University of Groningen iPS cell therapy for Parkinson’s disease … · 2016-03-09 · Oxidative stress is considered to play an important role in the specific loss of DA neurons

University of Groningen

iPS cell therapy for Parkinson’s diseasePeng, Suping

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IPSCELLTHERAPY FOR

PARKINSON’SDISEASE

Su‐PingPeng

Allresearchdescribedinthisdissertationwasconductedat:

Depatment of Neuroscience, Section Medical physiology, University Medical CenterGroningen, University of Groningen, the Netherlands; Center for Neuroscience, ShantouUniversityMedicalCollege,Shantou,GuangdongProvince,P.R.China.

Printingofthisthesiswassupportedby:SchoolofBehaviouralandCognitiveNeurosciences(BCN)UniversityMedicalCenterGroningen(UMCG)UniversityofGroningen(RUG)

Copyright 2015 by Su‐Ping Peng. All rights reserved. No part of this book may bereproducedor transmitted inany formorbyanymeanswithoutpriorpermissionof theauthor.

Coverdesign:Su‐PingPeng&IliaVainchtein

Printing:CPIKoninklijkeWöhrmann

ISBN:978‐94‐6203‐950‐6

iPScelltherapy

forParkinson’sdisease

PhDthesis

toobtainthedegreeofPhDatthe

UniversityofGroningenontheauthorityofthe

RectorMagnificusProf.E.Sterkenandinaccordancewiththe

decisionbytheCollegeofDeans.

Thisthesiswillbedefendedinpublicon

Monday16November2015at12.45hours

By

SupingPeng

bornon22March1983Guangdong,China

Supervisors:Prof.H.W.G.M.BoddekeProf.M.SchachnerCo‐supervisors:Dr.J.C.V.M.CoprayDr.Y.Q.ShenAssessmentcommittee:Prof.P.P.deDeynProf.E.M.HolProf.L.A.’tHart

CONTENT

CHAPTER1

Generalintroduction 7

CHAPTER2

Comparisonofhuman(foetal)primarywithhumaniPScell‐deriveddopaminergicneurongraftsintheratmodelforParkinson’sdisease 31

CHAPTER3

ComparisonofgeneexpressionprofilebetweeniPScell‐derivedandprimaryventralmesencephalicdopaminergicneurons 61

CHAPTER4

PotentialroleofcelladhesionmoleculesintheneuriteoutgrowthofiPScell‐deriveddopaminergicneurons 77

CHAPTER5

ParticipationofperforininmediatingdopaminergicneuronlossinMPTP‐inducedParkinson’sdiseaseinmice 97

CHAPTER6

ComparisonofAAV2andAAV5ingenetransferintheinjuredspinalcordofmice 109

CHAPTER7

Summaryanddiscussion 119

CHAPTER8

Nederlandsesamenvatting 129 Acknowledgments 137

CHAPTER1

GENERALINTRODUCTION

GENERALINTRODUCTION

9

1. PARKINSON’SDISEASE(PD)

AgeisthelargestriskfactorforthedevelopmentandprogressionofParkinson’sdisease(PD).Withtheincreasingagingofthepopulationinthelast3decades,PDhasbecomethemostprevalentneurodegenerativediseaseintheWesterncountries,affecting1in100peopleovertheageof60.Althoughinlaterstagesmentalandcognitivefunctionsbecomeaffected,thefirstdiagnosisrecognizesPDasamovementdisorder.ClinicalfeaturesforthediagnosisofPDare tremors, rigidity,akinesiaorbradykinesiaandpostural instability [1].The lossofmotorcontrolisduetothedegenerationofdopaminergicneuronsinthesubstantianigra(SN),withconsequentdenervationanddopamine(DA)leveldepletionofitsprojectionarea,theputamenandcaudatenucleusofthestriatum.Attheonsetofmotorsymptoms,almost60%of theDAneurons in theSNappear tobealready lostwith theputamenalDA leveldepletedby80%.TheDAneuronsstillpresentarecharacterizedbythepresenceofLewyBodies (LB), theproteinaceous cytoplasmic inclusions containingα‐synuclein aggregates.Theprogressof thedisease takes several years todevelop froma slight senseofmuscleweaknesstoapronenesstotremblingandeventuallytothelossofcontrolovermuscularactivity[2].MostPDpatientssufferfromaconsiderablemotoricdisabilityat5‐10yearsofdisease, evenwhen treatedwithpresent availablemedications [3].At that timemostPDpatientsalsomayhavestartedtodevelopnon‐motorrelatedfeaturessuchassleepdisorders,depression,psychosis,anddementia[1].DegenerationandLBformationarenotrestrictedtoSNDAneurons.Noradrenergic,serotonergicandcholinergicneuronsarealsoaffectedinmoresevereorlatestagesofthedisease[3].

Figure1.TheBraakhypothesisof progression of diseasepathology in Parkinson’sdiseasePathologystartsintheperipheryandprogresses into theCNSviathevagusnerveand/orolfactorynerve in a predictable,progressive manner. Numbersindicate proposed sequence ofprogression into various brainregions.Modifiedbasedon[4].

Basedonthepatternofα‐synucleinaggregateandLBformationandspreading,Braaketal.proposedaPD‐specificpatternfortheprogressionofLewybodypathology(Figure1).Lewybodiesfirstappearintheolfactorybulb,orthegastrointestinaltract,andlaterenterthe medulla oblongata and pontine tegmentum. At these stages, patients are pre‐symptomatic.Asthediseaseprogresses,Lewybodiesdevelopinthesubstantianigra,areasof the midbrain and basal forebrain, and finally in the neocortex [4]. This hypothesiscomprisesthemulti‐systemicnatureofthediseaseinwhichalsoinflammationandimmune

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reactivityplaysamajorrole.Inthatrespect,‐synucleinhasbeenfoundtobeabletoactivatebothinnateandadaptiveimmunecells[5].

2. PATHOGENESISOFPD

MostofthePDcasesaresporadicwithanasyetunknowncause,althoughsomeetiologystudies have correlated PD onset to chronic exposure to environmental toxins such asherbicidesandpesticides [6‐7].Only5%ofPDcasesare familiar.Genetic linkagestudieshaveidentifiedthemajorgenesinvolved,suchastheonesencodingfor‐synuclein,DJ‐1,PINK1,Parkin,andLRRK2.However,thedefinitionofgeneticorsporadicPDisgettinglesssharp,sincesomemutationsinthePDgeneswerealsofoundinpatientswithoutafamilyhistoryofPD[8].GeneticassociationstudiescomparingPDpatientgroupswithmatchedgroupsofhealthycontrolshavenowrevealedatleast20PDriskgenes.Molecularpathwaysinvolved inthepathogenicmechanismunderlying familialPDcanhelptounderstandthepathogenesis in sporadic cases. In general, the degeneration of DA neurons during PDdevelopment is the consequence of a number of cellular pathogenicmechanisms brieflysummarizedbelow(Figure2).

Figure2.OverallparadigmforPDpathologyEnvironmental/genetic factors may initiate 1) neuronal dysfunction via oxidative stress or proteinmisfoldingandaggregation;2)activationofmicrogliaand/orotherinnateimmunemechanismswhichcanamplifyoneanother.AntigenpresentationbymicroglialeadstoactivationofTcells,whichinturncanactivateBcellstoproduceantibodies.Continuedreleaseandpresentationofantigensfromdyingneurons or modified antigens allow the propagation of a specific, chronic inflammatory responsemediatedbytheadaptiveimmunesystemtowardstheneuronsthatdegenerateinPD.Modifiedbasedon[9].

2.1Cellularpathogenicmechanisms

MitochondrialdysfunctionisconsideredamajorplayerinthedegenerationofDAneurons.StudiesonneurotoxicanimalmodelsforPDusing6‐OHDA,rotenoneorMPTPhaveclearly

GENERALINTRODUCTION

11

demonstratedthemitochondriaasprimarytargets.MitochondrialcomplexIwasinhibitedbyhydrogenperoxideandhydroxyl radicalsproducedby theneurotoxins, leading to theviciouscircleofsuperoxidefreeradicalsproductionandeventuallycelldeath[10‐12].Thenormal function of PINK1 and Parkin in healthy neurons is to label andmark damagedmitochondriafordegradation[13];PINK1facilitatesthecreationofmitochondria‐derivedvesicleswhichcanseparatereactiveoxygenspeciesandshuttlethemtowardlysosomesfordegradation[14].ItisclearthatmutationsinthesegenesinPDpatientswilleventuallyleadtomitochondrialdysfunctionintheDAneuronsandsotoneuronaldegeneration.Proteinmisfoldingoraggregation,and impairmentoftheproteindegradationmachinery

are closely related scenarios that contribute to neuronal death in PD. The presence ofmisfolded proteins can be toxic to cells [15]. Missense mutations of ‐synuclein andduplicationsortriplicationsofthelocuscontaining‐synucleinleadingtotoxicoligomereformationorlargeaggregateshavebeenfoundindifferentgroupswithfamilialPD[16].PDmodelsbasedonover‐expressionorexpressionofthemutatedformof‐synucleincontainintracellularaggregateformationandshowcharacteristicSNdopaminergicneuronloss[17].The formation of Lewy bodies containing damaged or misfolded protein aggregates isinferred as a protective mechanism of the cells against accumulating misfolded andaggregatedproteins,eitherasaresultoftheimpairedubiquitin‐proteasomesystemorthedeclined ability of chaperones to refold proteins [3]. Mutations in ATP13A2 or β‐glucocerebrosidase (GBA) are also considered risk factors for PD by inducing lysosomalstorage disorder [18]. Parkin is identified as an E3 ubiquitin ligase in the ubiquitin‐proteasome system that identifies and targetsmisfolded proteins to the proteasome fordegradation [19]; mutations in the Parkin gene abolish the E3 ligase activity and causedysfunctionoftheubiquitin‐proteasomesystem.OxidativestressisconsideredtoplayanimportantroleinthespecificlossofDAneurons

inPD.Themetabolismofdopamineproduceshydrogenperoxideandsuperoxideradicals;auto‐oxidationofDAproducesDA‐quinone [20].Low levelsof intracellularROSarenowrecognized to have an important role in the maintenance of normal cellular function.However, excess of ROS results in oxidative stress, which involves damage to cellularcomponents, suchas lipids,proteinsandnucleic acids, and leads to the lossofbiologicalfunction.TheabilityofhandlingintracellularROSdeclineswithaging,andincreasedlevelsof oxidized lipids, proteins and nucleic acids have been found in PD brains [21‐23]. PD‐causingmutationshavebeenidentifiedinthegeneencodingforDJ‐1,aproteinimplicatedinthecellularmonitoringofoxidativestress[24].In addition to the abovementioned intracellular pathogenic processes involved in the

degenerationofDAneurons,increasingevidencesuggeststhattheprogressiveDAneuronaldeath is not autonomous, but promoted by the involvement of the innate aswell as theadaptiveimmunesysteminPDdevelopment.

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2.2InflammationinPD

Microgliaactivationand severeastrocytosis have been observed in postmortemPDbraintissueandinPDanimalmodels,andsuggestthatinnateneuroinflammatoryprocessesplayaroleintheprogressionofDAneurondeath.ThefirstindicationfortheinvolvementoftheinnateimmunesysteminPDpathogenesiscamefromtheobservationbyMcGeeretal.thatactivatedmicrogliaexpressedHLA‐DRintheSNofPDpost‐mortembrains[25].PostmortemstudiesonbraintissuetakenfromyoungPDpatientsintoxicatedwithMPTPconfirmedthepresenceofchronicactivatedmicrogliaexpressingHLA‐DRaroundthedegeneratingSNDAneurons [26]. Ithasbeenshownthatmicrogliaareattracted to localbrain injurybyATPgradients[27]orcalciumwaves[28]releasedfromthedegeneratingneurons.Microglia phagocytosis can contribute toDA neuron death in Parkinson’s disease. The

presenceofneuromelaninwithinactivatedmicrogliaintheSNindicatesthatfragmentsofdisintegratingdopaminergicneuronswerephagocytized[29].PhagocytosisofdegeneratingDAneuronsbyactivatedmicrogliahasalsobeenobservedinmicePDmodels[30].TheTNFreceptor,thedeath‐signalingreceptor,hasbeenfoundtobewidelyexpressedonDAneuronsin human SN [31]. This suggests that another harmful effect on SN DA neuronsmay bemediated by cytokines expressed by activated glia cells. It has been demonstrated thatactivatedastrocytesexpressedamajorportionofTNF‐ inSNofParkinsonianmacaques[32]. Microglia upon stimulation are also able to release the proinflammatory cytokinestumornecrosisfactor‐(TNF‐),interleukin‐(IL‐)1β[33]andinterferon(IFN)‐γ[32].IFN‐γ,withthesynergisticcontributionofTNF‐,hasbeenshowntomediatecellspecificmicroglialandastroglialactivationinexperimentalmodelsofParkinson'sdisease[34].Inadditiontothis self‐activation effect, cytokines such as IL‐1β, IL‐2, IL‐4, and IL‐6 were also foundelevated inbrain,blood,orCSFofPDpatients [35‐36]. It isknown that these circulatingcytokinescanalsoinduceglialactivationandleadtonewcytokinereleasebyactivatedglialcells.Theviciouscyclethusmaintainsalong‐terminflammatoryresponseandDAneuronprogressivedegenerationinParkinsonism.Moreover,theresidentglialcellsalsocontributetoPDpathologybythecross‐talkwith

peripheral immune cells. It has been shown that various neurological disorders orneurodegenerativeconditionsaffecttheproductionandreleaseofvariouschemokines,suchasCCL2,CCL3,andCCL5,byperivascularastrocytes,componentsoftheblood‐brainbarrier.Thesechemokinesarefundamentalfortheinfiltrationofmonocytesandlymphocytesinthebrainparenchyma[37‐38].Indeed,inPDpatientsandinexperimentalanimalmodelsofPD,infiltratingmonocytesandlymphocyteshavebeenobservedwithinthebrainparenchyma.Inaddition,alterationsinlymphocytesubpopulationshavebeendetectedintheperipheralbloodofPDpatients.Theaberrant compositionof theperipheralblood leucocytepopulationof PDpatients is

reflectedinanincreaseofneutrophilsandnaturalkiller(NK)cells(correlatedwithdiseaseseverity)[39],andadecreaseofCD4+TandCD19+B‐lymphocytes[40‐41].ThereductionofCD4+TcellsappearedtobecorrelatedwithUPDRSIIIperformanceinPDpatients[39].Inaddition,asfarasT‐cellsubpopulationsareconcerned,anincreaseof TcellsandCD45RO+

GENERALINTRODUCTION

13

memoryTcells,andadecreaseoftheCD45RA+subsethavebeenobservedinPDperipheralblood[42‐43];increasedproportionsofTcellswerealsofoundintheCSFofPDpatients[44].Infiltrationoflymphocytes,inparticularCD8+Tcells,wasfirstdetectedinthepostmortem

SNofapatientwithPD[45].RecentstudiesperformedinpostmortembrainsofPDpatientshave described a 10‐fold greater infiltration of CD4+ and CD8+ T cells in the brain,specificallyinfiltratingtheareaswithdegeneratingdopaminergicneuronsincomparisontoage‐matchedcontrols[46].SimilartoPDpatients,inparkinsonianmiceactiveCD4+Tcellscriticallyincreasedintheperipheralblood[43,47];anincreaseofCD4+andCD8+Tcellswasalsoobservedinthebrain[46,48].Importantly,theinfiltrationofCD4+TcellsseemstocontributetothedegenerationofdopaminergicneuronasattenuationofneurodegenerationhasbeendemonstratedinCD4+TcellsKOmice[46].ItislikelythatinfiltratingmonocytesarepresentamongtheactivatedmicrogliaintheSN

of PD patients but theymay have been difficult to distinguish so far from themicroglia.Infiltration of other leucocytes into the SN, such as NK cells or B cells has not yet beendescribed.However,depositionofIgGintheSNondopaminergicneuronsinPDpost‐mortembrainappears tobeplentiful [49], and IgGcollected fromserumofPDpatientshasbeenshowntoxictoDAneuronswheninjectedintotheratSN[50].

3. THERAPIESFORPD

3.1Dopaminereplacement

TheidentificationofstriataldopaminedepletionasamaincauseofmotorsymptomsofPDledtothefocusofpharmacotherapeuticsondopaminereplenishment.L‐DOPAhasbecomethegold‐standardtherapyfortreatingearlyPDmotorsymptomssinceitsfirstapplicationin1960s[51].ThetreatmentwithL‐DOPAclearlyimprovesdailyfunction,qualityoflife,andsurvivalofPDpatients.ThedopamineprecursorL‐DOPAcanpasstheblood‐brainbarrierand is converted in dopaminergic neurons by dopa decarboxylase into dopamine,compensating the decrease in DA level in the striatum due to loss of dopaminergicinnervation. Maybe due to the short half‐life of L‐DOPA, leading to fluctuations in thedopamineconcentration,themostsignificantsideeffectofchronictreatmentwithL‐DOPAis thedevelopmentofdyskinesias [52].Eversince, improvementshavebeenmade in thedopaminereplacementtherapy.Acombinationof inhibitorsfordopamine‐metabolisationenzymes such as catechil‐O‐methyltranferase (peripheral) and monoamine oxidase B(central)havebeenincludedtoreduceL‐DOPAmetabolizationoutsideDAneuronsandtoenhance the effect of L‐DOPA [53‐54]. Specific dopamine receptor agonists have beendevelopedthatbypassthedegeneratingdopaminergicneuronsanddirectlystimulatetheintact, although denervated, postsynaptic dopamine receptors in the striatum [53]. Thisalternative appeared to decrease the development of dyskinesias, however, increasedsomnolence,sleepattacks,REMsleepdisorder,andavarietyofpsychiatricsymptoms[55].

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3.2Surgeryapproach

InadvancedPD,directmodulationofbasalgangliaactivityviaablativetechniquestargetingtheinternalglobuspallidus(GPi)orthesubthalamicnucleus(STN),hasbeenshowneffectivetoreducesomeofthesymptoms[56].Deepbrainstimulation(DBS)involvesimplantationofelectrodesintotheinternalglobuspallidus(GPi)orthesubthalamicnucleus(STN),withanexternallyprogrammablestimulatorthatisconnectedtotheelectrodes[57].Thesystemdeliverscontinuoushigh‐frequencyelectricalstimulation(mostcommonlyinthe100–150‐Hzrange)totheimplantedbrainareas[57].Inmostpatients,DBSalleviatesparkinsonianmotor signs, shortens ‘off’ periods, and reduces drug‐induced dyskinesias, dystonia, andmotorfluctuations[58];combinationofDBSwithpharmacologicaltreatmentappearstobemosteffectiveinalleviatingmotordeficitsinpatientswithadvancedPD[59‐60].However,thereisevidencethatDBSaffectsverbalfluency,cognitionandemotionalstability,andevenworsendepressionandmania[61‐62].

3.3Dopaminergicneuronreplacement

Cell replacementofDAneuronshasbeenconsideredapromising therapy forPD. In thisapproach,replacementofthelostnigrostriataldopaminergicinnervationofthestriatumbyexogenousdopaminergic neurons is intended to restorebasic dopamine levelwithin thestriatum. In the 1970s and 1980s, experiments with the grafting of foetal ventralmesencephalic (VM) tissue in rodent andnon‐humanprimatemodels forPD,had shownsurvival of grafted DA neurons and reinnervation of the striatum [63‐64]; moreover,reduction in drug‐induced rotation behavior in ’Parkinsonian’ rats demonstrated thefunctional integrationof the graftedDAneurons [65‐67]. Clinical trials in the1980s and1990swiththestriatalimplantationofhumanfoetalDAneuronsprovidedproof‐of‐principleforthistreatment.PatientswithhumanfoetalVMgraftsrecoveredfromrigidityandtremorsinvariousdegrees,correlatedtotherestorationofthedopaminelevelasdetectedbyPETscans [68‐70]; they experienced an overall improvement in life quality [68‐69, 71‐72].However,thistherapeuticalapproachwascompromisedbythedevelopmentofseveregraft‐induced dyskinesias in a large number of patients, the limitation of tissue sources andconsiderablelogisticalandethicalissues.The ground‐breaking detection of induced pluripotent stem cells (iPS cells) generated

fromeasilyaccessiblesomaticcells(e.g.skinfibroblasts)[73‐74]andthedevelopmentofin‐vitro differentiation protocols for DA neurons have provided unprecedented novelautologous sources forhumanDAcell grafts. Presently, the functionalityof these in‐vitrogeneratedhumanDAneuronsisassessedafterintrastriatalimplantationinrodentandnon‐human primate models for PD. Apart from changes in motor behaviour, the majorhistological parameters in such analyses are the neuronal survival, the extent of neuriteoutgrowth,thecoverageofthestriatumwithafinenetworkofdopaminergicterminals,thereleaseofdopamineandgeneralfunctionalrecovery.

GENERALINTRODUCTION

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Proper differentiation of pluripotent stem cells into DA neurons in‐vitro implies theaccuraterecapitulationoftheembryonicdevelopmentoftheventralmesencephalon(VM)anditsDAneurons.Thisin‐vivoprocesswillbeelaboratedbelow.

4. DEVELOPMENTOFVENTRALMESENCEPHALICDANEURONS

Almost75%ofallDAneuronsintheadultcentralnervoussystem(CNS)resideintheventralmesencephalon.TheestimatednumberofDAneuronsinmouseVMis20,000‐30,000,andinhuman 400,000‐600,000. DA neurons are generated from the floor plate region of themesencephalon, which eventually give rise to three distinct clusters of DA neurons: A8(retrorubral field),A9 (substantianigra, SN)andA10 (ventral tegmental area,VTA).Theformation of VM DA neurons relies on specific patterns of induction signals along theanterior‐posterior and dorsal‐ventral axes of the neural tube (Figure 3) in a relativelynarrowtimewindow:betweenE10.5andE12inmouseandbetween6and8.5weekspostconception(PC)inhumans[75].Themostcrucialinductivefactorsaresonichedgehog(SHH)secretedbythenotochordand later the floorplateandfibroblastgrowthfactor8(FGF8)secretedfromtheisthmusorganizerinthemidbrain‐hindbrainboundary.FGF2alsoplaysanimportantroleintheregulationofproliferationanddevelopmentalcelldeathoftheDAneuralprogenitor(NP)cells[76‐77].TheformationofthedopaminergicVMregioninfactstartswhenfloorplateradialglial‐

likeNPsgiverisetoDANPs.AtE9inmice,thefirstsignofaDAphenotypeisindicatedbytheexpressionofLMX1a(expressedfromE9tillP180)andMSX1(onlypresentinDANPs).OncetheNPsarecommittedtotheDAneuronal fate, theygraduallybecomepost‐mitoticbetweenE10 andE14 inmice (E12‐16 in rats). Shortly after the final division, thepost‐mitoticcellsmigratefromtheproliferativezonetotheintermediatezone.ThesecellsstarttoexpressNURR1,whichcontrolstheDAneurontransmitterphenotype[79],andregulatestheexpressionoftyrosinehydroxylase(TH),atE10.5inmice[80‐81]andE14inrat[82].ThecombinationofNURR1,LMX1bandWNT1signalling insomecellsappears to induceearlyPITX3expressionpriortoTHexpression[83].Next,theTH+cells(TH+/PITX3‐cells,targetVTA)orPITX3+cells (TH‐/PITX3+cells, targetSN) start tomigrate: firstventrallyalong tenascin‐expressing radial glialprocesses to reach thebasalVM,and later laterallyalongL1CAM‐expressingfiberstoreachVTAorSN.Inthetargetregions,thesecellsundergoterminaldifferentiationbetweenE13‐14(mouse),afterwhich theyallwill co‐expressTHandPITX3.MostofthetranscriptionfactorsresponsibleforVMDAneuronpatterningarewell documented from studies in rodents, but corresponding descriptive studies in thehumanarescarce.Fromthefewstudiesthatexaminedandcomparedtheexpressionandpatterningofdopaminergictranscriptiongenes,itcanbeconcludedthatthespecificlocationand patterning of relevant regulatory genes was usually, though not always, faithfullyconserved between humans, primates and rodents [84‐86]. Therefore, most of ourunderstandingofthedevelopmentofhumanVMDAneuronsisbasedontheextrapolationofourknowledgeofrodentembryogenesis,adaptingittothedifferenttimescaleofhumanontogeny.

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Developmentaltimingamongspeciesisunique.Analysesof29humanembryonicbrains(PCweek4.0‐11.2)revealedthatthesequenceofdevelopmentaleventsisindeedsimilarinamoreprotractedontogenyperiodincomparisontorodents.THimmunoreactivitywasfirstseenincellsoftheventralmesencephalonatPCweek6.5adjacenttotheventricularzone.ThesecellsbegantomigrateventrallyatPCweek6.7.TH+neuronalextensionswerefirstidentifiedatPCweek8.0andTH+neuriteswereseeninitiallyinthedevelopingputamenatPCweek9.0.AtthelatterstagefromPCweek10.0‐11.2,allTH+neuronsappearedtomigrateout of the ventricular zone to their destination and a large number of DA neurons hadelaboratedneuralprocesses[87].Base on the current understanding of embryonicDAneurondevelopment, factors that

induce thedevelopmentofVMDAneuronphenotypeareapplied indifferentiationofDAneuronsin‐vitro.ThedifferentiationofDAneuronfromPSCsin‐vitrowillbeelaboratedinchapter2.

Figure3.SchemeoffactorsandstagesinmouseDA(mDA)neurondevelopmentRegionalisationof theneural tube (hindbrain (hb) brown,midbrain (mb)pink) establishesmidbraintissueidentityviatheinductivefactorSHHproducedinthenotochord(greycircle)andFGF8producedinthemidbrain‐hindbrainborder(blue)respectively.SpecificationofthemDAneuronalidentityoccurswithin the proliferative zone (grey) of the ventralmidline. Here,MSX1 and FOXA2 promote genericneurogenesisviaregulationofNGN2whilstLMX1a,supportedbyMSX1,specifiesmDAneuroncellfate.AsthesemDAneuronprogenitorsbecomepostmitoticandentertheintermediatezone(yellow),theybegintoexpressthepanneuronalmarkerTUJ1and,subsequently,theDAneurontransmitterregulator,NURR1;LMX1bandWNT1positivelycontrolearlyPITX3expressioninsomeNURR1+cells.ThelaststageinmDAneuronaldifferentiationproceedsasthePITX3+cellsandtheTH+cellsmigrateventrallyintotheperipheralzone(red).ThePITX3‐TH+cellgroupsettleinamedialpositiontoformtheVTA,whilethePITX3+TH‐expressingsubpopulationmigrateslaterallytomakeuptheneuralpopulationoftheSN(pinkcells)[78].

GENERALINTRODUCTION

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5. CELLADHESIONMOLECULESANDDOPAMINERGICNEURONDEVELOPMENT

Over‐expressionofmoleculesthatarecrucial ingeneralaspectsofneuronaldevelopmentmayenhancethesurvivalandfunctionalintegrationofDAneuronaftertransplantation.Celladhesionmolecules(CAM)oftheimmunoglobulinsuperfamilyplayanimportantroleinthedevelopmentandregenerationoftheCNS.PSA‐NCAMandL1‐CAMarethefirstmembersofthe immunoglobulin superfamily that have been described to modulate the migration,survival,axonguidanceandsynaptictargetingofneurons.

5.1PSA‐NCAM

NCAMisexpressedonthesurfaceofmostcellsthroughouttheCNS[88‐89].TherearethreemainsubtypesofNCAMwithsizesof120,140and180kDageneratedbyalternativesplicing.TheextracellularregionofallNCAMsisthesame.Itcomprisesfiveimmunoglobulins(Ig1–5) and two fibronectin type III (Fn1–2) domains. NCAM 140 has a shorter cytoplasmicdomainthanNCAM180butthesametransmembranedomain.NCAM120hasnocytoplasmicdomainandislinkedtothecellsurfacebyaglycosylphosphatidylinositolintermediate[90](Figure4).Duringtheembryonicformationoftheneuronalcircuitry,NCAMshouldprovidea dynamic type of cell adhesion allowing structural plasticity instead of a stable, morepermanentcellinteractionintheadultstage.ThisdynamicmodulationofNCAMduringCNSformation is provided by its polysialylation into PSA‐NCAM, a so called active form orembryonicformofNCAM.WithintheGolgibody,sialicacidispolymerizedtoPSA,alinearhomopolymerof a2‐8‐linkedN‐acetylneuraminic acid containingbetween8 toover100monomers, by thepolysialyltransferases ST8SiaIV (PST) andST8SiaII (STX) [91‐94]. It isattachedtotwoasparaginesintheIg5moduleoftheextracellularpartofNCAM[95].The classical view on PSA function refers to its ability to decrease NCAM‐mediated

membrane‐membraneadhesionthroughsterichindranceduetoahighdensityofnegativechargesthatcontributestothehydratedvolumeofNCAM[96‐97].Inthisway,PSAdecreasestherateofbindingamongreceptors, suchas IgCAM,L1CAM,cadherins,and integrins,onopposingcells,withoutaffectingtheintrinsicbindingpropertiesofthesereceptors[97‐101].Alternatively,PSA‐NCAMisalsothoughttoincreasetheconcentrationofsolublefactorssuchasBDNF[102]inthevicinityofcellmembranesbyitshydrophilicproperty[103].Althoughcommonlyconsideredasamoleculeabundantinthedevelopingnervoussystem,

PSA‐NCAM is absent during the early phases of neurogenesis [105]. In the parenchymasurrounding the germinative layers, a relatively faint, widespread distribution has beendescribedtooccurintheembryonicandearly‐postnatalCNS[105‐109].PSAappearstobestrongly expressed in bundles of growing axons [110]. PSA parenchymal stainingdramaticallydecreasesduringtheearlypostnatalperiod,andalmostdisappearsaroundtheendofthethirdweekoflifeintheCNS.AfterthisstagetheNCAMremainspolysialylatedonlywithinrestrictedCNSregionsand/orcellpopulations[108‐109,111‐116].

