University of Groningen

iPS cell therapy for Parkinson’s diseasePeng, Suping

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CHAPTER3

COMPARISONOFGENEEXPRESSIONPROFILEBETWEENIPSCELL‐DERIVED

ANDPRIMARYVENTRALMESENCEPHALICDOPAMINERGICNEURONS

Reinhard Roessler1, Su‐Ping Peng1,2, Jesse Veenvliet3,Petros Pechlivanoglou4, Koushik Chakrabarty5, MarianGroot‐Koerkamp6, JeroenPasterkamp5, ErikBoddeke1,MartenSmidt3andSjefCopray1

1DepartmentofNeuroscience,UniversityMedicalCenterGroningen,theNetherlands2CenterforNeuroscience,ShantouUniversityMedicalCollege,Shantou,GuangdongProvince,P.R.China3CenterforNeuroscience,SwammerdamInstituteforLife Science, Science Park Amsterdam, 1098XHAmsterdam,theNetherlands4Unit of Pharmacoepidemiology andPharmacoeconomics, Department of Pharmacy,UniversityofGroningen,theNetherlands5DepartmentofNeuroscienceandPharmacology,RudolfMagnus Institute of Neuroscience, University MedicalCenterUtrecht,Universiteitsweg100,3584CGUtrecht,theNetherlands6MolecularCancerResearch,UniversityMedicalCenterUtrecht, Universiteitsweg 100, 3584 CG Utrecht, theNetherlands

PublishedinStemCellReports,2014

ABSTRACT

Inducedpluripotent stemcells (iPS cells)harbor greatpromise for in‐vitro generationofdisease‐relevantcelltypes,suchasventralmesencephalicdopaminergic(VMDA)neuronsinvolved in Parkinson’s disease. Although iPS cell‐derived VM DA neurons have beengenerated,detailedgeneticcharacterizationofsuchneuronsisstilllacking.ThegoalofthisstudyistoexaminetheauthenticityofiPScell‐derivedDAneuronsobtainedbyestablishedprotocols.WeusedPitx3gfp/+transgenicmicetoFACS‐purifyVMDAneuronsderivedfrommouseiPScellsandprimaryVMDA(Pitx3gfp/+)neuronsinordertoanalyzeandcomparetheirgeneticfeatures.AlthoughiPScell‐derivedDAneuronslargelyadoptcharacteristicsoftheirin‐vivocounterparts,relevantdeviationsinglobalgeneexpressionwerefound.Genes,mainlyinvolvedinneurodevelopmentandbasicneuronalfunctions,consequentlyshowedreducedexpressionlevelsintheiPScell‐deriveddopaminergicneurons.Suchabnormalitiesshouldbeaddressedastheymightaffectunambiguouslong‐termfunctionalityandhamperthe potential of iPS cell‐derived DA neurons for in‐vitro disease modeling or cell‐basedtherapy.

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INTRODUCTION

The field of regenerative medicine has experienced a powerful impetus after thegroundbreakingdiscoveryofinducedpluripotency[1].NumerouspublicationshaveshownthatmouseaswellashumaniPScellshavethepotencytodifferentiateintovarious,clinicallyrelevantcelltypes,suchascardiomyocytes[2‐3],hepatocytes[4],hematopoieticprogenitors[5],oligodendrocytes[6]andspecificsubtypesofneurons[7‐8].Suchin‐vitrogeneratediPScell‐derived cell types provide new possibilities in terms of disease modeling and cellreplacement strategies. In particular, the generation of autologous iPS cell‐derived VMdopaminergicneuronsprovidesaveryinterestingtooltostudyandtreatParkinson’sdisease(PD) [9].However, future clinical applicationof iPS cell‐derivedDAneurons canonly beconsideredrealisticifthedesiredcellpopulationisstrictlypurifiedandcompletelydefined.SeveralgroupshavereportedthegenerationofDAneuronsfrommouseandhumaniPS

