CurrentStateofStemCellTreatmentsforCerebralPalsy:AGuideforPatients,Families,andServiceProviders

StephanieBeldickMichaelG.Fehlings

KrembilResearchInstitute,UniversityHealthNetworkInstituteofMedicalSciences,UniversityofToronto

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GlossaryPerinatal–Aroundthetimeofbirth

Centralnervoussystem—Thebrainandspinalcord

Neurons–Cellsinthebrainthatsendsignalsinordertocommunicatewithotherregionsofthebrainandthebody.

Axons–Longprocessesattachedtoneuronsthatrelayelectricalsignals.Analogoustowiresinanelectricalcircuit.

Oligodendrocytes–Producemyelin,afatthatinsulatesneuronalaxonssothatneuronscansendsignalsquickly.

Whitemattertract–Abundleofaxonsconnectingtwodifferentregionsofthecentralnervoussystem.

Regenerativetherapy—Atherapythatseekstorestoretheintegrityofdamagedtissue.Regenerativetherapiesusingstemcellsoftenseektoreplacedeadcellsandsupporttheremainingcells.

Differentiate—Theabilitytotransformintospecializedcelltypesfoundinthebody.Oncedifferentiated,thecellscannotgobacktotheirpreviousstate.

Pluripotent–Atermusedtodescribecertainstemcellsthatcandifferentiateintoallthedifferentcelltypesfoundinthehumanbody.

Somaticcell–Afullydifferentiatedcellfoundinthehumanbody(forexample,askincell).

Directlyreprogrammedcell–Asomaticcellthathasbeendirectlytransformedintothedesiredcelltype.Thiscellhasby-passedthepluripotentstatethatisrequiredwhenmakingiPSC-derivedcells(seesectiononinducedpluripotentstemcells).

Multipotent—Stemcellsthatcanonlydifferentiateintospecificcelltypes.Forexample,aneuralprecursorcellcanonlydifferentiateintothecelltypesfoundinthebrainandspinalcordandnotintocelltypesfoundintheskin.

Allogeneicstemcelltransplantation–Stemcellsthataretakenfromadonorandgiventoapatient.Posesahighriskofimmunerejection.

Autologousstemcelltransplantation–Stemcellsthataretakenfromapatientandafterculturing,aretransplantedbackintothepatient.Minimalriskofimmunerejection,butthepreparationofthecellstakesalongtime.

Endogenous–Residingwithinatissue.Thisisopposedto“exogenous”,whichmeansfrom“outside”thetissue.

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StemCellsandCerebralPalsy

Cerebralpalsy(CP)isanumbrellatermusedtodescribeadisorderthatresultsfromperinatalbraininjury.CPcanbecausedbyamultitudeoffactors,includingprematurebirth,infectionintheuterus,lackofnutritionalsupportduringdevelopment,lackofoxygenatthetimeofbirth,andgeneticaberrations.WhilethecauseofCPismultifactorial,theinjuryconsistentlyleadstovariousneuro-motordeficits,oftenaccompaniedbyothersymptoms,suchasvisualandcognitiveimpairments.

Whenaninsultoccurstothebrainduringthesensitiveperinatalperiod,theresidentbraincellsareunabletopromotepropergrowthanddevelopmentofthebrain.Neuronsandoligodendrocytesdieand/orfailtomature,andthewhitemattertractsthatconnectvariousbrainregionsbecomedamaged.Importantly,thecorticospinaltract(CST),whichconnectsthemotorregionsofthebraintothespinalcordandhelpscontrolmovement,isoftendamaged.WithoutfunctionalCSTconnections,motordeficitsensue.

Stemcelltransplantationisaregenerativetherapythathasthepotentialtoreplacethedamagedandnon-functionalcellsinthebrainsofCPpatients,aswellastoprovidesupporttotheremainingneuronsandoligodendrocytes.Therearemanykindsofstemcells,eachwithdifferentanduniquecharacteristics.