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Figure4.SchematicstructureofPSA‐NCAMsandL1CAMThe three major isoforms of NCAM, termed NCAM180, NCAM140 and NCAM120 all have fiveimmunoglobulin‐likedomains(Ig1toIg5)andtwofibronectintypeIIIrepeats(FN1andFN2).PolysialicacidcanbetransferredonN‐glycosylationsitesattachedtotheIg5domainofNCAM.L1CAMhassiximmunoglobulin‐likedomainsand four to five fibronectin type III repeats.The locationofproteolyticcleavagesitesisdenotedbyarrows.Baseon[94]and[104].

PSA‐NCAMmaybeinvolvedinthecontrolofmigrationandmaturationofdopaminergicneurons in thedevelopingmesencephalon [117].NCAM ispresentondopaminergic cellsisolatedfromthemesencephalonofthefoetalrat[118].CellsalongtheventralsurfaceofthemesencephaloninE13ratscontainanincreasedamountofNCAMandPSA‐NCAM.DuringthedevelopmentofVMDAneurons, theamountofPSA‐NCAMincreasesstrikinglyand itoutlinesTH+cellsofthemesencephalon.AtE19,PSA‐NCAMisalsopresentintheneuropilbetweencellsandimmunostainingforPSA‐NCAMinsectionsofthemesencephalonappearstobemuchmoreintensethanthatforNCAM.FromE19toE21,PSA‐NCAMisexpressedinboththedevelopingmesencephalonandthestriatumbutthelevelofexpressiongraduallydecreasesfromE21onwards.AtP3,NCAMnolongeroutlinesthecellbodiesbutislocateddiffuselythroughtheneuropilofthemesencephalon[117].ThepresenceofahighcontentofPSAonmesencephaliccellsandaxonsprojectingtothemesencephalonduringthe lateprenatalandearlypostnatalperiodsreflecttheroleofPSA‐NCAMinplasticityduringthedevelopmentofdopaminergicneurons.Inthedevelopingratstriatum,theexpressionofPSA‐NCAM is topographically and dynamically regulated, strategically associated with theformationofsynapticcircuitryduringP7toP25[111].PSA‐NCAMexpressionpersistsnot

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only during the ingrowth of cortical and nigral inputs but also during the formation ofdendriticspinesandsynaptogenesis[109].

5.2L1CAM

L1CAM is encoded by the L1CAM gene located at the long arm of the X chromosome.Alternative splicing give rise to neuronal isoforms including exon 2‐encoded sequence(YEGHH inhumanL1CAM) different from thenon‐neuronal isoforms [119]. It contains aregionofsiximmunoglobulin‐likedomainsandfivefibronectintypeIIIrepeats,followedbyasingle‐passtransmembraneregion,andashortcytoplasmicdomain[120](Figure4).ThefulllengthofL1CAMis220kD.Byproteolyticcleavageattheextracellularcleavagesites,ityieldsproductsof140kDand85kD(cleavageattheFn3site),or~200kDand32kDL1CAMfragments(cleavagedistaltotheFn5site)[121].The140kDand200kDfragmentcanbedepositedintotheextracellularmatrix[122]andcanalsoberecoveredfromtheCSF[123].Themulti‐domainstructureofL1moleculesenablescomplex interactionswithdiverse

receptors on neighbouring neurons, glial cells and the extracellular matrix, which mayfunctiontodynamicallymodulatethecellwithrespecttomigration,axonalfasciculationandpathwaysformation[124].L1CAMdoesnotaccomplishthisalone,butratheristhoughttorecruit other CAMs and signalling receptors at the neuronalmembrane to form amulti‐proteincomplexonopposingcellsurfacesviaitsextracellularregionand,atthesametime,organizecytoskeletalandsignallingproteinsviaitscytoplasmicregion[125].In thedevelopingCNS,postmitoticneuronsinmanyregionsof thecerebralcortex first

migrate away from theventricular zone along radial glia, but later alsoperpendicular toradialglialprocessesalongaxonaltracts[126].Guidancecuesthatdirectneuronalmigrationincludecelladhesionmolecules,extracellularmatrixproteins,andneurotrophicfactors.Theneural cell adhesionmoleculeL1CAM is a transmembraneglycoprotein that is expressedwidelyonthesurfaceofpostmitoticdevelopingneuronsandconcentratedingrowthconesandaxons,playinganimportantroleincelladhesion,migration,andaxongrowth[127‐129].DuringVMDAneurondevelopment,L1CAMisalsoexpressedinVMDAneurons,lessin

the soma andmore enriched in the axons; numerousTH+ fibres in themedial forebrainbundelwereshowntostainforL1CAM[130].IthasbeenreportedthatL1CAMcanaffectthedistribution [131] and survival of VM DA neurons [132]. L1CAM was also transientlyexpressedonthemedianpartoftangentialfibrescoincidentwiththelateralmigrationofDAneuronsfromE11toE13,whenneuronsmovealongthetangentialfibrestowardstheirfinaldestinations[133].L1CAM transmits signals that benefit neuronal survival. When triggered by a trans‐

interactingL1CAMmoleculefromaneighbouringcellorintheextracellularmatrix,L1CAMinducessignaltransductioninL1CAM‐expressingneurons,whichresultsincellsurvivalandneuritogenesis[134].Inprimarymesencephaliccultures,theadditionofL1CAMantibodytothecellculturemediaincreasedthesurvivalofTH+cells;L1CAMAb‐treatedVMtissuegraftsdemonstratedsignificantlygreaterareasofgraft‐derivedinnervationcomparedtocontrolgrafts[135].

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ItseemsthatbothPSA‐NCAMandL1CAMmaybeemployedtoenhancethesurvivalandoutgrowthof(iPScell‐derived)DAneuronsafterimplantation.Expressionofbothadhesionfactors in the implanted DA neurons or the surrounding brain parenchyma may bestimulated/inducedviaviraltransfectionofrelevantgenes(e.g.STX).Varioustypesofviralvectorsareavailableforthat.Wehavestudiedtheadeno‐associatedvirusinmoredetailandtesteditspotentialtodeliverspecificgrowth‐stimulatingfactorsininjuredtissue;forthatwehavechosenthetraumaticspinalcordinjurymodel.

6. TRAUMATICSPINALCORDINJURY

Traumaticspinalcordinjuryresultsfrombluntorpenetratingtrauma;causesincludemotorvehicleaccidents,violence,falls,andrecreationalactivities[136].Spinalcordinjurycausesmotorandsensoryimpairmentsdistalfromtheinjurylevel.Primarytissueinjuryiscausedbytractionandcompressionbyfractureswithdisplacedbonefragments,discmaterial,orligament injuries leading todamageof bloodvessels and axons.Microhaemorrhages andswelling happen within minutes, and lead to secondary ischaemia. Combined with thereleaseoftoxiccomponents,suchasglutamateandcalciumions,fromdamagedcells,axons,andbloodvessels,itattacksintactneighbouringcellsandtriggersasecondaryinjurycascadethatextendstheprimaryinjurytoregionsneighbouringthelocalinjurysite[137].The spinal cord‐compressionmodel recapitulates theprocessof traumatic spinal cord

injury[138].Theconsequentlydegeneratingspinalcordismarkedwithglialscar[139]andnecrosis[140],whichformrigidobstaclesfortheaxonalextensionanddistributionofthegenedeliveryvectors.Thus,thespinalcordinjurymodelprovidesaproperanimalmodeltostudygenedeliveryinaseverelydamagedCNS.

7. ADENO‐ASSOCIATEDVIRUS

Recombinantadeno‐associatedviruses(AAVs)arepromisingvectorstodelivermoleculescapableofmodifyingtheinjuredparenchymaoftheCNS.AAVsestablishalatentinfectionwithinthecell,eitherbysite‐specific integrationintothehostgenomeorbypersistinginepisomal forms [141]. AAV was first described as a satellite virus observed in electronmicrographs of adenovirus [142]. The profitable features of AAVs as a viral therapeuticinclude: it is replicationdefective, and requireshelperviruses forproductive replication;evenpresentinahighsero‐prevalencerate,AAVisconsideredasnon‐pathogenic[143‐144];theyareabletodirectlong‐termgeneexpressionwithoutdestructiveTcellresponses[145‐146]. One downside for the application of AAV is that the limitation for packaging isapproximately4700nucleotides[147].ElevennaturallyoccurringAAVserotypeshavebeen isolated.All theseAAVserotypes

shareconsiderablehomologyinbothrepandcapgenesexceptAAV5,whichisamoredistalmemberofthedependovirusfamily[148].ThedifferencesamongdistinctserotypesofAAVsare the usage of binding receptors, the methods for internalization, the release fromendosomes, the uncoating etc... These differences eventually result in tissue tropisms oftransduction,andinfluencegenetransductionefficiency.AAV2isknowntotransduceawiderange of tissue types, including liver, muscle, lung, and central nervous system, with

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moderateefficiency.AAV9exhibitsa similarprofilebutwithmuchhigherefficiency thanAAV2[149].AAV1andAAV7areknowntoperformwellwithrapidonsetandhighlevelsoftransductioninskeletalmuscle[150‐151].AAV6,whichdiffersfromtheAAV1capsidbyonlysix aminoacid residues, has also shownapropensity for transductionof skeletalmuscle[152].AAV5appearstobeasuitablevectorforgenedeliverytothebrain,liver,andairwayepithelialcells[148,153].

8. OUTLINEOFTHETHESIS

ThemajorfocusoftheresearchdescribedinthisthesisconcernsstudiesonthetreatmentofParkinson’s disease by the implantation of DA neurons generated from iPS cells. ThepotentialofintracerebrallyimplantedDAneuronstoameliorateanumberofsymptomsofParkinson’sdiseasehavebeendemonstratedinthe1990swiththeuseofabortion‐derivedfoetalDAneurons.Worldwideabout400PDpatientsreceivedanimplantationwithhumanfoetal DA neurons. The ground breaking phenomenon of iPS cell has provided anunprecedentedautologousalternativesourceforDAneurongrafts.Inchapter2,weaddressthequestionwhetherhumaniPScell‐derivedDAneuronsarefunctionallyidenticaltothehumanfoetalDAneurons.FunctionalityofDAneuronsfortransplantationcanbetestedinanimalmodelsforPD.Inanextensivereview,wecomparedtheperformanceofhumanfoetalVMtissueandhumaniPScell‐derivedDAneurongraftsintheratrotationmodelforPD,byanalysing the functional improvement of the grafted animals and the survival andcharacteristicsoftheimplantedDAneurons.Ingeneral, the iPScell‐derivedDAneuronsareconsideredsimilar to ‘real’primaryDA

neurons.However,itislikelythatduringreprogrammingandsubsequentdifferentiationanaberrant epigenetic landscapemayhavedevelopedwith consequences for their ultimateexpressionprofile.TodeterminepotentialdifferencesinexpressionprofilebetweeniPScell‐derivedDAneuronsandprimaryDAneurons,wemadeuseoftransgenicPtix3‐GFPmicetogeneratepuresuspensionsofiPScell‐derivedDAneuronsandtoisolatepureprimaryVMDAneuronsusingFAC‐sorting(chapter3).OfbothpureDAneuronsuspensionswehaveanalysed and compared in detail the genome‐wide expression profile. We found a fewaberrations in the gene expression of iPS cell‐derived DA neurons that may affect theiroutgrowthandmaturationaftergrafting.Inanattempttostimulatetheneuriteoutgrowthof iPScell‐derivedDAneurons,wehavestudied, inchapter4, theeffectof inducedover‐expressionofthecellularadhesionmolecules,PSA‐NCAMandL1CAM,bothin‐vitroandin‐vivo,i.e.afterimplantationinthestriatumof6‐OHDAlesionrats.DA neuron replacementmay indeed compensate the decreased dopamine level in the

striatum of PD patients; however, the trend for degeneration in the aged brainwill stillcontinueandmayevenaffectnewlyimplantedDAneurons.UnderstandingthecomponentsthatcontributetoneurodegenerationinthePDbrainiscrucial.Inchapter5,westudiedtherole of immune cells in the pathogenesis of PD. Whereas the role of microglia in PDpathogenesishasbeenwellestablished,onlyafewindicationssofarseemtosupporttheinvolvement of the peripheral immune cells in PDpathology. CD4+ andCD8+ cellswere

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foundinabundantnumbersinthedegeneratingSNofPDpatients.Perforin‐mediatedcellapoptosisisanimportanttoolforcytotoxiclymphocytestoeliminateabnormalcells.Inthischapter, we used perforin (Pfp) null mice and wild type mice to study the potentialinvolvementofperforin‐mediatedcytotoxicityintheSNafterPDinductionwithMPTP.In the somewhat detached final experimental chapter 6, we addressed the most

appropriateway to induce the expression of target genes in lesionedCNS areas by viraldelivery.Spinalcordinjurymodelischaracterizedbyglialscar,necrosisandtheinhospitableenvironmentsurroundingtheinjuredsitethatgeneratedifficultobstaclefortheviralvectortocarryontransduction.Westudiedtheefficiencyofdifferentserotypesofadeno‐associatedvirusindeliveringgenesintheinjuredspinalcord.Thefinalchapter7containsasummaryof themajor findings presented in this thesis and a discussion of their potential clinicalimplications.

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COMPARISONOFHUMAN(FOETAL)PRIMARYWITHHUMANIPSCELL‐DERIVEDDOPAMINERGICNEURONGRAFTSINTHERATMODELFORPARKINSON’SDISEASE

Su‐PingPeng1,2,andSjefCopray1

1DepartmentofNeuroscience,UniversityMedicalCenterGroningen,theNetherlands2Center for Neuroscience, Shantou University MedicalCollege,Shantou,GuangdongProvince,P.R.China

PublishedinStemCellReviewsandReports,2015

ABSTRACT

Neuronaldegenerationwithinthesubstantianigraandthelossofthedopaminergicnigro‐striatalpathwayarethemajorhallmarksofParkinson’sdisease(PD).Graftsoffoetalventralmesencephalic(VM)dopaminergic(DA)neuronsintothestriatumhavebeenshowntobeable to restore striatal dopamine levels and to improve overall PD symptoms. However,humanfoetus‐derivedcellgraftsarenotfeasibleforclinicalapplication.Autologousinducedpluripotent stem cell (iPS cell)‐derived DA neurons are emerging as an unprecedentedalternative.Inthisreview,wesummarizeandcomparetheefficacyofhumaniPScell‐derivedDAneurongraftstorestorenormalbehaviourinaratmodelforPDwiththatofhumanfoetalprimaryDAneurons.Thedifferencesweobservedintheefficacytorestorenormalfunctionbetweenthe2typesofDAneurongraftscouldbeascribedtointrinsicpropertiesoftheiPScell‐derivedDAneuronsthatcriticallyaffectedsurvivalandproperneuriteextensioninthestriatumafterimplantation.

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1. INTRODUCTION

Parkinson’sdisease(PD)isoneofthemostprevalentneurodegenerativediseases,affecting1in100peopleovertheageof60[1].Thedisturbancesintheinitiationandfine‐regulationofmovementresultingintremor,rigidity,akinesiaandposturalinstabilityinPDpatientsarecausedbytheprogressivelossofdopaminergic(DA)neuronsinthesubstantianigra(SN).ThelossofDAneuronsintheSNleadstoadisruptionofthenigro‐striatalcircuitryandadopaminergic depletion of the striatum. The current standard therapy, L‐DOPAadministration, only temporarily restores striatal dopamine levels whereas deep brainstimulationonlytransientlyreducesseveretremor[2‐3].Thesearchformoreefficientandlong‐termeffectivetreatmentstrategiesforPDisstill

ongoing.Theground‐breakingdevelopmentsinstemcellresearchinthelastdecadehaverevivedtheinterestforintracerebralcelltransplantationasatherapeuticalapproachforPD.In this approach, replacement of the lost nigrostriatal dopaminergic innervation of thestriatumbyexogenousdopaminergicneuronsisintendedtorestorebasicdopaminelevelswithinthestriatum.Experimentsonrodentandnon‐humanprimatemodelsforPDinthe1970sand1980s,hadshownthatembryonicDAneuronscouldsurviveandreinnervatethestriatum after stereotactic injection [4‐5]; moreover, reduction in drug‐induced rotationbehaviour in ’Parkinsonian’ rats demonstrated that the grafted DA neurons indeedfunctionallyintegratedintothestriatumandreleaseddopamine[6‐8].Basedonthefindingsin the experimental animalmodels, various clinical trialswere started in the 1980s and1990swith thestriatal implantationofhuman foetalDAneuronswithpromisingresults.Patientswithhumanfoetalventralmesencephalon(VM)graftshavebeenshowntorecoverfromrigidityandtremorstovariousdegrees,correlatedtotherestorationofintracerebraldopaminelevelsasdetectedbyPET[9‐11];manyofthesepatientsexperiencedanoverallimprovementinqualityoflife[9‐10,12‐13].ThoughtheoutcomeofstriatalimplantationofhumanfoetalDAneurons ina largegroupofPDpatientswasverypromising,reportsonsevere side effects, presumablydue to suboptimal, poorlydocumentedgraft compositionand/oradverseimplantlocalization,ledtoadiscontinuanceofthisapproach.Apartfromthecomplicatedlogisticstoobtainmultipledonorfoetusesofthesamedevelopmentalstage,theuse of abortion‐derived human foetal brain tissue to obtain a sufficient number ofhomogenousgraftableDAneuronsraisedconsiderableethicalconcern.SeveralothersourcesforimplantablehumanDAneuronshavebeenexploredeversince.

DAneuronshavebeensuccessfullydifferentiatedin‐vitrofromhumanembryonicstem(ES)cells andhumanneural stemcells (NSCs). Besides the fact that both sources still causedethical objections, the non‐autologous origin of these cells, demanding lifelongimmunosuppression after implantation, was considered a major obstacle. The ground‐breaking detection of induced pluripotent stem cells (iPS cells) generated from easilyaccessiblesomaticcells(e.g.skinfibroblasts)[14‐15]hasprovidedanunprecedentednovelautologoussourceforhumanDAneurongrafts.Thefunctionalityofthesein‐vitrogeneratedhumanDAneuronsafterintrastriatalimplantationhavebeenrecentlystudiedinrodentandnon‐humanprimatemodelsforParkinson’sdisease:ingeneral,apartfromtheassessment

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ofmotorbehaviour,parameterssuchasneuronalsurvival,theextentofneuriteoutgrowth,thecoverageofthestriatumwithafinenetworkofdopaminergicterminalsandthereleaseofdopaminehavebeenevaluated.Theaimofthisreviewistosummarizethebehaviouraleffects,thesurvivalandneurite

outgrowthofhumaniPScell‐derivedDAneuronsaftertheirintrastriatalimplantationintheParkinsonmodelsandcompare themwith thoseobserved for implantedhumanprimaryfoetalDAneurons.Wediscusspossibleexplanationsforthedifferencesinefficacy,survivalandoutgrowthbetweentheiPScell‐derivedDAneurongraftsandtheprimaryDAneurongrafts.

2. UNILATERAL6‐OHDALESIONRATMODELFORPARKINSON’SDISEASE

InordertostudythefeasibilityandefficacyofgraftedDAneuronsastherapyforParkinson’sdisease,aproperanimalmodelisrequired.AmongvariousanimalmodelsforPD(forreviewsee[16‐17]),6‐OHDA‐lesionedratsarethemostoftenusedPDanimalmodelinDAneurongraftresearch.Thetoxin6‐hydroxy‐dopamine(6‐OHDA)specificallyusesthecatecholaminetransport system of catecholaminergic neurons to enter the cells. It generates hydrogenperoxideandhydroxyl radicals anddisturbsmitochondrial complex I,which leads to theproductionofsuperoxidefreeradicalsandeventuallytocelldeath.Stereotacticalinjectionof6‐OHDAintothestriatumofanadultrat,willinduceretrogradedegenerationoftheSNDAneurons,usuallywithin2to3weeks[18].Whentheinjectionof6‐OHDAisdirectedintothenigrostriataltractorintheSNitself,acuteDAneurondeathwillbeinducedwithin24hrs[19].Thesizeofthelesioncanbeadjustedbyvaryingthedosageof6‐OHDA[18].Theefficacyoftheunilateral6‐OHDAinducedlesionandsotheeffectofaspecifictreatmentinratsarevalidatedbyipsilateralorcontralateralcirclingmotorbehaviouraftertheadministrationofamphetamineorapomorphine[20].Thenumberofrotationsperminutecanbetakenasameasure for the severity of unilateral DA loss in the striatum and the efficacy ofcompensationbyDA‐producinggraftedDAneurons[21].Besidesdrug‐inducedrotation,6‐OHDAunilaterallesionsoftherodentmeso‐telencephalicdopaminepathwaysalsoleadtopostural curvature, spontaneous rotation, contralateral sensory neglect and aberrantactivity[7,22].Variousactivitytests,suchastheadjustingsteptest[23]andthecylindertest[24],havebeenusedtostudythenon‐rotationalbehaviouralconsequenceofthe6‐OHDAunilaterallesionsandtheeffectsofaspecifictreatmentonthat.

3. HUMANFOETALDOPAMINERGICNEURONALGRAFTS

Sincethelate1970s,transplantationofembryonicVMtissuefromrodentsandprimatesinPD animal models has been extensively studied (for reviews see [25‐26]). From thesefundamentalstudies,protocolsfortransplantationwereestablishedasparadigmforstudieson human foetal tissue transplantation. It was established that only newly formedpostmitoticDAneurons (E13 ‐E15 in rats) are suitable forgrafting, since theyhave justreachedadopaminergicphenotypebutdonotyethave fullyoutgrownextensions,whichmakesitpossibletoisolatethemwithouttoomuchcellulardamage[21,27].ExtrapolatingthesedatatohumanDAneurongrafts,humanfoetalVMtissueofagestationalageof7to10


35

weekswasconsideredoptimal.Inalmostallrodentstudies,transplantationwasperformedright after tissue isolation and preparation, either dissociated into cell suspension ordissectedassmalltissuepieces.Inthisapproach,DAneuronsappearedtobestillviableandsurvivethegraftingprocedure[26,28].Based on the promising data of rodent and primate DA neuron‐rich VM tissue

transplantedintoPDmodels[4,8,21,29‐31],experimentalstudieswiththeimplantationofhumanfoetalDAneuronsinPDmodelswerestarted.

3.1Cellularcomposition

When isolating and dissociating the foetal human ‘DA’ VM region for Parkinson‐relatedtransplantation purposes, one should be aware of the actual cellular composition of thisregion.This isofconsiderable importancesinceinmostPDtransplantationstudiesfoetalhumanVMcellsuspensionshavebeenusedwithoutanyformofpurification.Inthehuman(foetal)VMregion,SNDAneuronsandventral tegmentalarea (VTA)DAneurons indeedmake up around70%of the neurons [32]. About 30%of the neurons areGABAergic; inaddition,2‐3%ofglutamatergic(Glu)neuronscanbefoundinthisregion[32].However,oneshouldbeawarethatneuronsaccountforonly5.6%ofthetotalnumberofcellsintheVM,whichisevenlessthaninmostotherbrainareas,forinstancethebrainstem;8%ofthecellsin this area are neurons [33]. Most of the cells in the VM region are nonneuronal andcompriseastrocytes,microglia,endothelialcellsetc…ThecontaminationofallthesespecificnonneuronalcelltypesintheultimategraftwillinevitablyaffectthesurvivalandfunctionoftheimplantedDAneuronsandthereactionofthehostintheenvironmentaroundthegraft.So,infact,onlyaminorityofcellsinthenon‐purifiedfoetalcellsuspensionfromthehumanVM region, isolated for grafting in PD animal models, can be actually considered SNdopaminergicneurons.WhenhumanVMtissuegraftsarecollectedfromabortedfoetuses,thecellularcompositionmaybeevenmoreuncertain,duetothemethodofabortionused.Inmanycases,theabortionproceduresledtoadestructionofthefoetus,whichmadeitdifficulttorecognizebraintissue,letalonetheverysmallspecificVMportion.Evencontaminatingserotonergic(!)neuronshavebeendemonstratedintheperipheryofthegraftsinthestudybyStrombergin1989[34]:many5‐HT‐immunoreactiveterminalswerefoundinthestriatalneuropilofthehostbrainarisingfromthegraft.MoststudiesdonotevenreportordiscussthecellcompositionoftheusedhumanfoetalDAneurongrafts.

3.2EfficacyofintrastriatallygraftedhumanfoetalDAneuronsinPDanimalmodels

3.2.1BehaviouralimprovementandDAneuronsurvival

InmoststudiesonhumanfoetalVMtissuetransplantationintheunilaterally6‐OHDAlesionratPDmodel(Table1),asignificantreductionindrug‐inducedrotationwasobservedeitheraftergraftingwithaVM‐derivedcellsuspensionorsmallVMtissuepieces.Intworats,inthestudybyBrundinetal.,rotationwascompletelyannihilatedandevencontralateralrotationwas induced at 12.5 weeks post grafting. Another two rats in this study reducedamphetamine‐inducedrotationby69%and92%atweek15.5postgrafting[35].Rotation

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reductionappeared tobecorrelatedwith thenumberofsurvivingDAneurons:completerecoverywasestablishedbyanaverageof1200DAneurons21weekspostgrafting,whilethe14to107DAneuronsfoundinsomeratswereunabletoinduceanybehaviouralchange[35].ThebehaviouralimprovementafterhumanDAgraftingoccurredmuchlaterthanwhatwasobservedwithratembryonicVMtissuegrafts(3‐4weekspostgrafting),indicatingthathumanneuronsrequiredmoretimetomatureandintegrateintothestriatum.Reductionindrug‐inducedrotationwasonlyobserved15.5weekspostgraftinginratsreceivingahumanVMcellsuspensionfromfoetusesofgestationalageof9weeks(PC9weeks),butnotfromfoetuses isolatedatgestationalagebetweenPC11‐19weeks [35]. Inanotherexperimentwithasimilarset‐up,intrastriatalgraftingofhumanVMtissuewithagestationalageof6.5weeks and 8 weeks resulted in a pronounced reduction in both amphetamine‐ andapomorphine‐inducedrotation,aswellasinspontaneousrotation18weekspostgrafting[36].Intheseanimals,300to4249(average1626)DAneuronswerefoundtosurviveinthegraftedstriatum.ThestudiesbyBrundinetal.indicatedthatingeneralbetween1‐10%ofthegraftedhumanfoetalVMcellsuspension(approximately40,000DAneurons)survivedthe implantation procedure [35‐36]. Additional studies have shown that the survival ofbetween 500‐700 human foetal VM DA neurons are needed for full reversal of theamphetamine‐inducedrotationbehaviourby19‐21weekspostgrafting[37‐38].TransplantationofsmallVMtissuepiecesinapremadecavityinthecortexalsoyielded

significantDAneuronsurvivalandledtoasignificantreductionofdrug‐inducedrotation.Inthis approach, even human VM tissue pieces of a gestational age up to 12 weeks couldactuallysurvivetransplantationandgaverisetoasimilarbehaviouralimprovementat3‐5monthspostgraftingasthecellsuspensiongrafts[34,39].ThegenerationofhumanVMDAneuronstakesplaceduringpostconceptionweek6.5to9;theystartextendingelaboratedprocessestowardsthestriatumfromweek10on[40].ItisapparentthattheDAneuronsinyoung explants (PC 6.5‐9weeks), since they do not have extensive axonal and dendriticoutgrowth,arehardlydamagedduringisolationanddissociation;incontrast,olderfoetalDAneurons(PC11‐19weeks)alreadyhaveextensiveaxons,dendritesandconnections,somostof them are severely damaged after dissociation andwill not survive after implantation.WhendissociationisomittedandsmallblocksoffoetalVMtissueareused,thestructureofmostcellswillbepreserved,sothateventissuesfromPC12weekscanbeused.Fullreversalofapomorphine‐inducedrotationwasseldomreported.Yet,Strombergetal.

showedthatthisispossiblewithprolongedtime,7monthspostgrafting.TheincreaseofD2andD3receptorsinthestriatumseemstobenormalizedatthistimepoint[41].Ithasbeensuggested that the function of SN also depends on dendritic DA release in the SN [42].Interestingly, transplantation of DA neurons in the SN can give rise to a reduction ofapomorphine‐induced rotation [38]. However, it is still controversial whether the DAneurons grafted close to SN are able to extend through the meso‐striatal pathway toreinnervatethestriatum;whereasRathetal. [38]wereunabletodetectsuchconnection,bothWictorinetal.[43]andGrealishetal.[79]showedthatgraftsofhumanfoetalVMof


37

differentdonoragescouldindeedreconstructthemesostriatalpathwaywhengraftedtoSNofadult6‐OHDAlesionedrats.