cells[8,10‐11].Inthesestudies,iPScell‐derivedneuronsdisplayedexpressionofcrucialDAmarkersandexhibitedtypicalneuronalelectrophysiologicalproperties.Furthermore,theseiPS cell‐derived DA neurons could functionally integrate into a rat PD model upontransplantation.Inprinciple,theseresultsindicatedthataDAneuronalpopulationcouldbeobtained from iPScellsandpresent importantVMDAneuronalcharacteristics.However,vitalgenome‐widestudies,comparinggeneticfeaturesofiPScell‐derivedDAneuronsversusprimaryDAneurons,arecurrentlylacking.SincereprogrammingofsomaticcellstoiPScellsresets their identity back to an embryonic stage, iPS cell‐derived differentiated neuronsshould be considered freshly formed ‘embryonic’ neurons. Accordingly, comprehensivecomparisonof iPS cell‐derivedDAneuronswith freshly formedembryonic andperinatalprimaryVMDAneuronsisself‐evident.WegeneratediPSClinesfromPitx3gfp/+knock‐inmouseembryonicfibroblasts.Pitx3isa

highly specific ventral mesencephalic dopaminergic (VM DA) neuron marker, which isrequired for DA neuron differentiation in the substantia nigra [12‐14]. Specific Pitx3‐associatedGFPexpressionallowedustostrictlyidentifyandFACS‐purifyDAneurons,eitheriPS cell‐derivedor from theventralmesencephalonat specificdevelopmental stages.Wesubjected these pure suspensions of VM DA neurons to genome‐wide gene expressionanalysiscomparingiPScell‐derivedDAneuronsandprimaryisolatedVMDAneurons.

MATERIALSANDMETHODS

Mice

Pitx3gfp/+embryosofseveraldevelopmentalstageswereobtainedbyintercrossingC57BL6/Jwith Pitx3gfp/gfp mice. Pitx3gfp/+ embryos are heterozygous for wild‐type Pitx3 and havenormalVMDAsystemdevelopment[15].OverlapofendogenousPitx3withGFPhasbeenshowntobe~100%[15].AllprocedureswereaccordingtoandfullyapprovedbytheDutchEthicalCommitteesforanimalexperimental(UMC‐UandUvA).

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iPScellgenerationandpropagation

MouseembryonicfibroblastswereisolatedfromE14.5embryosofPitx3gfp/+andNestin‐GFPmice.Bothmicehavebeendescribedandcharacterizedearlier [16‐17].Fibroblastswerecultured until passage 5‐8 and then retrovirally transfected with the four Yamanakareprogramming factors.SeparatevectorscontainingeitherOct4,Klf4,Sox2orcMycwereusedforpluripotencyinduction.RetroviruseswereobtainedfromPhoenixEcopackagingcells transfected with the reprogramming factors (for vector information: Addgene,Cambridge,www.addgene.org).Adetailedinductionprotocolhasbeendescribedearlier[6].iPSC clones were characterized by immunocytochemistry, reverse transcriptase PCR,WesternblotandbisulfitesequencingforthepromoterregionsofNanogandOct4(Figure1).

DifferentiationofPitx3gfp/+iPScellstowardsdopaminergicneurons

Wecombinedastromalfeeder‐basedprotocol[18]withadualBMPandTGFinhibition[19].Briefly, iPSCcoloniespriorculturedongelatinweremanuallypickedandseededonMS5stromalcellsinserumreplacementmedium,containingDMEM‐F12,15%knock‐outserum,glutamateandβ‐mercaptoethanol.Thecellswereallowedtosettlefor24hrs;Noggin(300ng/ml)andtheTGFinhibitorSB431541(10µM)wereaddedfor4‐5days.Neuralprecursors(Nes‐GFP)werecollectedafter14dayofdifferentiation.MediumwasgraduallychangedtoN2 containing SHH (200 ng/ml). After neural induction, neuronal patterning and DAdifferentiationwereinducedusingacombinationofBDNF,ascorbicacid,SHHandFGF8inN2medium.MaturationwasinitiatedbywithdrawingSHHandFGF8inthepresenceofBDNF,GDNF, ascorbic acid and cyclic‐AMP [20]. At about 4 weeks of differentiation, Pitx3gfp/+neurons reached a mature state as determined by morphology, marker expression,electrophysiologyanddopamineproduction.Continuousculturingintheseconditionscouldnotprovokeany furthermaturation/differentiation, i.e.no change in cellmorphology,nochangeinlevelandprofileofmarkerexpression,nochangeinelectricmembranepropertiesand no change in dopamine production; however, continuous culturing resulted in anincrease in celldeath.ForgeneexpressionandDNAmethylationanalyses,wehaveusedPitx3gfp/+VMDAneuronsderivedfromiPScellsafter4weeksofdifferentiation.CellsweresortedandimmediatelysubjectedtoRNAorDNAisolation.

