Bio Quark Invitro Manuscript

7/28/2019 Bio Quark Invitro Manuscript

1/42

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


2/42

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.


3/42

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


4/42

associatedtranscriptionfactorsrecognizedas markersofpluripotency.Furthermore,thisco

electroporationtechniqueachievedarelativelyhighlevelofreprogrammingefficiency.


5/42

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


6/42

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.


7/42

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


8/42

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


9/42

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


10/42

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.


11/42

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


12/42

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


13/42

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).


14/42

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


15/42

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


16/42

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).


17/42

Twohumancancercelllines,HeLaandMCF7,weresubjectedtocoelectroporationwith

Xenopuslaevis

oocytesfollowedbycocultureoniMEF.Thecellsderivedfromco

electroporationofthesetumorcellsshowedpartialdedifferentiation,withformationof

clustersandexpressionofOct3/4(HeLaderivedcellsandMCF7derivedcells.Figure9)and

Nanog(MCF7derivedcells;Figure9).Thecellclusterstendedtobesmallerthanthosederived

fromcoelectroporationofnontumorcells(datanotshown).


18/42

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.


19/42

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.


20/42

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


21/42

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


22/42

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


23/42

types.Thesystemalsoprovidesforeasyseparationofreprogrammedcellsfromoocytesand

subcultureofthesecellsintheabsenceoffeedercells.


24/42

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.


25/42

REFERENCES

1 LirasA.Futureresearchandtherapeuticapplicationsofhumanstemcells:general,regulatory,andbioethicalaspects.JTranslMed2010;8:131.

2 ZhouH,WuS,JooJY,etal.Generationofinducedpluripotentstemcellsusing

recombinantproteins.CellStemCell2009;4:381 384.

3 JeeMK,KimJH,HanYM,etal.DHPderivativeandlowoxygentensioneffectivelyinduces

humanadiposestromalcellreprogramming.PLoSOne2010;5:e9026.

4 JudsonRL,BabiarzJE,VenereM,etal.EmbryonicstemcellspecificmicroRNAspromote

inducedpluripotency.NatBiotechnol2009;27:459461.

5 LinSL,ChangDC,YingSY,etal.MicroRNAmiR302inhibitsthetumorigenecityofhuman

pluripotentstemcellsbycoordinatesuppressionoftheCDK2andCDK4/6cellcyclepathways.

CancerRes2010;70:94739482.

6 LinSL,ChangDC,LinCH,etal.Regulationofsomaticcellreprogrammingthrough

induciblemir302expression.NucleicAcidsRes2011;39:10541065.

7 AnokyeDansoF,TrivediCM,JuhrD,etal.HighlyefficientmiRNAmediated

reprogrammingofmouseandhumansomaticcellstopluripotency.CellStemCell2011;8:376

388.8 MiyoshiN,IshiiH,NaganoH,etal.Reprogrammingofmouseandhumancellsto

pluripotencyusingmaturemicroRNAs.CellStemCell2011;8:633638.

9 HockemeyerD,SoldnerF,BeardC,etal.Efficienttargetingofexpressedandsilentgenes

inhumanESCsandiPSCsusingzincfingernucleases.NatBiotechnol2009;27:851857.

10 MarkoulakiS,HannaJ,BeardC,etal.Transgenicmicewithdefinedcombinationsofdrug

induciblereprogrammingfactors.NatBiotechnol2009;27:169 171.

11 HuangfuD,OsafuneK,MaehrR,etal.InductionofpluripotentstemcellsfromprimaryhumanfibroblastswithonlyOct4andSox2.NatBiotechnol2008;26:12691275.

12 WernigM,LengnerCJ,HannaJ,etal.Adruginducibletransgenicsystemfordirect

reprogrammingofmultiplesomaticcelltypes.NatBiotechnol2008;26:916924.

13 YoshidaY,TakahashiK,OkitaK,IchisakaT,YamanakaS.Hypoxiaenhancesthegeneration

ofinducedpluripotentstemcells.CellStemCell2009;5:237241.