3.2.2CharacteristicsoftransplantedhumanfoetalDAneurons

ItwasobservedthathumanfoetalDAneuronsgraftedinthestriatumneededalongerperiodtomatureand integrate than those fromrodents.Two to threemonthspostgrafting, thehumanTH+neuronsstillhadanimmaturesmallroundedcellbody.Upto4months,theDAneuronsweremoresimilartoadultSNDAneuronswithTH+dendritesandoftenwithfinespine‐likelateralprocesses[39].TwomainpopulationsofTH+neuronswereobserved:themostfrequenttypewithsmallperikaryaofdiameterbetween12to25m,andtherestofthepopulationwithalargeperikaryadiameterupto50mlong[36‐37].Insomecases,TH+perikaryawerefoundupto1‐1.5mmfromthegraftbordersinthehoststriatum,indicatingtheabilityofthegraftedprematureDAneuroblaststomigrate[36].Mostoftheneuronsweremultipolarwithcoarseprocessesextending2.5‐3.0mm[34]intothehoststriatum,andinsomecase,upto6mm[36]oreven9‐10mm[38,43].Theextentofactualreinnervationofthestriatumcorrelatedtothepostgraftingtime.AsparseTH+fibreplexuswasseenadjacenttotheimplant11weekspostgrafting,whilearichstrongTH+fibrenetworkappearedtohavereinnervatedtheentirestriatumat20weeks(orlonger)postgrafting[37,41,44‐45].ThedemonstrationofreciprocalsynapticconnectivitybetweengraftedhumanfoetalDA

neuronsandthehoststriatalinterneuronsconfirmedtheintegrationofthegraftedhumanfoetalDAneurons.UltrastructuralstudiesidentifiedtheTH+coarseprocessesasdendrites[37],providingasite forhost input to thegraft:graft‐derivedTH+dendritesappearedtoreceivenon‐THlabelledsynapticcontactsinthehoststriatalneuropil.Moreover,TH+axonscontainingroundorovalvesiclesformedsymmetricsynapses,likethoseseeninthenormalmeso‐striatalDApathway,withdendriticshaftsandspinesandinanevenhigherincidenceonneuronalperikarya[34,37,46].The electrophysiological characteristics of grafted humanDAneuronswere similar to

thoseofDAneuronsinanintactrat,whichfirespontaneouslyinaslowfiringrate(1‐10Hz)[47].SomeofthegraftedhumanDAneuronsshowedthecharacteristicfeaturesofinitiallypositivelydeflecting,bi‐ortriphasicactionpotentialswithdurationsgreaterthan1.5msec.ThesustainedactivityrateoftheseDAneuronsrangedbetween0.1and10spikespersecond.Asalsofoundintheintactrat,some‘partiallyactive’graftedhumanDAneuronsexhibitedactionpotentialwaveformsthatwereconsistentwiththewaveformsrecordedfrominsituactiveDAneurons[44].ThefunctionalityofthesynapsesformedbythegraftedhumanDAneuronswiththehost

striatalinterneuronswasvalidatedwithstriatalrecordings.Thestriatalinterneuronsatthesiteofthehumanfoetalgraftdisplayedslowdischargerates(3.10.4spikes/sec)thatwerealmostidenticaltothosefoundinstriatalneuronsatthenonlesionedsite(mean3.00.4spikes/sec)[44].

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38

Table1‐SummaryofgraftingofhumanVMtissuesintoratPDmodel

Tobecontinued

[Ref],Donorage,(mode)/graftsite

Functionalrecoveryreductionofrotation

Tissuesurvival(numberofDAneurons)

[35],PC9‐16w,(cells);PC16‐19w,(pieces)/CPu

Amphetamine:PC9w:69‐92%(15.5WPG)>100%(20WPG)PC11‐19w:<0%

PC‐9w:300‐1800(10,21WPG)PC‐11w:14‐74(6,12.5WPG)PC15‐19w:0‐2(5,20WPG)

[39],PC9‐12w,(pieces)/CPu

Apomorphine:50‐80%(4‐5MPG)

Moderatetolargenumbers

[36],PC6.5‐11.5w,(cells)/CPu

Amphetamine:PC6.5‐8w:>94%in6outof7rats(18WPG)PC11.5w:13‐52%in4rats(19.5WPG)Apomorphine:PC8w:58‐79%(18WPG)Spontaneousrotation:>100%

PC6.5‐8w:402‐4249PC‐11.5w:0‐198NosurvivingofDAneuronswithoutimmunosuppression

[37],PC6.5‐9w,(cells)/CPu

Apomorphine‐inducedrotation:>100%(19‐21WPG)

500‐700

[46],PC7‐12w,(pieces)/CPu

n.d. n.d.

[34],PC7‐12w(pieces)/CPu

n.d.1.LargeamountofTH+neurons2.Numerous5‐HT+neurons


39

CharacteristicsofgraftedDAneuronNeuriteoutgrowth&striatum

reinnervation(%)

Morphology:1.Bipolarappearance2.Longcoarseprocesses3.Diameterofcellbody50m

Extend2‐3mm,denseclosegraftsite;sparseaway

Morphology:1.2‐3MPG:smallroundedcellbodies2.4MPG:similartoadultSNneurons

40‐60%ofthedorsalstriatumFibersalsoreachingventralparts

lntracerebraldialysi:1.BasalextracellularDAlevels(intactstriatum):0.26±0.08(0.19±0.06)pmol/25l2.ActivatedDAlevels:Amphetamine,3.7(15.12)folds;nomifensine,3(5.79)foldsMorphology:Diameterofcellbody:50m;mean2519.5m;coarseTH+processesMigration:1‐1.5mm

Extensivenetworkreinnervated100%oftheheadofthecaudate‐putamenExtendinguptoatleast6mm

Morphology:Diameterofcellbody:50m;mean12x16mMultipolarandofirregularshapeCoarseTH+dendritesSynapseformation:8WPG:TH‐synapse11WPG:somefewTH+synapses20WPG:TH+synapsesthroughouttheneostriatumTH+boutons0.6‐1.2mindiameter

8WPG:nofiberoutgrowth11WPG:sparseTH+fiber20WPG:richfibernetwork,reinnervated100%ofneostriatum

Electrophysiology(localappliedK+):50%amplitudesofelectrochemicalsignalsofnormalcontrol;2foldsofdenervatedareaMorphology:TH+processescontainedvesicles;formedsymmetricandasymmetricsynapseswithhostdendriticspinesandshafts

TH+neuriteextendedafewmillimetersfromthegraft

Morphology:1.Ovalorroundedcellbody,diameters40m2.>1primarydendrites3.ThickTH+fibers4.Vesicle‐filledaxonsmadeclosesymmetric/asymmetricalsynapseswithdendriticshaftsanddendriticspinesElectrophysiology(localappliedK+):Signalsamplitudeof0.5±0.1mM(fornormalcontrol2.0±0.1mM)

1.TH+fiberextendto2.5‐3mm2.Innervationsof5‐HTfibers

2

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40

Continued

[Ref],Donorage,(mode)/graftsite

Functionalrecoveryreductionofrotation

Tissuesurvival(numberofDAneurons)

[44],PC7‐12w(pieces)/CPu

78%(5MPG)1.TH+cellswerefound2.Few5‐HT+cells

[43],PC6‐8w,(cells)/Rostralmes;IC;CPu

n.d.13‐20WPG,laughnumber

[41],firsttrimesterfetus,(pieces)/lateralventricle

Apomorphine:Completelyeliminatedin3rats;93%decreasein1rat(7MPG)

n.d.

[45],firsttrimesterfetus,(pieces)/lateralventricle

Apomorphine:86%(4MPG)

NumerousTH+neurons

[38],PC7‐9w,(cellsorpieces)/CPuorSN

Apomorphine:Part/metal/CPu=75%≈Full/glass/CPu≈Full/glass/SN>Full/metal/CPu>Sham/glass/SN=Sham/metal/CPuAmphetamine:Part/metal/CPu>100>Full/glass/CPu=Full/metal/CPu>Full/glass/SN=Sham/glass/SN=Sham/metal/CPu

Pieces/metal/CPu:2,183±449Cells/metal/CPu:892±378Cells/glass/CPu:657±199Cells/glass/SN:1548±362

Abbreviate:PC(post‐conception);CPu(caudat‐putamen);SN(substantianigra);mes(mesencephalon);glass(glasscapillary);WPG(weekspostgrafting);MPG(monthspostgrafting)


41

CharacteristicsofgraftedDAneuronNeuriteoutgrowth&striatum

reinnervation(%)

Morphology:Oval,fusiformortriangularcellbodyThickTH+fibersElectrophysiology:1.Actionpotentials:bi‐ortriphasic,>1.5msecdurations(similartonormalDAneurons)2.Patternsofspikeactivity:‘activate’in14/41cellsrecorded;‘partiallyactive’in27/41cellsrecorded3.Slowdischargerate(2.9±0.4spike/sec)(normalcontrol3.0±0.4spike/sec)

TH+neuritereinnervated100%ofthestriatumNoincreasein5‐HT‐positivefibers

n.d.

Rostralmes&IC:Neurites(majorityTH+)extendedalongthemedialforebrainbundle,evenuptotheolfactorybulb(10mm);neuritesramifiedwithinthecaudateputamenCPu:Mostneuritesramifiedandterminatedwithinthecaudalcaudateputamen,afewextendedcaudally

Striatalrespondstograft:ReversedtheincreasedofD2receptorsThedecreasedofD1receptorremainNormalizedtheincreasedofD3receptor

hNF+cover100%ofstriatum

MPTPrespond:TH+reinnervatedfiberindorsalstriatumweredegeneratedTH+reinnervatedfibersinolfactorytuberclewerespared

TH+fiberdenselyinnervated100%ofstriatum

VMAT+/TH+co‐labeling

Cells/metal/CPu:reinnervatedthroughouttheentirestriatum,exdendeduptocaudalbrainregions(5–9mm)Pieces/glass/SN:hNF70+fibersexclusivelyconfinedtotheSNandtheneedletract

IC(internalcapsule);cells(singlecellsuspension);metal(metalcannula);pieces(tissuepieces);

2

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Intracerebraldialysis showed that thebasal level ofdopamine in the grafted striatum(0.26±0.08pmolper25lofsampleperfusate)ofthe6‐OHDAlesionratwasrestoredtothelevelobservedinintactstriatum(0.19±0.06pmolper25lofsampleperfusate).GraftedhumanDAneuronswere able to respond to amphetamine stimulationwith a significantincreaseintheDAlevelofthegraftedstriatum(3.7timesincrease),althoughthisincreasewaslesspronouncedthanthatobservedinintactstriatum(15.1timesincrease)[36].

3.3ApplicationofhumanfoetalVMtissuetransplantationinParkinsonpatients

ThepromisingresultsofintracerebralimplantationofhumanfoetalVMDAneuronsintheratmodelforParkinson’sdiseaseledtoopenclinicaltrialsinParkinsonpatients.Intheseopenclinicaltrials,manypatientswithhumanfoetalVMgraftsrecoveredfromrigidityandtremors in various degrees, correlated to the restoration of the striatal dopamine leveldetectedbyPET[9‐11];thepatientsexperiencedanoverallimprovementinlifequality[9‐10,12‐13].OccasionalpostmortemstudiesshowedthesurvivalofDAneuronsatthegraftsites (between 80,000 and 135,000 TH+ neurons), and extensive reinnervation of thestriatum[48‐49].Thepositiveresultsoftheseinitialopenclinicaltrialssharplycontrastedwith theresultsofdouble‐blindcontrolNIHstudies (reviewedby [50‐52]): thesestudiesfailedtoshowanysignificantbenefit ingraftedpatientsup to1and2years in follow‐upstudies.Moreover, approximately15%of thegraftedpatients inNIHstudy I and56% instudyIIexperiencedseveregraft‐induceddyskinesias[53‐54].Themajordifferencesintheoutcomeofgrafttreatmentbetweenthemoreaccuratedouble‐blindcontrolNIHstudiesandthenon‐standardizedopentrialscouldbeascribedtomajordifferencesintheselectionofpatients, in thepreparation, thestorage, the final compositionand thesizeof thehumanfoetalcellgraft,thesiteofintrastriataldepositionandtheassessmentmethods.AlthoughtheimpurecompositionofthehumanfoetalgraftsdidnotseemtopreventsignificantrotationreductionsintheUngerstedtratmodelforParkinson,itmaybeofmajorrelevanceforclinicalapplication.Apparently,thecomplexarchitecture,composition,sizeandregionaldifferencesin functionality of the human striatum represent a completely different niche than thesmaller, less complicated striatum of the rat. Recently, a number of post‐mortem brainanalyseswereperformedonParkinsonpatientsthatreceivedafoetalDAgraftfrom3upto16 years ago andmore of such analyses are expected in the near future. Such analysesrevealedtheextentofsurvivalandthecellularcompositionofthegraft.Thefirstdataclearlyshowedthatthegrafteddopaminergicneuronswereintegratedinthestriatalcircuitry;inaddition,interestingly,someofthegraftedDAneuronsappearedtohavedevelopedaLewybodypathologypointingtoaprion‐liketransferfromsurroundingcellsoftheresponsiblepathogen,mostlikelyalpha‐synuclein[55‐57].It is clear that thedevelopmentof autologous inducedpluripotent stemcellshasnow

provided an unrestricted source of graftable humanDA neurons that can be extensivelycharacterizedandstringentlypurifiedbeforeactualimplantationintheParkinsonpatients.ClinicalapplicationoftheseiPScell‐derivedDAgraftsinthenearfuturecanbenefitfromtheamplepracticalexperiencewithhumanfoetalDAneuronintrastriatalimplantations,despite


43

theirmuch‐debatedoutcome.However,beforeclinicalapplication,itneedstobeestablishedthat DA neurons differentiated from human iPS cells show similar survival and neuriteoutgrowth after implantation in the PD animal models with a similar (or even a morepronounced)behaviouralimprovementastheprimaryfoetalhumanDAneurons.

4.HUMANIPSCELL‐DERIVEDDOPAMINERGICNEURONGRAFTS

Reprogramming of human fibroblasts towards iPS cells involves a complete epigeneticresetting and a concomitant complete cellular rejuvenation. Subsequent in‐vitrodifferentiationandmaturationofhumaniPScellstowardsadopaminergiccelllineagewillyield foetalDAneurons,withamaturationalstagecomparableto thehumanDAneuronsisolated from aborted foetuses. Proper DA differentiation largely depends on thecompleteness of the reprogramming process and on the in‐vitro protocol used fordifferentiation. Such a protocol should meticulously recapitulate the normal embryonicdevelopment of DA neurons, mimicking the local micro‐environment. Extensivecharacterization and purification should lead to a much better defined DA neuron cellsuspensionforimplantationincomparisontothehumanfoetalprimaryDAcellgrafts.

4.1CellularcompositionofhumaniPScell‐deriveddopaminergicneuronalgraft

In order to generate proper VM DA neurons in‐vitro from hiPS cells, three differentdifferentiationstrategieshavebeenappliedthathavebeendevelopedforembryonicstemcells(ESCells):theembryonicbody(EB)method[58],thestromalcellco‐culturemethod[59]andthedefined‐mediumfloorplateinducedmethod[60](forreviewssee[61‐65]).Therepresentative procedure of each differentiation method and the expression of VM DAneuronalmarkersindifferentiatedcellsaresummarizedinFigure1.Recently,severalhiPSDA‐differentiationprotocolshavebeenforwardedbutingeneraltheyarejustmodificationsofthe3mentionedstrategies[66‐68].The EB‐based differentiation protocol starts with the spontaneous differentiation ofculturediPScellsintoEBscontainingthe3germlineagesandisfollowedbytheselectiveenrichmentoftheneuroectodermandtheneuralprecursors[58].Thestromalcellco‐culturemethodisbasedonthestromalcell‐derivedinducingactivity(SDIA)thathasbeenshowntopromoteneuraldifferentiationofESCs[69].Inbothmethods,neuralrosettestructuresareformed, which are composed of neuroepithelial cells [70‐71]. The defined‐medium floorplateinducedmethodemploysdualinhibitionofSMADsignallingbyNogginandSB431542toforcethedifferentiationofhumanESCellsandiPScellsintoaneuralcelllineage[72].EarlyexposuretoSHHfromday1ofdifferentiationdirectstheiPScellstoafloorplatefate[73],and in combinationwith theWNTsignallingactivatorCHIR99021 (CHIR), typicalVMDAneuronprecursorsthatco‐expressFOXA2andLMX1aaregenerated[69].Withthismethod,more than 70% of the cells can be committed to a floor plate cell fate [73‐74]. TheneuroepithelialcellsandfloorplateprecursorscanbeinducedtoaVMDAneuronalfatebycombinationoftheinductionfactorsSHHandFGF8.

2

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44

Figure 1. Schematic summary of the conditions for DA neuron differentiation in therepresentativeprotocolsRespectivelyfortheEBmethod,thestromalcellco‐culturemethodandthefloorplatemethod.Basedon[58](EB),[59](co‐culture)and[60](floorplate).

The in‐vitro properties of the generated DA neuronswere shown to vary between thedifferentdifferentiationmethods.AlldifferentiatedDAneuronswerereportedtobeTH+andtoco‐expressmajorVMDAneuronmarkersNURR1,GIRK2,PITX3etc.[72,74‐75];dopaminereleaseandslowautonomouspace‐makingspontaneoussynapticactivitywasalsoreported[67,76].However,incontrasttothefloorplateinducedprotocol[77‐78],boththeembryonicbody(EB)methodandthestromalcellcoculturemethod,failedtoinduceFOXA2expressionduringneuroepithelialcelldifferentiation[79].ThislaterdevelopedprotocolwasdesignedtofirstlydirectpluripotentcellstowardsfloorplatecellsbeforeinductionofVMDAneuronswhich ismoresimilar to the in‐vivoVMDAneurondevelopment [80]. It seems the floorplate‐derivedDAneuronsaremoreauthentictoprimaryVMDAneurons[79].Apartfrommore precise specification and higher efficiency, direct comparison of rosette‐neuroepithelial cell‐derived and floor plate cell derived DA neurons post grafting alsofavouredfloorplate‐derivedDAneurons[60].UnliketransplantationstudieswithhumanfoetalVMtissue,thecompositionoftheiPS

cell‐derivedDAneurongraftshasbeenwelldescribed.Bytheendofin‐vitrodifferentiation,thepercentageofTH+DAneuronsderivedfromiPScellsvariedconsiderably,dependenton

2


45

thedifferentiationmethodused[60,74‐76,81].InthestudybyHargusetal.,5‐10%ofthetotalnumberofdifferentiatedcellswereneuronspositivefortyrosinehydroxylase(TH):lessthan1%oftheTH+neuronsco‐expresseddopamine‐β‐hydroxylase(DBH),indicatingthatthevastmajorityoftheTH+neuronswereDAandnotnoradrenergicneurons[82].Rheeetal.reportedthat50‐60%ofalldifferentiatedcellswereTUJ+,ofwhich35‐45%wereTH+[75], whereas Kikuchi and colleagues described that, with their approach, most of thedifferentiated cells were TUJ+, with 85% being TH+ [83]. Other studies reported TH‐expression in 18% to 54% of FOXA2+ cells after differentiation [60, 74]. A smallcontamination with serotonergic, GABAergic and glutamatergic neurons and GFAP+astrocyteswereoftendetected[60,75‐76].Ingeneral,onlysporadically,undifferentiatediPScells(indicatedbytheexpressionofSSEAs,typicalpluripotencycellmarkers)weredetectedafterlongtermdifferentiation[76,82]andalsoafewKi67+proliferatingcells[60,74,82].

4.2EfficacyofintrastriatallygraftedhumaniPScell‐derivedDAneuronsinPDanimalmodels

4.2.1BehaviouralimprovementandDAneuronsurvival

ThestudiesofhumaniPScell‐derivedDAneurontransplantationintheratPDmodelaresummarizedinTable2.Thereductioninrotationbehaviourintheunilaterallylesioned6‐OHDAratsstronglydependsonthenumberofsurviving,integrating,dopamine‐producinggraftedneurons.Whereasbetween500‐700survivingfoetalVMgraftderivedDAneuronshavebeenshowntobesufficient tocompletelyannihilateamphetamine‐inducedrotationactivity,asmanyas29,000survivinghumaniPScell‐derivedDAneuronsappearedtobeableto induceonlya50%reductionindrug‐inducedrotation4weekspostgrafting; lessthan1,500survivinghumaniPScell‐derivedDAneuronsresultedinnotanysignificantchangeinrotationbehaviour.Rheeetal.reportedthataround27,000survivingTH+cellswerefoundin the 6‐OHDA‐lesioned rats that exhibited a 47.5% reduction in rotations 8weeks postgrafting [75]. In the study byHargus et al., the survival and integration of around5,000humaniPScell‐derivedDAneuronsledtoareductioninrotationofonly56%16weekspostgrafting [82]; these rats still didnot showany improvement in the cylinder test and theadjustment stepping test [82]. The same study showed that rats containing only 350surviving iPS cell‐derived TH+ cells did not show any functional improvement inapomorphine‐inducedrotation[82].Similarly,thesurvivalofaround6,700humaniPScell‐derivedDAneuronsreducedrotationbehaviourto78%16weekspostgraftinginastudybyDoietal. [74].Kriksetal. showed that15,000 survivinghuman iPS cell‐derivedTH+DAneurons could fully compensate or even reverse amphetamine‐induced rotation andimprovedtheperformanceincylindertestandsteppingtest20weekspostgrafting[60].Ingeneral,amuchgreaternumberofsurvivingTH+DAneuronsderivedfromhumaniPScellsisneededtogiverisetosignificantfunctionalimprovementincomparisontohumanfoetalDAneurons.

CHAPTER2

46

Table2‐SummaryofgraftingofhumaniPSC‐derivedDAneuron

[Ref],source(protocol:stageofgraft)/graftsite

Totalcellnumber,[cellcomposition]

FunctionalrecoverySurvivalofgrafts(W/Mpostgrafting)

[60],hiPSClines2C6,SeV6((1)floorplate:d25(12dayspostwithdrawalofCHIR);(2)MS5:14dayspostwithdrawalofSHH&FGF8)/CPu

2.5105cells[(1):18%TH+/FOXA2+,40%NUUR1+/FOXA2+,95%LMX1A+/FOXA2+(2):30%TH+,20%Nurr1+,lowinFOXA2,LMX1A]

(4.5MPG)Amphetamine:(1):>100%reductionSteppingtest:(1):30%increasecylindertest:(1):>10%reduction(2):alltestsn.s.

(4.5MPG)(1):15,000TH+/FOXA2+cells;serotongergicandGABAnergicneurons<1%(2):FewTH+neurons

[74],hiPSCline836B3(floorplate,(1)d28,(2)d42(withdrawalofpurmorphamine,FGF8atd7;CHIR99021atday12))/CPu

4105cells(CORIN+sortingatd12,ornot)[(1):70–75%FOXA2+,27.3%NURR1+cells,2.1%TH+cells(2):70–75%FOXA2+,19.9%NURR1+cells,42.0%TH+cells]

(16WPG)Amphetamine:(1):78%reduction(2):n.s.

(1)Sorted:6,747TH+cells;2.5%serotonin+(1)Unsorted3,4364TH+cells;1.2%serotonin+(2)Sorted:1,900TH+cells(2)Unsorted2,000TH+cells.

[75],Lenti,retro,proteinhiPSCs,hESCs(MS5:(1)NPCsP1‐P2;(2)NPCsP4;(3)day5postwithdrawofSHH&FGF8)/CPu

(1):7.5105cells(2):3105cells(3):3105cells[(3):5%GFAP+,60‐70%TUJ1+,35–45%TH+inTUJ1+cells,5‐10%serotonin+,afewGABAergicandglutamatergicneuronalcells]

(8WPG)Amphetamine(1):76.43%reduction(2):47.54%reduction(3):0%reduction

(1)54,418TH+cells(halfsizeofstriatum)(2)26,882TH+cells(3)NoTH+cells

[82],PDiPSC(MS5:day42,5dayspostwithdrawofSHH&FGF8)/CPu

(1):4105unsortedcells(2):4105NCAM+sortedcells5‐10%TH+cells

(16WPG)Amphetamine:(1)56%reduction(2)52%reductionApomorphine:(1)37%reduction(2)0%

(1)4,890TH+cells(2)350TH+cells

Abbreviate:CPu(caudate‐putamen);WPG(weekspostgrafting);MPG(monthspostgrafting);n.d.(notdetermined/notdescribed)


47

CharacteristicsofgraftsofDAneurondifferentiatedculture

Neuriteoutgrowth&striatumreinnervation

Sideeffects

n.d.TH+/NCAM+fibresextendedintostriatum.

(2):NeuralovergrowthinNOD‐SCIDIL2Rgcnullmice

Co‐expressionofTH,FOXA2,NURR1andPITX3;50%oftheTH+cellsco‐expressedGIRK2.

Moreneuriteoutgrowthinthegraftsderivedfromtheday12CORIN+sortedcells

Largegraftsizeofunsortedgrafts

CoexpressionofTH,TuJ1andVMAT2;Nurr1andEN1Oct4+insomelentivirus‐basedhiPSCs‐derivedneurons

n.d.Tumorformation

(12WPG)53.4%DAneuronsGIRK2+;6.6%DAneuronscalbindin+4.7%forebrainDAneurons5.8%andhypothalamicDAneurons0.09±0.02%Ki‐67+83.1±8.2%hNCAM+NoSSEA‐4‐noroct4+;noubiquitin+andα‐synuclein+inclusionbodies

AfewTH+fiberextendedtowardthestriatum

n.d.

n.s.(nosignificantimprovement);hiPSC(humaniPSC);PDiPSC(PDpatientiPSC);

2

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48

In most studies, between 200,000‐400,000 human iPS cell‐derived cells werestereotacticallytransplantedinthedenervatedstriatumoftheunilaterally6‐OHDAlesionedrats. Cells were transplanted mainly at two stages of differentiation. TransplantationsexecutedwithcellsatthelatestageofterminaldifferentiationresultedinahighlyvariablepercentagesofsurvivingDAneurons:from0%[75],toonly1.1%[74],and5‐10%[82].Insome studies, human iPS cell‐derived cells were transplanted at an early stage ofdopaminergicdifferentiation, containingpredominantlyneuralprecursorcellsandonlyaveryfewTH+cells[74].Suchgraftedneuralprecursorcells,alreadydestinedtowardsaVMDAneuronfate,couldstillproliferatebeforefinaldifferentiationintomatureDAneuronin‐situ and gave rise to amuch larger (yet unpredictable) number of TH+ cells: 6700 [74],15,000 [60] and 27,000 [75] TH+ cellswere foundwithin grafts. The graft size in thesestudieswasusuallymuchbigger than thatof terminaldifferentiationstage implantations[74‐75].Thecontinuingproliferationoftheneuralprecursorcellscouldresultinovergrowth[75],andtheearlystageofdifferentiationcouldleadtothepresenceofundifferentiatediPScellsandsoteratomaformation.

4.2.2CharacteristicsoftransplantedhumaniPScell‐derivedDAneurons

AnalysisofthehumaniPScell‐derivedgraftsinthedenervatedstriatumofthe6‐OHDAlesionratsrevealedTH+DAneuronsthatwerealsopositiveforspecificVMDAneuronmarkers,such as EN1, VMAT2, DAT [75] NURR1 [75], GIRK2, PITX3 [74, 83], and FOXA2 [60]. AdetailedcharacterizationofsurvivinghumaniPScell‐derivedDAneuronsshowedthat53.4±6.6%oftheTH+cellswereGIRK2+VMDAneurons,4.7±1.0%wereGABA+forebrainDAneurons,and5.8±1.2%wereNKX2.1+hypothalamicDAneurons[82].However,itshouldbenotedthatinmoststudiestheTH+DAneuronswereactuallyonlyasmallportionofthesurvivinghumaniPScell‐derivedcells.ThemajorityoftheiPScell‐derivedneuronsappearedtobenon‐DA[75‐76,82];somewereidentifiedasserotonergic,GABAergicneurons[60,75‐76].GFAP+astrocyteswerealso foundwithin thegrafts [75,82].Oligodendrocyteswerenever found, probably due to their very specific late generation during embryonicdevelopmentandtheconsequentlylongspecificin‐vitrodifferentiationprocedurerequired[75]. Ki‐67+ proliferating cells could still be found in the intrastriatal graft [82].Contaminationofthesecellsmayleadtograftovergrowth[75]orteratomaformation.WhendopaminergicneuralprecursorcellswereenrichedbysortingforNCAM[84]orCORIN[74],graftovergrowthandteratomaformationweresignificantlyreduced.Noubiquitin‐positiveand/orα‐synuclein–positiveinclusionbodiescouldbedetectedingraftediPScell‐derivedDA and non‐DA neurons generated from fibroblasts of PD patients up to 12weeks postgrafting[82].Remarkably,exogenouspluripotentgene(OCT4,NANOGandSOX2)expressionpersisted in subpopulations of TUJ1+ neuronal cells and TH+ DA neurons derived fromlentivirallyreprogrammedhiPScellsevenafter terminaldifferentiation[75]. In thestudydescribedbyHargusetal. engraftedhuman iPS cell‐derivedDAneurons showed intensearborisationandbranching,yetmostoftheDAneuronssentTH+fibresonlytowardsothercellswithinthegraftsanddidnotextendouttothesurroundingparenchyma[82].Krikset


49

al. reportedthat thegraftcorewassurroundedbyahaloofTH+fibres,withsomefibresoccasionally extendingup to3mm from the graft [60].Non‐TH+neuriteswere found toprojecttotheirtargetareasaccordingtotheirintrinsicphenotypicdeterminationinspecificandreproduciblepatterns[82].Inacuteorganotypicslicespreparedfromgraftedrats,alltransplantedneuronsrecordedshowedspontaneousactionpotentialcurrents.