Asresearchhasadvanced,wehavediscoveredthatstemcellscanbeinducedtobecomemorespecializedcelltypes,andwhentransplantedintothebody,theycanprovidesupporttoadamagedenvironment.Inaddition,wecanmodifythesestemcellstoexpresscertainfactorsthatcanenhancethisability.Wecanalsomodifythecellstoexpressscar-degradingfactorsthatcanhelptoreducescarringinthebrainandpromoterecovery.

ThereisaplethoraofresearchinthefieldofstemcelltransplantationforCP,andthiswillbediscussedbelowinthecontextofbothpreclinicalanimalresearchandclinicaltrials.

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WhatAreStemCells?

Therearetwocharacteristicsthatmakestemcellsuniquefromothercellsinthebody.First,theyhavetheabilitytodivideandmakecopiesofthemselvesoverextendedperiodsoftime.Second,theycandifferentiateintomorespecifiedfunctionalcelltypes.Thismeanstheycantransformintospecializedcelltypesofthebodysuchasheart,lung,orbraincells.Thetwocharacteristicsoflong-termcelldivisionanddifferentiationcapacityhavesomeveryimportantimplicationsforregenerativetherapeuticstrategies.Theabilityofstemcellstocreatecopiesofthemselvesoverlongperiodsmeansthattheirsupplyistheoreticallylimitless;underoptimalconditions,astemcellcanbegrowninaculturedishandendupformingcoloniesofstemcells.Aftergeneratingalargepoolofstemcells,theycanbedirectedtotransformintomorespecializedcelltypes,assumingthescientistknowsthecorrectfactorsthatwillleadtothedesiredchange.Below,wewilldiscusssomeofthemostimportantstemcelltypesrelevanttothetreatmentofCP,aswellastheirprosandconsforuseinresearchandtheclinic.

Embryonicstemcells(ESCs)arefoundinthedevelopingembryo.Thesecellsareknownaspluripotent,meaningtheyhavethepotentialtodifferentiateintoallthecelltypesfoundinthehumanbody.ESCshavebeenstudiedextensivelyinordertounderstandthemolecularpathwaysthatregulatetheir“stemcell-ness”,alongwiththosepathwaysthatguidedifferentiationtomorespecializedcelltypes(forexample,howdoesoneESCbecomeaneuronandanotheraskincell?).ESCshaveuniquepotentialforrepairingdamagedtissuebecauseof

theirversatiledifferentiationcapacity.ThismeansthatESCscouldbeusedtotreatavastnumberofdisorders.However,scientistsarestillworkingtopiecetogetherthecorrectbiologicalcodesthatcanfullydifferentiateanESC

intothedesiredcelltype.AconsequenceofnotyetunderstandingthefullmolecularpictureisthatESC-derivedcellscansometimesrevertbackintopluripotentcellsandformtumours.AnotherdrawbackisthatthederivationofESCsrequirestheuseofcellsfromtheembryo,thusraisingmoralandethicaldilemmas.

In2008,ProfessorShinyaYamanakawontheNobelPrizefordiscoveringfourbiologicalfactorsthatcouldbeusedtoturnanysomatic(fullydifferentiated)cellintoapluripotentstemcell.Thesenew,ESC-likecellsweretermedinducedpluripotentstemcells(iPSCs).Followingthisdiscovery,therewasagreatexcitementinthescientificcommunity.Beingabletocreatepluripotentstemcellsessentiallyeliminatestheneedforembryonictissueinordertoobtainpluripotentstemcells.Thiswasnottheendoftheexcitementthough;Normally,ifapatientis

