bio quark invitro manuscript
TRANSCRIPT
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CoelectroporationwithXenopuslaevisOocytesReprogramsNormalandCancerousHuman
CellstoResembleReprogrammingNormalandCancerousHumanCellsto
ResembleInducedHumanPluripotentStemCells
SergeiPaylian*
*Bioquark,Inc.,P.O.Box46686,Tampa,FL336466686,USA
Correspondingauthor:SergeiPaylian;LaboratoryPhone/Fax:8138887307
email:[email protected]
Length
Abstract: 173words(limitof175)
Text:4079words
References:45
Tables:1
Figures:9
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ABSTRACT
Objective:Toinvestigatereprogramminghumancellsintoinducedpluripotentstemcells(iPSc)
usingcoelectroporationwithXenopuslaevisoocytes.
Methods:Humanbonemarrowstromalcells(BMSC),BJcells,preadipocytes(HPA),CD4+T
lymphocytes(CD4TLs),buccalmucosacells,andHeLaandMCF7 cellswerecoelectroporated
withmatureXenopuslaevisoocytes,cultured,andassessedforpluripotencymarkerexpression
usingfluorescentimmunohistochemistry.
Results: Thecoelectroporatedhumancellsformedcoloniesonirradiatedmouseembryonic
fibroblastcells(allstudycells)andStemAdhereTMsubstrate(assessedforcoelectroporated
buccalmucosacells).CellsderivedfromBMSC,BJcells,HPA,CD4TLs,andbuccalmucosacells
expressedOct3/4,Nanog,SOX2,Rex1,TRA160,andSSEA1.Cellsderivedfromco
electroporatedHeLacellsexpressedOct3/4;cellsderivedfromcoelectroporatedMCF7cells
expressedOct3/4andNanog.ReprogrammingefficacyforCD4TLswas23.43.5%.Co
electroporatedHPAtransdifferentiatedintoneuralprogenitorcellsincultureconditionsthat
fosterneuraldifferentiation.Controlexperimentssuggestedthattheelectroporateconveyeda
reprogrammingfactor(s).
Conclusions:HumancellscoelectroporatedwithXenopuslaevisoocytesresemblediPScin
colonyformationandpluripotencyassociatedmarkerexpression.
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INTRODUCTION
Inducedpluripotentstemcells(iPSc)constituteapotentialsourceofcellsforstemcelltherapy
thatavoidsthebioethicalconcernssurroundingtheuseofembryonicstemcells(ES).1 Recent
advancesinnonviralreprogrammingmethodologyincludetheuseofrecombinantproteins,2
DHPderivative(novelantioxidant)andlowoxygentensionconditions,3microRNAs,
46,7,8zinc
fingernucleases,9drugs,
10,11,12 hypoxia,
13silencingthep53pathway
14andEScellderived
proteinextracts.15
Unfortunately,contemporarymethodsarehamperedbythelowefficacyof
reprogramminghumansomaticcellsintoiPScandthenonautologousnatureofthefinal
product.16
17,18
Manyspecieshaveevolvedmechanismsforcellularreprogramming;pathwaysforinducing
cellulardedifferentiationandredifferentiationexistawidevarietyoforganismsincludingsome
speciesofbacteria,19
plants,20,21
andloweranimals.22
Thepoorefficacyofreprogramming
achievedusingcurrentapproachesmaybeduetheuseofmethodsthatlackvital
reprogrammingcomponentsnaturallypresentinsomelivingorganisms.
Inthepresentstudy,Xenopuslaevisoocyteswerechosenasasourceofnaturalreprogramming
factor(s)basedonthesuccessfulreprogrammingeventsreportedformammaliansomaticcells
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associatedtranscriptionfactorsrecognizedas markersofpluripotency.Furthermore,thisco
electroporationtechniqueachievedarelativelyhighlevelofreprogrammingefficiency.
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MATERIALSANDMETHODS
Thehumansubjectswhoprovidedbuccalmucosatissuesamples(usinganoninvasive
technique)gavewritteninformedconsent.ProceduresinvolvingXenopuslaeviswere
conductedinaccordancewithpublishedlaboratorystandards.24
PreparationofXenopuslaevisOocytes
Female,eggbearingXenopuslaevis(NASCO)wereatkeptat18Cusinga12/12hour
light/darkcycleincarbonfilteredwatersupplementedwith13.3g/gallonNaCl,whichwas
changeddaily.24
Priortosurgicalremovalofoocytes,frogswereanesthetizedinaplasticbeakercontaining1L
of0.2%tricanesolution(Sigma)forupto20minand,then,placedonadissectingpanfilled
withice.Aftera0.5cmincisionthroughtheskinandmusclelayers,thebagsofovarieswere
removedandplacedintooocytewashing(OW)solution(82.5mMNaCl,5.0mM2[4(2
hydroxyethyl)piperazin1yl]ethanesulfonicacid[HEPES],2.5mMKCl,1mMMgCl2,1.0mM
Na2HPO4,and0.5%penicillin/streptomycin[pen/strep]atpH7.4[penicillinandstreptomycin
fromGibco;othersfromSigma]).Bagscontainingovarieswereopenedwithfineforceps,the
ovarieswererinsedseveraltimesinOW,andtreatedwitha0.2%collagenasetypeIIsolution
(WorthingtonBiochemicalCorporation)for 1houratroomtemperature.Thedefolliculated
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serum(Sigma)titratedtopH7.4.Recoveredoocytesinthefinalstageofmaturitywere
collectedinsterile6wellcellcultureclusters(Costar)prefilledwithanHBsolutionand
incubatedat17Cfor24hoursbeforeelectroporationexperiments.