FACS

FreshlydissectedPitx3‐GFPpositiveventralmesencephalonsfromseveraldevelopmentalstages,undifferentiatedanddifferentiatediPScell‐derivedcellsweredissociatedusingthePapaindissociationKit(WorthingtonBiochem,Products,LK003150).DissociatedcellswerecollectedincolorlessDMEM(Gibco)with20ng/mlBDNF,20ng/mlGDNF,0.2mMascorbicacid, 1 ng/mlTGFb3 and0.1mMdbcAMP, and sortedon aMoFlo‐XDPorMoFlo‐Astriossorterwitha100umnozzleatapressureof15‐17psibasedonGFPexpression.CellswerecollectedinN2mediumwithBDNF,ascorbicacid,GDNF,TGFb3anddbcAMPorcellswerecollectedinPBS,pelletedandusedforRNAandDNAisolation.

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Electrophysiology

Forpatchclampexperiments,FAC‐sortediPScell‐derivedPitx3gfp/+cellswerespundownat300rcffor10min,resuspendedandplatedonmouseastrocytefeeders.ElectrophysiologicalrecordingswereperformedonPitx3gfp/+neurons asdescribedearlier [1] oneweekafterplating.Briefly,astandardwhole‐cellpatchclamptechniquewasusedtomeasurecurrentsatroomtemperature(22–24°C)usingAxopatch200Bamplifier,Digidata1320A,andpClampversion 8.2 software (Axon Instruments, Foster City, CA). Extracellular bath solutioncontained (inmmol/L): 140NaCl, 4 KCl, 5 glucose, 2 CaCl2, 1MgCl2, and 10 HEPES, pHadjustedto7.4withNaOH.Pipettesolutioncontained(inmmol/L)140KCl,10EGTA,1CaCl2,1MgCl2and10HEPES,2Na2ATP,pHadjustedto7.2withKOHandhadanosmolalityof290‐300mOsm.MicroelectrodesweremadeofGC120F‐10borosilicateglass(HarvardApparatus)andpulledonaP‐87puller(SutterInstruments,Novato,CA)havingafinalresistanceof4–5MΩ.Seriesresistancewascompensatedby80%–85%.Currentswerefilteredat5kHzanddigitized at 10 kHz. Data were analyzed using Clampfit (Axon Instruments) and Excel(Microsoft). In the current clampmode cellmembrane potentialwas held at −50mVbyinjectingasteadyholdingcurrent.Cellswereactivatedwitha500mspulseofdepolarizingcurrent.

Microarrayanalysis

Total RNA was isolated from embryonic ventral mesencephalic tissue at variousdevelopmental stages (4 biological replicates each) and iPS cell‐derived neurons (3biological replicates) using Trizol (Invitrogen) and purified using RNA‐easy columns(Qiagen).Microarrayanalysiswasperformedinbiologicaltriplicates.Foreachexperimentalsampleadyeswapwasperformedtocorrectfordyeeffects.Agilentwholemousegenomemicroarray(Agilent,G4122F)setswereusedforallhybridization.Thearraysetiscomprisedof60‐meroligonucleotideprobesrepresentingover41,000mousegenesand transcripts.HybridizedslideswerescannedonanAgilentscanner(G2565AA)at100%laserpower,30%PMT.AfterdataextractionusingImaGene8.0(BioDiscovery),print‐tipLoessnormalizationwas performed on mean spot intensities. Data were analyzed using ANOVA (R version2.2.1/MAANOVAversion0.98‐7;http://www.r‐project.org/).P‐valuesweredeterminedbyapermutationF2test,inwhichresidualswereshuffled5000timesglobally.

Ratrotationmodel

TotestfunctionalityofiPScell‐derivedDAneurons,wehaveusedthewell‐establishedratrotationmodel[21].AdultfemaleSpragueDawley(Harlan)rats(180–230g)werehousedunderstandardconditionswithfreeaccesstofoodandwater.AllanimalexperimentswerecarriedoutaccordingtotheDutchRegulationsforAnimalWelfare.Protocolswereapprovedby the InstitutionalAnimalCareandUseCommitteeof theUniversityofGroningen.Ratswereanaesthetizedwithketamine(90mg/kg)andxylazine(4mg/kg).Unilateralretrogradedestructionofdopaminergicneuronsinthesubstantianigrawasinducedby2stereotaxicinjectionsof2.5ul6‐OHDA(3mg/mlin0.2%ascorbicacidand0.9%saline,Sigma)inthe