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toreceiveastemcelltransplantation,adonorforthesecellsisrequired(allogeneictransplantation).iPSCs,however,canbederivedfromthepatient’sownsomaticcells.First,abiopsyofsomaticcells(e.g.skincells)wouldbetaken;thecellswouldthenbeinducedintoiPSCsinaculturedishandallowedtodivide.Followingdivision,theiPSCscouldbedifferentiatedintoanycelltypeandthenplacedbackintothepatient(autologoustransplantation)Notneedingadonortoacquirethecellssignificantlyreducestheriskofrejectionfollowingtransplantationintothepatient.Despitetheenthusiasmsurroundingthesecells,iPSC-derivedcellsstillpresentariskoftumourformation,similartoESCs.Furtherresearchisneededtobetterunderstandthemolecularpathwaysunderlying“stemcell-ness”anddifferentiationprocessessothatthesafetyandefficacyofthesecellscanbeoptimizedforregenerativetherapies.Interestingly,inrecentyears,newtechnologyhasallowedfortheinvestigationofdirectlyreprogrammedcells.Inthiscase,somaticcellscanbedirectlytransformedintothedesiredcelltype,essentiallyby-passingthepluripotentstagethatisrequiredwhenusingiPSCtechnology.Onebenefitofthesedirectlyreprogrammedcellsisthereducedriskfortumourformation;however,furtherresearchisneededinordertofully

understandhowwecangeneratethesecellsinasafeandefficientway.

WhileESCsandiPSCsareexcellentsourcesforversatilecells,theyarenotconvenientfortransplantationontheirown(duetotumourformation),andsotheyarenearlyalwaysinducedtodifferentiateintomorespecializedcelltypesbeforetransplantation.

Neuralprecursorcells(NPCs)arestemcellsthatarefoundinhumanswithinthecentralnervoussystem(CNS).Thesestemcellsaremoredifferentiatedthanpluripotentstemcells,inthattheycanonlydifferentiateintothecellsthatarefoundintheCNS.Accordingly,theyarecalledmultipotentstemcells.NPCscanbetakenfromadonororthepatient,growninaculturedishandthentransplantedintothepatient.NPCshaveauniquebenefitoverothercelltypestotreatCP;theirabilitytodifferentiateintobraincellsprovidesthemwiththegreatestpotentialtoappropriatelyintegrateintotheinjuredbrainandreplacethedamagedandnon-functionalcells.However,thedrawbacktousingNPCsisthatthereisalimitedsupplyofthesecells,andwhiletheycanbegrowninculture,themethodofretrievalfromthebrainorspinalcordisveryinvasive.Inaddition,thesecellsareincrediblycomplexandthereislimitedresearchontheirusefortreatingCP.Conversely,somebenefitsinclude:noethicaldilemmaswiththeirderivation,theycanbetakenfromthepatientsotheriskofrejectionisminimizedupontransplantation,andthetimerequiredtoculturethecellsisshorterthanforpluripotentstemcells,sincetheyarealreadyinthedesiredformat.

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Mesenchymalstromalcells(MSCs)areanotherkindofstemcellthathavebeenusedinresearchsurroundingCP.Thesecellshavetwomainorigins,eitherfromthebonemarrow(BM-MSCs)orfromtheumbilicalcordblood(UC-MSCs).LikeNPCs,MSCsaremultipotent,andtheyhavethepotentialtodifferentiateintovariouscelltypesthatplaystructuralandimmunerolesinthebody.MSCshavebeenusedinresearchandtheclinicfordecades.Theirbenefitslieintheireasyaccessibilityandtheirimmune-modulationproperties.However,MSCslackthefullpotentialtofullydifferentiateintofunctionalcellsoftheCNS,andforthisreason,theyareunlikelytobethemajorfocusoffuturemultipotentstemcellresearchforCP.

SourcesofStemCellsforTransplantation

Whendiscussingtransplantationstrategies,allstemcells,regardlessoftheirtype,caneitherbeclassifiedasallogeneicorautologous.Allogeneiccellsarethosetakenfromadonorandgiventoapatient,whereasautologouscellsarederivedfromthepatient.Allogeneicstemcellshavebenefitsinthatlargestemcellbankscanbecreatedthatcanfreezeandstorestemcelldonationsforextendedperiodsoftime.Whenneeded,thecellscanbethawedandgiventothepatientinashortamountoftime.Adownsidetoallogeneiccells,however,isthatthereisahighriskofimmunerejectionupontransplantation,sincethecellscomefromadonor.Conversely,autologousstemcellsposeminimalriskforimmunerejection,sincetheycomefromthepatient.However,theprocessforextractingthecells,andculturingthemcanbelengthy,andifcellsareneededwithinashorttimeframe,thismethodisinconvenient.