CellLines
HumanbonemarrowstromalCells(BMSCs)andstablytransfectedGFPexpressingBMSCs
(BMSCGFP)wereprovidedbyTulaneUniversityCenterofGeneTherapy.Priortoreleasefrom
thesource,twotrialsoffrozen,passage1cellswereanalyzedoverthreepassagesforcolony
formingunits,cellgrowth,anddifferentiationintofat,bone,andchondrocytes.TheBMSCand
BMSCGFPwereculturedinDulbeccosmodifiedEaglesMedium(DMEM;Sigma),supplemented
with10%fetalbovineserum(FBS;Gibco)and1%streptomycin/penicillin(Gibco)andcultured
in25cm2flasksat37Cwith5%CO2.Atday4,thecultureswerewashedwithphosphate
bufferedsaline(PBS;Sigma)toremovethenonadherentcellsandfurtherexpandeduntil
80%confluence,whentheywereharvestedandexpandedin75cm2flasks.
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Humansubcutaneouspreadipocytes(HPA)fromScienCellResearchLaboratorieswerecultured
at37Cand5%CO2inT25flaskscoatedwith0.01%polylysine(Sigma)andcontaining5mLof
speciallyformulatedpreadipocytemedium(PAM;ScienCells);PAMwassupplementedwith5
%FBS,1mMsodiumpyruvate,0.1mMNEAA,and1%pen/strep.
HumanperipheralbloodCD4+Tlymphocytes(CD4TLs)fromLonzaGroup,Ltd.(pathogenfree
poieticsCD4TLs)weremaintainedasacellsuspensioninT25cultureflasksat37Cand5%CO2
in5mLoflymphocytegrowthmedium3(LGM3,LonzaGroupLtd.)supplementedwith
10%FBS,1mMsodiumpyruvate,0.1mMnonessentialaminoacids,1%pen/strep,and50
ng/mLrecombinanthumanInterleukin4(R&DSystems).
Humanbuccalmucosacellswereobtainedfromhealthyhumansubjectsapproximately1hourbeforethecoelectroporationprocedure.Subjectsabstainedfromdrinkingcoffeefor1hour
beforecollection.SubjectsmouthswererinsedtwicewithListerinefollowedbysterile
distilledwaterbeforeswabbing.Cellswerecollectedbyswabbingfirmlyontheinsideofthe
cheek20timesonbothsidesusingaMasterAmpBuccalSwabBrush(Epicentre
Biotechnologies).Thebrushholdingcheekcellswasplacedintoa50mLcentrifugetubefilled
with20mLofsterilefilteredPBS(Sigma)containing1%pen/strep.Thesamplewasvigorously
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Humancervicalcarcinoma(HeLa)cells(routinelymaintainedattheBioquark,Inc.facility)were
grownat37Cand5%CO2inT25flasksfilledwith5mLofEaglesessentialmedium(ATCC)
supplementedwith10%FBS,1mMsodiumpyruvate,0.1mMNEAA,and1%pen/strep.
Humanbreastadenocarcinoma(MCF7)cellsfromATCCweremaintainedinEaglesMinimum
EssentialMediumsupplementedwith10%FBS,1mMsodiumpyruvate,0.1mMNEAA,1%
pen/strep,and0.01mg/mLrecombinanthumaninsulin(EliLilly;agiftfromNorthSuburban
Pharmacy,Skokie,IL)
Irradiatedmouseembryonicfibroblasts(iMEF;AmericanR&DSystems)weregrownat37C
and5%CO2innonpyrogenic,sterile25cm2,0.2mventilatedcellcultureflasks(T25;Corning)
containing5mLofhighglucoseDMEM(Millipore)supplementedwith10%FBS,1mMsodium
pyruvate,0.1mMNEAA,and1%pen/strep.
CoelectroporationofXenopuslaevisOocyteswithHumanCells
ElectroporationparametersforXenopusLaevisoocytesweredevelopedfromseveralpublished
studiesofelectroporation.2527
Fortytofiftyfreshoocytesfromsuspensionswith 90%viability
(oocytesshowingabnormalpigmentdistributionorsignsofdamageofequatorialband,patchy
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serumfreeDMEMandthenplacedintotheshockingchamber.Coelectroporationoffrog
oocyteswiththesuspensionofhumancellswasconductedusingthefollowingparameters:
150v/cm/25F/7pulses,withtimeconstantat0.5 0.7msec.Afterelectroporation,
cuvettescontainingoocytesandthehumancellswereincubatedat17oCforthreehoursto
recover.ThehumancellsweretransferredtoT25cultureflaskscontainingiMEFfeedercellsfor
culturing.