14 HongH,TakahashiK,IchisakaT,etal.Suppressionofinducedpluripotentstemcell

generationbythep53p21pathway.Nature2009;460:1132 1135.

15 ChoHJ,LeeCS,KwonYW,etal.Inductionofpluripotentstemcellsfromadultsomatic

cellsbyproteinbasedreprogrammingwithoutgeneticmanipulation.Blood2010;116:386

395.

16 OhSI,LeeCK,ChoKJ,LeeKO,ChoSG,HongS.Technologicalprogressingenerationof

inducedpluripotentstemcellsforclinicalapplications.ScientificWorldJournal2012;2012:


26/42

20 ZhaoJ,MorozovaN,WilliamsL,LibsL,AviviY,GrafiG.Twophasesofchromatin

decondensationduringdedifferentiationofplantcells:distinctionbetweencompetenceforcell

fateswitchandacommitmentforSphase.J

Biol

Chem2001;276:2277222778.21 KimYD,KangSM,MinJY,etal.Productionoftropanealkaloidsduringdedifferentiation

ofScopoliaparvifloracalli.JNatProd2010;73:147 150.

22 StraubeWL,TanakaEM.Reversibilityofthedifferentiatedstate:regenerationin

amphibians.ArtifOrgans2006;30:743755.

23 MiyamotoK,FurusawaT,OhnukiM,etal.Reprogrammingeventsofmammaliansomatic

cellsinducedbyXenopuslaeviseggextracts.MolReprodDev2007;74:12681277.

24 RandM,KalishmanJ.Xenopuscare,health&disease:Abriefoverview.NewYork,NY:

ColumbiaUniversity,2001.

25 BuonoRJ,LinserPJ.Transgeniczebrafishbyelectroporation.BioRad.EU/EGBulletin1354,

1992.

26 HostetlerHA,PeckSL,MuirWM.Highefficiencyproductionofgermlinetransgenic

Japanesemedaka(Oryziaslatipes)byelectroporationwithdirectcurrentshiftedradio

frequencypulses.TransgenicRes2003;12:413424.

27 GrannemanJG,LiP,LuY,TilakJ.Seeingthetreesintheforest:selectiveelectroporation

ofadipocyteswithinadiposetissue.AmJPhysiol

Endocrinol

Metab2004;287:E574E582.

28 DoetschmanT.GeneTargetinginEmbryonicStemCells.ALab.Handbook.SanDiego,CA:

AcademicPress,2002.

29 PetersonS,LoringJF,WesselschmidtRL,Schwartz.PH.HumanStemCellManual,A

LaboratoryGuide:AcademicPress,2007.

30 AxellMZ,ZlatevaS,CurtisM.Amethodforrapidderivationandpropagationofneural

progenitorsfromhumanembryonicstemcells.JNeurosciMethods2009;184:275284.

31 OkitaK,YamanakaS.Inducedpluripotentstemcells:opportunitiesandchallenges.Philos

TransRSocLondBBiolSci2011;366:21982207.

32 StroncekDF,BergerC,CheeverMA,etal.Newdirectionsincellulartherapyofcancer:a

summaryofthesummitoncellulartherapyforcancer.JTranslMed2012;10:48.

33 ChunYS,ByunK,LeeB.Inducedpluripotentstemcellsandpersonalizedmedicine:current

progressandfutureperspectives.AnatCellBiol2011;44:245255.

34 MiyoshiK,TsujiD,KudohK,etal.Generationofhumaninducedpluripotentstemcells

fromoralmucosa.JBiosciBioeng2010;110:345350.

35 FrensterJH,HovsepianJA.Modelsof

embryonic

gene

induced

initiation

and

reversion

of

adultneoplasms.PresentedattheAACRNCIEORTCInternationalConference:Molecular

TargetsandCancerTherapeutics:Discovery,Biology,andClinicalApplications,p.2589,

October25,2007.