4.3HumaniPScell‐derivedDAneuronsforclinicalstudies

BasedontheanimalstudiesoncellreplacementtherapyforParkinson’sdisease,thereisnodoubtthatiPScellsareaprominentsourcefortransplantableDAneurons,buttheprocedureto generate VM DA neurons from iPS cells in‐vitro is as yet not acceptable for clinicalapplication. A reliable quality check for human iPS cell‐derived DA neurons is yet to beestablished. iPSC lines vary from one another both in pluripotency and differentiationefficiency [85], which may also dependent on passage number [86]. Although iPS cellsresembleESCs,acomparativestudyshowedthatsomeiPSClineshaveadelayedformationofEBs incomparisontoESCs[86].Differentreprogrammingmethodsmayalsoaffect thequalityof iPS cell‐derivedDAneurons [87].Rheeetal. found thatexogenouspluripotentgeneswerenotcompletelysilencedattheendofthedifferentiationprocedure.OCT4,aswellas NANOG, was still detectable in subpopulations of TUJ1+ neuronal cells and TH+ DAneuronsderivedfromlentivirallyreprogrammedhiPScells,butnotinthosederivedfromretrovirally reprogrammed hiPS cells or protein‐transfer reprogrammed hiPS cells [75].Furthermore,unlikethosefromprotein‐transferreprogrammedhiPScells,NPcellsderivedfromvirally‐reprogrammedhiPScellsexhibitedearlysenescenceandapoptoticcelldeathduringpassaging,whichwasprecededbyabruptinductionofp53[75].ThevariationiniPSClinesmay affect the efficiency in both DA neuron differentiation and their survival postgrafting.GeneralstandardsforhumaniPScell‐derivedfunctionalandauthenticVMDAneuronsare

yettobeestablished.ItisapparentthatthemuchhighernumberofhumaniPScell‐derivedDAneuronstoachievethesamedegreeoffunctionalimprovementasthatofhumanfoetalVMDAneuronsisrelatedtothequalityoftheTH+cellsderivedfromhumaniPScells.The(asyetnotgenerallystandardized)validationofhumaniPScell‐derivedVMDAneuronsisbasedontheexpressionof(acombinationof)specificVMDAneuronmarkers,suchastheco‐expressionofTH,LMX1AandFOXA2[60],theco‐expressionofTHandGIRK2[81],theco‐expressionofTHandEN1,NURR1andPITX3, the lackofdopaminebeta‐hydroxylase(DBH)coexpressionwithTH+cells[82],etc.Inafewcases,thereleaseofdopamineandtheelectrophysiologyofiPScell‐derivedDAneuronswereusedtoconfirmtheresemblancewithprimaryVMDAneurons[60,75,88].ContaminationofothercelltypeswithintheiPScell‐derivedcultureisamajorconcern,inthatrespectsimilartothehumanfoetalVMcellgrafts.TheestablishmentofaVMDAneuronpurificationmethodwillbeabreakthroughforcellreplacement therapy for PD, for it will eliminate the presence of proliferating cells andundifferentiated iPScellsandreducetheriskofgraftovergrowthor tumorformation.Byreducingthepresenceofothercelltypes,potentialcellularinteractionsandunknowneffects

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ofotherneuronalcelltypeson(rotation)behaviourwillbeavoided.PSA‐NCAM,asurfacemarkerforneuralstem/precursorcells,hasbeenusedwithFAC‐sortingtoenrichforneuralprecursors[89]andfloorplate‐specificcellsurfacemarkerCORINhasbeenusedtoenrichfor hiPS cell‐derived dopaminergic neuronal precursors [74]. FAC‐sorting onNCAM+/CD29lowcellswasusedbySundbergetal.toenrichVMDAneurons[84].Neitherofthese cell sortingmethods resulted in a pure population ofmature VM DA neurons. AnalternativeapproachcouldbetogeneratereporterlinesforDAneuronaldifferentiationandusereporterexpressionforpurificationorselectionviaFACsortingofhumandopaminergicneuronsbyclinicallyacceptablegenemodificationmethods[90].ItisimportanttonotethatFAC‐sortingatthelatestageofdifferentiationmayhampertheviabilityofcellspostgrafting.ThenumberofsurvivingDAneuronswasreducedbymorethan10timesinthestudybyHargusetal.[82].

5.COMPARISONOFHUMANFOETALWITHHUMANIPSCELL‐DERIVEDDOPAMINERGICNEURONGRAFTS

Transplantation of human foetal VM tissue in PD animal models has provided proof‐of‐principleforDAneuronsurvival,outgrowthandintegrationaswellasfunctionalrecoveryofthegraftedanimal.ItmayserveasagoldstandardforevaluatingthequalityofDAneuronsderivedfromhiPScells.WesummarizedtheperformanceofhiPScell‐derivedDAneurongraftsandcompareditwiththatofhumanfoetalVMtissuegraftsinthe6‐OHDAlesionratmodel (Table 3). The number of surviving implanted TH+ neurons ranged from a fewhundredtoaround4000pergraftinratsgraftedwithhumanfoetalVMtissue.About500‐700 of viable functional DA neurons appeared to be sufficient to reverse drug‐inducedrotationunderoptimal surgeryconditions [37‐38].On thecontrary, survivalofTH+cellsfromiPScell‐deriveddopaminergicneurongraftsrangedfromafewto29,000.IncontrasttothehumanfoetalDAgrafts,thousandsofiPScell‐derivedTH+cellsappearedtobeneededtofullyreversedrug‐inducedrotation[60,74].PrimaryDAneuronsfromfoetalVMtissuegaverisetoextensiveneuriteoutgrowthupto

6mm[36]andreinnervatedthewholevolumeofthestriatum(Figure2A)[37,44].NeuriteoutgrowthofiPScell‐derivedDAneuronsvariesfromcasetocasebut,ingeneral,itremainedrelativelylimited[82]orextendedonly2‐3mmintothestriatum,reinnervatingonlyaround10%ofthestriatum,despitethelargenumberofsurvivingTH+neurons(Figure2B)[60,74].It seems likely that themore limitedsurvivalandneuriteoutgrowthofgraftedhiPScell‐derivedDAneuronsincomparisontotheprimaryhumanfoetalDAneurons,isresponsiblefor a lower degree of recovery of the 6‐OHDA lesion rats in the drug‐induced rotationbehaviour.IncontrasttohESCderivedDAneurons[92],dataaboutdopaminereleaseortheelectrophysiological characteristics of hiPS cell‐derived DA neurons integrated in thestriatumaftergraftingarenotyetavailable.


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Table3–Comparisonofmajorparametersof(non‐purified)humanfoetalandhumanstemcell‐deriveddopaminergicgraftsinratPDmodel

humanfoetalDAgraft humaniPSC‐derivedDAgraft

CompositionDAneurons,5‐HTneurons,GABAergicneurons,astrocytes,microglia[32,34]

DAneurons,NSCs,undifferentiatediPSCsserotonergic,GABAergicneuronsastrocytes[74‐76,81‐82]

PercentageDAneuronsinoriginalgraft

5.6%[32‐33] 5‐85%[60,74‐76,81‐82]

Survival++++1‐10%[35‐36]

+++0‐12%[74‐75,82]

Graftovergrowth ‐ ++[75]

Migration +[36] notdetected

NeuriteoutgrowthLongextensions[34,37‐39,44]

Shortextensions[60,82]

Synapsformation ++[34,37] notdetected

Recovery(rotationreduction,othertests)

++++[35,37‐38] ++[74‐75]

MinimalnumberofTH+cellsrequiredtofullreversionofrotationbehaviour

500‐700[37‐38] 15,000[60]

Co‐expressionofVMDAmarkersinsurvivingDAneurons

FOXA2+,LMX1a+,EN1+,NURR1+,GIRK2+,TH+[68,79]

TH+,GIRK2+,EN1+,NURR1+,LMX1A+,andPITX3+[60,74‐75]

IntrastriatalDArelease ++[34,36] nottested

Suppressionofstriatalinterneurons

+++[44] nottested

CharacteristicDAneuronfiringin‐vivo

++++[44] nottested

6.CONCLUSIONS

Although the human iPS cell‐derived DA neurons seem to fulfil the major criteria forfunctional dopamine‐releasing neurons, it is clear that theywere not as effective as thehumanprimary (foetal)DAneurons in restoring drug‐induced rotation behaviour in theunilaterally 6‐OHDA lesion rats. A much higher number of surviving DA neurons wererequiredtoobtainthesamebehaviouraleffectsoftheprimaryfoetalDAneurongrafts.ThehumaniPScell‐derivedDAneuronsshowedalowersurvivalrateafterstriatalimplantation

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Figure2.ComparisonofgraftedhumanfoetalVMtissueandhiPScell‐derivedDAneurons(A)OverviewofTH+neuritesinacoronalsectionofthestriatum4monthsaftergraftinghumanfoetalVMtissuefroma10‐week‐oldfoetus.Thegraft,filledwithTH+cellbodiesandfibresisseenatthefarright,TH+processesradiateintohoststriatumandformanetworkthatcoversthewholeareaofstriatum.(B)OverviewofTH+iPScell‐derivedDAneurons(unsorted)4monthsafterintrastriataltransplantation.Higher magnification showing TH‐positive somata and some innervation of the surrounding hoststriatumbygraft‐derivedneurites(b)Scalebar=200m,CC=corpuscallosum,V=ventricle.Adaptedfrom[44,74].

andtheirneuriteoutgrowthwasnotasextensiveasthatofthehumanprimaryfoetalDAneurons.Apparently,thehumanDAneuronsobtainedviadifferentiationfromiPScellswerenotidenticaltotheprimaryDAneuronsisolatedfromtheVMregionoftheabortedfoetuses.Indeed, after the reprogramming‐associatedcomplete juvenation, the iPS cell‐derivedDAneuronmayalsobeconsideredfoetal,butthedifferentiationprotocolmost likelydidnotresultintheveryspecificpopulationoffoetalA9DAneuronsthathavethespecificintrinsicpropertiestoinnervatethestriatum.Itislikelythat,afterthedrasticiPScell‐reprogrammingandthesubsequentdopaminergicdifferentiation,fibroblast‐derivedepigeneticmarkswerestillpresentinterferingwithapropercompletereprogrammingandDAdifferentiation.Inthatrespect,itisofconsiderableinteresttonotethatDAneuronsdifferentiatedfromhumanESCs(withasimilarprotocolusedforhiPScells)recentlyshowedlong‐termsurvival,efficacyinrestorationofmotorfunctionaswellaslongextensiveneuriteoutgrowthandwidestriatalinnervationafterimplantationintheratPDmodelwithapotencycomparabletothatseenwithhumanfoetaldopamineneurons[91].Recently,weperformedanin‐depthcomparativeanalysisof theexpressionprofileofpuremouse iPScell‐derivedDAneuronswiththatofpuremouseprimaryDAneurons[93].WeusedPtix3‐GFPmicetoenabletheisolationofpureiPScell‐derivedDAneuronsandpureprimaryVMDAneuronswithGFP‐basedFAC‐sorting.Besidescomparativeglobalgeneexpressionmapping,weperformedcomprehensiveDNAmethylationprofilingbyReducedRepresentationBisulfiteSequencing(RRBS).ThemouseiPScell‐derivedDAneuronsindeedlargelyappearedtoadoptcharacteristicsoftheirinvivocounterparts,inmorphology,dopamineproduction,globalgeneexpressionandCpGisland(CGI)methylationprofiles,thoughsomefibroblastgenesappearedtobeexpressedstill[93].


53

However,wealsofounddeviationsinCGImethylationforasubpopulationofgeneswhichclearly effected predominately the expression of genes associated with functionalannotations such as ‘nervous system development’, ‘neurogenesis’ and ‘neurondifferentiationandoutgrowth’ [93]. It is likely that similar iPS reprogramming ‘artefacts’mayberesponsibleforthelimitedoutgrowthandsurvivalofthehumaniPScell‐derivedDAneuronsafter implantationintheratParkinsonmodel.Extensivecomparativeexpressionprofilingcombinedwithgenomewideepigenomeanalysis,likewehavedoneforthemouse,is needed to examine in more detail the differences between human primary foetal DAneuronsandthehumaniPScell‐derivedDAneurons,althoughproperpurificationofhumanDAneuronsmaystillbeabottleneckforthat.Inviewoftheconsiderableundeniableclinicalpotential of (autologous) human iPS cell‐derived DA neuron grafts, fundamental andpreclinicalresearchisstillprogressingwhichwilleventuallyleadtoafirstclinicaltrialonasmall number of Parkinson patients, presumablywithin 2 years. Important intermediatesteps towards such trial have been taken in recent studies on the transplantation ofautologous iPSC‐derived dopamine neurons in non‐human primatemodel of Parkinson'sdisease[94,95],showingtheredundancyoftheuseofimmunosuppression.

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68. Sundberg,M., Bogetofte,H., Lawson,T., Jansson, J., Smith, G., Astradsson,A., et al. (2013).ImprovedcelltherapyprotocolsforParkinson'sdiseasebasedondifferentiationefficiencyand safety of hESC‐, hiPSC‐, and non‐humanprimate iPSC‐derived dopaminergic neurons.StemCells31(8):1548‐1562.

69. Kawasaki,H.,Mizuseki,K.,Nishikawa,S.,Kaneko,S.,Kuwana,Y.,Nakanishi,S.,etal.(2000).InductionofmidbraindopaminergicneuronsfromEScellsbystromalcell‐derivedinducingactivity.Neuron28(1):31‐40.

70. Elkabetz,Y.,Panagiotakos,G.,AlShamy,G.,Socci,N.D.,Tabar,V.,&Studer,L.(2008).HumanES cell‐derived neural rosettes reveal a functionally distinct early neural stem cell stage.GenesDev22(2):152‐165.

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72. Chambers, S.M., Fasano,C.A., Papapetrou,E. P., Tomishima,M., Sadelain,M.,&Studer, L.(2009).Highlyefficientneural conversionofhumanESand iPS cellsbydual inhibitionofSMADsignaling.NatBiotechnol27(3):275‐280.

73. Fasano,C.A.,Chambers,S.M.,Lee,G.,Tomishima,M.J.,&Studer,L.(2010).Efficientderivationoffunctionalfloorplatetissuefromhumanembryonicstemcells.CellStemCell6(4):336‐347.

74. Doi,D.,Samata,B.,Katsukawa,M.,Kikuchi,T.,Morizane,A.,Ono,Y.,etal.(2014).Isolationofhuman inducedpluripotentstemcell‐deriveddopaminergicprogenitorsbycellsorting forsuccessfultransplantation.StemCellReports2(3):337‐350.

75. Rhee,Y.H.,Ko,J.Y.,Chang,M.Y.,Yi,S.H.,Kim,D.,Kim,C.H.,etal.(2011).Protein‐basedhumaniPS cells efficiently generate functional dopamine neurons and can treat a rat model ofParkinsondisease.JClinInvest121(6):2326‐2335.

76. Sanchez‐Danes,A.,Consiglio,A.,Richaud,Y.,Rodriguez‐Piza,I.,Dehay,B.,Edel,M.,etal.(2012).EfficientgenerationofA9midbraindopaminergicneuronsbylentiviraldeliveryofLMX1Ainhumanembryonicstemcellsandinducedpluripotentstemcells.HumGeneTher23(1):56‐69.

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77. Sonntag,K.C.,Pruszak,J.,Yoshizaki,T.,vanArensbergen,J.,Sanchez‐Pernaute,R.,&Isacson,O. (2007). Enhanced yield of neuroepithelial precursors and midbrain‐like dopaminergicneuronsfromhumanembryonicstemcellsusingthebonemorphogenicproteinantagonistnoggin.StemCells25(2):411‐418.

78. Perrier,A.L.,Tabar,V.,Barberi,T.,Rubio,M.E.,Bruses,J.,Topf,N.,etal.(2004).Derivationofmidbraindopamine neurons fromhuman embryonic stem cells. ProcNatlAcad Sci U SA101(34):12543‐12548.

79. Cooper,O.,Hargus,G.,Deleidi,M.,Blak,A.,Osborn,T.,Marlow,E.,etal.(2010).DifferentiationofhumanESandParkinson'sdiseaseiPScellsintoventralmidbraindopaminergicneuronsrequiresahighactivityformofSHH,FGF8aandspecificregionalizationbyretinoicacid.MolCellNeurosci45(3):258‐266.

80. Ono,Y.,Nakatani,T.,Sakamoto,Y.,Mizuhara,E.,Minaki,Y.,Kumai,M.,etal.(2007).Differencesin neurogenic potential in floor plate cells along an anteroposterior location: midbraindopaminergic neurons originate from mesencephalic floor plate cells. Development134(17):3213‐3225.

81. Kirkeby, A., Grealish, S., Wolf, D. A., Nelander, J., Wood, J., Lundblad, M., et al. (2012).Generationof regionally specifiedneuralprogenitorsand functionalneurons fromhumanembryonicstemcellsunderdefinedconditions.CellRep1(6):703‐714.

82. Hargus,G.,Cooper,O.,Deleidi,M.,Levy,A.,Lee,K.,Marlow,E.,etal. (2010).DifferentiatedParkinsonpatient‐derivedinducedpluripotentstemcellsgrowintheadultrodentbrainandreducemotorasymmetryinParkinsonianrats.ProcNatlAcadSciUSA107(36):15921‐15926.

83. Kikuchi,T.,Morizane,A.,Doi,D.,Onoe,H.,Hayashi,T.,Kawasaki,T.,etal.(2011).Survivalofhumaninducedpluripotentstemcell‐derivedmidbraindopaminergicneuronsinthebrainofaprimatemodelofParkinson'sdisease.JParkinsonsDis1(4):395‐412.

84. Sundberg,M., Bogetofte,H., Lawson,T., Jansson, J., Smith, G., Astradsson,A., et al. (2013).ImprovedcelltherapyprotocolsforParkinson'sdiseasebasedondifferentiationefficiencyand safety of hESC‐, hiPSC‐, and non‐humanprimate iPSC‐derived dopaminergic neurons.StemCells31(8):1548‐1562.

85. Boulting,G.L.,Kiskinis,E.,Croft,G.F.,Amoroso,M.W.,Oakley,D.H.,Wainger,B.J.,etal.(2011).Afunctionallycharacterizedtestsetofhumaninducedpluripotentstemcells.NatBiotechnol29(3):279‐286.

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88. Hartfield,E.M.,Yamasaki‐Mann,M.,RibeiroFernandes,H.J.,Vowles,J.,James,W.S.,Cowley,S.A.,etal.(2014).PhysiologicalcharacterisationofhumaniPS‐deriveddopaminergicneurons.PLoSOne9(2):e87388.

89. Freed,W. J., Chen, J., Backman, C.M., Schwartz, C.M., Vazin, T., Cai, J., et al. (2008). GeneexpressionprofileofneuronalprogenitorcellsderivedfromhESCs:activationofchromosome11p15.5andcomparisontohumandopaminergicneurons.PLoSOne3(1):e1422.

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94. Hallett, P.J., Deleidi, M., Astradsson, A., Smith, G.A., Cooper, O., Osborn, TM, et al. (2015).SuccessfulfunctionofautologousiPSC‐deriveddopamineneuronsfollowingtransplantationinanon‐humanprimatemodelofParkinson'sdisease.CellStemCell.16(3):269‐274.

95. Morizane,A.,Doi,D.,Kikuchi,T.,Okita,K.,Hotta,A.,Kawasaki,T.,Hayashi,T.,Onoe,H.,Shiina,T., Yamanaka, S., & Takahashi, J. (2013). Direct comparison of autologous and allogeneictransplantationofiPSC‐derivedneuralcellsinthebrainofanon‐humanprimate.StemCellReports1(4):283‐292.

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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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CHAPTER4

POTENTIALROLEOFCELLADHESIONMOLECULESINTHENEURITE

OUTGROWTHOFIPSCELL‐DERIVEDDOPAMINERGICNEURONS

Su‐PingPeng1,2,MelittaSchachner2,ErikBoddeke1,andSjefCopray1

1DepartmentofNeuroscience,UniversityMedicalCenterGroningen,theNetherlands2Center for Neuroscience, Shantou University MedicalCollege,Shantou,GuangdongProvince,P.R.China

Submited

ABSTRACT

Intrastriatal transplantationof dopaminergic neuronshas been showna potentially veryeffective therapeutical approach for the treatment of Parkinson’s disease (PD).With thedetectionof inducedpluripotent stemcells (iPS cells), anunlimited sourceof autologousdopaminergic(DA)neuronsbecameavailable.AlthoughtheiPScell‐deriveddopaminergicneuronsexhibitedmostofthefundamentaldopaminergiccharacteristics,theyshowedsomeaberrations in the expression of genes involved in neuronal development and neuriteoutgrowth. The limited outgrowth of the iPS cell‐derivedDA neuronsmay hamper theirpotential application in cell transplantation therapy for PD. In the present study, weexaminedwhethertheforcedexpressionofL1CAMandPSA‐NCAM,viagenetransduction,promotedtheneuriteformationandoutgrowthofiPScell‐derivedDAneurons.Inculturesonastrocyte layers,bothadhesionfactorssignificantlyincreasedneuriteformationoftheadhesionfactorover‐expressingiPScell‐derivedDAneuronsincomparisontocontrol iPScell‐derivedDAneurons.However,whenplatedonpostnatalorganotypicstriatalslicesthatalready intrinsically expressed high levels of L1CAM and PSA‐NCAM, no significantdifferencesinmaximalneuriteformationbetweenthecontrolandtheadhesionfactorover‐expressing iPS cell‐derived DA neurons could be detected. We examined the neuriteoutgrowthoftheL1CAM‐orPSA‐NCAM‐over‐expressingiPScell‐derivedDAneuronsafterimplantation in the striatum of unilaterally 6‐OHDA‐lesioned rats; unfortunately, only asmallnumberofthesortedandvirallytransducedcellssurvivedthestressfulimplantationprocedure.No apparent increasedneurite outgrowthof these survivingDAneuronswasobservedpresumablydue to their reducedviabilityandhealth,annihilating thepotentialgrowthpromotingeffectsoftheadhesionfactors.

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INTRODUCTIONThespecificandprogressivelossofdopaminergic(DA)neuronsinthesubstantianigra(SN)is a major hallmark of Parkinson’s disease (PD) [1]. At the time point that patients arediagnosedwithPD,thelossofSNDAneuronsisusuallyalreadymorethan60%,resultinginamorethan80%depletionofdopamineinthestriatum.NeitherDAneuronlossnorstriatumdenervation is reversible. However, intrastriatal transplantation of DA neurons and thesubsequent restoration of the striatal dopamine level have proven to be an effectivesymptom‐reducingtreatmentforPD[2‐3].Nowadays,inducedpluripotentstemcell(iPSC)‐derivedDAneuronsareconsideredthe

most promising source for DA neuron grafts for PD patients. iPS cells are obtained byreprogrammingofsomaticcellssuchasskinfibroblasts[4‐5],whichcanbecollectedfromthePDpatientviaasmallskinbiopt;theautologousoriginoftheiPScell‐derivedDAneurongraftmakesimmunosuppressionsuperfluous.SinceiPScellshaveanunrestrictedcapacityforself‐renewal,aprincipallyunlimitednumberofcellscanbeobtainedfortransplantationpurposes. Various strategies have been developed to differentiate the pluripotent cellstowardsaventralmesencephalic(VM)DAneuronfate[6‐8].DAneuronsderivedfromiPScellsshowpropertiesresemblingprimaryVMDAneurons,suchastheexpressionofspecificmarkers LMX1a,NURR1,TH,PITX3,GIRK2 [9], the spontaneous or stimulated release ofdopamineandthespontaneousactionpotentialfiringwithaslowpace(1‐10Hz)[10‐11].However,whengraftedintothestriatum,theneuritesfromtheiPScell‐derivedDAneuronsappeartoberestrictedwithintheinjectiontracks[9]oronlyshortlyextendingfromthesiteofthegraft[11].Toachievesignificantfunctionalimprovement,asubstantialreinnervationoftheaffected

putamenandcaudateisessential.Incontrasttopluripotentstemcell‐derivedDAneurons,primaryfoetalVMDAneuronshavebeenshowntobeabletoextensivelyreinnervatethegraftedstriatum[12],providingtheproof‐of‐principleofthebeneficialfunctionalityofDAneurongraftsinPDpatients[2,13].Evenwhen10timesmoreTH+cellswereimplanted,pluripotent stem cell‐derived DA neurons gave rise to a much lesser extent of striatalreinnervationthanprimaryfoetalVMDAneurons[14].Anapproach to improveandpromote theoutgrowthof implanted iPS cell‐derivedDA

neuronscouldbetheforcedover‐expressionofcelladhesionmoleculesthatarerichinthedeveloping foetal brain, in particular L1CAMandPSA‐NCAM. L1CAMandPSA‐NCAMarecrucial cell adhesion molecules for embryonic neuron development and axon guidance.DuringthefoetaldevelopmentofVMDAneurons,thePSA‐NCAMcontentincreasesstrikinglyinboththedevelopingstriatumandthemesencephalon,withTH+cellsclearlyoutlinedbyPSA‐NCAM[15].L1CAMisalsoexpressedinfoetalVMDAneuronsandparticularlymostlyenriched in the TH+ fibers in the medial forebrain bundle (MFB) [16]. It has beendemonstratedthatspecificstimulationoftheL1CAMsignalingpathwayinVMtissuegraftsbyL1Ab(Neomarkers,LabVision,Fremont,CA)resultedinasignificantlygreaterareaofgraft‐derivedinnervationcomparedtocontrolgrafts[17].

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In the present study, we aimed to examine the effect of L1CAM or PSA‐NCAM (bytransductionof the geneencoding for STX, the enzyme that addsPSAontoNCAM)over‐expressionontheneuriteoutgrowthofmouseiPScell‐derivedDAneuronsin‐vitroaswellasin‐vivoaftertransplantationintheParkinsonratmodel.BygeneratingiPScellsfromthefibroblasts of Pitx3gfp/+ transgenic mouse, we were able to get a pure population of DAneuronsaftertheirspecificdifferentiationbyFAC‐sorting.

MATERIALSANDMETHODS

iPScellcultureandDAneurondifferentiation

MousePitx3gfp/+ iPSCclonesCI,CII,CIII(passage15–52)weremaintainedon‐irradiatedmouse embryonic fibroblasts in ES medium (Knockout DMEM, 15% knockout serumreplacement, 1% nonessential amino acids (all Invitrogen, Breda, The Netherlands,www.invitrogen.com),2mML‐Glutamine,100U/mLpenicillin,100µg/mLstreptomycin(allPAA,Germany,www.paa.com),100µMβ‐mercaptoethanol)supplementedwith1000U/mLLeukemia inhibitory factor (LIF; Millipore, Amsterdam, The Netherlands,www.millipore.com).TheDAdifferentiationprotocolformouseiPScellswasbasedontheMS5protocol[18].

iPSCcoloniesweremanuallydissectedandplatedonMS5cellsinKSRmedium(DMEM,15%knockoutserumreplacement,100µMβ‐mercaptoethanol,100U/mlpenicillin,100µg/mlstreptomycin) fromday2–5. SHH (200ng/ml,Peprotech),FGF8 (100ng/ml,Peprotech),wereaddedtothemediumfromday6–8ofdifferentiation.MediumwaschangedintoN2medium (DMEM/F12 (Invitrogen), 2% N2 supplement (PAA), 2 mM L‐Glutamine,penicillin/streptomycin,1%sodiumpyruvate(Invitrogen))supplementedwithSHH,FGF8,combined with FGF2 (100 ng/ml, Peprotech) from day 9‐11. Followed by a finaldifferentiation step in the presence of BDNF (20 ng/ml, Peprotech), GDNF (20 ng/ml,Peprotech),TGFβ3(1ng/ml,Peprotech),ascorbicacid(200M)anddbcAMP(100M)untiltheappearanceofdopaminergicneuronalcelltypes.

RT‐PCRandq‐PCR

TotalRNAwasextractedfromtransducediPScellsordifferentiatediPScellsusingstandardRNA easy kit (Qiagen). Primerswere used for L1, STX, Nestin, Pax6,Map2, β‐III‐tubulin,Nurr1, Pitx3, TH, and DAT. Primer sequence information is presented in SupplementaryTable1.RelativegeneexpressionlevelsweredeterminedbyqPCRreal‐timepcr(Lightcycler)using the QuantiTect™ SYBR® Green PCR (QIAGEN) LightCycler® kit (Roche, IdahoTechnologies)accordingtothemanufacturer’sinstructions.Foreachreaction0.1ngtotalRNAwasusedasinput.RelativeexpressionlevelswerenormalizedtohousekeepinggeneHMBS.


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QuantitatificationofdopaminereleasebyLC‐MS/MS

Thedopamineproductionby the iPS cell‐derivedDAneuronswasanalyzedasdescribedearlier[19].Weanalyzed100µlofsupernatantfromterminallyDA‐differentiatediPScellsunderstimulationby56mMKCl.

Immunostaining

CellculturesofiPScell‐derivedcellswerefixedwith4%paraformaldehydefor15minutesandwashed3 timeswithPBSbefore staining.Antigen retrievalwasperformedonbraincryosections with 10 mM Na citrate solution for 10 min heated in a microwave.Immunostainingwasperformedforthefollowingmarkers:rabbitanti‐tyrosinehydroxylase(1:500,AB152,Chemicon),mouseanti‐GFP(1:500,MAB3580,Millipore),mouseanti‐L1CAM(1:400,ab24345,Abcam),mouseanti‐PSA‐NCAM,IgM(1:400,MAB5324,Millipore).Sampleswere incubated for 24 hrswith primary antibodies at 4°C and primary antibodiesweredetectedwithappropriatesecondaryantibodies,coupledtoAlexa594orAlexa488(1:800,JacksonImmunoResearch,WestGrove,USA)for2–3hrsatroomtemperature.Sampleswerecounterstainedfor10minwithHoechsttovisualizecellnuclei.

Westernblotting

SDS‐PAGEgelelectrophoresisandWesternBlotanalysiswereperformedtodetectNanog,Oct3/4,Sox2,UTF1andL1CAMexpressioniniPScells.Thefollowingantibodieswereused:rabbitanti‐Nanogantibody(1:1000,ab80892,Abcam,Cambridge,UK),mouseanti‐Oct3/4antibody (1:500, sc‐5279, Santa Cruz), rabbit anti‐Sox2 (1:4000, ab15830, Abcam,Cambridge,UK),rabbitanti‐UTF1(1:2000),mouseanti‐L1CAM(1:1000,ab24345,Abcam)and mouse anti‐β‐Actin (ab6276, Abcam, Cambridge, UK) at 1:10,000 dilution. Thehousekeeping gene β‐actin has been used as loading control. Primary antibodies weredetectedusingfluorescentlylabeledsecondaryantibody:donkeyanti‐mouse(IRDye®680,LI‐COR, Biosciences) and donkey anti‐rabbit (IRDye® 800CW, LI‐COR, Biosciences)accordingtomanufacturesinstructions.

