Thinking out of the dish: What to learn about cortical development using pluripotent stem cells

Thinking

out

of

the

dish:

what

to

learn

about

cortical

development

using

pluripotent

stem

cells

Jelle

van

den

Ameele

,

Luca

Tiberi

,

Pierre

Vanderhaeghen

,

and

Ira

Espuny-Camacho

1

Universite´LibredeBruxelles(ULB),InstituteforInterdisciplinaryResearch(IRIBHM),andULBInstituteofNeuroscience(UNI),

B-1070Brussels,Belgium

2

DepartmentofNeurology,GhentUniversityHospital,9000Ghent,Belgium

3

WELBIO,B-1070Brussels,Belgium

4_{VIBCenterfortheBiologyofDisease,3000Leuven,Belgium}

5_{CenterofHumanGenetics,KULeuven,3000Leuven,Belgium}

The development of the cerebral cortex requires the tightly coordinated generation of dozens of neuronal subtypes that will populate specific layers and areas. Recentstudieshaverevealedhowpluripotentstemcells (PSC),whetherofmouseorhumanorigin,can differen-tiateintoawiderangeofcorticalneuronsinvitro,which can integrate appropriatelyinto the brainfollowing in vivo transplantation.Thesemodelsarelargelyartificial but recapitulate a substantial fraction of the complex temporalandregionalpatterningeventsthatoccur dur-ing invivo corticogenesis.Here, we reviewthese find-ingswithemphasison thenewperspectivesthat they havebroughtforunderstandingofcorticaldevelopment, evolution,anddiseases.

Oneforall?Corticaldiversityandstemcellpluripotency The cerebral cortex is among the most complex of all biologicalstructures,andthemajorsiteofhighercognitive functions specifictoourspecies.Themechanisms under-lyingitsdevelopmentandevolutionareatthecoreofwhat makesushumans,andcouldhavemajorimplicationsfora varietyofhuman-specificdiseases[1].Incorrelationwith itselaboratefunctions,thecerebralcortexdisplays multi-ple levels of complexity. It contains dozens of different types of neurons populating specific cortical areas and layers, and cortical neuron number and diversity are thought to be at the core of its powerful computational capacities.Afirstsubdivisionamongcorticalneurons dis-tinguishestwomaincellclasses.Pyramidalneurons con-stitute>85% ofcorticalneurons,theyareglutamatergic, andsendlong-rangeprojectionstoothercorticalor subcor-tical targets. The remaining 15% ofcortical neuronsare GABA-ergic interneurons that display onlylocal connec-tivity.Pyramidalneuronsandinterneuronscanbefurther

subdividedintodozensofsubtypes,characterisedby spe-cificmolecularandfunctionalproperties[2,3].

Pluripotentembryonicstemcells(ESC;seeGlossary)[4]

haveemergedasapromisingtoolforneurobiology, allow-ingthein vitrorecapitulationofmanyevents thatoccur duringbrainorganogenesisaswellasthedirected differ-entiationofspecificcelltypes[5].Inparallel,theadventof inducedPSC(iPSC)[6,7]hasprovidedtheopportunityto use ESC or iPSC-based neural differentiation to model humanbraindiseases[8,9].

Glossary

Embryonicstemcell(ESC):pluripotentcelllines,typicallyderivedfromthe innercellmassoftheearlyembryo(blastocyst),capableofindefinite self-renewaland differentiationinto thederivativesof allthreeprimary germ layers:ectoderm,endoderm,andmesoderm.

Induced pluripotent stem cell (iPSC): a pluripotent (ESC-like) cell thatis obtained throughreprogramming of differentiated cells, typically through ectopicre-expressionofadefinedsetoftranscriptionfactors.

Intermediateprogenitorcells(IPC):neurogeniccorticalprogenitorsthatmostly undergosymmetricdivisionstogeneratetwoneurons,orsometimestwoIPC. IPCarelocatedinthesubventricularzone(SVZ)andlackadefinedpolarity. Neuroepithelial(NE)cells:non-neurogeniccorticalprogenitorsthatundergo symmetricdivisionsleadingtotheamplificationoftheinitialprogenitorpool. NEcellsare located intheventricularzone(VZ)attheearliest stagesof corticogenesis(Figure2,maintext).