CulturingofHumanCellsFollowingCoelectroporation
Thecoelectroporatedhumancellswereculturedat37oConiMEFfeedercellsin0.1%gelatin
coated(gelatinfromSigma)T25cultureflaskscontaining5mLofspeciallyformulated
EmbryomaxDMEMculturemedium(Millipore).Mediumwassupplementedwith15%FBS,1
mMsodiumpyruvate,0.1mMNEAA,1%pen/strep,100 Mbetamercaptoethanol(Gibco),
and1000U/mLESGRO(Millipore).Tomaintainthecellsinanembryonicstemcelllikestate,
1000UESGROper1.0mLoftissueculturemediawasrequired.Afterformationofclusters,
thehumancellswereseparatedfromthefeedercellsusingthedifferentialsedimentation
techniquepreviouslydescribedbyDoetschman,28
whichremoved>99%ofcontaminating
feedercellsfromtheelectroporatedhumancellsuspension.Trypsinized(trypsinfromSigma)
humancellculturescontainingiMEFswerecentrifugedat200g,resuspendedin10mLof
completeESculturemedium,andtransferredtoanewT25cellcultureflaskfor30minutesat
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andthecellswerecounted,centrifugedagainat200g,andresuspendedintheESculture
medium.
Subculturing
Afterseparationfromthefeedercells,thehumancellswereplatedonT25cultureflaskscontaining
eitheriMEFfeedercellsorfeederfreeStemAdherepluripotencysubstrate(Primorigen
Biosciences).SubculturedhumancellsweregrowninNutriStem(StemGent).
CalculationofReprogrammingEfficacy
FluorescentimmunohistochemicallydetectableexpressionoftheNanoggenebycellsderived
fromCD4TLsoccurredbetween12h24hfollowingcoelectroporationwithXenopuslaevis
oocytes.ThisexpressionprecededtheformationoftightiPSclikeclusters,makingitpossibleto
determinetheefficiencyofreprogrammingbycalculatingtheproportionofcellsexpressing
Nanoggene.Themeanforthereprogrammingefficiencywascalculatedbycountingthetotal
numberofNanogpositivecellsperspecimenineachT25flask(34times),subtractingthe
numberofnonspecificbindingsitesinthecontrolflasks,dividingbytheoriginalnumberofcells
havingundergonecoelectroporationandmultiplyingby100%.Thestandarddeviationofthe
meanwasalsocalculated.
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medium(60%FBS,20%hEScellculturemedium,and20%dimethylsulfoxide.Cryovialswere
transferredto5100Cryo1CFreezingContainer(Nalgene),refrigeratedat 80Covernightand
thenrapidlytransferredtoliquidnitrogenrefrigerationunits.
TransdifferentiationintoNeuronalProgenitorCells
Afterformationofclusters,reprogrammedcellsderivedfromHPAwereseparatedfromthe
feederlayerusingtheDoetschmandifferentialsedimentationtechnique,28
andwere
dissociatedenzymaticallyusingcollagenaseIV(Sigma;200U/mL)for30minat37oCgenerating
acellsuspensioncontainingsmallcellaggregatesandsinglecells. Cellcultureconditionsfor
growingneuralprogenitorcells(NPs)fromembryonicstemcellswereemployed.30
Thecells
werewashedinwarmNeurobasalAmedium(GibcoBRL/Invitrogen),pelletedandresuspended
inprewarmed(37C)standardhumanembryonicstemcellculturingmedium(hESC)
supplementedwithfollowinggrowthfactorsandneuronalandothersupplements:fibroblast
growthfactor2(10ng/mL),epidermalgrowthfactor(20ng/mL),1%B27,1%N2,1%pen/strep,
1%lglutamine,1%nonessentialaminoacids(NEAA),0.2%betamercaptoethanol,and20%
KnockoutSerumReplacement(allmediacomponentsfromGibco BRL/Invitrogen).TheHPA
derivedcellsinsuspensionwerethenseededathighcelldensity(150200103cells/cm2)onto
BDBioCoatandlaminincoated150mmpetridishes(BecksonDickenson),andthemedium
wassupplementedwithhESCmediumand4ng/mLfibroblastgrowthfactor2.Proliferating
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thesameconditions,thusgeneratingamonolayerpopulationofproliferatingneural
progenitors.
QualitativeAssessmentofColonyMorphology
Assessmentofcolonymorphology(resemblancetoiPSccolonies)wasperformedbyDr.Nikolai
Strelchenko,PhDofthehESCResearchLabatReproductiveGeneticsInstitute,Chicago,IL,USA
andDr.ArshakAlexanian,VMD,PhD,oftheDepartmentofNeurosurgery,Neuroscience
ResearchLaboratories,ZablockiVeteransAffairsMedicalCenterandofMedicalCollegeof
Wisconsin,Milwaukee,WI,USA.