FACS

Differentiated iPS cell‐derived cells were dissociated using the Papain Dissociation Kit(WorthingtonBiochem,Products,LK003150).DissociatedcellswerecollectedincolorlessDMEM(Gibco)withBDNF,ascorbicacid,GDNF,TGFβ3anddbcAMP(seeabove),andsortedonaMoFlo‐XDPorMoFlo‐Astriossorterwitha100µmnozzleatapressureof15‐17psibasedonGFPexpression.CellswerecollectedinN2mediumwithBDNF,ascorbicacid,GDNF,TGFβ3anddbcAMP.Cellswerespundownat300rcffor10minandresuspendedinPBS.

Genetransduction

SortedGFP+iPScell‐derivedDAneuronsweretransducedwithlentiviralvectorscontaininggenesencoding forhumanL1CAM,humanSTX(ST8Sia II)under theEF‐1apromoter;anemptyvectorwasusedascontrol.

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Lentiviral particles were produced by co‐transfecting HEK 293T cells (Invitrogen,Carlsbad,CA)withthelentiviralvectorpCMVΔ8.91andvesicularstomatitisvirusGprotein(VSV‐G)plasmidusingFugene(Fugene‐HD;Roche,Almere,TheNetherlands)inasix‐wellplate following the procedure provided by themanufacturer. The ratio usedwas 3 μl ofFugene to 1 μg of DNA for each well. After overnight transfection, culturemediumwaschangedtoN2medium;24hrslater,mediacontainingvirusand4μg/mlpolybrene(Sigma‐Aldrich, Zwijndrecht, TheNetherlands)weremixed and filtered through a 0.45‐μm filter(Whatman, The Netherlands). The viral particles were further concentrated 100‐fold byultracentrifugation(20,000g,20min).ThepelletwasresuspendedinN2medium.For transduction, N2 medium containing lentiviral particles was added to the

resuspended sorted cells in a prelubricant EP tube. After 4 hrs transduction, cells werewashedtwicebyaddingN2medium,spundownat300rcffor10minandresuspendedinPBSforgraftingin6‐OHDAratsorinN2mediumforculturingonanastrocytelayer(4,000cells/well)oronorganotypicstriatalslices(400cellsperslice).After1weekofculturing,samplesweretakenforqPCRanalysisorforimmunocytochemistrystaining.

Cultureofprimaryastrocytes

BraintissuewasexplantedfromP1‐P3mice,meningeswerepeeledoffandthebraintissuewaschoppedintosmallpiecesandincubatedwithTEat37◦Cfor30minutes.Gliamedium(DMEM,with10%FBS,1%sodiumpyruvate(Invitrogen),100U/mLPen/Strep)wasaddedtostopthedigestionandtissuewaspipettedintocellsuspensionandculturedat37◦C,5%CO2.Mediumwaschangedevery3‐4days.Theflasksofcellculturewereshakenat150rpm,37◦Cfor1hr,for3timeseveryotherdaytoremovemicroglia.Theremainingastrocyteswere passaged into a 24well culture plate asmonolayer for sorted iPS cell‐derived DAneurons.

Cultureoforganotypicstriatalslices

Organotypic striatal slice cultures were performed with a modification of a previouslypublishedmethod [20]. Striatawere dissected from P1‐P3mice and cut on a vibratomecoronallyat250mmandcollectedinice‐coldF12medium.Sliceswereculturedattheliquid–air interface on Millicell CM culture inserts and maintained in Neurobasal mediumsupplementedwith0.5%B27supplement,2mMl‐glutamine,25%horseserum,25mg/mlgentamycin,at35◦C,5%CO2.Mediumwaschangedeveryotherday.

Neuritetracingandanalysis

Neurite tracing was performed with ‘simple neurite tracer’ in Fiji(http://fiji.sc/Simple_Neurite_Tracer).Thetotalneuritelengthrepresentedthesumofthelengths of all neurites of one TH+ neuron. The number of primary neurites per neuronrepresentedthenumberofneuritesdirectlyextendingoutfromaTH+soma.Thenumberofbranchesperneuroncomprisedthetotalnumberofneuritestracedminusthenumberofprimaryneurites.TheeffectiveareacomprisedtheareaofthepolygoncreatedbylinkingupthefarendsofallneuritesofoneneuronusingthepolygonselectionoptioninFiji.


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Lesions,rotationbehaviourandtransplantationsurgery

Adult female Sprague Dawley (Harlan) rats (180–230 g) were housed under standardconditionswithfreeaccesstofoodandwater.Ratswereanaesthetizedwithketamine(90mg/kg)andxylazine(4mg/kg).Unilateralretrogradedestructionofdopaminergicneuronsinthesubstantianigrawasinducedby2stereotaxicinjectionsof2.5l6‐OHDA(3mg/mlin0.2% ascorbic acid and 0.9% saline, Sigma) in themedial forebrain bundle of the nigro‐striatalpathway(stereotaxiccoordinates1:AP‐4.0,ML−0.8andV−8.0;toothbarsetat+3.4.and coordinates 2: AP ‐4.4, ML −1.2 and V −7.8; toothbar set at −2.4). The unilateraldestructionofthenigro‐striatalpathwaywasconfirmedbytherecordingofd‐amphetamine(5mg/kg)‐inducedrotationbehavior2weekspostlesion.Amphetamine‐inducedrotationscoreswereobtainedusingRota‐8software(UMCG).Ratsthatrotatedmorethan5rpmwereincluded. Under ketamine/metedomedine anesthesia, 2‐3×104 cells were stereotacticallytransplantedintothestriatumofeachanimal(coordinates:AP+0.9mm,ML−2.6mmandV−4.0mm;toothbarsetat0.0);controlratsreceivedasimilarinjectionwithmouseembryonicfibroblasts(MEF).Rotationbehaviorwasevaluated4and8weekspostgrafting.Dailys.c.injectionsofcyclosporineA15mg/kg(Sigma‐Aldrich)weregiven,starting24hrsbeforecellgrafting(doubledosage),andcontinueduntiltheratsweresacrificedandperfusion‐fixatedat9weeksaftercellgrafting.AllanimalexperimentswerecarriedoutaccordingtotheDutchRegulationsforAnimal

Welfare.TheprotocolwasapprovedbytheInstitutionalAnimalCareandUseCommitteeoftheUniversityofGroningen.

Tissuefixationandhistology

Under ketamine/metedomedine anesthesia, rats were transcardially perfused with 4%paraformaldehyde(PFA)inPBS.Brainswereexplanted,post‐fixedin4%PFAfor24hoursandsoakedina20%sucrosesolutionfor1day.Theyweresectioned(14µmsections)onacryostatafterembeddinginO.C.T.compound(SakuraFinetek,Torrance,USA).

Statisticalanalysis

Alltestswereperformedandanalyzedinablindedmanner.Throughoutthetextandinthefigures,allvaluesareexpressedas themean±SEM.SPSSanalysisofdifference inmeansbetween2groupsofindependentsampleswereanalyzedusingstudent’sttest.Differencesinmeansamongmultipledatasetswereanalyzedwithone‐wayANOVAbyTukeypost‐hocanalysis.Inallanalyses,Pvaluesoflessthan0.05wereconsideredsignificant.

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RESULTS

DopaminergicneurondifferentiationofiPSclines

iPScellsweregeneratedfromembryonicfibroblastsisolatedfromPitx3gfp/+transgenicmice.Thelineage‐specificdifferentiationofthreeiPSCclonesintoVMDAneuronswasperformedaccording to previously described protocol [18]. The appearance of Pitx3‐expressing DAneuronsinthedifferentiatingiPScellscouldbedetectedbytheirexpressionofGFP;atday20ofdifferentiationmostofthePitx3‐GFP+cellsco‐expressedtyrosinehydroxylase(TH)(Figure 1A). The green fluorescent protein (GFP) expression under control of the Ptix3promoterallowedpurificationofthedifferentiatedDAneuronswithFAC‐sorting[21].OfthethreeiPSCclonesused,cloneIIIshowedthehighestyieldofGFP+cells(1.78%)aftersorting(Figure1B).Ofall3sortedPitx3GFP+cellsuspensions,mRNAwascollectedandanalyzedwithRT‐PCR.OnlythepurifiedPitx3‐GFP+cellsofCIIIappearedtoexpresstheproperprofileof DA neuronmarkers, i.e. both neuronalmarkers (Map2 and β‐III‐tubulin) and VM DAneuron markers (Nurr1, Pitx3, TH and DAT) (Figure 1C); apparently, despite strongimmunostaining(seeFigure1A),THmRNAexpressionappearedlowinthisstageofmaturityoftheDAneurons.MeasurementofKClstimulatedsecretionofdopaminerevealedamuchhigherdopaminereleaseintheCIII‐derivedPitx3GFP+cellsthanthosefromtheother2iPSCclones(Figure1D).Inallourfollowingexperiments,weonlyusedthePitx3GFP+DAneuronsderivedfromiPSCcloneIII.

Over‐expressionofL1CAMandPSA‐NCAMiniPScell‐derivedDAneurons

ThesortedPitx3‐GFP+DAneuronsweretransducedwithalentiviralvectorcontainingthegenes encoding for human L1CAM or STX; an empty vector was used as control. Thetransducedcellswerethoroughlywashedbeforetheywereplatedontoprimaryastrocytelayers. The expression of L1CAM and STX was analyzed using q‐PCR. There was basalexpressionofmouseL1CAMandmouseSTXinDAneuronstransducedwithcontrolvectors;andthelevelofendogenousL1CAMandSTXexpressioninastrocytesappearedtobeverylow(Figure2A,C).Oneweekposttransduction,specificexpressionofhumanL1CAMmRNAcouldbedetectedata4timeshigherlevelintheL1CAMvectortransducedgroup(Figure2A).L1CAMwasalsodetectedbyimmunofluorescentstainingbothinthesomaandalongtheneuriteofDAneurons inbothgroups(Figure2B).TheexpressionofhumanSTXwasabout 10 times higher in the STX vector transduced group in comparison to the controlvectorgroup(Figure2C).ImmunostainingshowedthepresenceofPSA‐NCAMbothinthesomaandtheneuriteofDAneuronsinbothgroups(Figure2D).BothL1CAMandPSA‐NCAMwerenotdetectableintheprimaryastrocytelayer(Figure2B,D).

L1CAM and PSA‐NCAM over‐expression stimulates neurite outgrowth of iPS cell‐derivedDAneuronsin‐vitro

TransducedDAneuronswereplatedontoaprimaryastrocyte layer tocreateanoptimalsupportingenvironmentforoutgrowingneuronsenablingaclearanalysisoftheeffectsofL1CAMandPSA‐NCAMover‐expressiononcellmorphology.DAneuronsco‐culturedontop


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Figure1.Dopaminergicdifferentiationof3Pitx3gfp/+iPSCclones(A)TheappearanceofPitx3‐expressingDAneuronsinthedifferentiatingiPScellscouldbedetectedbytheirexpressionofGFP;atday20ofdifferentiationmostof thePitx3‐GFP+cellsco‐expressedTHasdemonstrated with immunostaining. (B) Three clones (CI, CII, CIII) of iPS cells reprogrammed frommouse embryonic Pitx3gfp/+ fibroblasts were differentiated into VM DA neurons. At day 20 ofdifferentiation, thePitx3‐GFP+ cells of each iPSC clonewere sorted. (C)mRNAexpression forneuralprecursor(NP)markers,neuronal(N)markersandVMDAneuron(DA)markerswasanalysedusingRT‐PCR.(D)Secretionofdopaminebythe3Pitx3‐GFP+cellculturesuponKClstimulationwasmeasuredwithHPLC.ItisclearthatthePitx3‐GFP+cellsdifferentiatedfromiPSCcloneIII,bestfulfilledthecriteriaofDAneurons.

of a primary astrocyte layer had an oval or trigonal somaof approximately similar sizes(Figure 3A). Tracingsmade ofDAneurons from the control group and the L1CAMover‐expressinggrouprevealedbipolaror tripolarneurite formation,whileDAneurons in thePSA‐NCAMgroupoccasionallyalsohad5or6primaryneurites(Figure3B).However,this

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differencewasnotsignificantamongthethreegroups.Themorphologyoftheneuritesinboth L1CAM and PSA‐NCAM groups was much more complex (Figure 3A), with morebranches than in the control group (Figure 3C). The longest neurite from each TH+ DAneuronwasalsomeasured.BothL1CAMandPSA‐NCAMover‐expressingcellsextendedtheirneuritesignificantlylongerthanthecontrolones(Figure3D).Withlongerneuritesandmorebranches,thetotalneuritelengthinthesetwogroupswassignificantlylonger(Figure3E)andcoveredalargerarea(Figure3F).

Figure2.Over‐expressionofhL1CAMandhSTXiniPScell‐derivedDAneurons(A,C)TransductionoftheiPScell‐derivedDAneuronswithhL1CAM,hSTXorcontrol(empty)vectorsresulted,afterculturingfor1weekonanastrocytecelllayer,intheover‐expressionofhL1CAMandhSTX.TheexpressionofhumanandmouseL1CAMorhumanandmouseSTXmRNAwasanalyzedbyq‐PCR.(B,D)ExpressionofL1CAMandPSA‐NCAMwasverifiedbyco‐immunostainingofL1CAMandPSA‐NCAMwithTH.

L1CAMandPSA‐NCAMover‐expressiondoesnotpromoteneuriteoutgrowthof iPScell‐derivedDAneuronsinorganotypicslices

To provide an environmentmimicking the striatal‐in‐vivo situation, transduced iPS cell‐derived DA neurons from all three groups (L1CAM or PSA‐NCAM over‐expressing andcontrols)wereplacedontomouseorganotypicstriatalslicesandculturedfor1week.TH+DAneuronswereeitherclusteredasgroupsorscatteredontheslice(Figure4A).Analysisofthenumberofprimaryneuritesrevealednodifferencesbetweenthethreegroups(Figure


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4C).Ingeneral,allDAneuronsonthestriatalslicesgrewmuchlongerneuritesthanthoseculturedontheastrocytelayers(Figure4Bv.s.Figure3D).Duetotheneuronalclusteringandthesevereintertwiningoftheneurites, itwasdifficulttotraceallbranchesfromoneneuron.Therefore,weonlytracedthelongestneuritedistinguishablefromeachneuron.NosignificantdifferenceswereobservedinthelongestneuritelengthbetweentheDAneuronsover‐expressingL1CAMorPSA‐NCAMorthecontrolgroup(Figure4B).ThismaybeduetothehighlevelofcelladhesionmoleculessuchasL1CAMandPSA‐NCAMalreadypresentinpostnatalorganotypicstriatalslices(SupplementaryFigure1),alreadypromotingmaximaloutgrowthofneuritesfromDAneurons.Remarkably,clusteredDAneuronsoftheL1CAMandPSA‐NCAMgroupstendedtoextendtheirneurites inaparalleldirection(Figure4A),unlikethoseofthecontrolgroup(Figure4A).ThisphenomenonmaypointtothefactthatL1CAMandPSA‐NCAM‐over‐expressingDAneuronsaremoreresponsivetopatterningcuesinthestriatalslice.

Figure 3. Over‐expression ofL1CAM and STX stimulated theoutgrowthofiPScell‐derivedDAneuronsonanastrocytelayer(A) iPS cell‐derived DA neuronsover‐expressing L1CAM or STXwere cultured on a primaryastrocyte layer for 1 week.Immunostaining forTHshowsthemorphology of DA neurons fromthe three groups (includingcontrols). Tracings of exemplaryneurons are shown. The tracingswere used to determine (B) thenumber of primary neurites, (C)the number of branches, (D) thelength of the longest neurite perneuron,(E)thetotalneuritelengthand (F) the effective area (arealinked‐upbythefarendofneuritesfrom one neuron).. Data arepresentedasmean±SEM,n=10to15pergroup,*P<0.05,**P<0.001,*** P = 0.000, one‐way ANOVA,Tukeyposthocanalysis.

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Figure4.Outgrowthof iPScell‐derivedDAneuronsonorganotypicstriatalslices(A) iPS cell‐derived DAneurons over‐expressingL1CAM or PSA‐NCAM orcontrol ones were culturedonorganotypicstriatalslicesfrom P0‐P3 mice. Soma andneuritesofDAneuronswerestained with TH. (B) Thelongest neurite fromscatteredDAneuronsorcellson the edge of clustered DAneuronsweremeasured. (C)The number of primaryneuritespercellwascounted.Datawerepresentedasmean±SEM,n=15pergroup.

ImplantationofiPScell‐derivedDAneuronsin6‐OHDAlesionrats

SortedcontroliPScell‐derivedDAneuronsandiPScell‐derivedDAneuronsover‐expressingL1CAMorPSA‐NCAMweretransplantedintounilaterally6‐OHDAlesionrats,accordingtotheproceduredescribedbefore.Twoweekspost6‐OHDAinjection,ratsthatrotatedmorethan5rpmafteramphetamineinjectionwereincludedintheexperiments.At8weeksafterimplantation, a small reduction in the number of rotations was observed in the groups(Figure 5A). Immunohistochemical examination of the implanted striata revealed thesurvivalofonly,onaverage,423(±108)implantedTH+neuronsinthedenervatedstriatum.ThepresenceofonlyaverysmallnumberofDAneuronsmayaccountforthesmallreductionin rotationbehavior.Apparently, the subsequent stressfulproceduresofFAC‐sortingandviraltransductionhadmadetheDAneuronsvulnerableforthestressassociatedwiththe


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implantation procedure, resulting in the survival of only about 2% of the injected cells.Accurate analysis of the 3D outgrowth of the newly‐formed neurites extending fromindividualimplantedneuronsappearedtobedifficult,butthemaximallengthoftheneuritesineachofthegroupsofimplantediPScell‐derivedDAneuronsdidnotexceedthe500µm(Figure5B).

Figure5. Intrastriatal transplantationof iPS cell‐derivedDAneurons inunilaterally6‐OHDA‐lesionedrats(A)Amphetamine‐inducedrotationofthe6‐OHDA‐lesionedratstransplantedwiththeiPScell‐derivedDAneuronsat4or8weeksaftergraftingshowedasmallnonsignificantreductionafter8weeks;mean± SEM, n = 3. (B) Immunohistochemical staining for TH shows the intact contralateral side of theunilaterallylesionedratstriatumwiththepunctuatedopaminergicterminals(1),whichiscompletelygoneattheipsilateralsideafterdopaminergicdenervationby6‐OHDAlesioningoftheSNDAneurons(2); in this denervated striatum a cluster of TH+ implanted neurons can be detected at the side ofimplantation.Individualneuronalcellbodies(arrow)fromtheseclustersextendtheirthin,curlyneuritesinthestriatum(3,4).Bar=100µm(1&3)or50µm(2&4).

DISCUSSION

ConsideringtheoptionofacellreplacementtherapyforParkinson’sdisease, iPScellsarepresentlythemostpromisingsourceforanunlimitednumberofautologousDAneuronstobegraftedintracerebrally.Thein‐vitrodifferentiated,iPScell‐derivedDAneuronshavebeendemonstrated toadopt thecharacteristicsof true(ventralmesencephalic)DAneuronsasassessedbycelltypespecificmarkerexpression,electrophysiologyanddopaminerelease[11,22].However,comparisonofthegenomewideexpressionprofileofiPScell‐derivedDAneuronstoprimaryDAneurons,showedasubsetofgenesthatweredown‐regulatediniPScell‐derived VM DA neurons. Gene annotation analysis showed that these genes wereinvolved in nervous system development, neuron differentiation, and neurogenesis [21].ThismayexplainthefactthatiPScell‐derivedDAneuronsarenotasefficientasprimaryDAneuronsinreinnervatingtheDAdepletedstriatuminParkinson6‐OHDAratsandgivingrisetofunctionalrecovery(reviewedinchapter2).Extensivestriatum‐widereinnervationisanabsolutepre‐requisiteforaDAneuronreplacementtherapyforPDtobesuccessful.Yetthe

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shortneuritesextendingfromgraftediPScell‐derivedDAneuronsappearedtoberestrictedwithinashortrangearoundtheinjectiontrack[22‐23].Inthepresentstudy,weattemptedto improve the neurite outgrowth of iPS cell‐derived DA neurons by forcing their over‐expressionofL1CAMandPSA‐NCAM,both crucial adhesionmolecules foraxonguidanceduringembryonicneuronaldevelopment.iPScellswerereprogrammedfromfibroblastsofPitx3gfp/+mice[24],andsubsequently

differentiated into DA neurons. The various iPSC lines used demonstrated a differentefficiency in in‐vitro DA neuron differentiation, not only as far as the yield of Pitx3‐GFPpositiveDAneuronsisconcerned,butalsowithregardtotheexpressionprofileofVMDAneuronalmarkersinthesePitx3‐GFPpositivecellsandtheirproductionofdopamine.ThetranscriptionfactorPitx3hasbeendemonstratedtobeoneofthemoststringentmarkersforfullydifferentiated, functionalVMDAneurons [25‐26].Yet, apparently, theexpressionofPitx3 in the iPS cell‐derived DA neurons did not guarantee uniformity in the fulldifferentiation of DA neurons derived from different iPSC lines. Thismust be related todifferencesinthecompletenessofreprogrammingandtheremnantofepigeneticmarkersinterfering with complete DA neuron differentiation. These findings emphasize theimportance and need for extensive detailed analysis of PD patient iPS cell‐derived DAneurons,ultimatelypreparedforclinicalusage.Inducingover‐expressionofL1CAMandPSA‐NCAMinDAneuronsderivedfromiPScells

appearedtobecumbersome.OurfirstapproachwastotransducetherelevantgenesintheiPScellsandtoestablishstablytransducediPSClines(SupplementaryFigure2)thatweresubsequentlydifferentiatedintoDAneurons.However,L1CAMexpressionwaslostduringdifferentiation and could only be detected in the undifferentiated cells (SupplementaryFigure3).This is in accordancewithpreviousobservationson transgene silencingwhentransducedstemcellsdifferentiateintospecializedcelltypes[27‐28].Moreover,theyieldofPitx3+/gfp DA neurons was reduced to 0.7% due to the iPSC transduction procedureemployingpuromycin(SupplementaryFigure3).WedecidedtoperformlentiviralhL1CAMandhSTXgenetransductiononasuspensionofPitx3‐GFPFAC‐sortedDAneuronsandwereable to show stable up‐regulation of both hL1CAM and hSTX in the transduced iPS cell‐derived DA neurons. These L1CAM and PSA‐NCAM over‐expressing iPS cell‐derived DAneuronsallowedustoevaluatetheeffectofthesemoleculesonneuriteoutgrowthin‐vitro,on top of a feeder layer of astrocytes (which did not express these molecules). Wedemonstrated that, indeed, theneurites inbothL1CAMandPSA‐NCAMgroups showedamuchmore complex, branchedmorphology and overall length than those in the controlgroup.WhentheiPScell‐derivedDAneuronswereculturedontopoforganotypicslicesofpostnatalstriatum,bothL1CAMandPSA‐NCAMover‐expressingiPScell‐derivedDAneuronsas well as control iPS cell‐derived DA neurons equally demonstrated extensive neuriteoutgrowthandcomplexbranching.AsthepostnatalstriatalslicesareveryrichinL1CAMandPSA‐NCAM expression, we speculate that the iPS cell‐derived DA neurons were alreadypromotedtogrowtheirneuritestotheirmaximum.Only, thepatternofoutgrowthoftheL1CAM or PSA‐NCAM over‐expressing iPS cell‐derived DA neurons appeared slightly


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differentthanthecontrolones,indicatingadifferentresponseofthesecellstothecuesinthesubstrate.WeintendedtoexaminetheeffectofL1CAMandPSA‐NCAMontheneuriteoutgrowthof

iPScell‐derivedDAneurons in thedenervatedstriatumof6‐OHDA lesionParkinsonrats.Unfortunately, the subsequent stressful procedures of FAC‐sorting and lentiviraltransduction made the DA neurons vulnerable for the stress associated with theimplantationprocedure,resultinginthesurvivalofonlyabout2%oftheinjectedcells.Lowsurvival(<3%)ofimplantediPScell‐derivedDAneuronshavebeendescribedbefore[29‐30]andsortingbeforetransductionhasbeenshowntofurtherreducethesurvivalratetolessthan 0.06% [22]. Although our in‐vitro experiments clearly revealed the outgrowthstimulatingabilityofL1CAMandPSA‐NCAM,pronouncedoutgrowthstimulationcouldnotbedetectedinthefewsurvivingL1CAMandPSA‐NCAMover‐expressingiPScell‐derivedDAneuronsinthedenervatedstriatumoftheunilaterally6‐OHDAlesionrats.ThisincontrasttorecentfindingsbyBattistaetal.[31]whodemonstratedthatPSA‐NCAMoverexpressionwasabletoenhanceneuriteoutgrowthofstablytransducedmESC‐derivedDAneuronsin‐vivo.Moreover,experimentalstudieswiththespinalcordinjurymodelclearlydemonstratedthatL1CAM[32]andPSA‐NCAM[33]wereabletoenhanceaxonregenerationandextension.Itseemslikelythatthesorting,thelentiviraltransductionofthesortedDAneuronsandtheimplantation protocol affected the viability and health of the surviving DA neuronsannihilatingthegrowthpromotingeffectsoftheadhesionfactors.Inconclusion,wehavedemonstratedthatL1CAMandPSA‐NCAMsignificantlystimulate

theneuriteoutgrowthofiPScell‐derivedDAneuron,bothneuriteextensionandbranchingin‐vitro.WefailedtodemonstrateactualneuriteoutgrowthstimulationiniPScell‐derivedDAneuronsafterimplantationinthedenervatedstriatum,butweassumethatthisisonlydue to the general poor health of the cells as a consequence of the subsequent stressfultreatmentsofthesecellsbeforeandduringimplantation.

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31. Battista,D.,etal.,Enhancementofpolysialicacidexpression improves functionofembryonicstem‐deriveddopamineneurongraftsinParkinsonianmice.StemCellsTranslMed,2014.3(1):p.108‐13.

32. Chen, J., et al.,Adeno‐associated virus‐mediatedL1 expressionpromotes functional recoveryafterspinalcordinjury.Brain,2007.130(Pt4):p.954‐69.

33. Zhang,Y.,etal.,Lentiviral‐mediatedexpressionofpolysialicacidinspinalcordandconditioninglesionpromoteregenerationofsensoryaxonsintospinalcord.MolTher,2007.15(10):p.1796‐804.

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SupplementaryFigure1.OrganotypicstriatalslicecultureStriatal sliceswerecut from the striatumofP0‐P3micewitha thicknessof250m.Thesliceswereculturedfor2weeksbeforesortedDAneuronswerelayeredonthem.Striatalsliceswerecharacterizedby immunostaining for (A) the neuronalmarker β‐III‐tubulin, (B) themicrogliamarker IbaI, (C) theastrocytemarkerGFAP,and(D)thecelladhesionmoleculesL1CAM,(E)andPSA‐NCAM.


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SupplementaryFigure2.TransductionofL1CAMiniPScellsAfter lentiviral transduction, L1CAMwas found to be stably expressed after selection by puromycinexposure for several passages. L1CAMwas detected by both (A) IF staining, and (B)WB. (C, D) Thepluripotent markers were detected in all three iPSC lines: non‐transduced, L1CAM transduced andcontrolfactortransducedones.

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SupplementaryFigure3.ReductionofL1CAMexpressioniniPScells(A)SchemeoftheDAneurondifferentiationprocedure.ThetimepointsofsamplingforIFstainingwereindicated. (B) L1CAM expression was gradually reduced during differentiation. In the presence ofpuromycin,L1CAMexpressioncanbemuchmorepronouncedduringdifferentiation,yetitismostlyintheroundandcompactpluripotent‐likecells,insteadofincellswithaneuralmorphology.Bytheendofdifferentiation,L1CAMwasmostlynotco‐localizedintheclustersofTH+cells.

SupplementaryTable1.PrimersequenceforRT‐PCRandq‐PCR

CHAPTER5

PARTICIPATIONOFPERFORININMEDIATINGDOPAMINERGIC

NEURONLOSSINMPTP‐INDUCEDPARKINSON’SDISEASEINMICE

Su‐Ping Peng1,2, Ye Zhang1, Sjef Copray2, MelittaSchachner1andYan‐QinShen1

1Center for Neuroscience, Shantou University MedicalCollege,Shantou,GuangdongProvince,P.R.China2DepartmentofNeuroscience,UniversityMedicalCenterGroningen,theNetherlands

ABSTRACTParkinson’s disease (PD) is gradually recognized as a neuroinflammatory disease. BothresidentinnateandperipheralimmuneaberrationshavebeenobservedinPDpatientsandtheyhavebeendemonstratedtoinfluencediseaseprogressioninanimalmodels.However,it is still enigmatic how and which immune components are lethal to the dopaminergicneuroninPD.Hereweshowedthatperforinwassignificantlyincreasedintheserumofwild‐type mice 4 weeks after an i.p. injection of 1‐methyl‐4‐fenyl‐1,2,3,6‐tetrahydropyridine(MPTP),atoxinusedtoinducePD‐likesymptomsinmice.Wemadeuseofperforin‐knockoutmice and demonstrated that perforin‐deficiency attenuated the acute striatal dopaminereduction in theMPTP‐treatedmiceby33%andabatedmicrogliaactivation3dayspostMPTP‐injection.Perforin‐deficiencyappearedtoretarddopaminergicneurondeathintheMPTP‐mice resulting in striataldopamine recovery twiceashighas forwild‐typemice4weekspostMPTP‐injection.OurstudysuggeststhatperforinplaysaroleindopaminergicneuronlossinPD.