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medialforebrainbundleofthenigro‐striatalpathway(stereotaxiccoordinates1:AP‐4.0,ML−0.8andV−8.0;toothbarsetat+3.4.andcoordinates2:AP‐4.4,ML−1.2andV−7.8;toothbar set at −2.4). Unilateral destruction of the nigro‐striatal pathway was established byrecordingofd‐amphetamine(5mg/kg)‐inducedrotationbehaviorafter3,6and9weeks.Ratswereselectedfortransplantationifthenumberofrotationsexceeded4perminat6–8weeks post injection. Under ketamine/metedomedine anesthesia, 2‐3104 cells werestereotacticallytransplantedintothestriatum(coordinates:AP+0.9mm,ML‐2.6mmandV−4.0mm;toothbarsetat0.0);controlratsreceivedasimilarinjectionwithmouseembryonicfibroblasts. Daily intraperitoneal injections of cyclosporine 15mg/kg (UMCG pharmacy)weregiven,starting24hrsbeforecellgrafting(doubledosage),andcontinueduntiltheratswere sacrificed and perfusion fixated at 9 weeks after grafting. Underketamine/metedomedine anesthesia, rats were transcardially perfused with 4%paraformaldehyde(PFA)inPBS.Brainswereexplanted,post‐fixedin4%PFAfor24hrsandsoakedina20%sucrosesolutionfor1day.Brainsweresectioned(14umsections)onacryostatafterembeddinginO.C.T.compound(SakuraFinetek,Torrance,USA).

Immunocytochemistry

Cellswerefixedin4%paraformaldehydefor15minandblockedwith5%normalgoatserum,2% foetal calf serum in 0,1% Triton X in PBS. To characterize iPSC colonies, primaryantibodies forNanog(1:500,Abcam,ab80892),Oct3/4(1:500,sc‐5279,SantaCruz),Klf4(1:500,Abcam, ab72543), Sox2 (1:500,Abcam, ab15830),UTF1 (1:1000,mUTF1 custommaderabbitpolyclonal,Eurogentec,(VandenBoometal.,2007))andalkalinephosphatase(1:500, Abcam, ab65834) were used. To characterize iPS cell‐derived neurons, primaryantibodies forMap2 (1:500, Millipore, AB5622), TH (1:1000, Millipore, ab152) and GFP(1:500, Abcam, ab290)were used. To visualize primary antibodies, fluorescently labeledsecondary anti‐mouse and anti‐rabbit antibodies were applied. Nuclear staining wasperformedusingHoechst.

Westernblot

SDS‐PAGEgelelectrophoresisandWesternBlotanalysiswereperformedtodetectNanog,Oct3/4, Sox2 andUTF1.The following antibodieswereused: rabbit anti‐Nanog antibody(1:1000,ab80892,Abcam,Cambridge,UK),mouseOct3/4antibody(1:500,sc‐5279,SantaCruz),rabbitanti‐Sox2(1:4000,ab15830,Abcam,Cambridge,UK),rabbitUTF1(1:2000)andmouseanti‐β‐Actin(ab6276,Abcam,Cambridge,UK)at1:10000dilution.Thehousekeepinggene β‐Actin has been used as loading control. Primary antibodies were detected usingfluorescently labeled secondary antibody: donkey anti‐mouse (IRDye® 680, LI‐COR,Biosciences) and donkey anti‐rabbit (IRDye® 800CW, LI‐COR, Biosciences) according tomanufacturesinstructions.

QuantitativePCR(q‐PCR)

RelativegeneexpressionlevelsweredeterminedbyqPCRreal‐timepcr(Lightcycler)usingtheQuantiTect™SYBR®GreenPCR(QIAGEN)LightCycler®kit(Roche,IdahoTechnologies)

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accordingtothemanufacturer’sinstructions.Foreachreaction0.1ngtotalRNAfromFAC‐sorted neurons was used as input. Relative expression levels were normalized tohousekeepinggene18s.

QuantitativedopaminedeterminationbyLC‐MS/MS

Dopamine production was determined as described earlier [22]. Instead of plasma weanalyzed100µlofsupernatantorcellhomogenatefromterminallyDAdifferentiatediPScellsorundifferentiatedDopamineproductionwasdeterminedasdescribedearlier [22]. Insteadofplasmawe

analyzed100µlofsupernatantorcellhomogenatefromterminallyDAdifferentiatediPScellsorundifferentiatediPScells(3technicalreplicateseach)forthederivationstep.