AlkalinePhosphataseStainingandFluorescentImmunocytochemistry
Histochemicalstainingforalkalinephosphatase(AP)wasconductedusingtheVectorBlue
AlkalinePhosphataseSubstrateKitIII(VectorLaboratories,Inc.).Expressionofseveral
pluripotencyfactorswasassayedusingfluorescentimmunohistochemistryconductedatroom
temperature.Samplesfromall populationsofhumancellsinT25cultureflaskswentthrough
thefollowingsteps:(a)thegrowthmediumwasremoved,(b)washedthreetimeswithPBS,c)
fixedin 10 Cmethanol,c)washedthreetimeswithPBS,d)incubatedfor20minin10%
normalserum,e)incubatedfor60min.inprimaryantibodydilutedin1.5%normalserum,f)
washedthreetimeswithPBS,g)incubatedfor45min.inthedarkwithsecondaryantibody
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reagent2%DABCO(Sigma),andk)processedT25flaskswithspecimensweresealedwith
parafilm,wrappedinaluminumfoilandstoredat4 C.
Theprimaryandsecondaryantibodiesandnormalsera(2.5g/mL)includedpolyclonalgoat
antiOct3/4IgG,polyclonalgoatantiNanogIgG,polyclonalgoatantiSox2IgG,monoclonal
mouseantiTRA160IgG,monoclonalmouseantiSSEA1IgM,polyclonalgoatantiRex1IgG,
goatantimouselgMTR,donkeyantimouselgGFITC,donkeyantigoatIgGFITC,donkeyanti
goatIgGTR,normaldonkeyserum,andnormalgoatserum(allfromSantaCruzBiotechnology,
Inc).Antiseratothefollowingwereusedtoanalyzeformationofneuralprogenitorcells:nestin
(1:500dilution,BDPharmingen),beta3tubulinmonoclonalantibody(B3T;10g/mL;Pierce
antibodies),neuralcelladhesionmolecule(NCAM),1:500dilution(Abcam),glialfibrillaryacidic
protein(GFAP,1:250dilution(Abcam).DNAstainingwasperformedusing4',6diamidino2
phenylindole,4',6diamidinophenylindole(DAPI;SantaCruzBiotechnology,Inc.)
ControlExperiments
ThecontrolsdescribedinTable1wereusedtotestfortheeffectofthepresenceofhuman
cells,oocytes,feedercells,coelectroporation,andtheelectroporateonreprogramming
(expressionofNanog;detectedusingfluorescentimmunohistochemistry).
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RESULTS
Controls
Table1listsfindingsfromthecontrolexperimentsconductedonallhumancelltypesusedin
thisstudy.Nanogwasnotdetectedinhumancellsfromcontrolsa,b,c,andfAsmall
numberofhumancellsfromcontrold,inwhichnonelectroporatedhuman cellswere
exposedfor3hourstoelectroporate,expressedtheNanoggene(reprogrammingefficiencyof
0.4%;calculatedonlyforCD4TLs). Asimilarlylownumberofhumancellsfromcontrole
expressedtheNanoggene(0.9%efficiency,calculatedonlyforCD4TLs);inthiscontrol,human
cellswereelectroporatedintheabsenceofoocytesandthenwereexposedtoelectroporate
for3hours.
BMSCandBMSCGFP
WithinoneweekofcoelectroporationwithXenopuslaevisoocytes,cellsderivedfromhuman
BMCSstronglycoculturedwithiMEFcellsexpressedthepluripotencyassociatedtranscription
factorsOct3/4,SOX2,Nanog,Rex1,andSSEA1andformedcoloniesresemblingthoseknown
toformbyiPScincultureinculture(Figure1).
Inseparatestudies,BMSCGFPwerecoelectroporatedwithXenopusoocytesandgrownoniMEF
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BJCells
CoelectroporationinthepresenceofXenopusoocytes,followedbycocultureoniMEFfeeder
cells,resultedinreprogrammingofBJcells,evidencedbyahighlevelofalkalinephosphatase
activityandresemblancetoiPScincolonymorphologyandtheexpressionofOct3/4,Nanog,
SOX2,TRA160,Rex1,andSSEA1(Figure2).
HPACellsReprogramming,Cryopreservation,andTransdifferentiation
AftercoelectroporationofHPAandcocultureonfeedercells,thehumancellsformedcolonies
morphologicallysimilartothoseofiPSc(Figure3).ThereprogrammedHPAderivedcells
displayedstrongalkalinephosphataseactivity(Figure3).Thecellsinthesecoloniesstrongly
expressedOct3/4,Nanog,SOX2,TRA160,Rex1,andSSEA1(Figure3).