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PreclinicalStemCellResearch

CPresearchershavebeenutilizingvariouscelltypes,includingESC-andiPSC-derivedNPCs,MSCs,aswellasmultipotentNPCsfoundinthebrain.Resultshavebeenpromisinginthatstemcelltransplantationhasresultedinmodestfunctionalimprovementsandpositiveanatomicalchangeswithinthebrain.

AsapartoftheKidsBrainHealthNetworkpreclinicalresearchteam,Dr.MichaelFehlingsandhislaboratory(KrembilResearchInstitute,UniversityHealthNetworkandUniversityofToronto)arecurrentlyinvestigatingNPCsandhumaniPSC-derivedNPCsforthetreatmentofCP.TheteamisdevelopingreproducibleanimalmodelsofCPandinjectingthesestemcellsintothebrain.Importantly,thefocusofthisresearchistousestemcellsasatreatmentinachronicinjurycontext.Thismeansthatwearedesigningstemcelltherapiesthatwillbeefficaciouslongafterthebraininjuryhasoccurred.

Inpreclinicalmodelsafewofthehurdlesthatneedtobeovercomeare:improvingcellsurvivalinthebrainfollowingtransplantation,reducingtheriskoftumourformation(inthecontextofpluripotent-derivedcells),andunderstandinghowwecanforceourtransplantedcellstointegratebetterintotheexistingneuronalpathways.Despitethesechallenges,theknowledgegleanedfromhowstemcellsfunctionandincorporateintothedamagedCNSisincrediblyuseful.Thisinformationhashelpedresearcherstoadvancetheirstrategiesforcelltransplantation,aswellastobetterunderstandtherolesofendogenousNPCs(stemcellsfoundwithinthebrain)followingbraininjury.

StemCellTherapyClinicalTrials

Currentlythereare12clinicaltrialsaroundtheworldusingstemcellstotreatCP.Fourofthesearerecruiting,oneisactivebutnotrecruiting,andsevenhavebeencompleted.Manyofthesetrialsaremakinguseofbonemarrow/blood-derivedmononuclearcells(acombinationofcellsoriginallyfoundinthebonemarrowthatcontainhematopoieticstemcells(stemcellsthatgiverisetoredandwhitebloodcells,alsotermedHSCs)andBM-MSCs).OthersareusingpurifiedHSCs,orumbilicalcordbloodcontainingUC-MSCsandHSCs.Manyofthesetrialshavepassedtheinitialphasesofclinicaltrialsthatonlyseektoevaluatethecorrectdosageandsafetyoftheintervention.ThesetrialsareactuallybeginningtotesttheefficacyofthesestemcellsastreatmentsforCP.

Todate,thefindingsofonlyonestudyhavebeenpublished.Thisstudyhadthreegroupsofchildparticipants:Thosereceivingconventionalrehabilitationtherapyonly,thosereceivingadrugcallederythropoietin(showntohavepromiseintreatingCP)alongwithconventionalrehabilitationtherapy,orthosereceivingumbilicalcordblood(containingstemcells)+erythropoietin+conventionalrehabilitationtherapy.Thestudyfoundthatthegroupwhoreceivedstemcellsshowedgreaterimprovementsoncognitiveandmotorassessmentswhencomparedtotheothertreatmentarms.