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INTRODUCTIONAftermanydecadesofresearch,themechanismsunderlyingthepathogenesisofParkinson’sdisease(PD)arestillnotfullyunderstood.Amongthemanycellularmechanismsproposed,the contribution of neuroinflammation in PD pathogenesis has gained more and moreattention. Microglia activation, as resident innate immune reaction, has been welldemonstratedinPD[1].The changes in peripheral lymphocyte constitution are significant in PD patients and

animalmodels[2].IncludinganincreaseinδTcells,CD45RO+memoryTcells[3]andNKcells[4]aswellasadecreaseinthetotalcountofTcellsandBcells[5‐6].InfiltrationofCD4+TcellsandCD8+Tcells in thebrainparenchymahasbeendescribed[7‐9]; inparticular,CD8+Tcellsappeartobeprominentwiththeirnumber4.8timesashighasCD4+Tcells[9].TheseresultsclearlyindicatetheinvolvementofthesecellsinPDdevelopment.Recent studies revealed several molecular mechanisms, in which these immune cells

contribute to dopaminergic (DA) neuron loss. It was proposed that IFN‐γ is critical inmicroglia‐mediated loss of DA neurons in 1‐methyl‐4‐fenyl‐1,2,3,6‐tetrahydropyridine(MPTP)‐intoxicatedmice[10].ThedeleteriousactivityofinfiltratedCD4+TcellsseemstoinvolvetheFas/FasLpathway[9].Althoughnotpresentinthebrainparenchyma,BcellsmaycontributetoDAneuronlossbysecretingIgGthatbindstotheFcreceptorandmodulatesthemicroglialresponse[11].AnotherpotentialfactorinvolvedinDAneuroninjurycouldbeperforin(Prf1).Perforinisapore‐formingproteinandisemployedmostlybycytotoxicCD8+TandNKcellstoeliminatetargetcellsviathedeliveryofgranzymes[12];alsomurineCD4+Th1cellsseemtoexpressPrf1mRNA[13].IthasbeenshownthatperforinfromNKcells,CD8+TcellsandCD4+Thcellscanmediateneuritedamage[15‐16]orneurondeath[17]in‐vitroandithasbeensuggestedtoparticipateinneuronaldegenerationinmultiplesclerosis[18].Inthisstudy,wesetouttoobtainfirstevidenceforthepotentialinvolvementofperforin

in dopaminergic degeneration. We applied MPTP to induce DA neuron degeneration inperforin‐deficientmice(Pfp‐/‐)aswellasinwild‐type(WT)controlmice.AfterassessingtheincreaseintheperforinserumlevelduetoMPTP,wehavemonitoredthestriataldopaminelevelandtheactualdopamineneuronlossduring4weeksinbothgroups.Inaddition,wecomparedthechangesinMPTP‐inducedmicrogliaactivationintheperforin‐deficientmice(Pfp‐/‐)aswellasinthewild‐type(WT)controlmice.

MATERIALANDMETHODS

Animals

Eight‐ to twelve‐week‐oldmalemice,weighing22–26gwereused.The followingstrainswere obtained from Taconic, Borup, Denmark: Pfp‐/‐ (B6.129S6‐ Prf1tm1ClrkN12) andcorrespondingWTinbredC57BL/6J.Theanimalsweremaintainedunderstandardspecificpathogen‐freeconditionsandallowedaccesstofoodandwateradlibitum.Allexperimental

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procedureswereapprovedbytheAnimalEthicsCommitteeofShantouUniversityMedicalCollege(People’sRepublicofChina).

Treatmentandtissuepreparation

Groupsofmicereceived4timesofi.p.injectionsofMPTP‐HCl(15mg/kg,at2‐hourintervals)andweresacrificedfrom3to28daysafterthelastinjection;controlmicereceivedPBSonly.Bloodsamplesweredrawnfromtheheartafterisofluraneanesthesia.Forobtainingfreshstriatum, mice were transcardially perfused with PBS only. The striata were quicklydissected, frozen on dry ice and stored in ‐80C. For immunohistochemistry, miceweretranscardially perfused with PBS followed by 4% paraformaldehyde (PFA) in 0.1 Mphosphatebuffer,pH7.3.Tissueswerepostfixedin4%PFAat4Covernight,keptin20%sucrose at 4C for 1 day, embedded in optimal cutting temperature compound (SakuraFinetek,Torrance,USA),andsnap frozen in2‐methyl‐butane (isopentane)precooled to–70C.Twenty‐fivemicrometerthickcross‐sectionswerecutusingacryostat(CM1850,Leica,Nussloch,Germany).Seriesofsectionswith10‐sections‐intervalswerecollected.

Measurementofstriataldopamine

DopamineResearchELISAkit(DopamineResearchRIA,LaborDiagnostikaNordGmbh&Co.KG,Germany)wasusedtomeasurethedopaminelevelinstriatum.Onthedayofanalysis,striatawerehomogenizedin0.01NHClwith1mMEDTAand4mMsodiummetabisulfite.Protein concentrations were measured using BCA protein assay (Pierce). Samples wereprocessedaccordingtothemanufacturer’sinstructionswhileensuringproperstandardsandcontrols.

Immunohistochemistry

Afterantigenretrieval,sectionswereincubatedinPBScontaining0.2%v/vTritonX‐100,0.02%w/vsodiumazideand5%v/vnormalserum(accordingtosecondaryantibodyused)for1hr.Primaryantibodies(anti‐TH1:800,Millipore;anti‐IbaI1:800Millipore)werethenapplied for24hrsat4°C.Fordetectionof theprimaryantibodies,appropriatesecondaryantibodies,coupledtoCy2orCy3(1:800,JacksonImmunoResearch,WestGrove,USA)wereused.Sectionswerecounterstainedfor10minuteswith50μg/mlDAPI(Sigma)tovisualizecellnuclei.Specimenswereexaminedwithanepifluorescencemicroscope(AxioImagerZ1,Zeiss,Oberkochen,Germany).

ImageandTH+cellcounting

FluorescentsectionswereanalysedonaZeissAxioImagerZ1.Foreachanimal,oneseriesofsectionswasstainedwithTHandDAPI.TheTH+neuronswerecountedintherightandleftsubstantia nigra (SN) of every tenth section throughout the entire extent of the SN. Anepifluorescencemicroscope(AxioImageZ1,Zeiss,Oberkochen,Germany)thatwasequippedwith a motorized stage and a Stereo Investigator software‐controlled computer system(MicroBright‐Field,Magdeburg,Germany)wasusedforquantitativeanalysis.TheborderofSNandventral tegmentalarea(VTA)wasdelineatedat lowermagnificationbasedonTH

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immunostaining.UsingrandomsamplingintheSN,cellcountswereperformedaccordingtotheopticaldissectorprincipleatamagnificationof400×.

Cellcountingofmicroglia

ImagingwasperformedwithaZeissAxioImagerZ1microscope. Sectionsof comparativelevelsinonesectionserieswereselected.SiximagesofIbaIstainingweretakenfromboththeleftandrightsidesofthedorsalstriatumat200×magnification.ThenumberofIbaI+cellsineachimagewascounted,anddatawerepresentedasnumberofcellsperimage(450µm×337µm).

Analysisofperforinlevel

Bloodsamplesweredrawnandkeptatroomtemperaturefor30minbeforecentrifugationat3,000rpmfor10min.Serumwastakenandstoredat–70Cuntilfurtheranalysis.Theconcentrationof perforin in serumwas analysedwithmouseperforin1 (PRF1)Elisa kit(MyBioSource, Inc. SanDiego,California,USA); sampleswereprocessed according to themanufacturer’sinstructions.

Dataacquisitionandstatisticalanalysis

Alltestswereperformedandanalysedinablindedmanner.Throughoutthetextandinthefigures,allvaluesareexpressedasthemean±SEM(standarderrorofthemean).Differenceinmeansbetween2 groups of independent sampleswas analysedusing student’s t test.Differences inmeans amongmultiple data setswere analysed using one‐wayANOVA byDunnett’s post‐hoc analysis. In all analyses, P values of less than 0.05 were consideredsignificant.

RESULTS

Perforinlevelisup‐regulatedinMPTP‐lesionedwildtypemice

WefirstanalysedtheperforinlevelinserumfromMPTP‐intoxicatedWTmiceandcompareditwiththeserumlevelinPBS‐treatedmice(Figure1).AbasallevelofperforinwasdetectedinthePBSgroupaswellasintheMPTP‐treatedgroupsatthetimepointof3days,1and2weekspost injection.A significant increaseofperforinwasdetected4weekspostMPTPintoxication.Sinceperforinispredominantlyexpressedbyactivatedcytotoxiclymphocytes(and not by naive ones), our data suggest that MPTP induce activation of cytotoxiclymphocytesleadingtotheproductionofperforinanditsreleaseintheserum.

Perforin‐deficiencyabatedtheMPTP‐inducedstriataldopaminelevelreduction

To test thepotential involvementofperforin inMPTP‐inducedPDdevelopment,perforinknockout(Pfp‐/‐)micewereused.Pfp‐/‐andWTmicewereeitherintoxicatedwithMPTPorreceivedPBSviaintraperitonealinjections.Thestriataldopaminelevelsofeachgroupweredeterminedatdifferenttimepoints(i.e.3days,1,2,4weeks)postinjection.ThedopaminelevelsinthestriatumoftheMPTP‐treatedgroupswerenormalizedtothatoftherelatedPBS‐treatedgroup(Figure2.).Asexpected,thestriataldopamineleveldramaticallydecreasedin

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both thePfp‐/‐ andWTmice3days postMPTP injection. In theMPTPmousemodel,DAneuronsareinitiallydistressedbytheneuraltoxinMPP+,derivedfromtheinjectedMPTP[24].Theacutedropinstriataldopaminelevel3dayspostMPTPinjectionmaybeascribedtothisfirststressrespondofDAneuronsandnotyettoactualneuronalloss.ThesignificantlyhigherdopaminelevelinPfp‐/‐mice(41%)at3daysincomparisontotheWTmiceindicatestheinvolvementofperforininearlydistresstoDAneurons.

Figure 1. Increased concentration ofperforin in the serum of MPTP‐intoxicatedWTmiceTheconcentrationofperforin inserumofwild‐typemicewassignificantlyincreased4 weeks after MPTP treatment incomparison to PBS‐treated control. * P <0.05(onewayANOVA,Dunnettt‐test.n=4‐6ineachgroup).

ThedopaminelevelsinWTmicetreatedwithMPTPchangedovertime:itdecreasedtothe lowestat3daysandincreasedslowlytoaround40%by4weekspost injection.Thisrecovery to about 40% presumably points to a stabilization of stress and the level ofdegenerationofDAneurons.IncomparisontotheWTmice,perforin‐deficientmiceshowedaslowerpatternandlessreductionofthestriataldopaminelevel.Thelowestdopaminelevelwas foundat1‐weekpost injectionandwas around20%ofPBS‐treatedPfp‐/‐mice.ThedopaminelevelinthePfp‐/‐micequicklyincreasedto80%at4weeksafterMPTPinjection.Atthistimepoint,thedopaminelevelinperforin‐deficientmicewassignificantlyhigherthaninwildtypemice.TheseresultssuggestlessstressandabettersurvivalofSNDAneuronsinperforin‐deficientmiceafterMPTPintoxication.

Figure2.ReductionofstriataldopaminelevelinMPTP‐intoxicatedmiceStriatal dopamine levels dramaticallydecreased3dayspostMPTPtreatmentandrecovered over time, most prominently inthe Pfp‐/‐ mice up to 4 weeks post MPTPtreatment. Each MPTP‐treated group iscomparedwithitsrespectivePBScontrol*P< 0.05, ** P < 0.001 (One way ANOVA,Dunnett’spost‐hocanalysis.n=6‐8ineachgroup.).

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Figure 3. Perforin‐deficiencyabates MPTP‐induced SN DAneurondegeneration

(A) RepresentativestainingofTH in theSNofwildtype and Pfp‐/‐ mice. Shown arePBS control and MPTP‐treatedmicesacrificedat3days,1,2and4 weeks post MPTP injection.Scalebaris200μm.(B)SurvivalofTH+ cells in theSNofMPTP‐lesionedwildtypeandPfp‐/‐mice.TH+ cells were quantified withunbiased Stereo Investigatorsoftware‐controlled computersystem.*P<0.05(Student’sttest;OnewayANOVA,Dunnettt‐test.n=3or4ineachgroup).

Perforin‐deficiencyreduceddopaminergicneurondegeneration intheSNofMPTP‐intoxicatedmice

Toexaminetheeffectofperforin‐deficiencyonthesurvivalofSNDAneurons,weanalysedthedynamicchangesof thenumberofDAneurons inSNofPfp‐/‐andWTmice followingMPTPintoxication(Figure3).TH+cellswerecountedbyStereoInvestigator,andtheborder

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betweentheSNandVTAwasdefinedunderthemicroscope.InWTmice,thenumberofDAneurons gradually decreased over time from 3 days up to 4weeks postMPTP injectioncomparedtoPBS‐treatedwild‐typemice;thedifferencebetweentheMPTPgroupandthePBScontrolgroupwassignificantat4weekspostMPTPinjection.However,thenumberofSNDAneuronsinthePfp‐/‐micetreatedwithMPTPdidnotsignificantlychangewithintheperiodof timestudied.Byweek4, thenumberofDAneurons in theMPTP‐treatedPfp‐/‐groupwassignificantlyhigherthanthatintheMPTP‐treatedWTmice(Figure3B).Theseresults demonstrate the involvement of perforin‐mediated DA neuron loss and thesubsequentreductionindopamineproductioninMPTP‐treatedmice.

Figure4.Microgliaactivation in thestriatumof theMPTP‐intoxicatedmiceStainingofIbaIinthestriatumofwild‐type(A‐E)andPfp‐/‐(F‐J)mice.PBS‐injectedmice(A,F)weresacrificedatday 3 as control. Dynamic changes of IbaI+ cells areshownat3days(B,G),1week(C,H),2weeks(D,I)and4weeks(E,J)postMPTPinjection.Microgliaactivationwasdetected3daysafterMPTP‐injectionevidencedbyretractedpseudopodiaandalargercellbody(b,g).Thescale bar is 100μm in A‐J, and is 10μm in a‐j. (K) Thedynamicchangesinthenumberofmicroglia(numberofcells/imageareasize(450µm×337µm)).InWTmice,the number of microglia at MPTP 3D group issignificantlydifferentincomparisontoallothergroups;

inPfp‐/‐mice,thenumberofmicrogliaintheMPTP3Dgroupissignificantlydifferenttotheothergroupsindicated.*P<0.05(Student’sttest;OnewayANOVA,Dunnett’spost‐hocanalysis.N=3ineachgroup).

MicrogliaactivationinPfp‐/‐miceandwildtypemiceafterMPTPtreatment

Microgliaactivation inthestriatumandSNhasbeendemonstrated inpost‐mortembraintissueinPDpatientsaswellasinanimalmodelsforPD[8,25].Theactivationofthemicrogliainthemeso‐striatalpathwayisthoughttobeinducedbythe(stress)signalsreleasedbytheendangered,degeneratingSNDAneuronsduetoMPTPintoxication.WeanalysedtheextentofactivationofmicrogliainthestriataofthePfp‐/‐andWTMPTP‐treatedmicebasedonthemorphology(e.g.presenceofretractedpseudopodia)andthenumberofmicroglia(detected

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byIbaIimmunostaining)(Figure4).AsignificantmicrogliaactivationwasdetectedinboththePfp‐/‐and(evenhigher)intheWTmiceonlyat3dayspostMPTPintoxication(Figure4K); this appeared to be consistentwith previous findings thatmicroglia are transientlyactivatedintheacutephaseofMPTP‐lesionedmice[8].Theactivationstateofthemicrogliaat day 3was also reflected in the activated cellmorphologywith retracted pseudopodia(Figure3b&g),andalsointhenumberofIbaI+microglia(Figure4K).InWTmice,thenumberofIbaI+cellswassignificantlyhigheratday3incomparisontoPfp‐/‐mice(Figure4K).

DISCUSSION

Our experimentswithMPTP toxification in perforin‐knockoutmice strongly suggest thatperforinisinvolvedintheinjuryandlossofdopaminergicneuronsinthisanimalmodelforParkinson’sdisease.MPTPinjectionledtoanincreaseintheserumlevelofperforininwild‐typemice,butitisunlikelythatthissystemicincreaseisresponsibleforthedeleteriouslocaleffectsonthedopaminergicneuronsinthesubstantianigra.ThefactthatcytotoxicCD8+T,NKandevenCD4+Thcellsarethemostprominentsourceofperforin[12,13]combinedwiththefindingsthatcytotoxicCD8+TcellsandCD4+ThcellsinfiltratebrainparenchymainPDanimalmodels[7‐9],makeitverylikelythattheseinfiltratingcellsareresponsiblefortheperforin‐mediatedeffectsonthedopaminergicneurons.Moreover, ithasbeenshownthatperforinfromNKcells,CD8+TcellsandCD4+Thcellscanmediateneuritedamage[15‐16] or neurondeath [17] in‐vitro. CD8+T cells have been found in close proximitywithactivated microglia and degenerating neurons [28]. NK cells have been demonstratedcapabletokillneuronsthroughperforin‐mediatedcytotoxicity[29].Interestingly,perforinmayparticipate inPDdevelopmentby inducingblood‐brainbarrier (BBB)disruption, asperforinhasbeenshowntobeanimportantcomponentininducingBBBdisruption[21‐22];in thisway,perforinmayevencontribute toan increaseof the infiltrationofdeleteriouslymphocytes.As far as the acute decrease in striatal dopamine level is concerned: It has been

demonstratedthatlyticgranulesisolatedfromCD4+Tcells,CD8+TcellsandNKcellsareable to induce axonal microtubule destabilization independent of apoptosis [16].Microtubulesprovideplatformsforintracellulartransportandareinvolvedinavarietyofcellular processes, including the movement of secretary vesicles [27]. Perforin, as animportantcomponentoflyticgranules,mayparticipateinthegranule‐inducedmicrotubuledestabilizationinSNDAneuronaxons,impairingDAsecretioninstriatumasobservedintheinitialstageafterMPTP‐intoxication.FourweekspostMPTPlesion,DAneuronsrecoveredfromtheacuteMPP+shockandso

the striatal dopamine level in both groups at this time point represents the capacity ofsurvivingDAneuronstoproducedopamine.Thestriataldopaminelevelgraduallyrecoveredto40%inwildtypemiceandto80%inPfp‐/‐mice.Accordingly,significantDAneuronlossinSNwasfoundinwild‐typemice(around40%)4weekspostMPTPlesion;DAneuronlosswasnotsignificantinPfp‐/‐mice.TheattenuationofDAneuronlossbyperforin‐deficiencystronglydemonstratestheinvolvementofperforininthisprocess.

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ItisevidentthatdoubleimmunostainingoftheMPTP‐lesionedmicebrainsforperforinandoneofthespecificlymphocytemarkerscansimplyconfirmthestrongsuggestionthattheseinfiltratedcellsareresponsiblefortheperforin‐mediatedDAneurondamage.Practicalproblems,sofar,havepreventedustosuccessfullyperformtheseexperiments.Ourpresentstudy,anyway,advocatesforthedevelopmentofaperforin‐targetingtherapyandthetestingofcurrentlyexistinganti‐perforinantibodies[30‐31]inourPDanimalmodel.

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (81072622,31271580), LiKa Shing Foundation (LD030601) Jiangsu Six Talents PeakProject (2013‐SWYY‐041),Bi‐InnovationPlanofJiangsuProvince(2014‐27).

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CHAPTER6

COMPARISONOFAAV2ANDAAV5INGENETRANSFERINTHEINJUREDSPINALCORD

OFMICE

Su‐PingPeng1,SebastianKügler2,Zhi‐KuiMa1,Yan‐QinShen1andMelittaSchachner1

1Center for Neuroscience, Shantou University MedicalCollege,Shantou,GuangdongProvince,P.R.China2DeutscheForschungsgemeinschaftResearchCenterforMolecular Physiology of the Brain, Department ofNeurology,UniversityofGöttingen,Göttingen,Germany

PublishedinNeuroReport,2011

ABSTRACT

Recombinantadeno‐associatedvirus(AAV)vectorsarepromisingtoolsforgenetherapy.Inspinal cord injury where extensive damage occurs, vectors with high diffusion andtransductionabilitiesare required.Wecompared thediffusioncapacityand transductionefficiency ofAAV2 andAAV5vectors using amouse spinal cord injurymodel.Our studydemonstratedthatAAV5wasmoreeffectivethanAAV2fordeliveringgenesintotheinjuredspinal cord tissue. AAV5 diffused 6.9 mm from the injection site, transduced with anapproximately 2‐fold increase in total cell number and yielded an approximately 3‐foldincreaseingeneexpressionincomparisontoAAV2.

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INTRODUCTION

Traumaticspinalcordinjuryisacentralnervoussystem(CNS)lesionthatresultsfrombluntorpenetratingtrauma,whichcausesmotorandsensoryimpairmentsdistalfromtheinjurylevel.Spinal injurycausesseverephysical,psychologicalandeconomicdifficulties for thepatientsandtheirfamilies,sinceunlikeintheperipheralnervoussystem,neuronsintheCNSdonotregenerateingeneral[1].ThelackofadevelopmentalprogramtorecapitulategrowthintheCNSisprimarilyduetorestrictionsontheadultCNSneurons,whichpreventtheseneuronsfromexpressingthegenesnecessaryforneuralrecovery,andbecauseoftheoverallnon‐permissivepropertiesoftheglialscartissueandCNSmyelin[2].Forseveraldecades,extensiveresearchhasfocusedonpromotingregenerativegrowthof

theinjuredaxonsinthedamagedspinalcord.Genetherapyprovidesonepotentialtreatmenttoovercomethelackofregenerativeabilityoftheinjuredneuronsandmayalsobeeffectiveinmodifying the environment at the injury site by providing factors to promote axonalregrowth.Up‐regulationofthecAMPsignallingpathway[3],reductionoftheactivityoftherhoAGTPase[4‐5],increasedlevelsofpolysialicacidwhichisthemostconduciveneuriteoutgrowthpromotingformoftheneuralcelladhesionmoleculeNCAM[6‐7],anddeliveryofneurotrophins,includingneurotrophin‐3[8]andbrain‐derivedneurotrophicfactor[9],areabletopromotetheregrowthofdifferenttypesofinjuredaxons.Todelivermoleculescapableofmodifyingtheinjuredparenchymaofthespinalcordfrom

inhibitorytogrowthpromoting,genetherapyvectorsbasedontherecombinantAAVaremostpromising.ElevennaturallyoccurringAAVserotypeshavebeenisolated.AAV2,thefirstserotypetobediscovered[10],hasbeenthemostextensivelystudiedinclinicaltrials[11].However,otherAAVserotypeshaveproventobemoreeffective forgenedelivery.AAV5,originally identified from a human clinical sample [12], was considered a promisingcandidateforuseasagenetransfervectorintheCNS[13].BothAAVvectorshavebeenusedforgenetransferintothespinalcord[14‐15],buttheyhavenotbeendirectlycomparedintheinjuredspinalcord.Inthisstudy,wequantifiedthediffusioncapacityandtransductionefficiencyofAAV2and

AAV5usingamousespinalcordinjurymodel.ThespinalcordcompressionmodelcreatesatraumaticenvironmentthatallowscomparingdiffusionandtransductionpropertiesoftheAAV2andAAV5vectors,whichcontainedthesamegenomewithAAV2invertedterminalrepeats(ITRs)andtheenhancedgreenfluorescentprotein(EGFP)reportergene.Ourresultsdemonstrate thatAAV5wasmore effective thanAAV2 in the injured spinal cord inbothdiffusionandgenetransduction,warranting furtherdevelopmentof thisvector forspinalcorddirectedgenetherapyapproaches.

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MATERIALSANDMETHODS

Animals

Female C57BL/6Jmicewere purchased from theGuangdongMedical LaboratoryAnimalCenter.Theanimalsweremaintainedunderstandardconditionsandallowedaccesstofoodandwaterad libitum. All experimental procedureswere approved by theAnimal EthicalCommitteeofShantouUniversityMedicalCollege.

AAVvectorproduction

AAV2andAAV5vectors containing the samegenomewere constructed.TheEGFP cDNAunder thecontrolof thesynapsin‐1genepromoterwascloned intoaplasmidcontaininginvertedterminalrepeats(ITRs)fromAAVserotype2.TheplasmidwaspackagedintothecapsidsofeitherAAV2orAAV5.InfectioustitersofbothAAVvectorswere2.8×108tu/μl.

Surgicalprocedures

Forsurgery,3‐month‐oldmiceweredeeplyanaesthetizedusinganintraperitonealinjectionofketamineandxylazine[125mgofketanesthydrochlorideand12.5mgofxylazineperkgbodyweight].ThreemicewereusedforeachAAVserotypevector.SurgerywasperformedasdescribedbyApostolovaetal.[16],withminormodifications.Inbrief,alaminectomywasperformed atT7–T9.Themouse spinal cord compressionwasperformedbyholding thespinalcordbetweenapairoftweezers(tweezerdumont#5,WorldPrecisionInstrument,Sarasota,US),closingthetweezersasquicklyaspossibleandwithafullcompressionlastingfor 5 seconds. This procedure yields results that are as reproducible as those with themagneticdeviceusedbyApostolovaetal.[16].Next,1μloftheviralconstructs,AAV2orAAV5,wasinjectedslowlyintothespinalcord

2mmrostralandcaudaltothelesionsite.Theinjectionandwithdrawalofthesyringetookapproximately5minsoastoavoidleakingbackoftheinjectedfluid.Themuscleandskinweresurgicallyclosedusing6‐0stitches.Topreventhypothermia,themiceweremaintainedinawarmroom(35°C)forseveral

hours after the operation [16]. Thereafter, themicewere singly housed under standardconditionswithwater and standard food providedad libitum. During the post‐operativeperiod,theirbladdersweremanuallyvoidedtwicedaily.

Tissuepreparation

Four weeks after spinal cord compression and AAV vector injection, the mice weretranscardiallyperfusedwith4%paraformaldehydein0.1Mphosphatebuffer,pH7.3(4%PFA).A3.6‐cmlongsectionofeachspinalcordcontainingtheinjurysiteinthemiddlewasremoved.Thetissuewaspost‐fixedin4%PFAat4°Covernight,keptin20%sucroseat4°Cfor1day,embeddedinOCTcompound(SakuraFinetek,Torrance,USA),andsnapfrozenin2‐methyl‐butane(isopentane)precooledto‐70°C.Twentyfiveμmthickcross‐sectionswerecutonacryostat(CM1850,Leica,Nussloch,Germany).

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Cellcountingandfluorescenceintensitymeasurement

Serialcross‐sectionsspaced400μmapartwerecollectedononeglassslide.Foreachanimal,oneseriesofsectionswasstainedwithDAPI,andthecellspositiveforEGFPwerecalculatedaccordingtotheOpticalFractionatormethod(MicroBright‐Field,Magdeburg,Germany).Anepifluorescencemicroscope(AxioImageZ1,Zeiss,Oberkochen,Germany)thatwasequippedwith a motorized stage and Stereo Investigator software‐controlled computer system(MicroBright‐Field)wasusedforquantitativeanalysis.ForquantitativeEGFPfluorescenceintensity evaluation, the NIH software ImageJ (http://rsbweb.nih.gov/ij/index.html) wasused.

Dataacquisitionandstatisticalanalysis

All tests were performed and analyzed by a blinded observer. Student’s t‐test forindependentsampleswasusedtocomparethegroups.Throughoutthetextandinthefigures,dataarepresentedasthemeanswithstandarderrorsofthemean(SEM).

RESULTS

MaximumdiffusiondistanceofAAVvectorsfromtheinjectionsite

WithEGFPasareportergene,wefirstanalyzedthediffusiondistanceoftheAAV2andAAV5vectorsawayfromtheinjectionsite.Inseriallyspacedcross‐sections,EGFPpositivesectionsfurthest from the injury site rostrally and caudallywere recorded. The distance of thesesectionsfromtheinjurysiteminus2,000μm(distanceoftheinjectionsitefromtheinjurysite)isshownasthemaximumdiffusiondistance.DeterminationofthemaximumdiffusiondistanceofeachAAVvector(Figure1)theinjuredspinalcordshowedthatAAV5(6.9±0.41mm)hadasignificantlyhigherdiffusioncapacitythanAAV2(3.5±0.61mm,P=0.001).

Figure1.ThemaximumdiffusiondistanceofAAVvectorsfromtheinjectionsiteintheinjuredmousespinalcordTherostralandcaudalcross‐sectionsfurthestfromtheinjurysitethatwerepositiveforEGFPwererecorded.The distance of these sections from the injury siteminus2,000μm(distanceoftheinjectionsitefromtheinjurysite)isshownasthemaximumdiffusiondistanceoftheviruses(n=3).MeanvaluesSEMareshown,*P<0.01(Student’st‐test).

DistributionofAAVvectorsintheinjuredspinalcordofmice

TofurthercharacterizethediffusioncapacityofAAV2andAAV5,thedistributionprofileofEGFP (Figure 2A) was determined. The distribution profile was composed of the

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fluorescenceintensityofEGFPforeachseriallyspacedcross‐section.Thesectionswiththesite of injury, site of injection, site of maximum EGFP labelling intensity and site of thefurthestdiffusiondistanceofEGFPexpressionwererecorded(Figure2B‐M).Thesectionsatthe injurysiteshoweddenseDAPIstainingwith littleEGFPexpression

(Figure2B,F),indicatingthatadensecellularcorehadformedattheinjurysite[15]thatwasduetotheinfiltrationofmeningeal,vascularendothelialandimmunesystem‐derivedcellswhichdonotexpresssynapsin‐1andthereforedonotactivatethepromoterofthisgeneforEGFPexpression.Atthesiteofinjection,whichwas2mmfromthesiteofinjury(Figure2C,G),sections

fromtheAAV5injectedmiceshowedalargerareaofEGFPexpression(Figure2G)comparedtoAAV2.However,duetothelimiteddiffusionofAAV2,theviralparticlesaccumulatedinthe site of injection. When analyzing the expression profile, the average fluorescenceintensitywashigherforAAV2atthesiteofinjectioncomparedtoAAV5(Figure2A).