OnemonthaftercryopreservationofthereprogrammedHPAderivedcells,thereprogrammed
cellswerethawed,resultingin78%viability. Byday4aftersubculturingonfreshfeedercells
thereprogrammedHPAderivedcellsformedsecondaryclustersresemblingthoseformedby
iPSc(datanotshown).
SubculturingcellsderivedfromHPAfollowingcoelectroporationinconditionsthatpromote
the neural differentiation of embryonic stem cells resulted in formation of cells expressing
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CD4TLsReprogrammingandEfficiency
Within3to5daysaftertransfertofeedercelllayersfollowingcoelectroporationwithXenopus
laevisoocytes,thehumanCD4TLsformedcoloniessimilartothoseformedbyiPSc.Cellsin
thesecolonieshadhighlevelsofalkalinephosphataseactivity(Figure5)andstronglyexpressed
Oct3/4,Nanog,SOX2,TRA160,Rex1,andSSEA(Figure6).
Within12to24hoursaftercoelectroporationwithXenopuslaevisoocytes,thecellsderived
fromhumanCD4TLscoculturedwithiMEFstartedtoexpresstheNanoggene.Duringthistime
period,singlecellsandsmall iPSclikeclustersinwhichindividualcellscouldbecountedwere
present(datanotshown).TheproportionofcellsexpressingNanogandthetotalnumberof
cellswerecountedforcalculationofreprogrammingefficacy,whichwas23.43.5%.
HumanBuccalMucosaCells
Freshlyobtainedhumanbuccalmucosacells,coelectroporatedinthepresenceofXenopus
oocytesandculturedoniMEFandonfeedercellfreeStemAdheresubstrate,gaverisetocells
thatformedcoloniessimilartothoseofiPSc(Figure7).Cellsinthesecoloniesexpressed
Oct3/4,Nanog,SOX2,TRA160,Rex1,andSSEA1(Figure8).
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Twohumancancercelllines,HeLaandMCF7,weresubjectedtocoelectroporationwith
Xenopuslaevis
oocytesfollowedbycocultureoniMEF.Thecellsderivedfromco
electroporationofthesetumorcellsshowedpartialdedifferentiation,withformationof
clustersandexpressionofOct3/4(HeLaderivedcellsandMCF7derivedcells.Figure9)and
Nanog(MCF7derivedcells;Figure9).Thecellclusterstendedtobesmallerthanthosederived
fromcoelectroporationofnontumorcells(datanotshown).
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DISCUSSION
Thelimitedabilityofmanyhumantissuestoregeneratehasspurredinterestinmethodsto
produceiPScfortherapeuticapplications.Weevaluatedanewmethodologyforthenonviral
reprogrammingofcellsintoiPSc.UsingcoelectroporationoflivingXenopuslaevisoocyteswith
varioushumannormalandcancercelllines,weobtainedcellsresemblingiPScasevidencedby
colonymorphologyandexpressionofhumaniPScmarkers.
HumanbonemarrowstromalcellsshowedsignsofreprogrammingintocellsresemblingiPSc,
withcolonyformationandstrongexpressionthepluripotencyassociatedtranscriptionfactors
Oct3/4,Nanog,SOX2,TRA160,Rex1,andSSEA1.TheisolationofhumanBMSCsrepresents
aroutineprocedureatmanyhospitals,andthisnewmethodforthegenerationofhuman
BMSCderivediPScmaypresentopportunitiesfortheirtherapeuticapplicationsinhumans.
Becauseofthepioneeringstudiesonsuccessfulretroviralvectormediatedreprogrammingof
fibroblastcelllines,31
wetestedreprogrammingofBJcellsusingXenopuslaevisoocyteco
electroporation. CulturedBJcellswerereprogrammedintocellsthatresemblediPSc.These
cellsformedclusterswithhighAPactivityandstrongexpressionofmajorstemcellmarkers,
namelyOct3/4,Nanog,SOX2,TRA160,Rex1,SSEA1.ThesuccessfulreprogrammingofBJ
cellsprovidedfurtherevidenceoftheeffectivenessofthisreprogrammingmethod.
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50gofhumanadiposetissueobtainedfromaclinicthatperformsliposuction.
CoelectroporationwithXenopuslaevis
oocytesresultedinreprogrammingtheHPAintocells
resemblingiPSc.WealsoshowedthattheseHPAderiveddeprogrammedcellscouldbe
cryopreserved,thawed,subcultured,andtransdifferentiatedintocellsexpressingneuraland
neuralprogenitormarkers.Thesedatasuggestthatthisreprogrammingtechnologymayhave
thepotentialforlargescaleproductionofinexpensivehumaniPScfromadiposetissue.
ReprogrammingofTcellstoproducestemcellsforadoptivetransferconstitutesanimportant
areaofinterestinimmunebasedoncologytherapy.32
WhilehumanCD4TLcanbeeasily
isolatedfromaslittleas5mLofperipheralblood,thisspecificcelltypehasnotbeenacommon
researchtargetforproducingiPSc.33
Wedemonstratedthatourprotocolreprogrammed
CD4TLsintocellsresemblingiPScwithformationofclustersoniMEFfeedercells,highalkaline
phosphataseactivity,andexpressionofpluripotencyassociatedtranscriptionfactors.