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Whilethegrowthinthenumberofclinicaltrialssincethelasteditionofthismanuscriptin2011(4vs.thecurrent12)isexcitingandholdspromiseforthefutureofstemcelltherapiesforCP,thereisstillalongwaytogo.NoneoftheclinicaltrialstodatehaveusedNPCsoriPSC-sourcedcells,which,asdiscussedabove,haveuniqueadvantages.NPCshavethegreatestpotentialtointegrateintothedamagedbrain,andbeingabletouseiPSC-derivedcellswillheavilyreducetheneedforstemcelldonors,therebyminimizingtheriskofrejectionupontransplantationintothepatient.iPSCsalsoholdthepotentialtobedifferentiatedintoanycelltype.ThisversatilitywillbequiteusefulasmoreresearchisconductedondifferentcelltypesforthetreatmentofCP.However,learninghowtofullycontrolbothNPCsandiPSCsandmaximizetheirpotentialisanongoingareaofresearchthatrequiresalotoftimeandmoney.Researchershavebeenworkinghardthough,andwiththehelpofextensivecollaborations,funding,anddedication,theworld’sfirstiPSCclinicaltrial(RIKENtrialforage-relatedmaculardegeneration),putonholdinearly2015,hasresumed.

ManufacturingandRegulatoryIssues

Despitetheprogressseenwithclinicaltrialsinrecentyears,therearesomehurdlesthatneedtobeovercomeinorderforstemcellstobecomeawidelyaccessibletreatment.Oneofthemajorissuescurrentlyfacingthestemcellcommunityistheproblemof“scaleup”.Inthelaboratory,stemcellsareculturedinPetridishes.Itisfeasibletousethisapproachtotreatoneortwopatients,butasstemcelltherapiescomeclosertotheclinicthereisanincreasingneedtodevelopstrategiestomanufacturecellsonalargescale.Toolscalledbioreactorsareapromisingapproachtosolvethisproblem,however,withnewlarge-scaleproductionstrategies,theprotocolsforcellproductionwillneedtobere-evaluatedandtheefficienciesofcell

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productionwillchange.Moreresearchisneededinordertobetterunderstandhowwecanproduceourdesiredstemcellsinmassquantities.

Theotherproblemisthatregulatoryagencies,suchastheFDAandHealthCanadaarehavingdifficultydevelopingstandardizedguidelinesfortheproductionanduseofstemcells.Notonlywillindividualregulationsberequiredforeachuniquecelltype,butalsocellsareacompletelydifferentbiologicaltherapeuticwhencomparedtoconventionaldrugs.Cellsarelivingentitiesandtheireffectsonthehumanbodywillnotbeaswelldefined.Thereisanurgentneedtobetterestablishtheseguidelines,aswellastostandardizethemanufacturingprocessinordertoreducevariabilityamongthecellsduringproduction.Atthebasicsciencelevel,someofthesechallengeswillbeovercomebyimprovingourunderstandingofthemechanismsunderlyinghowthecellswork.Basicscientists,clinicians,industrypartners,regulatoryagencies,andpatientadvocatesallneedtoworktogethertohelpstemcelltherapiesmoveforward.

StemCellTourism

WhiletheNorthAmericanmedicalcommunityisworkingtobetterestablishstemcelltherapiesforCP,itisimportanttobecautiousofunregulatedoverseasstemcellclinics.Theactofgoingtotheseunregulatedclinicsiscalled“stemcelltourism”.Whileitmayseemtemptingtodoso,andthetestimonialsontheseclinics’websiteslookpromising,takeheed;Stemcelltransplantationisstillanunproventreatmentandwhiletherearecertainlycasesofreportedrecovery,therearealsomanyincidencesofincreasingdisability.Manyoftheclinicsusethesametypesofstemcellstotreatahostofdifferentdisordersdespitethelackofevidencetosupporttheiruse.Thetreatmentsarecostlyandtherisktopatientsisenormous.Ifyouareinterestedinstemcelltherapiesforyourselforalovedone,itisstronglyadvisedtogothroughreputableroutesandaccessregisteredclinicaltrials.