ReversetranscriptasePCR

Briefly, RNA was isolated using the RNA‐easy kit (Qiagen). To characterize endogenousexpressionofpluripotencymarkersinPitx3gfp/+iPSCclones3and5,thefollowingprimerpairswereused:Nanog,Oct,Sox,andcMyc.ExpressionlevelswerecomparedbetweentwoiPSCclones,EScelllineIB10andmouseembryonicfibroblasts(C57BL/6).TocharacterizesortedPitx3‐GFPpositivecellsandtocomparethemtotheGFP‐negativepopulation,primersfor nestin, Pax6, β‐III‐tubulin, Map2, Nurr1, Pitx3, TH and DAT were used (for primersequencessee[23]).

Geneontologyanalysis

GOanalysisontranscriptomedatawasperformedusingtheBiNGO2.44plug‐in(Cytoscape2.8.2;Hypergeometrictest;FDRcorrection)usinggeneswithanatleast2‐foldup–ordown‐regulationiniPScell‐derivedDAneuronsascomparedtotheirprimarycounterpartatanydevelopmentaltimepointasinput,andallannotatedgenesonthemicro‐arrayasreference.

RESULTS

iPScell‐derivedpurifiedPitx3gfp/+neuronsexpresscrucialDAmarkers

iPS cells were generated from embryonic fibroblasts of Pitx3gfp/+ transgenic mice asdescribed in [24] and characterized (Figure 1). DA‐specific differentiation followingpreviouslydescribedprotocols[18‐19]resultedinMap2‐andTH‐positivePitx3‐expressingneurons exhibiting typical morphology of mature DA neurons (Figures 2A and B). Acomparative FACS profile showed absence of GFP‐expressing cells within theundifferentiated iPSC population, whereas the DA‐differentiated population contained adistinguishablesubgroupofGFP‐expressingcells(Figure2C).BothGFP‐negativeandGFP‐positivepopulationswere subjected to an initial transcript analysis (Figure2D).All fourselectedDAmarkers(Nurr1,TH,Pitx3,DAT)werefoundinGFP‐positivecells,indicatingthatGFP‐positivecells,inadditiontoacharacteristicmorphology,alsoacquiredaVMDA‐specificexpressionprofile.TheGFP‐negativefractionnotonlyexpressedneuralprecursormarkers(Nestin and Pax6) but also some neuronal markers (Tuj1 and Map2). As expected, noexpressionofthehighlyVMDA‐selectivemarkerPitx3andthematureDAneuronmarker

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dopaminetransporter(DAT)couldbedetected.Surprisingly,however,expressionofNurr1andTHwasobservedinthisPitx3‐GFP‐negativepopulation.Upon lineage‐specificdifferentiation, iPS cell‐derivedDAneuronswereable to secrete

dopamine(Figure2E).WeexaminedthefunctionalityofiPScell‐derivedPitx3‐GFPsortedVMDAneuronsafterintrastriatalimplantationinunilaterally6‐OHDAlesionrats,therodentmodelforParkinsondisease.iPScell‐derivedDAneuronsuspensionsfunctionallyintegratedin 6‐OHDA‐lesioned rat brains and were able to reduce amphetamine‐induced rotationbehavior in these rats (Figure 2F‐G). Finally, patch clamp recordings revealed bona fideelectrophysiologicalpropertiesofiPScell‐derivedVMDAneurons(Figures3A‐C).OurdatashowsuccessfuldifferentiationofiPScellstowardsfunctionalPitx3‐expressing

VMDAneuronsthatcouldbepurifiedfromanundefinediPScell‐derivedcellpopulation.Next,we set out to analyze the global gene expression profile of the iPS cell‐derivedDAneuronsandtocorrelatethatwiththeexpressionprofileofprimaryVMDAneurons.

Figure1.GenerationofiPScellsfromPitx3gfp/+MEFs(A)Characterizationof iPSCclone3and5usingimmunereactivityforselectedpluripotencymarkers(=red;Hoechstnuclearstaining=blue).(B)EndogenousexpressionofpluripotencymarkerscomparingiPSC clones 3 and 5 with embryonic stem cells (ES) and MEFs. (C) Protein expression of selectedpluripotencymarkerscomparingiPSCclones3and5withEScells.(D)Bisulfitesequencingofpromoterregions ofNanog andOct4 comparingPitx3gfp/+ iPS cellswithPitx3gfp/+MEFs. Open circles representunmethylatedCpGdinucleotides,whereasclosedcirclesindicatemethylatedCpGdinucleotides.