Theoralmucosacontainscellsthatcanbeobtainedwithoutinvasivetechniques,butthe
literatureonreprogrammingofthesecellsislimited.MiyoshietalreportedproductionofiPSc
fromoralmucosacells(obtainedfromoralbiopsytissue)usingretroviraltransferofOct4,Sox
2,cMyc,andKLF4.34
UsingcoelectroporationwithXenopuslaevisoocytes,wewereableto
reprogramcellsfromthebuccalmucosa(obtainednoninvasively)toresembleiPSc.
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suggeststhepossibilityofgenerationoflargeamountsofhumanautologousstemcellsfrom
thiseasilyobtainedtissue.
Theprospectforconvertingcancercellsintonormalorbenignquiescentcellsusinga
reprogrammingapproach,whichcanaltercellulartranscriptionprograms,iswidelydiscussedin
thescientificliterature.Experimentalapproachesincludesrevertingadultneoplasms,35
epigeneticreprogrammingofbreastcancercellsbyvalproicacid,36
miRNAreprogrammingof
humanskincancer,37
reprogrammingofhumancancercellsinthemousemammarygland by
exposuretomammaryepithelialcells,38
andviralmediatedtransferofstemcelltranscription
factorstoreprogramcolorectalcells.39
UsingcoelectroporationwithXenopuslaevisoocytes,
weobservedthatcellsfromthesehumancervicalcarcinomaandbreastadenocarcinomacell
linespartiallydedifferentiated.ThecellsformediPSclikeclusters,withsomecellsexpressing
Oct3/4.Thispartiallyreprogrammingmayprovideatransitionalpointforpotential
redifferentiationintonormalizedcells.Partiallyreprogrammedcellsmayalsobeamenableto
transdifferentiationalreprogramming.40
Therelativelyhighrateofreprogrammingachievedusinghiscoelectroporationmethod,
23.4%,isofnote. Theefficiencyofreprogrammingreportedintheliteratureincludes0.5%with
standard,fourfactorretroviralmethodology,41,42
0.98%2.34%whenaddingtwomore
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welimitedtheevaluationofreprogrammingefficacytohumanCD4TLs,whichcouldbe
evaluatedforNanogexpressionatearlystagesbeforeformationoftightlyclusteredcolonies.
Modificationsofthemethodmayimprovethereprogrammingefficiency.Forexample,
preliminarystudiessuggestthatthereprogrammingefficiencycanbemodulatedby
fluctuationsinbarometricpressureandenvironmentaltemperature(BioquarkInc.,dataon
file).
Theabsenceofnaturalreprogrammingsignalsmayresultinfailureorinconsistencyofcellular
reprogramming.Compromiseofsignalingfactorsmayoccurduringthepreparationofcrude
extractsfromXenopuslaevisoocytes,inwhichpotentiallyvitalnuclearandcytoplasmic
componentspresentinlivingeggscouldbedisrupted.Ganieretalobservedsimilarlylow
efficacyofnucleartransferandreprogrammingofmouseembryonicfibroblastsusing
pretreatmentwithXenopus
laevis
oocyteextractsandwithviralmediatedexpressionofOct4,
Sox2,Klf4,andcMyc(OSKM).However,reprogrammingefficiencywasimproved
approximately10foldwhenextractpretreatmentandviraltransferofthetranscriptionfactors
werebothperformed.44 Usingaprocessthatpromotesthenaturalorderofreprogramming
signalsalsoappearsimportant.Gradetalreportedthatreprogrammingthatdeviatesfrom
whatisknownofthenormalsequenceofevents(inductionofNanogbeforeOSKM)produced
abnormalcells.45
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thehumancells.Thisinterpretationissupportedbylowbutdetectablelevelofreprogrammimg
incontrolsdande,inwhichhumancellsthatwerenotcoelectroporatedwithXenopus
laevisoocyteswereexposedtoelectroporatefromXenopuslaevisoocytes.Thepossibilityof
multiplefactorsbeingnecessaryislogicalconsideringsuchabiologicallysignificantcellular
eventasreprogrammingwouldberegulatedthroughmultiplefactorsandpathways.In
addition,successfulretroviralandothermolecularreprogrammingtechniquesrequiretransfer
ofmultipletranscriptionfactors.31
Identification,purification,andamplificationofactive
reprogrammingcomponentstransferredduringcoelectroporationmayprovideopportunities
forinvestigationoftherapeuticpotential.
Theresultspresentedaboveneedtobeconfirmedbyindependentstudies,andfurther
researchisneededforfullproofofconceptofreprogrammingcellstopluripotency.Ongoing
activitiesincludeassessmentofredifferentiationandtransdifferentiation,molecular
karyotyping,DNAfingerprinting,andteratomaformation.Biochemicalandmolecularanalysis
oftheintrinsicmolecularmechanismsunderlyingtheXenopusoocytemediatedreprogramming
phenomenaareinprogress.