RisksandLimitationsofStemCellTherapy

ItmustbecautionedthatstemcelltherapyisstillanexperimentaltechniquethatisnotreadytobeadoptedasthestandardofcareforpatientswithCP.Asmentionedpreviously,theriskoftumourformationisahurdlethatmustbeovercome.Thisisanimportantissuetoaddress,becauseonceinjectedintothebody,stemcellscannotberemoved.Atpresentitisstillnotwellknownastowhetherornotthe“reprogrammingprocess”ofESCsandiPSCstomoredifferentiatedcelltypesisaleadingcauseoftumourformation.Severalresearchprojectsarecurrentlyunderwaytoreduceandifpossibleeliminatethegenerationofroguestemcellsthatcouldbetumour-forming.Theissuesof“scale-up”andcreatingwell-definedregulatoryguidelinesfortheuseofstemcellsintheclinicmustalsobeaddressed.

Thepromiseofstemcelltherapyisgreatandheraldsapotentialrevolutioninmedicinebyprovidingnewtherapiesforpreviouslyuntreatableconditions.However,itisunlikelytoprovideaone-stopmagicbullettoalleviateallclinicalsymptoms.InthecaseofCP,stemcelltherapyislikelytoresultinsmallincrementalimprovementsinfunctionthatinturnwillleadtonoticeableimprovementsinthequalityoflifeformostpatients.Additionally,atpresentitisnotknownfor

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howlongthebeneficialeffectsofstemcelltransplantationwilllast,andifmultipletransplantationsofstemcellsoverseveralyearswillberequiredtoimproveormaintainfunctionalrecovery.

Asacommunity,itisimportantthatwemanageourexpectationsofstemcelltherapies.Asmentionedabove,thistreatmentisstillundergoingdevelopmentandisnotyetwidelyacceptedintheclinic.WhilestemcelltherapieshavehugepotentialforthefuturetreatmentofCP,thestudyofthesecellsisalengthyprocessthatrequiresextensiveinvestmentoftimeandmoney.Supportoftheresearchthatismovingstemcelltherapiesforwardwillhelptofast-trackthistherapeutictotheforefrontofmedicinewithinthenext20years.

NextSteps

Nowthatyouhaveabasicunderstandingofthestateofstemcelltherapiesinthelaboratoryandclinic,wewouldliketoprovideyouwithsomereputablesourcestohelpyoulearnmore.Formoredetailsonstemcelltherapies,pleaseseethefollowinglinks:

Basicsofstemcells:http://stemcellfoundation.ca/en/;https://stemcells.nih.gov/info/basics/1.htm

StemcellsinCP:http://stemcellfoundation.ca/en/diseases/cerebral-palsy/

Informationonclinicaltrials:https://clinicaltrials.gov/;http://stemcellfoundation.ca/en/toward-treatments/clinical-trials/

Stemcelltourism:http://stemcellfoundation.ca/en/tag/stem-cell-tourism/;https://www.cirm.ca.gov/patients/stem-cell-tourism;http://www.nytimes.com/2016/06/23/health/a-cautionary-tale-of-stem-cell-tourism.html

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Table1:TheProsandConsofDifferentStemCellTypes

Pros ConsESC-DerivedCells • Candifferentiateinto

anycelltypetheuserdesires

• Well-studied

• Destructionofanembryo

• Riskoftumourformation

• Longprocesstoobtaindesiredcelltype

iPSC-DerivedCells • Candifferentiateintoanycelltypetheuserdesires

• Canbederivedfrompatient’sowncellssothereisminimalriskofrejectionupontransplantation

• Riskoftumourformation

• Longprocesstoobtaindesiredcelltype

MultipotentStemCells(e.g.NPCs,MSCs,HSCs)

• Alreadyexistinthedesiredformatwithinthebody

• Lowriskoftumourformation

• Potentiallyinvasiveprocesstoretrievethecells

• Limitedsupply• Ifthepatienthas

geneticriskfactors,thesegeneswillpersistinthesecellsiftakenfromthepatient.Canbeovercomebyusingadonor,butthisincreasestheriskofrejection