforgrowthfactorexpressedbyEScells,primitiveectoderm,anddevelopingcardiomyocyte

GATA4gene

Endoderm

ExpressionincreasesasESdifferentiatesintoendoderm

GCTM2

ES,EC

AntibodytoaspecificextracellularmatrixmoleculethatissynthesizedbyundifferentiatedPSCs

Genesis

ES,EC

TranscriptionfactoruniquelyexpressedbyEScellseitherinorduringtheundifferentiatedstateofPSCs

Germcell
nuclearfactor

ES,EC

TranscriptionfactorexpressedbyPSCs

Hepatocyte
nuclearfactor4
(HNF4)

Endoderm

Transcriptionfactorexpressedearlyinendodermformation

Nestin

Ectoderm,neuraland
pancreaticprogenitor

Intermediatefilamentswithincellscharacteristicofprimitiveneuroectodermformation

Neuronalcell
adhesion
molecule(N
CAM)

Ectoderm

Cellsurfacemoleculethatpromotescellcellinteractionindicatesprimitiveneuroectodermformation

OCT4/POU5F1

ES,EC

TranscriptionfactoruniquetoPSCsessentialforestablishmentandmaintenanceofundifferentiatedPSCs

Pax6

Ectoderm

TranscriptionfactorexpressedasEScelldifferentiatesintoneuroepithelium

Stagespecific
embryonic
antigen3
(SSEA3)

ES,EC

GlycoproteinspecificallyexpressedinearlyembryonicdevelopmentandbyundifferentiatedPSCs

Stagespecific
embryonic
antigen4
(SSEA4)

ES,EC

GlycoproteinspecificallyexpressedinearlyembryonicdevelopmentandbyundifferentiatedPSCs

Stemcellfactor
(SCForcKit
ligand)

ES,EC,HSC,MSC

MembraneproteinthatenhancesproliferationofESandECcells,hematopoieticstemcell(HSCs),and
mesenchymalstemcells(MSCs)bindsthereceptorcKit

Telomerase

ES,EC

AnenzymeuniquelyassociatedwithimmortalcelllinesusefulforidentifyingundifferentiatedPSCs

TRA160

ES,EC

AntibodytoaspecificextracellularmatrixmoleculeissynthesizedbyundifferentiatedPSCs

TRA181

ES,EC

AntibodytoaspecificextracellularmatrixmoleculenormallysynthesizedbyundifferentiatedPSCs

Vimentin

Ectoderm,neuraland

Intermediatefilamentswithincellscharacteristicofprimitiveneuroectodermformation

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Vimentin

Ectoderm,neuraland
pancreaticprogenitor

Intermediatefilamentswithincellscharacteristicofprimitiveneuroectodermformation

SkeletalMuscle/Cardiac/SmoothMuscle
MyoDandPax7

Myoblast,myocyte

Transcriptionfactorsthatdirectdifferentiationofmyoblastsintomaturemyocytes

Myogeninand
MR4

Skeletalmyocyte

Secondarytranscriptionfactorsrequiredfordifferentiationofmyoblastsfrommusclestemcells

Myosinheavy
chain

Cardiomyocyte

Acomponentofstructuralandcontractileproteinfoundincardiomyocyte

Myosinlight
chain

Skeletalmyocyte

Acomponentofstructuralandcontractileproteinfoundinskeletalmyocyte

AppendixD|TableofContents|AppendixF
Historicalcontent:June17,2001

Pagecitation:AppendixE:StemCellMarkers.InStemCellInformation[WorldWideWebsite].Bethesda,MD:NationalInstitutesofHealth,U.S.Departmentof
HealthandHumanServices,2015[citedThursday,June23,2016]Availableat<http://stemcells.nih.gov/info/scireport/pages/appendixE.aspx>
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