CONCLUSION
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types.Thesystemalsoprovidesforeasyseparationofreprogrammedcellsfromoocytesand
subcultureofthesecellsintheabsenceoffeedercells.
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ACKNOWLEDGMENTS
WethankIraPastor,VPatPhytomedics,Inc.(Jamesburg,NJ)andCEOofBioquark,Inc.(Tampa,
FL),forhisfundraisingeffortsandoutstandinghelpinallaspectsofthisproject.WethankDr.
NikolaiStrelchenko,PhD,formerlyofthehESCResearchLabattheReproductiveGenetics
Institute,Chicago,IL,andcurrentlyatNewYorkUniversityandDr.ArshakAlexanian,DMD,PhD
attheZablockiVeteransAffairsMedicalCenterandattheMedicalCollegeofWisconsin,
Milwaukee,WI,fortheirscientificadviceandprofessionalhelp. AnnetteF.Skorupa,PhDof
EnlightenMed,LLCprovidedacriticalreadingofthemanuscriptandwritingassistance.
DISCLOSUREOFPOTENTIALCONFLICTSOFINTEREST
TheauthorisanemployeeofBioquarkInc.
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TABLES
Table1.ControlExperiments
ElectroporationConditions PostElectroporationIncubation
Conditions
Control Human
cells*
Oocytes iMEF
cells*
Electroporation Human
cells*
Other Nanog
Expression
(a) Negative
(b) Negative
(c) Negative
(d) Electroporatefrom
oocytes
0.4%
(e) (humancells
electroporated
separately)
Electroporatefrom
oocytes
0.9%
(f) iMEFcells;
completeES
growthmedia
Negative
*Approximately105ofthefollowing:bonemarrowstromalcells,BJcells,humanpre
adiposites,CD4TLs,humanbuccalmucosacells,HeLacells,MCF7cells(controlexperiments
conductedseparatelywitheachhumancelltype);oocytesremovedfromtheelectroporate
priortoincubation;calculatedusingCD4TLs.
CD4TLs,humanCD4+Tlymphocytes;ES,embryoniccell;iMEF,irradiatedmouseembryonic
fibroblasts.
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FIGURES
FigureTitles
Figure1.Expressionofpluripotencymarkersbycellsderivedfromhumanbonemarrow
stromalcellsond7followingcoelectroporationwithXenopuslaevis
oocytes.
Figure2. ExpressionofpluripotencymarkersbycellsderivedfromBJcellsfollowingco
electroporationwithXenopuslaevisoocytes.
Figure3.Expressionofpluripotencymarkersbycellsderivedfromhumanpreadipositecells
followingcoelectroporationwithXenopusoocytes.
Figure4.Expressionofneuralmarkersbycellsderivedfromhumanpreadipositesfollowing
cultureunderconditionsthatpromoteneuralprogenitordifferentiationbyembryonicstem
cells.
Figure5.ReprogrammedCD4TLsexpressionofalkalinephosphatasefollowingco
electroporationwithXenopuslaevisoocytesandcoculturewithfeedercells.
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Figure6.ExpressionofpluripotencymarkersbycellsderivedfromhumanCD4+TLymphocytes
followingcoelectroporationwithXenopuslaevisoocytes.
Figure7.Coloniesofcellsderivedfromhumanbuccalmucosacellson6afterco
electroporationwithXenopuslaevisoocytes.
Figure8.Expressionofhumanpluripotencyassociatedfactorsbycellsderivedfromhuman
buccalmucosacellsfollowingcoelectroporationwithXenopuslaevisoocytes.
Figure9..PartialdedifferentiationofHeLaandMCF7cellsfollowingcoelectroporationwith
Xenopuslaevisoocytes.
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A B C D
______500 m
E F G H
______500 m
I J K L
______500 m
Figure1.Expressionofpluripotencymarkersbycellsderivedfromhumanbonemarrow
stromalcellsond7followingcoelectroporationwithXenopuslaevisoocytes.(A)(D)same
field;(A)DAPI;(B)Oct3/4;(C),Sox2;(D),DAPI,Oct3/4,andSox2combined;(E)(H)same
field;(E)DAPI;(F)Oct3/4; (G)Nanog;(H)DAPI,Oct3/4,andSox2combined;(I)(L),same
field;(I),DAPI;(J)Rex1;(K)SSEA1;(L)DAPI,Rex1,andSSEA1combined.