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Figure2.CharacterizationofPitx3gfp/+iPScell‐derivedVMDAneurons(A,B)Confocalimageofco‐labeledMap2+GFP+cells;TH+andGFP+cells.Scalebar=40μm.(C)FACSprofilecomparingundifferentiatedanddifferentiatedPitx3gfp/+iPScells.(D)RT‐PCRshowsdifferencesforaselectionofspecificmarkers forneuralprecursors(Nestin,Pax6),neurons(Map2,Tuj1)andDAneurons(Nurr1,TH,Pitx3,DAT).(E)QuantitativedopaminemeasurementcomparingundifferentiatedanddifferentiatedPitx3gfp/+ iPScells;valuesaremean±S.D. fromthreeindependentexperiments.(F)ImmunohistochemistryforgraftedratstriatumshowinginjectedPitx3‐GFP‐positiveiPScell‐derivedVMDAneuronsandadetailoftheirmorphology9weekspostgrafting.(G)Amphetamine‐inducedrotationinpurifiedPitx3gfp/+neurongraftedanimals(n=7)vs.controls(onlylesioned,n=2andgraftedMEFs,n=3).

Comparativeexpressionprofiling:primaryVMDAneuronsv.s. iPScell‐derivedDAneurons

We performed genomewide comparative gene expression analysiswith iPS cell‐derivedPitx3‐GFP‐positivecellsandprimaryisolatedVMDAneuronsfromseveraldevelopmentalstages(E12.5tillP0)(seeFigure4Aforexperimentalscheme).FAC‐sortingwithanefficiencyof98%allowedpurificationofiPScell‐derivedPitx3‐GFP‐positivecellsandprimaryPitx3‐GFP‐positive VM DA neurons (Figure 4B). Telencephalic brain homogenate served asnegative control for primary cells (Figure 4B) and undifferentiated iPS cells served asnegativecontrolforiPScell‐derivedGFP‐positivecells(asshowninFigure2C).

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Figure3.Electrophysiologicalpropertiesof iPS cell‐derivedDAneurons(A) Representativephasecontrastimageofarecordedneuron.(B)Currentclamprecordingofanin‐vitrogeneratedVMDAneuron.(C) I‐V curve indicative for voltage‐independent Ca2+ channelsandvoltage‐dependentNa+channels.

CorrelationofgenomewideexpressionprofilesofPitx3gfp/+VMDAneuronsatdifferentdevelopmentalstages(Figure4C)revealedhighestsimilaritiesbetweenembryonicVMDAneurons(r=0.92to0.98).Incomparison,thegeneexpressionprofileofiPScell‐derivedDAneurons is less correlated (highest correlation (r = 0.66) with E14.5 VM DA neurons),howevertheweakestcorrelationwasfoundbetweeniPScell‐derivedDAneuronsandP0VMDAneurons(r=0.55).We performed gene ontology (GO) term analyses to annotate genes that we found

differentiallyexpressediniPScell‐derivedDAneuronsincomparisontoprimaryembryonicVMDAneuronsandfoundthatgenesmostdown‐regulatediniPScell‐derivedDAneuronsbelong to GO terms such as nervous system development, neuron differentiation andneurogenesis(Figure4D).WeanalyzedtheexpressionofaVMDAspecificsubsetofgenesinmoredetailandfound

highsimilaritiesbetween iPS cell‐derivedDAneuronsandmostprimaryVMDAneurons(Figure4E).Hierarchical clusteringrevealed severalVMDAspecific genesequallyup‐ordown‐regulatedinprimaryisolatedandiPScell‐derivedDAneurons.Sampleclusteranalysisrevealed strongest gene expression correlation of iPS cell‐derived VM DA neurons withembryonic primary neurons,whereas postnatal stage primary VMDA neurons clusteredseparately. Several keyDAgenes, suchasOtx2,FoxA1,FoxA2,Nr4a2 (Nurr1),Lmx1a andLmx1baresimilarlyexpressediniPScell‐derivedVMDAneuronsandinembryonicVMDAneurons.OtherDAgenes,suchasEn1andEn2,showedexpressionlevelsiniPScell‐derivedVMDAneuronscomparabletothoseinpostnatalVMDAneurons.InviewoftheoriginofiPScell‐derivedDAneurons,wealsoanalyzedthetranscriptprofile

forpluripotencygenesandfibroblastrelatedgenes,visualizedbydendrograms(Figure4F).Geneexpressionofasetofpluripotencymarkerswassubjectedtoclusteranalysis,showingsimilartranscriptlevels(e.g.Nanog,Oct4(Pou5f1),Zfp42andNr0b1(alsoknownasDax1))inprimaryandiPScell‐derivedcelltypes(Figure4F).Thisresultdoesnotonlysubstantiatesuccessfultransitionawayfromapluripotentstatebutitalsoindicatesappropriatesilencing