Figure2.ThedistributionoftheAAVvectorsintheinjuredmousespinalcord(A) The profile of EGFPexpression in cross‐sectionsof theinjuredmousespinalcordisshownforAAV2andAAV5.Theprofile iscomposed of the fluorescencelabellingintensityofEGFPfromtheserially spaced cross‐sections (n =3).(B‐I)ExpressionofEGFPatthesiteofinjury(B,F),siteofinjection(C, G), site of maximum EGFPfluorescence intensity (D, H) andsite of the furthest diffusiondistance (E, I) are shown.Representative images for AAV2(B‐E)andAAV5(F‐I)weretakenata 25× magnification. Scale bar is500μm.

ThesiteofmaximumEGFP labelling intensity(Figure2D,H)and the furthestdiffusiondistance (Figure 2E, I) observed for the AAV5 (5.2 mm, 9.6 mm, respectively) injectedanimalswereseenatagreaterdistancethanthoseseenfortheAAV2injectedanimals(2.8

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mm,6.8mm,respectively).Thus,AAV5diffusedmoreextensivelyalongthespinalcordwithpeaksofEGFPfluorescenceintensitybeinghigherandfurtherawayfromthesiteofinjectionthanAAV2.

TransductionefficiencyofAAVvectorsintheinjuredspinalcord

ThetransductionefficiencyoftheAAVvectorscanbedividedintotwoaspects:thenumberofcellsthataretransducedwiththeAAVvectorsandthelevelofproteinexpressionofthetestedgene,consideringthatseveraltransductioneventscouldoccurinonecell.AnunbiasedstereologicalestimationofthenumberofEGFPpositivecells(Figure3A)was

used to determine the number of cells that were transduced with AAV vectors. AAV5(198,792±17,736)transducedasignificantlyhighernumberofcellsintheinjuredspinalcord,beingapproximately2‐foldhigherthanthosetransducedwithAAV2(98,171±3,191,P=0.005).ThelevelofexpressionoftheEGFPreportergenecanbedeterminedbyitsfluorescence

intensity.The total fluorescence intensityofEGFP inserialcross‐sectionsspaced400μmapartalongthe3.6‐cmlongspinalcordtissuewasmeasured. IntheAAV5(100±4.31%)injectedspinal cordsEGFPexpressionwasapproximately3‐foldhigher than in theAAV2injectedones(29.6±3.85%,P<0.001)(Figure3B).ThisindicatesthatAAV5yieldsamorepotentgeneexpressionthanAAV2intheinjuredspinalcord.

Figure 3. The transductionefficiency of AAV vectors in theinjuredmousespinalcord(A) DeterminationofthenumberofEGFPexpressingcellsisshownascounted in serially spaced cross‐sections throughout the 3.6‐cmspinal cord tissue (n = 3) with theOpticalFractionatormethod.(B)Thetotal fluorescence labelling intensityof EGFP was measured in seriallyspaced cross‐sections (n = 3).Meanvalues+SEMareshown,*P<0.01,**P<0.001(Student’st‐test).

DISCUSSION

Traumaticspinalcordinjuryimmediatelyleadstoirreversibleprimaryinjury.Attheinjurysite,bloodvesselsaredamaged,axonsdisrupted,astrocytesandmicrogliaareactivatedandimmune system cells infiltrate into the lesioned tissue. Haemorrhages occur rapidly andspreadaxially[17].Subsequently,alterationsinthelocalionconcentrations[18],productionof free radicals [19], and release of cytotoxic neurotransmitters [20] contribute to thedamageofhealthy,neighboringcells.Inthechronicphases,thespinalcordsustainsmajortissuelosswithoftenlargecavitations,andsitesrostralandcaudaltothelesionshowwell‐demarcatednecroticareas[21],indicatingextensivedamageatandfurtherawayfromthelesionsite.Thus,usingAAVgenetherapy,vectorswithhighdiffusioncapacityandpotent

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transductionandexpressionefficienciespromisetobeaneffectivetreatmentoncetheviruscodesforexpressionofaregeneration‐conducivemolecule.In this study, we used a spinal cord compression model in mice to mimic a form of

traumatic spinal cord injury. The AAV capsids of AAV2 and AAV5, containing the samegenomeandtiterofinfectiousunits,wereusedtocomparethediffusioncapacityaswellastransductionandexpressionefficiencies.Inpreviousstudies,thetransductionofAAV2inthebrain[22]orothertissues[13]ledto

geneexpressionbeingconfined to the injectionsite,whereasAAV5wasdispersedoveralargerarea[22].However,whencomparingtheAAV2andAAV5expressionintheuninjuredspinalcordofrat,AAV5wasnotfoundtobestatisticallymoreeffectivethanAAV2for,both,the number of cells transduced and the transduction volume [23]. In the present study,examiningtheinjuredmousespinalcord,AAV5hadasignificantlyhigherdiffusioncapacityand a larger number of transduced cells compared to AAV2. Hence, AAV5may bemoreeffectiveatdiffusingthroughthanAAV2alsoinotherconditionsoftraumaticinjury.WiththegeneswithintheAAV2andAAV5capsidsbeingidentical,genedeliverybyAAV5

yielded significantly higher EGFP expression compared to AAV2. The capsid is not onlyimportantforthevectortobindandenterthetargetcells,but isalso importantforpost‐entry steps, such as the pathways involved in cellular processing, intracellular transit,nuclearentry,andvectorgenomeprocessing[24].For instance,AAV5transducedretinascontained 30‐fold more genome copies per cell than those transduced with AAV2 [25].Similarly,ourresultsindicatethattheAAV5capsidshaveincreasedexpressionofthetargetgeneintheinjuredspinalcordcomparedtotheAAV2capsids.

CONCLUSIONS

Inconclusion,vectorswithhighdiffusioncapacityandtransductionefficiencyarerequiredforpotentialtherapyafterspinalcordinjuryorothertypesoftraumaticandchronicinsultstothecentralnervoussystemofmammals.Inthisstudy,thespinalcordcompressionmodelprovidedatraumaticenvironmenttoprobeforthetwomostcommonlyusedAAVvectors.WedemonstratethatAAV5wasamoreeffectiveoptionafterspinalcordinjurybecauseofitshigherdiffusioncapacityaswellastransductionandexpressionefficiencies.

ACKNOWLEDGEMENTS

TheauthorsaregratefultoProfessorAndreyIrintchevforexcellentadviceandsupport,andthankHaoZhang, Jin‐FeiLinandXuan‐JunZhaofortheirhelpwiththeexperiments.ThisstudywassupportedbytheCenterforNeurosciencestart‐upfundsatSUMC(LB0103)toMelittaSchachnerandtheproject‐sponsoredNNSFChina(81072622)toYan‐QinShen.

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GENERALDISCUSSION

CurrentanddevelopingtherapiesforParkinson’sdisease(PD)aimtorestorethedopaminelevelinthestriatumandtostopthedegenerationofthedopaminergic(DA)neuronsinthesubstantianigra(SN).Intrastriatalimplantationofyoungdopaminergicneuronsseemstobeaverypromisingapproachtoachievethefirstaim.Byre‐establishingcircuitrybetweenthestriatalinterneuronsandthegraftedDAneurons,thecontinuousreleaseofdopaminebythegrafted cells may be even regulated and modified. The replenishment of the striataldopaminelevelcanleadtotherestorationofmovementcontrolandareductionofthemajormotoricproblemsobserved inPDpatients.Abreakthrough in this approachwas thatnolonger human dopaminergic neurons from the ventral mesencephalon (VM) had to beisolatedandcollectedfromabortedfoetusesbutthatVMDAneuronscouldbegeneratedin‐vitrofrom(embryonic)stemcells[1‐2].Thediscoverylateronofinducedpluripotentstemcells(iPScells)[3]promisedanunlimited,autologoussourceofin‐vitroderivedDAneuronswithoutethicalconcernsfortransplantation.Asfarasthesecondaimisconcerned,stoppingthe process of DA neuron degeneration, latelymuch interest has been addressed to thecontribution of inflammatory processes in the onset and development of PD [4‐5].Discovering the relevant immune components may lead to the development of newtherapeuticapproaches todecelerateorevencease thedegenerationofDAneurons.Themajor focusof theresearchdescribed in this thesis concernsstudieson the treatmentofParkinson’sdiseasebytheimplantationofDAneuronsgeneratedfromiPScells.TheimmunepathogeniccomponentofPDandpossiblegenedeliverytoolsforthecentralnervoussystem(CNS)werealsoaddressed.After a broad Introduction in chapter1, we reviewed previously published DA graft

studieswiththe6‐OHDAratmodelforPDinordertocomparetheperformanceofhumaniPScell‐derivedDAneuronswiththatofhumanprimary(foetal)VMDAneuronsinchapter2. Human foetal VM DA neurons have set the standard for a beneficial outcome afterintrastriatal implantation. Invariousclinical trials, ithasbeendemonstrated thatgraftedhumanfoetalVMDAneuronscansurvivemorethan10yearsinthestriatumofPDpatients[6‐7]andthatthesepatientssignificantlyimprovewithareduceddependencyonL‐DOPA[8‐11].TheimportantquestioniswhethergraftedhumaniPScell‐derivedDAneuronswillhavethesamebeneficialeffectasthehumanprimaryfoetalDAneurons.Indeed,theyexpressalltypicalventralmesencephalonmarkersandDAneuronmarkers[12‐13],theyshowthesamespontaneous firingpatternandtheyareable toreleasedopamine[14‐15];buthowwellwilltheysurviveandkeepastableVMDAneuronphenotypeaftertransplantationintheadult striatum?Howwellwill they integrate in thehostbrainandprovide functionalrecoveryforthehost?ComparisonofthebehaviourandefficacyofhumanprimaryfoetalDAneuronswiththoseofhumaniPScell‐derivedDAneuronsintheunilaterally6‐OHDAlesionratmodelforPDmayofferaresponsetothesequestions.Ourreviewshowsthatindeedbothprimary and iPS cell‐derived DA neurons are able to survive and induce functionalimprovement in the grafted rats. However, a larger number of iPS cell‐derived TH+ DAneuronsappearedtoberequiredtoachievethesamereductioninrotationbehaviourasthe

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primary foetalDA neurons [12, 15‐16]. Analysis of the expression of theVMDA neuronmarkersNURR1, EN1, and FOXA2, aswell as DAT andVMAT, revealed that the iPS cell‐derivedDAneuronsindeedkepttheirDAsignatureaftergraftingintothestriatum[12,18‐20]. Itbecameevident,however, that theactualneuriteoutgrowthof iPScell‐derivedDAneurons in the host striatumwas limited [17], particularly in comparison to that of thehuman primary DA neurons. Apparently, the iPS cell‐reprogramming and subsequentdifferentiationprocessmusthavecauseddisturbancesintheexpressionofgenesinvolvedinneuronaldevelopmentandoutgrowth.Toelucidatethedifferencesbetweenin‐vitrogeneratediPScell‐derivedDAneuronsand

primaryDAneurons,wecompared,inchapter3,theexpressionprofileofiPScell‐derivedDA neurons with that of primary DA neurons. Ptix3gfp/+ mice were used to enable theisolationofpureiPScell‐derivedDAneuronsandpureprimaryVMDAneuronswithGFP‐basedFAC‐sorting [21].Wedifferentiated themousePtix3‐GFP iPScells intoDAneuronsusing generally accepted protocols [2, 22‐23]. Their ventralmesencephalon identitywasconfirmedbytranscriptanalysisofVMDAneuronspecificmarkers.Moreover,themouseiPScell‐derived DA neurons fulfilled most other DA‐neuron criteria, such as the release ofdopamine, thetypicalslowspontaneous firingpatternand, inparticular, thereductionofamphetamine‐inducedrotationaftertransplantationintheunilaterally6‐OHDAlesionrats[12,17].Subsequently,weanalysedandcomparedthegenome‐wideexpressionprofileofthe iPS cell‐derived DA neurons and primary pure DA neuron suspensions isolated atdifferentstagesduringembryonicandpostnataldevelopment.ItisrelevanttocomparetheiPScell‐derivedDAneuronswithembryonicandperinatalprimaryDAneurons,since iPScell‐derived DA neurons should also be considered embryonic: during iPS cell‐reprogramming adult fibroblasts become completely rejuvenated and the cell typesdifferentiatingfromthemshouldbeconsideredembryonic,despitethelongcultureperiodrequired for their differentiation and maturation. Gene analysis showed that thepluripotency genes in iPS cell‐derived DA neurons were properly silenced, yet somefibroblastgenesappearedtobeexpressedstill.iPScell‐derivedDAneuronsappearedtohavethestrongestgeneexpressioncorrelationwithembryonicprimaryDAneuronsintheVMDAspecificgeneclusters.Remarkably,afewaberrationsintheexpressionofgenesassociatedwith functional annotations such as ‘nervous system development’, ‘neurogenesis’ and‘neurondifferentiationandoutgrowth’wereevidentiniPScell‐derivedDAneurons.Theseaberrant gene expression profiles may account for the poor neurite outgrowth capacityobservedafterintrastriatalimplantationofiPScell‐derivedDAneurons.ExtensiveneuriteoutgrowthiscrucialforthesuccessfulimplantationofDAneuronsand

thebeneficialeffectsonPDpatients.iPScell‐derivedDAneuronscanbemanipulatedin‐vitrotoincreasetheirabilitytoformextensiveneuritesinthestriatumaftertransplantation.Inchapter4,we induced the (over‐) expressionof PSA‐NCAM (by transfectionof the geneencodingforPSA‐transferaseSTX)andL1CAMiniPScell‐derivedDAneuronsandstudiedtheeffectofthesetwocellularadhesionmoleculesonneuriteoutgrowthofiPScell‐derivedDAneurons.TransductionwasperformedinapuresuspensionofPitx3+/gfpiPScell‐derived


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DAneuronsafterFAC‐sorting.TheforcedexpressionofL1CAMandSTXwasdemonstratedbytheincreaseintheirmRNAlevel.TheDAneuronsover‐expressingL1CAMandPSA‐NCAMdevelopedamorecomplexmorphologyduringcultureonanastrocytelayerthanthecellsthatweretransducedwithacontrolvector.Moresecondarybrancheswereformedandthetotal neurite length was significantly higher. Plating the L1CAM or PSA‐NCAM over‐expressingDAneuronsontopostnatalstriatalslicesdidnotenhanceneuriteformation,butresultedintypicalpatternsofparallelneuriteoutgrowthindicatingadifferentinteractionwiththesubstrate.WetestedtheperformanceofL1CAMandPSA‐NCAMover‐expressingiPScell‐derived DA neurons after intrastriatal implantation into 6‐OHDA lesion rats.Unfortunately, only a few of the implanted DA neurons survived the implantationpresumablybecauseoftheirincreasedvulnerabilityduetothelong,traumaticproceduresofviraltransductionandFAC‐sorting.ThefewsurvivingDAneuronsshowedlimitedneuriteoutgrowth(TH+neuriteslessthan500m).Itisclearthatthetimingoftransductionandthe transfection method used should be improved taking into account that transgeneexpressioninthestemcellstagewilldeclineduringdifferentiation[24‐25].Understandingthefactorsthatcontributetothespecificneurodegenerationandlossof

DAneurons in thePDbrain remains crucial; these factorsmayeventually also affect thenewlygraftedDAneuronsinthestriatum.Oneofthesefactorscouldbeperforin,apowerfulcell death‐mediating component of the immune system: by drilling holes in the cellmembraneandmediatingthereleaseofgranzymes,itcaninducecelllysisorapoptosis[26].Inchapter5,we showed thatMPTP‐intoxicationofmice (awell‐accepted animalmodelmimickingDAneuronlossandPDpathology),resultedinanincreaseoftheperforinlevelinserum.To study the roleofperforin in thepathogenesis of PD,wemadeuseofperforinknock‐out (Pfp‐/‐)mice and induced PD by the i.p. injection ofMPTP.We found that thestriataldopaminelevelinPfp‐/‐micewaslessaffectedbytheacuteMPTPshockandthatthedecrease was less pronounced than that in MPTP‐treated WT mice. In accordance, thenumberofVMDAneuronswasnotsignificantlychangedintheMPTP‐treatedperforin‐nullmiceafter4weeks,while itwassignificantlyreducedinMPTP‐treatedwild‐typemice. Inaddition,microgliaactivationinthestriatumofMPTP‐treatedPfp‐/‐micewasalsoattenuatedincomparisontoMPTP‐treatedwild‐typemice.Ourstudyisthefirstonetoshowthatlackof perforin can alleviate the MPTP‐initiated toxicity to the nigrostriatal pathway. TheseresultssuggestperforincanmediatedirectorindirectdamagetoVMDAneuronsduringPDpathogenesis.Sinceperforin ispredominantlyproducedbyspecific lymphocytesubtypes,whichhavebeenshowntoinfiltratethePDbrain,itislikelythatthesecellsareresponsiblefor the local perforin‐mediated effects on the DA neurons. Unfortunately, practicalimmunohistochemicalproblemspreventedusfromactuallydemonstratingthepresenceofperforin‐producinglymphocytesclosetotheVMDAneuronsortheirprojectingaxonsinthebrainoftheMPTP‐treatedmice.Sinceperforinhasbeenshowntodisrupttheblood‐brainbarrierinotherdiseases[27‐28],itmayhaveindirectlycontributedtoPDpathogenesisbyfacilitating lymphocyte infiltration through adysruptedblood‐brainbarrier.Our findingsthat inhibiting perforin activity is beneficial for VMDAneuron survival,may lead to the

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development of new specific therapeutical approaches for PD based on the selectiveblockageofperforin.Modification of the micro‐environment of specific cells by the forced expression of

relevant factors may reduce inflammation and stimulate neuroprotection andneuroregenerationasexemplifiedinchapter4withL1CAMandPSA‐NCAM.Inchapter6,weexaminedtheappropriatewaytoinducetheexpressionoftargetgenesinlesionedCNSareas.Inthedegeneratingand/orlesionedCNS,glialscar[29]andnecrosis[30]formrigidobstacles for the distribution of the gene delivery vectors. The spinal cord injurymodelprovides a proper animal model to study gene delivery in the hostile environment of aseverelydamagedCNS.Adeno‐associatedvirus(AAV)hasbeenextensivelystudiedinclinicaltrials[31‐32],yettheefficiencyofdifferentserotypesishighlyvariableandtissue‐specific[33].We studied the efficiencyofdifferent serotypesofAAVs (different capsidswith thesamegenome)indeliveringgenesintheinjuredspinalcordofmice.WithGFPasareportergene,itwasdemonstratedthatAAV5,incomparisontotheclinicallyusedAAV2,cantravelmuch further from the injection site through the traumatic spinal cord.More cellsweretransducedbyAAV5thanAAV2andthetotaltransgeneexpressionwasalsomuchhigherinAAV5transducedspinalcord.InviewofthenatureofParkinson’sdisease,theoptimaltherapyforPDshouldcombine

DAneuronreplacement,toreplenishstriataldopaminelevel,withtheblockageoffurtherlosstothestillremainingDAneurons.Withthediscoveryofinducedpluripotencyin2006,anamplesourceofautologousDAneuronfortransplantationpurposesbecameavailable.ImportantdevelopmentsinthisfieldhavemadetheiPSCtechnologysaferbyreplacingvirus‐mediatedreprogramming[3]bynew,viral‐freeorat leastintegration‐freestrategies[34‐35]. Dedicated studies were carried out to deepen the understanding of the embryonicdevelopment of human VM DA neurons [36‐38] and consequent improvement in theprocedures of in‐vitro DA neuron differentiation have increased the yield of safe, well‐defined human iPS cell‐derived VM DA neurons almost identical to their primarycounterparts[12,39].Recentstudieshavebeendedicatedtoimproveeverypossiblestepintransplantationtherapy,includingoptimalsafecultureproceduresforiPScells[40],furtherincreaseintheyieldofDAneurons[41‐42],eliminationofharmfulcontaminatedcellsinthefinal cell grafts [16], andmodification of themicro‐environment of the niche of the cellimplant[43].Moreover,theongoing‘TransEuro’programaimstoaddressremainingissuesaroundclinicaltrialsonhumanfoetalVMtissuetransplantationforPD[44].ItwillprovideknowledgeandpracticalexperienceforthealmostinevitableclinicalapplicationofiPScell‐derivedDAneurons.As far aspotential immune‐modification therapies forPD is concerned, attemptshave

beenmadetoreduceDAneuronlossbytargetingdeleteriouslymphocytephenotypesorbyenhancingbeneficialphenotypes forParkinson’sdisease.Passive immunization inanimalmodelswithanti‐‐synucleinantibodieshasbeeninvestigatedasapotentialmechanismforclearing accumulations of ‐synuclein; it was shown that it improves behaviouralperformance and, indeed, promotes degradation of accumulated ‐synuclein [45‐46].


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AdoptivetransferofTcellsfromacopaxone‐immunizedanimalappearedtobeprotectiveagainstneurodegeneration inanMPTPmodel [47].Copaxone isused in the treatmentofmultiplesclerosissinceitappearedtobeabletoalterthecytotoxicTcellresponsetoamoreanti‐inflammatory phenotype [48]. Adoptive transfer of Tregs also attenuatedneurodegenerationinanMPTPmodel[49],bytheirsuppressivecapacitiestowardsTcellover‐reaction[50].In conclusion, in this thesis,we compared iPS cell‐derivedDA neurons to the current

standard,primaryfoetalVMDAneurons,notonlybyreviewingtheirperformanceinvarioustransplantationstudies,butalsobyanalysisof theirgenome‐wideexpressionprofile.WedemonstratedthatcombiningtheiPSCtechnologywiththetransgenicmodificationoftheiPS cell‐derived cells can provide iPS cell‐derivedDA neuronswith an enhanced neuriteextensioncapacity.FuturetreatmentofPDwilleventuallyconsistentofreplacementoflostDA neurons by new DA neurons, which aremodified to establish optimal dopaminergicreinnervationofthestriatumcombinedwithapproachestoblockthepathologicalprocessescausingtheinjuryanddegenerationofDAneurons.

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43. Tornqvist, N., et al., Implantation of bioactive growth factor‐secreting rods enhances fetaldopaminergic graft survival, outgrowthdensity, and functional recovery in a ratmodelofParkinson'sdisease.ExpNeurol,2000.164(1):p.130‐8.

44. Rath, A., et al., Survival and functional restoration of human fetal ventralmesencephalonfollowingtransplantationinaratmodelofParkinson'sdisease.CellTransplant,2013.22(7):p.1281‐93.

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46. Masliah,E.,etal.,Passiveimmunizationreducesbehavioralandneuropathologicaldeficitsinanalpha‐synucleintransgenicmodelofLewybodydisease.PLoSOne,2011.6(4):p.e19338.

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CHAPTER8

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Dehuidige ennog in ontwikkeling zijndebehandelingsmogelijkhedenvoorde ziekte vanParkinson zijn er enerzijds op gericht om het dopamine‐niveau in het striatum zo goedmogelijk teherstellenenanderzijdsomdedegeneratievandopaminergeneuronen indesubstantianigraeenhalttoeteroepen.Intrastriatale implantatie van jonge dopaminerge neuronen lijkt een zeer beloftevolle

benadering om het eerste doel te bereiken. Door ervoor te zorgen dat er functionelesynaptische verbindingen kunnen ontstaan tussen de geïmplanteerde dopaminergeneuronen en de interneuronen in het striatum, is het zelfs mogelijk om de afgifte vandopaminedoordegeïmplanteerdeneuronenenigszinsteregulerenenmodificeren.Herstelvan het striatale dopamine‐niveau kan leiden tot verbetering van de sturing van fijnemotoriek en tot een vermindering van de motorische problemen karakteristiek voorParkinsonpatiënten. Een belangrijke doorbraak in de toepasbaarheid van dopaminergeceltransplantatie als een therapeutische optie was de mogelijkheid om dopaminerge celimplantatenteverkrijgenvia in‐vitrodifferentiatievanhumaneembryonalestamcelleninplaats van isolatie uit het ventrale mesencephalon van geaborteerde foetussen. Debelangwekkende ontdekking van iPS cellen (induced pluripotent stem cells) door ShinyaYamanaka in 2006 bracht met zich de belofte op een onbeperkte, autologe bron vandopaminerge neuronen zonder de ethische en praktische bezwaren verbonden aan hetgebruikvanuitabortusverkregenfoetalecellenofvanembryonalestamcellen.Wat betreft het tweede doel van een Parkinsontherapie, het stopzetten van het

degeneratie‐procesindopaminergeneuronen,iserrecentelijksteedsmeeraandachtvoorde rol van ontstekingsprocessen in het ontstaan en het ontwikkelen van de ziekte vanParkinson.Hetidentificerenvanrelevanteimmuuncomponentendiedaarbijeenrolspelenkan leiden tot nieuwe therapeutische benaderingen waarmee de degeneratie vandopaminergeneuronenvertraagdofeventueelgeheelgestoptworden.Hetonderzoekbeschreveninditproefschriftbetreftmetnamestudiesnaardemogelijke

toepassingvanuitiPS‐cellenverkregendopaminergeimplantatenindebehandelingvandeziektevanParkinson.Daarnaastkomeneennieuweimmuno‐pathogenecomponentvoordeziektevanParkinsoneneenmogelijkeviralevectorvoorgentransfectievanhersencellenaandeorde.Na een inleiding over de stand van zakenwat betreft kennis over de pathogenese en

behandelingsoptiesvoordeziektevanParkinsoninhoofdstuk1,hebbenwij,indevormvaneenReviewartikel,eenoverzichtgegevenvandestudiesdiegedaanzijnmethetimplanterenvandopaminergeneuroneninhet6‐OHDAratmodelvoordeziektevanParkinson,waarbijhetgedragendeeffectenvanprimairedopaminergeneuronenvergelekenwerdenmetdieverkregenuitiPS‐cellen(hoofdstuk2).Humanefoetaledopaminergeneuronengeïsoleerduithetventralemesencephalongeldenalsdegoudenstandaardvoordepositieveeffectenvan intrastriatale cel‐implantatie. Verscheidene klinische trials hebben aangetoond datParkinsonpatiënten na transplantatie met deze cellen significant verbeterden in hunmotoriek en minder afhankelijk waren van L‐DOPA; postmortem onderzoek opParkinsonpatiënten 10 jaar na implantatie liet zien dat de getransplanteerde foetale

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dopaminergeneuronen,duidelijkherkenbaar,geïntegreerdwareninhetstriatalecircuit.Decruciale vraag is of de dopaminerge neuronen verkregen uit iPS‐cellen na intrastriataleimplantatienetzoeffectiefzijnalsdeprimairefoetaledopaminergeneuronen.Zijvertonenweliswaar dezelfde karakteristieke eigenschappen van dopaminerge neuronen, o.a.dopamine‐productieenreleaseeneenkarakteristiekspontaanvuurpatroon,enzijbrengendezelfdemarker‐eiwitten tot expressie,maar de vraag is of zij op vergelijkbarewijze detransplantatieprocedureoverleven,uitgroeienenintegrerenenstabielzijnnaimplantatie.Vergelijking van hun gedrag en hun effectiviteit met die van primaire humane foetaledopaminergeneuronennaimplantatieinhetratmodelvoordeziektevanParkinson(meteenunilateralelaesieindesubstantianigram.b.v.6‐OHDA)kaneenantwoordopdievragenbieden.InhetReview,gevenwijaandatzoweldeprimairealsdeuitiPS‐cellenafkomstigedopaminergeneuroneneenfunctioneleverbeteringgevenvanhet‘Parkinson’rotatie‐gedragvan de experimentele ratten. Het blijkt echter dat een veel groter aantal uit iPS‐cellenafkomstigedopaminergeneuronennodigisomeenzelfderotatie‐reductieteverkrijgenalsmet primaire foetale dopaminerge neuronen. Hoewel het dopaminerge karakter van degeïmplanteerde,uit iPS‐cellenafkomstigeneuronenbehoudenbleefna implantatie inhetstriatum, bleek hun uitgroei velemalen beperkter dan die van geïmplanteerde primairedopaminerge neuronen. Blijkbaar had het iPS‐reprogrammeringsproces en hetdaaropvolgende differentiatie‐proces geleid tot verstoringen in de expressie van genenbetrokkenbijdeontwikkelingenuitgroeivandedopaminergeneuronen.TeneindeverschillentussendeuitiPS‐cellengedifferentieerdedopaminergeneuronenen

primairedopaminergeneuronenvasttestellen,hebbenwij,inhoofdstuk3,hetexpressie‐profielvanbeidecelpopulatiesindetailvergeleken.DaarvoorhebbenwijtransgenePtix3‐GFP muizen gebruikt, zodat we met behulp van GFP‐FAC‐sorting zuivere dopaminergeneuronen kregen. We hebben de Ptix3‐GFP iPS‐cellen tot dopaminerge neuronengedifferentieerdmetbehulpvanalgemeengeaccepteerdeprotocollendaarvoor.Doormiddelvan de analyse van de transcriptie van, voor ventrale mesencephalon specifieke,dopaminerge marker‐eiwitten hebben wij de correcte identiteit van deze dopaminergeneuronen bevestigd. Bovendien voldeden de uit iPS‐cellen afkomstige dopaminergeneuronen aan een aantal meer algemene criteria voor dopaminerge neuronen, zoals deproductie en secretie van dopamine, het typische patroon van spontaneactiepotentiaaltreinenende reductie vanamfetamine‐geïnduceerd rotatie‐gedragnahunimplantatie in het Parkinson ratmodel met een unilaterale 6‐OHDA laesie. Vervolgenshebbenwijgenome‐widehetexpressie‐profielvandeuitiPS‐cellenverkregendopaminergeneuroneninkaartgebrachtenvergelekenmetdatvanprimairedopaminergeneuronendiegeïsoleerdwarentijdensverschillendestadiavanembryonaleenpostnataleontwikkeling.Vergelijkingmetdopaminergeneuronenuitverschillendeontwikkelingsstadia is relevantomdat de uit iPS‐cellen verkregen dopaminerge neuronen eigenlijk ook als embryonaalbeschouwdkunnenworden: immers,hetprocesvan iPS‐reprogrammering induceert eenvolledigere‐settingenverjongingvanderesulterendedopaminergeneuronenondanksderelatieflangekweekduurnoodzakelijkvoorhuncorrectedifferentiatieenmaturatie.Uitde