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A B C
______ ______ ______
1000 m 500 m 500 m
D E F G
______
500 m
H I J K Phase(Rex
______500 m ______1000 m
L M
______500 m
N O
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Figure2.ExpressionofpluripotencymarkersbycellsderivedfromBJcellsfollowingco
electroporationwithXenopuslaevisoocytes.(A)controlcells(nocoelectroporation);(B)(C)
samefield,d5;(B)phasecontrast;(C)alkalinephosphatase; (D)(G)samefieldond5;(D)DAPI;
(E)Oct3/4;(F)Nanog;(G)DAPI,Oct3/4,andNanog;(H)(I)samefield,d9;(H)phasecontrast,
(I)TRA160; (J)(K)samefield,d9; (J)phasecontrast;(K)Rex1;(L)(M) same,field,d11;(L)
phasecontrast;(M)SSEA1;(N)(O)samefield,d5;(M)phasecontrast;(N)Sox2.
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Nocoelectroporation10x 6h 20x 18h 20x 24h 20x
A ______250 m B ______500 m
C D E F ______500 m
G H ______500 m I J ______500 m
K L ______250 m M N ______500 m
(SSEA1) __x DAPI(SSEA4) __x SSEA4,d__ __x
Figure3.Expressionofpluripotencymarkersbycellsderivedfromhumanpreadiposites(HPA)
followingcoelectroporationwithXenopusoocytes.(A)clusterofcellsond5usingphase
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phasecontrast,(L)Rex1;(M)(N)samefield,d10;(M)phasecontrast,(N)SSEA1.
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Nestin B3T
NCAM
DAPISox-2
DAPI
______500 m
Figure4.Expressionofneuralmarkersbycellsderivedfromhumanpreadipositesfollowing
culturefor8daysinconditionsthatpromoteneuralprogenitordifferentiationbyembryonic
stemcells.
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A B C ______1000 m D ______500 m
______500 m _ _____500 m
E F G H
______500 m ______250 m ______500 m ______250 m
Figure5. CellsderivedfromhumanCD4+Tlymphocytesfollowingcoelectroporationwith
Xenopuslaevisoocytes.(A)control,nocoelectroporation;(B)nocoelectroporation,cultureon
irradiatedmouseembryonicfibroblasts;(C)(D)cellcultureond5followingco
electroporation;(E)(F) lowerpartofcluster in(D);(G)(H)alkalinephosphataseond9.
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A B ______500 m C D ______1000 m
E F G H ______500 m
I
J
K
L
______250 m ______500 m
Figure6. ExpressionofpluripotencymarkersbycellsderivedfromhumanCD4+TLymphocytes
followingcoelectroporationwithXenopuslaevisoocytes.(A)(B),samefield,d10;A,phase
contrast;(B) Oct3/4;(C)(D)samefield,d10;(C)phasecontrast;(D)Nanog;(E)(H)same
field,d5;(E)DAPI;(F)Rex1;(G)Sox2;(H)DAPI,Rex1,andSox2;(I) (J)samefield,d9;(I)
phasecontrast;(J)TRA160;(K)(L),samefield,d10;(K)phasecontrast;(L)SSEA1.
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6days ______500 m ______500 m
A ______ B_
Figure7.Coloniesofcellsderivedfromhumanbuccalmucosacellson6afterco
electroporationwithXenopuslaevisoocytes.(A)grownonirradiatedmouseembryonic
fibroblastsubstrate;(B)grownonStemAdheresubstrate.
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E F G H
Phase(SSEA4) 40x SSEA4,d11 40x
_
Figure8.Expressionofhumanpluripotencyassociatedfactorsbycellsderivedfromhuman
buccalmucosacellsfollowingcoelectroporationwithXenopuslaevisoocytes.(A)(B)same
field,96h; (A)phasecontrast;(B)Oct3/4;(C)(D)samefield,d10;(C)phasecontrast;(D)
Nanog;(E)(F) samefield,d10;(E)phasecontrast;(F)Sox2;(G)(H)samefield,d9,(G)phase
contrast;(H)TRA160;(I)(J),samefield,d11;(I)phasecontrast;(J)Rex1;(K)(L)samefield,
______250 m
______250 m ______250 m
A B ______500 m C D ______500 m
I J ______250 m K L
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A B C C D
______500 m ______500 m ______1000 m ______250 m
E F G H
______500 m ______500 m
I J K L
______250 m ______250 m
Nanog,d11 40x
41
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Figure9.PartialdedifferentiationofHeLaandMCF7cellsfollowingcoelectroporationwithXenopuslaevisoocytes.(A),HeLacells,
nocoelectroporation;(B)HeLacellsgrownonirradiatedmouseembryonicfibroblastcells,nocoelectroporation;(C)MCF7cells,
nocoelectroporation;(D)MCF7cellsgrownonirradiatedmouseembryonicfibroblastcells,nocoelectroporation;(E)(H)cells
derivedfromHeLacellsfollowingcoelectroporationwithXenopuslaevisoocytes;(E)(F),samefield,d11;(E)phasecontrast;(F)
Oct3/4;(G)phasecontrast;(H)Oct3/4;(I)(L)MCF7cellsfollowingcoelectroporationwithXenopuslaevisoocytes;(G) (H)
samefield,d11;(G)phasecontrast;(H)Oct3/4;(I)(J)samefield,d11;(I)phasecontrast;(J)Nanog.
42