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of pluripotency genes upon in‐vitro differentiation. However, a similar cluster analysisperformed on a subset of fibroblast specific markers revealed differential expression inprimaryandiPScell‐derivedneurons,suggestingremnantsofastillactivefibroblastgeneprograminiPScell‐derivedDAneurons.

Figure4.Geneexpressionprofiling:mesodiencephalicPitx3+neuronsversus iPS cell‐derivedPitx3+neurons(A) Schematic experimental setup. (B) FACSprofileof telencephalonandmesencephalonofPitx3gfp/+mice (E14.5) compared to iPS cell‐derived Pitx3+ neurons. (C) Correlation matrix of global geneexpressioncomparingallPitx3gfp/+purifiedneurons.(D)GOtermanalysischartofgenesmostreducediniPS cell‐derived VM DA neurons. (E) Expression levels of selected VM DA‐specific genes, comparingdevelopmentalmesencephalon stageswith iPS cell‐derived Pitx3+ neurons. R=correlation coefficientrelative to iPS cell‐derived DA neurons. (F) Hierarchical clustering for a subset of pluripotency andfibroblastmarkers.

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In order to validate our genomewide expression profiling data, we performed qPCRexperimentsforasetofselectedkeyDAgenes(Pitx3,Nurr1,Dlk1,En1andEn2),forSox9andDesmin.ExpressionlevelsoftheseparticulargenesappearedtobeinlinewiththeresultsobtainedbymicroarrayanalysisandindicatethatDA‐specificmarkerexpressioniniPScell‐derivedDAneuronsmostcloselyresemblesthatofterminallydifferentiatedstageVMDAneurons(Figure5A);asfarastheexpressionofPitx3,Nurr1,En1andEn2wasconcerned,thehighest similaritywas found between iPS cell‐derivedDA andE16.5VMDAneurons(Figure5B).Summarizing, comparative gene expression profiling of purified iPS cell‐derived DA

neuronsandembryonicVMDAneuronsrevealedaclearcorrelation.FewersimilaritieswerefoundbetweeniPScell‐derivedDAneuronsandP0DAneurons.Down‐regulatedgenesiniPScell‐derivedDAneuronsweremainlyassociatedtobiologicalfunctionssuchasnervoussystemdevelopment,neurogenesisandneurondifferentiation.

Figure5.GeneexpressionvalidationbyqPCRforasetofselectedkeyDAgenes(A)qPCRprofilecomparingtranscriptlevelsofPitx3,Nurr1,Dlk1,En1/2,Sox9anddesmininembryonicVMDAneuronsandiPScell‐derivedDAneurons(bothpurifiedonPitx3gfp/+expression).OnewayANOVA:***P<0.001;**P<0.01;*P<0.05;valuesaremean±S.D.fromthreeindependentexperiments.(B)Net‐plotsforPitx3,Nurr1,En1,En2andDlk1indicateclosestsimilaritiesbetweenE16.5VMDAneuronsandiPScell‐derivedDAneuronsformostmarkers.AxesrepresentrelativefoldexpressionandsimilarityinprimaryVMDAneurons(E12.5,E14.5andE15.5)vs.iPScell‐derivedDAneurons.