36 TravagliniL,VianL,BilliM,GrignaniF,NerviC.Epigeneticreprogrammingofbreastcancer


27/42

39 DewiDL,IshiiH,HaraguchiN,etal.Dicer1,ribonucleasetypeIIImodulatesa

reprogrammingeffectincolorectalcancercells.IntJMolMed2012;29:10601064.

40 SareenD,SvendsenCN.Stemcellbiologistssureplayameanpinball.Nat

Biotechnol2010;28:333 335.

41 MeissnerA,WernigM,JaenischR.Directreprogrammingofgeneticallyunmodified

fibroblastsintopluripotentstemcells.NatBiotechnol2007;25:1177 1181.

42 CondicML,RaoM.Regulatoryissuesforpersonalizedpluripotentcells.StemCells2008;

26:27532758.

43 HakelienAM,LandsverkHB,RoblJM,SkalheggBS,CollasP.Reprogrammingfibroblaststo

expressTcellfunctionsusingcellextracts.NatBiotechnol2002;20:460466.

44 GanierO,BocquetS,PeifferI,etal.SynergicreprogrammingofmammaliancellsbycombinedexposuretomitoticXenopuseggextractsandtranscriptionfactors.ProcNatlAcad

SciUSA.2011;108:1733117336.

45 GradI,HibaouiY,JaconiM,etal.NANOGprimingbeforefullreprogrammingmay

generategermcelltumours.EurCellMater2011;22:258274.


28/42

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.


29/42

FIGURES

FigureTitles

Figure1.Expressionofpluripotencymarkersbycellsderivedfromhumanbonemarrow

stromalcellsond7followingcoelectroporationwithXenopuslaevis

oocytes.

Figure2. ExpressionofpluripotencymarkersbycellsderivedfromBJcellsfollowingco

electroporationwithXenopuslaevisoocytes.

Figure3.Expressionofpluripotencymarkersbycellsderivedfromhumanpreadipositecells

followingcoelectroporationwithXenopusoocytes.

Figure4.Expressionofneuralmarkersbycellsderivedfromhumanpreadipositesfollowing

cultureunderconditionsthatpromoteneuralprogenitordifferentiationbyembryonicstem

cells.

Figure5.ReprogrammedCD4TLsexpressionofalkalinephosphatasefollowingco

electroporationwithXenopuslaevisoocytesandcoculturewithfeedercells.


30/42

Figure6.ExpressionofpluripotencymarkersbycellsderivedfromhumanCD4+TLymphocytes

followingcoelectroporationwithXenopuslaevisoocytes.

Figure7.Coloniesofcellsderivedfromhumanbuccalmucosacellson6afterco

electroporationwithXenopuslaevisoocytes.

Figure8.Expressionofhumanpluripotencyassociatedfactorsbycellsderivedfromhuman

buccalmucosacellsfollowingcoelectroporationwithXenopuslaevisoocytes.

Figure9..PartialdedifferentiationofHeLaandMCF7cellsfollowingcoelectroporationwith

Xenopuslaevisoocytes.


31/42

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.


32/42

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


33/42

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.


34/42

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


35/42

phasecontrast,(L)Rex1;(M)(N)samefield,d10;(M)phasecontrast,(N)SSEA1.


36/42

Nestin B3T

NCAM

DAPISox-2

DAPI

______500 m

Figure4.Expressionofneuralmarkersbycellsderivedfromhumanpreadipositesfollowing

culturefor8daysinconditionsthatpromoteneuralprogenitordifferentiationbyembryonic

stemcells.


37/42

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.


38/42

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.


39/42

6days ______500 m ______500 m

A ______ B_

Figure7.Coloniesofcellsderivedfromhumanbuccalmucosacellson6afterco

electroporationwithXenopuslaevisoocytes.(A)grownonirradiatedmouseembryonic

fibroblastsubstrate;(B)grownonStemAdheresubstrate.


40/42

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


41/42

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


42/42

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

Bio Quark Invitro Manuscript

Documents

Transcript of Bio Quark Invitro Manuscript