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uitgebreidegen‐analysesbleekdatdeexpressievanpluripotentie‐genenindeuitiPS‐cellenverkregendopaminergeneuronen,naarverwachting, vollediggeremdwas,maarookdatenkelefibroblastspecifiekegenentochnogtotexpressiekwamen.Desterkstegen‐expressiecorrelatie tussen de uit iPS‐cellen verkregen dopaminerge neuronen en de primaireembryonaledopaminergeneuronenbleekmetnameaanwezig indegenclustersspecifiekvoorventraalmesencephaledopaminergeneuronen.OpmerkelijkgenoegbleekdatdeuitiPS‐cellenverkregendopaminergeneuroneneenafwijkendeexpressievangenenhaddendiebetrokken zijn bij globale transcriptieprogramma’s coderend voor de fundamenteleontwikkelingvanhetzenuwstelsel,voordebasale,normaleontwikkelingvanneuronenennog specifieker voor de differentiatie en neuriet‐uitgroei van neuronen. Het is zeerwaarschijnlijkdatdezekleineafwijkingen ingen‐expressieverantwoordelijkzijnvoordegeringe neuriet‐uitgroei van de uit iPS‐cellen verkregen dopaminerge neuronen naintrastriatale implantatie. Een zeer uitgebreide en ver reikendeuitgroei vanneurieten iscruciaalvooreensuccesvolleimplantatievandopaminergeneuronenenvoordegunstigeeffectenopdesymptomenvandeziektevanParkinsoninpatiënten.Hetismogelijkom,aanheteindvande in‐vitrodifferentiatievan iPS‐cellen totdopaminergeneuronen,decellenzodanig (genetisch) te modificeren dat hun capaciteit tot vorming van uitgebreideneurietbomenspecifiekgestimuleerdwordt.Inhoofdstuk4,beschrijvenwijexperimentenwaarinwijde(over)expressievan2adhesie‐factoren,PSA‐NCAM(doortransfectievanhetgencoderendvoorhetPSA‐transferaseSTX)enL1CAM,specifiekinducereninuitiPS‐cellenverkregendopaminergeneuronen.Naverificatievandegeïnduceerdeover‐expressievanPSA‐NCAM en L1CAM, onderzochten wij de effecten op de neurietvorming in dezedopaminerge neuronen. Het bleek dat de over‐expressie van beide adhesie‐moleculenresulteerde in een significante toename in de uitgroei en complexe morfologie van deneurietbomenindedopaminergeneuronen,tenminstealszegekweektwerdenopeenlaagvanastrocyten.Echter,alsdePSA‐NCAM/L1CAM‐getransfecteerdeuitiPS‐cellenverkregendopaminergeneuronenuitgeplaatwerdenopslicesvanpostnataalmuizenstriatumkongeenenkeleffectmeeropneuriet‐uitgroeigedetecteerdworden;slechtseenopvallendpatroonvanparallel‐uitgroeiendeneurietenwaszichtbaarduidendopeenspecifiekeinteractiemethetsubstraat.OoknaimplantatievandePSA‐NCAM/L1CAM‐getransfecteerde,uitiPS‐cellenverkregendopaminergeneuroneninhetstriatumvanrattenmeteen6‐OHDAlaesieindesubstantienigra,wasdeneuriet‐uitgroeinietopvallendgestimuleerdenvergelijkbaarmetde uitgroei op de striatale slices in‐vitro. Daar kwam bij dat het aantal overlevende,geïmplanteerdedopaminergeneuronenuitermategeringwas,waarschijnlijktoeteschrijvenaandesterkverzwakteconditievandecellennadelangetraumatischeproceduresvanviraletransfectie en van (Pitx3‐GFP) FAC‐sorting. Het is duidelijk dat voor een succesvollegenetische modificatie van de uit iPS‐cellen verkregen dopaminerge neuronen, detransfectie/transductie‐methodiekengeoptimaliseerdmoetenworden.Het is vancruciaalbelangom inzicht tekrijgen inde factorendieeen rol spelen inde

degeneratie en sterfte van dopaminerge neuronen in het brein van Parkinsonpatiënten;immers deze factoren kunnen uiteindelijk ook de nieuwe geïmplanteerde dopaminerge

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neuronenweergaanaantasten.Een interessantenieuwe factor isperforin,eenuitermatekrachtigecytotoxischecomponentvanhetimmuunsysteem:perforinkanporesvormeninde celmembraan en granzymes uitstoten, die direct leiden tot cellysis of apoptosis. Inhoofdstuk5latenweziendatintoxicatievanmuizenmetMPTP,eenveelgebruiktemethodeominmuizenspecifiekedegeneratievandopaminergeneuronenen‘Parkinson‐pathologie’natebootsen,resulteerdeineentoenameinhetserumniveauvanperforin.Metbehulpvantransgeneperforin‐knockout(Pfp‐/‐)muizenhebbenwederolvanperforininde(MPTP‐geïnduceerde) pathogenese van de ziekte van Parkinson onderzocht.Wij vonden dat deacute effecten van MPTP op het dopamine‐niveau in het striatum van Pfp‐/‐ muizensignificant minder waren dan in dewild‐type muizen; ook was het aantal dopaminergeneuronenindesubstantianigranaMPTP‐behandeling,inschrilletegenstellingtotdewild‐typemuizen,nietsignificantgereduceerdindePfp‐/‐muizen.Daarbijbleekdatdemicroglia‐activatie inhetstriatumvanMPTP‐behandeldePfp‐/‐muizenveelminderwasdanindeMPTP‐behandeldewild‐typemuizen. Onze studies laten voor het eerst zien dat perforin‐deficiëntie, MPTP‐geïnduceerde toxiciteit aan het nigrostriatale systeem voorkomt. DezeresultatensuggererendatperforineenrolspeeltindedirecteofindirectebeschadigingvandopaminergeneuronentijdensdepathogenesevandeziektevanParkinson.Perforinwordtmet name geproduceerd in specifieke cytotoxische lymfocytensubpopulaties waarvan isaangetoonddatzeParkinsonherseneninfiltrerenenhetisdanookwaarschijnlijkdatdezecellenbetrokkenzijnbijdeperforin‐effectenopdedopaminergeneuronen.Jammergenoegzijn we tot nu toe niet in staat geweest om de aanwezigheid van perforin‐positievelymfocytenindesubstantianigravanMPTP‐behandeldemuizenonomstotelijkaantetonen.Daarnaastisinandereaandoeningenaangetoonddatperforindebloed‐hersen‐barrièrekanaantastenenwellichtspeeltperforinookeenindirecterolinParkinsonpathogenesedoordathetzohetbinnendringenvancytotoxische lymfocyten faciliteert.Het lijktzeerdemoeitewaardomdetherapeutischemogelijkhedenvan(anti‐)perforinvoordeziektevanParkinsonverderteonderzoeken.Modificatievandemicro‐omgevingvanspecifiekecellendoormiddelvandegeforceerde

expressievanrelevanteextracellulairematrixcomponentenkaninflammatiereducerenenneuroprotectieenneuroregeneratiestimuleren.Methetoogopdezetoepassing,hebbenwein hoofdstuk 6 onderzocht wat de meest geschikte manier is om bepaalde genen totexpressietebrengenineenbeschadigderegiovanhetcentraalzenuwstelsel.Eenprobleemdaarbij is dat door glia‐littekenvorming en necrose een rigide barrière is ontstaan dieverspreiding van virale transfectievectoren belemmert. Een geschikt experimenteeldiermodel om de afgifte van genen via transfectie in de vijandige omgeving van hetbeschadigde zenuwstelsel te bestuderen, is het muismodel met een gestandaardiseerdegeïnduceerde ruggenmerg‐beschadiging. Het adeno‐associated virus (AAV) is veelvuldiggetestinklinischetrials,maardeefficiëntievanverschillendeserotypesiszeervariabelenweefselspecifiek. Met GFP als reportergen, hebben wij de efficiëntie van verschillendeserotypesvanAAV’s (d.w.z.verschillendecapsidesmethetzelfdegenoom)onderzocht inmuizenmet een beschadigd ruggenmerg. In vergelijkingmet het in de kliniek gebruikte


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AAV2,bleekhetAAV5zichveelverderteverspreidenvandeinjectie‐plekinhetbeschadigderuggenmerg.Meer cellenwaren getransduceerd door AAV5 dan door AAV2 en de totaletransgen‐expressiewasveelhogerinhetAAV5‐getransduceerderuggenmerg.

CONCLUSIES

GeziendeaardvandeziektevanParkinsonzoueenoptimaletherapiemoetenbestaanuiteen combinatie van dopaminerge neuronvervanging en een blokkering van het verdereverliesvannogresterendedopaminergeneuronen.Met de ontdekking van iPS‐cellen in 2006, is er een onuitputtelijke bron van autologe

dopaminergeneuronenvoorceltransplantatiebeschikbaargekomen.Meteenaanzienlijketoenameinhetbegripvanonderliggendereprogrammeringsmechanismes,zijn inrecentejarentalvanverbeteringenindeiPS‐protocollendoorgevoerd,metnamemethetoogopeenveiligeklinischetoepassing,die indenabije toekomstonafwendbaar lijkt.Onsonderzoeklaat zien dat de in‐vitro processen van iPS‐reprogrammering en daaropvolgendedifferentiatie leidt tot kleine afwijkingen in het (epi)genoom van de resulterendedopaminergeneuronendiemetnameconsequentieshebbenvoorhunneuriet‐uitgroeinatransplantatie;verdereoptimalisatievanhetreprogrammeringsprotocolentoepassingvanadditionelegenetischemodulatiezouhierineenverbeteringkunnenbrengen.Wat betreft het stoppen of beperken van het verdergaande verlies van dopaminerge

neuronenbijParkinsonpatiënten,kanimmuunmodificatie‐therapieindetoekomstwellichtnieuwe mogelijkheden bieden, b.v. door schadelijke lymfocyten uit te schakelen en/ofprotectieve lymfocytenselectieftestimuleren.Zobleekpassieveimmunisatievanmuizenmetanti‐‐synucleineantilichamendegradatievan‐synucleine‐aggregatentebevorderenendopaminergeneuronenteredden.DetransfervanT‐lymfocyten,geïsoleerduiteenmuisdie behandeld was met copaxone, bleek neuroprotectief in MPTP‐behandelde muizen.CopaxoneisinstaatcytotoxischeT‐lymfocytenteveranderenineenmeeranti‐inflammatoirphenotype.Ook de transfer van een specifiekeT‐lymfocytsubpopulatie, de regulatoireT‐cellenofTregs,leiddetoteenverminderdeneurodegeneratieinMPTP‐muizenwaarschijnlijkdoordatzedeoverreactiviteitvanT‐lymfocytenonderdrukken.

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Dearall,finallyitcomestomyfavoritepartofeverythesis,theacknowledgments!Ifthisisthefirstpartofthisthesisyouread,don’ttakemewrong,Ihaveseriousworkinthepreviouschapters.Themainpurposeofthisbookistorecordthefindingsfromthebenchwork,andtherelevantscientificthinkingduringmyPhDlife,whichhasbeenleadingmetowardsthePhDtitlebygivingvariousquestionsandproblems.LookingbackatmyPhDtime,Iseeyouguys,itwasyouwhohelpedmetofindtheanswerstothequestionsandgivemethestrengthtodealwiththeproblems.ItwasyouwhomademyPhDlifeenjoyable.

IwouldliketothankmysupervisorsProf.ErikBoddeke,Prof.MelittaSchachner,Dr.SjefCoprayandProf.Yan‐QinShen.Iamreallygratefulthatyoutookmeintoyourgroupsand gave me the chance to work on the cutting‐edge projects. As a master student inMicrobiologywhowasveryinterestedinNeuroscience,IwassoluckytogettheofferfromMelittaandShen,whilenothavingtherelevantbackground.ThankyouforopeningthedoortothefieldofNeuroscienceforme.Iwasluckyagaintwoyearslater,whenErikandSjefopenedanotherwindowforme,guesswhat,thistimewasiPScells!Andmostimportantly(joking)inthisnewplaceIworked,Google,Facebook,YouTube,Dropbox…workperfectly.

SjefandShen,youarebothgreatsupervisors,alwayskindandpatient!You listenedtotroublesanduncertaintynomatteritwasrelatedtoworkornot.DearShen,intheearlydaysofmy PhD study, you not only shared your experience of being a PhD in Bordeaux andpostdocinMichigan,butalsoyourcommunicationswithotherscientistsandreviewers.Eventillnow,I’mstill learningalot fromyourexperienceofbeingadeputydean.Andbesidestheseseriousandbusyduties,youareawonderfulmothertoalovelygirlandpuppy.DearSjef,thankyousomuchforallthehelpwiththeanimalexperiments!AllthoselongdaysinCDP,evenafterIleftGroningen,youwerestilltakingcareofmyanimals.IremembertherewasatimewhenwewereworkingtogetherintheCDPandyoutoldmethatyoursonfinallybecamea‘pirate’.Itsoundedsocooltome,becauseIalwayswantedtobeapiratewhenIwasachild.Butitturnedouttobe‘pilot’.Ithinkthiswasasignalreadyofthedifficultythatwewouldfacewhenwritingthisthesis.Withoutyou,itcouldnothavebeenfinishedinaniceway!

ErikandMelitta,itwasagreatexperiencetoworkwithyou!Iwassoimpressedbybothofyourschedules.DearMelitta,wheneveryouarrivedinShantou,thefirstplaceyouwouldgo is theoffice, evenaftera long flight fromHamburgorRutgers.Thedaysyou spent inShantoualwaysstartedwithsaying‘Hi’inthemorningandsaying‘Goodnight’lateintheevening to the students who were working in the lab. You are the best example of ahardworkingperson.Idobelievethatthehardmomentswilleventuallyshowtheirvalue.Thanksfortellingmethat‘stillwaterrunsdeep’.AndIlikeyourstoriesabout‘mushrooms’and‘candles’atourEnglishhour.DearErik,Iappreciatethemeetingsanddiscussionsthatwehad,theyaresoimportantformakingtheappropriatechangesinstrategiestokeepmyprojectsontherighttrack.ThankyousomuchforaskingmetobeyourChineselanguageteacherwhenIfirstarrivedinGroningen.Ialwaysthinkthatitwasyourtricktomakemefeelmoreathomeabroad,andtoteachmehowtocorrectmysupervisor(Chinesestudent

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tendtosay‘yes’moreoften).Butyoureallyhaveatalentforlanguages;Ibelievewhenyoufinallyhavetimeoneday,youwillbeabletomastertheChineselanguageveryfast.

IwouldliketoacknowledgethemembersofthereadingcommitteeProf.P.P.deDeyn,Prof.E.M.HolandProf.L.A.’tHartforreadingandapprovingmythesis.

GreetingstothenewPIsinthedepartment:Prof.JonLamanandDr.ArmaganKocer.Goodluckwithyourcareer,hopeyouenjoyworkingintheMedFys.

Iwould like to thank the collaborators,Prof.SebastianKügler from theUniversityofGöttingen forprovidingus theAAVvectors; andProf.Andrey Irintchev fromFriedrich‐Schiller‐UniversityJenaforhelpingmetosetupthespinalcordinjurymodel.

Being a foreign student who knows little Dutch, it would be impossible to deal withadministrativeworkoftheuniversityandthecity.SpecialthankstoGerryforhelpingmeoutwithfillingintaxformsandmanyotherforms.ThankyouHarryandHenkforyourhelpwithmyUMCGguestaccount.ThankstoDianaofBCNgraduateschoolforarrangingsomanyactivities,andhelpingtokeeptrackofmyPhD.ManythankstoProf.GuofSUMC;Prof.Huo,DrZhangandZhengMinofthegraduateschoolofSUMC.Thankyouforyourworkonthejoint‐trainingPhDprogramofUMCG‐SUMC.

IwouldliketoexpressmygratitudetotheFACSfacilityteam:Geert,Henk,RoelofJanforthe long sorting and jokes; and also the animal facility staff, thanks to Catriene forsupervisingtheproject,Natascha,fortakingcareofmyanimals;MichelandAnnemiekefortechnicalsupport;Juulforgivingmethesurplusmice.

ManymanythankstoallthecurrentandformermembersoftheCenterforNeuroscience,SUMC.Dr.Zhao, itwasalotoffuntoworkwithyou!Youwerealwayscheerful,eventhecomplainssoundedcheerful.Yourdedicationtosciencewasagreatinspirationformeatmydownmoments.Dr.Yu,thanksforsharingyourexperienceasaPhDinJapan.XuanjunandPeizhi,itwasnoteasytotakecareofeverythingandeveryoneinthelabasasecretaryandatthesametimetakecareofthemiceintheanimalcenter.Butyoubothdidawonderfuljob!Hongchao, thezebrafishguy,wegot recruitedat thesame time.Wehavequitedifferentpersonalitiesandviewsonrunningalab,butthatwasneveraproblemforustobefriends.ZhangHao,Jinfei,Zhikui,WangYanandYongjian,Ithinkyouwillagreewithmethatitwas great fun to work in a newly established lab. We made our own rules and workregulations,weenjoyedthefreeatmosphereatjournalclubs,workmeetingsandshareouropinionsoneveryissuerelatedtoworkandthelab.AndIreallyappreciateyourhelpwithmyanimalexperiments.ZhangHao,youbecomeaclosefriendalltheseyears.Italwaysfeelsgreatnomatterwhatwedotogether,yoga,fitness,movies,travelling…Itissonicethatwecantalkabouteverythinginourlife,andbenefitfromthetalks.Jinfei,goodluckwithyourcompany,buttakesometimetothinkaboutotheraspectsoflife,notjustwork.Zhikui,youwerealwayswillingtohelpeveryonearoundyou.WishyouandXuanjunlotsofhappiness.WangYan,youwereaguyoffewwords.Buteveryconversationwithyouwasaninterestingone.Yongjian,nicethatyouwentbacktothemedicalworkthatyoulike.Youarethefirstoneamongustostartafamily,wishyougoodluckandlotsofhappiness.FangPing,XieLin,

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Qinghui,Danyang,itwasreallynicethatyoujoinedthelab.Youbroughtmorediversitytothe labandalsoverynicecompanyforbasketball,daytrips,BBQ,hotpotandtryingnicerestaurants in Shantou.FangPing, nice thatwe still keep in touch, good luckwith yourresearchinMunich.ZhangYe,YuYang,LiuYang,Tianli,andLiuDan,youcametothelabwhenIwasalmostleavingforGroningen.ItisnicethatwestillkeepintouchthroughourNSCWechatgroup.ZhangYe,thanksalotfortakingovermyprojectafterIleftShantou,Iappreciate your contribution. And best wishes to the new members at the Center forNeuroscience,Prof.Yang,Dr.Wei,Grace,Zhihua,Chunjie,Houde,andmanyothernewstudents,goodluckwithyourwork!GreetingstothecolleaguesinJiangnanUniversity.Ihadagreattimethere!Dr.Cui,thanksforthediscussiononiPSCsandyourhelpwithmycoverdesign.Dr.ZhaoandDr.Sun,nicethatIcanalwaysgettheChinesemedicalhelpfromyouwhenIhaveanallergyoracold.DrChu,thankyouforyourhelpduringmystayinJiangnanUniversity.GoodlucktotheresearchassistantsYaoLiandYuhong; themasterstudents,Shixiao,Shubin,LinfangandYida,hopeyourprojectswillgowell.

ManymanythankstothecurrentandformermembersofMedFys.,UMCG.Trix,Icannotimagine how the 8th floor would be without you. You are always cheerful and work soefficiently.Ihavebeenwonderinghowyoucanhandleeverythingwithin3daysperweek.ThatanswermayhavehelpedmetosavehalfthetimeofmyPhD.Thanksforsharingtheofficewithusandmakingitaverycozyplacewithcoffee,teaandcandies.Bart,thankyouforhelpingmeoutwiththecloningproblem.AndIenjoyedthefuntalksduringthecoffeebreaksandtheBerlintrip.Evelyn,thanksalotforyourhelpwithcloningandq‐PCR.Ireallylikedtoplaybadmintonwithyouatthecompanycompetition.Michel,thanksforshowingmehowtodoconfocalandperformingtheelectrophysiologyforme.Ietje,itwasgreatthatyouwerealwaystherewhenIneededhelpwithslicingandstaining.Loes,Iwassoimpressedbythewayyouorganizedthecellculturelabs,everythingweneededwaswithinreach,onlysometimesthelazy(tired,stressed)PhDstudents(alsome)forgottodotheirtasks.Nieske,thanksforalwaysremindingandhelpingmetodotheregistrationfortheanimalsontime.Youwereright,itsavestimetodoitwhenitisstillafreshmemory.Tjalling,thankyouverymuchforfixingtherota‐8system,theopticalmicroscope,andalsomybike.Rob,Inge,andWieb, it is nice to have you at theworkmeetings and sectionmeetings, your questionsalwaysmademetothinkaboutmyresearchinabroaderview.Kumar,Ming‐San,Zhilin,Thaiany,Koen,Javier,Chaitali,mydear(former)officemates,itwasreallynicetosharetheofficewithyou!Imissthetalkswithyouguysaboutyourhometowns,yourtraditionalculture, and our language lessons.Kumar, thanks a lot for helping me with the animalexperiments;alsothekindwordswhenIwassodown.Ming‐San,Istillhavethelistofnamesofpeopleinthelabthatyoumadeformeonmyfirstdayinthelab.Thatreallyhelped,itwasmyfirsttimeabroad,verydifficulttorememberthefacesandnames.Zhilin,youwerethe‘present’thatErikbroughtformefromChina!Itmademefeelathome,becauseIcouldfinallytalkinChinese.Wehaveverydifferentpersonalities,butIappreciateseeingtheworldfromyourangle.Thaiany,youarethesweetestone!Missyoualot!Iloveour‘girls’talk,andthankyouforthemake‐upanddancinglessons.Koen,thanksforthecandiesthatyousharedwith

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us.ItwasalsoalotoffuntogotoanArnoldSchwarzneggermoviewhenyouandWandertwerethere.JavierandChaitali,thankyouformakingitcomfortableandeasyintheofficewhenIwassonervouslypreparingforthedefense.

Reinhard, I cannever thankyouenough for teachingmethe iPSCreprogramminganddifferentiationstepbystep!Iloveyourjoke:thatthefirstDutchwordyoulearnedwhenyoucametotheNetherlandswas‘BIER’.Iwillneverforgetitbecauseitwastoldsomanytimes;)Itwas very ‘nice’ of you andRomy not to stopme fromordering the Irish coffee out ofcuriosity,IhavenoideahowIgothomethatnight.Marcin,therewasalwaysthecomfortableandfunnyatmospherewhenyouwereinthelab.Thankyouforhelpingmewithanimalwork.Weweretheonlycelltransplantationteaminthelabatthattime.IthinkwewerealsoagoodteaminbasketballwithLasse.GoodlucktoyouandKasiawithyourchild,careerandstudyinPoland.Arun,itwassonicethatwecoulddoalotofsportstogether,basketball,squash,badminton with the Chinese group, Dutch group, company competition... Your squashscheduleisimpressive!Duco,thanksforcalling‘lunch’at11:50everyday;)Divya,thankyouforbeingniceandwarm.TheIndiandinnersandChinesedinnerswedidtogetherweregreat!Wecouldhavespentmoretimetogether if IwouldhavecomebacktoGroningenearlier.Zhuoran,thankyouforbeingmyparanymph!Imisstheexcitingboardgameevenings.WishyouandKaitao lotsofhappiness.Inge,youaresuchanicepianist,yourmusicbringssomuchfunatourparties.Ialsoenjoytalkingwithyou!Yourwayoftalkingaboutthesubtlepolitical issues of Chinawere very interesting. That helpedme to better understand the‘western’opinionaboutChinaandtotakearelaxedattitudewhentalkingaboutsubtleissues.Wandert,thankyoufortellingmethatitispossibletoswitchthemindfromasadmoodtoahappymood,youjustneedtorememberthefeelingwhenyouarehappy.Thais,Corien,andRianne,yourofficeisthecenteroffuninthelab.Theteabreakstherewerealotoffun.Ithelpedmetoworkefficientlyinthelateafternoon.Xiaoming,yourvegetablecuttingskillsareimpressive.IloveallthedishesXuPingandyoumadeforusinthe2014springfestivaldinner.Sabrina,yourshiningsmileandlaughwarmuptheatmosphereinthelab.Ria,youarethemostprofessionalbasketballplayerinourgroup,itwasapitythatyoudidnotcomeoftentoplaywithus.Goodluckwithyourdefense!IthinkIwillgetprettynervouswhenyouaredefending,becausemineisshortlyafteryours.Vishnu,relaxandcheerup!HopeyouaredoingwellinIndia,Goodluck!Sham,wemetwhenyoucamebackforashortstayforyourdefense.ThatwasthefirstRUGPhDdefenseIhaveeverbeento,muchmorefascinatingthantheChinesedefenses.GoodluckintheUSA!Estherfromthegeneticsdepartment,itwasfuntolearniPSCworktogetherwithyou.Khayum, itwasalotoffunandlaughterwhenyouwereplayingbasketballwithus,andsometimeabitoftearswhenIdidnotmanagetoescapefromyourmovements.Greetingtothenewcolleagues,Susanne,Hilmar,Clarissa,Alain,ClaudioandHiske,thelabdaythisyearwasfun!

Manythankstothe(former)masterstudents,LauraandSilvia,howareyouinSpain?Missyoualot!Thedinners,partieswehadtogetherweregreat!ItwasonlywithyoucrazygirlsthatIdrankalcohol.Can’tbelieveyoumademedrinktequila,andthesangriayoumadewasfantástico.Marissa,Anna,Chiwan,Michelle,Christina,Katja,Lasse,Elisa,Marisela,and

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Ossian,althoughwedidn’tworkmuchtogether,itwasfuntoknowyou,wishyouallthebestwithyourcareer.

Iwouldliketothankmyperfectformerflat‐mates,YangJing,CuifengandEdyta.Itwasgreattolivewithyou.WeallworkinUMCG,sothatweperfectlyunderstoodeachother’scomplains.Thankyouallforbeingsosupportive.ThankstoAiHui,Yixian,Xiaolei,QinJing,Liwen,WangQi,forthegirls’nights,shopping,dancing,SushiMalltimeandboardgames!ThankstomydearfriendsfromShantou,WuRui,Xueting,ZengXiang,Junjun,nicetoworkwithyoutogetherinthejoint‐trainingPhDprogramofUMCG‐SUMC,itwasimportanttohaveyoursupportduringthedifficulttimes.Manythankstothehouse‐matesinBlekerslaan4.Missyou,Yana,Daniela,Luke,Uli,andJamie.

IwouldliketothankmyfriendsatBCGO!,Ingemarie,Gab,Stefan,Mark,Arno&Ada,ZhangLu,Rudolf,Kristborg,Wart,Garrelt,Sander;andintheChinesebadmintonteam,Zetao,Xiaobao,SunZe,HeTao,DingNing,Qibing,Kexin,Shuhai,Chengyong…,Ilovetoplaybadmintonwithyou,andIenjoyedthecompetitionsthatweplaytogetherandalsotheotheractivitieslikegoingoutandhotpot.

ManythankstothefriendsofSanguoshagroup,CaoQi,Hanbing,JiangYi&ZhaoTing,LiJun,LiMing…EverySanguoshaeveningwasgreat,especiallywehaddumplingandhotpotsectionsnexttoit.

IwouldliketothankShu,Liufei,Zhenran.Itwasnicetotravelwithyouguys.Youareallwonderfulguides!Withthelocalhistory,mapsandtrainschedulesreadyinyourmind.Inez&Rob,theTangocoursewasawesome.

感谢一起长大，一路相伴的老朋友们，叶婵，旭曼，丽敏，妙纯，楚姗，楚伟，锦素；

还有袁姐，晓玲姐，谦豫，谦豫爸爸，写论文的日子里有你们的陪伴特别幸福！

IwouldliketothankIlia,thiscouldnothavehappenedwithoutyou!Thankyouforbeingso supportive and patient duringmy seemingly endlessmedium changes, CDP, confocalwork…andthesiswriting.Thankyouforyourhelpwithq‐PCRdataanalysis,graphmaking,cover design, and being my paranymph for the defense. It was also great fun to playbadminton,basketball,boardgames,gotomovies,tangocourseswithyouanddinnerswithyour family. Thank youLiia,Rita andDavid for the nice dinners, help and constructivesuggestionsforthethesis.

Finally,Iwouldliketothankmyfamily.我特别感谢爸爸妈妈这么多年来的支持和谅解。

从初中开始我就是个贪玩不着家的小孩，直到最近才发现你们一直默默期盼着我回家，才

知道什么是‘在家千日好’。往后的日子，希望有更多的时间视频，回家，带你们出去玩。

谢谢弟弟，阿绵，多多，妹妹在我离家的日子里照顾着我们的家。

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University of Groningen iPS cell therapy for Parkinson’s disease … · 2016-03-09 · Oxidative stress is considered to play an important role in the specific loss of DA neurons