DISCUSSION

In view of their potential use in cell‐based therapy for Parkinson patients, ventralmesencephalicDA(VMDA)neuronshavebeenoneofthefirstcelltypesgeneratedfromiPScells.ResemblanceofiPScell‐derivedDAneuronswithtrueDAneuronshasbeenstudiedbasedonanumberofmainlymorphological and functionalproperties aswell asVMDAspecificgenesets[8,25‐26].Despitealltheserecentadvances,theriskoftumorgrowthandheterogeneousmolecular backgrounds inES‐ and iPS cell‐deriveddopaminergic neurons

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makeitstillhardlyapplicableastherapyinhumans[27‐28].Detailedunderstandingofthegenetic (and epigenetic) signature of ES‐ and iPS cell‐derived dopaminergic neurons isthereforeacriticalsteptoestablishcell‐basedtherapyasaviabletreatmentforParkinson'sdisease.Inthisstudy,wepresentacomparativegenome‐wideprofilingofgeneticfeaturesofiPS cell‐derived dopaminergic neurons, generated using established protocols, and theirprimary counterparts. Although iPS cell‐derived dopaminergic neurons met thecharacteristics widely used to classify functional VM DA neurons [26, 29‐30], causedreduction in rotationbehavior ina ratmodel forParkinson'sdisease, andadoptedmanygenetic featuresof theirprimarycounterparts,alsomajordeviationswereobserved, thatmayinterferewithproperfunctionalityaftergrafting.Since DA neurons obtained via induced pluripotency undergo fairly defined

developmentalstagesin‐vitro,wecomparedthemwithprimaryembryonicandpostnatalVMDAneurons.ForthegenerationofiPScellsandfortheirdifferentiationintoDAneurons,wehaveappliedacombinationofcurrentlywidelyacceptedandstandardizedprotocols[18‐19,24].WemadeuseoftransgenicPitx3gfp/+mice[15]forisolationofprimaryVMDAneuronsand for reprogramming of embryonic fibroblasts to iPS cells that were subsequentlydifferentiatedintoDAneurons.ThetranscriptionfactorPitx3hasbeendemonstratedtobeoneofthemoststringentmarkersforfullydifferentiated,functionalVMDAneurons[14]andexpressionofthePitx3‐GFPreporterallowedustostrictlyidentifyandFACS‐purifyiPScell‐derivedandprimaryVMDAneurons.OurglobalgeneexpressionanalysisshowedthatiPScell‐derivedVMDAneuronswere

highlysimilartoembryonicprimaryVMDAneurons,particularlywhenfocusingonVMDAspecificgenes.However,weidentifiedasubsetofgenestobedown‐regulatedin iPScell‐derivedVMDAneuronsincomparisontoembryonicprimaryVMDAneurons.Geneontologyanalysisrevealedthatthesegeneswerelinkedtotermssuchasnervoussystemdevelopment,neurondifferentiationandneurogenesis.While transcript analysis indicated that proviral gene expression introduced during

reprogrammingwas silenced in iPS cell‐derived VMDA neurons, we still found residualexpressionoffibroblastmarkers.IthaspreviouslybeenshownthatiPScellsarepronetodifferentiate along their somatic parental lineages, because they maintain a parentalepigenetic memory [31‐33]. Regardless of the notion that such epigenetic memory isrestricted to early passage iPS cells [34] there are reports about persistent parentalepigeneticstatesalsoinlatepassageiPScells[35].Ourfindingsraiseanimportantquestion.DodeviationsofexpressionprofilesiniPScell‐

derivedVMDAneuronsfromtheirin‐vivocounterpart,includingcontinuingexpressionofsome fibroblast genes, interfere with unambiguous long‐term functionality? Althoughcomprehensivestudieshavebeenperformedtotestin‐vivofunctionalityofhumanneurons[26,29]theseremainopenquestionsthatneedtobeaddressed,specificallywhenin‐vitrogenerated cells are to be considered for application in disease modeling and cell basedtherapy for Parkinson’s disease. Furthermore, it remains to be studied whethermodificationsinthereprogrammingprocessasrecentlyreportedforthehumansystem[36]

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as well as refined differentiation procedures may diminish these deviations. Increasedfundamental understanding about the genetic and epigenetic signature of dopaminergicneurons in‐vivo [37‐39] could offer new leads to generate safe and transplantabledopaminergicneuronsin‐vitro.

ACKNOWLEDGEMENTS

WethankN.BrouwerandM.Meijerforexcellenttechnicalsupport.WeacknowledgethehelpoftheFACScorefacility,UMCG.WethankR.Wichmannforexcellenthelpperforminganimalsurgeries.R.R.waspartiallysupportedbytheHazewinkel‐BeringerfoundationandtheJanKorneliusdeCockStichting.ConfocalimaginghasbeenperformedattheUMCGMicroscopyand Imaging Center (UMIC), which is sponsored by NWO grants 40‐00506‐98‐9021 and175010‐2009‐23.ThisworkwassupportedbyaVICI‐grant(no.865.09.002)toM.P.S.

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