Outerradialglial(oRG)cells:neurogeniccorticalprogenitorsthatcanundergo symmetricorasymmetricdivisionstogenerateneuronsandself-renew.oRGC reside in the outer SVZ (OSVZ). They display a distinctive polarised morphology characterised by a basal process towards the pial surface, whereastheytypicallylackanapicalprocess.Theyareparticularlyprominent inhighermammals,suchasprimates(Figure2,maintext).

Outer/innersubventricularzone(OSVZ/ISVZ):proliferativecompartmentthat islocatedatthebasalsideoftheSVZ,whereoRGcellsreside.Itisparticularly prominentinprimatespecies.

Pialsurface:theoutersurfaceofthecortexclosesttothemeninges. Radial glial cells (RG): neurogenic cortical progenitors that can undergo symmetricorasymmetricdivisionstogenerateneuronsandtoundergo self-renewal. RGcellsarelocatedinthecortical VZ.Theydisplayadistinctive polarised morphology characterised by an apical process towards the ventricule and a long basal processtowards the pial surface (Figure 2, maintext).

Subventricularzone(SVZ):proliferativecompartmentthatislocatedatthe basal(i.e.,awayfromtheventricle)sideoftheVZ,wheretheIPCreside. Telencephalon:mostanteriorpartofthecentralnervoussystem.Itcomprises thedorsaltelencephalon(pallium)andtheventraltelencephalon(subpallium). Ventricularzone(VZ):themostapical(i.e.,closesttotheventricle)proliferative zoneinthedevelopingcerebralcortex,whereNEandRGcellsreside. 0166-2236/

ß2014ElsevierLtd.Allrightsreserved.http://dx.doi.org/10.1016/j.tins.2014.03.005

Correspondingauthor:Vanderhaeghen,P. (pierre.vanderhaeghen@ulb.ac.be).

*_{Theseauthorscontributedequallytothisarticle.}

Here,we review recentprogress on the generation of corticalneuronsfromPSC,illustratingthatmuch,butnot allofthecomplexityofcorticaldevelopmentcanbe reca-pitulated with surprisingly simple in vitro conditions, providingnovelinsightsintocorticogenesis.

Forebrainidentity:lessismore

Thecerebralcortexisformedwithinthetelencephalon,the anterior-most part of the forebrain. Forebrain or telen-cephalon identity is thought to constitute a primitive patternofneuralidentity,whichisacquiredandretained throughlocalinhibitionofcaudalisingmorphogensignals

[10].In vitrostudies usingESC haveconfirmed and ex-tendedthismodelinbothmiceandhumans(Figure1A). WhenESCareculturedassinglecellsinaminimal medi-umdevoidofanyaddedextrinsiccues,theystarttoexpress neuralmarkerswithinhoursand,afterfewdays,mostof them adopt a forebrain identity [11–16]. Telencephalic and/orcorticalidentityisbestachievedwhenthemedium is supplemented with inhibitors of bone morphogenetic protein(BMP)/NodalandWntpathways[17–20], demon-strating how little extrinsic information is needed for telencephalic induction in vitro. The telencephalon then undergoespatterningalongthedorsoventralaxis, primar-ilythroughinductionofventralidentitiesbythe morpho-gen Sonic Hedgehog (SHH) [21]. This regionalisation

process is intimately linked to the specification of the twomainpopulationsofcorticalneurons:pyramidal neu-ronsandinterneuronsaregeneratedfromdistinct popula-tionsofprogenitorslocated inthedorsalandtheventral partofthetelencephalon,respectively[21–23].Thesame binarylogicisobservedduringESC-derivedtelencephalic induction(Figure1A).DuringmouseESCdifferentiation, SHH inhibitionleads tothegenerationofdorsal telence-phalic progenitors, which subsequently generate mostly pyramidalneurons[11,17,24,25].Intriguingly,SHH inhi-bitionisnotstrictlyrequiredduring humanESC cortico-genesis,whichislikelytobeduetolowerendogenousSHH signalling levels [18,26,27]. Conversely, specification of ventral telencephaliccells from both human and mouse ESCdoesrequireSHHstimulation,aloneortogetherwith Wnt inhibition [17,25,26,28–32], suggesting a conserved pathway togenerate ventral-likeidentity from forebrain progenitors.Theconcentrationandtiming(onsetand du-ration) ofexposure toSHH willleadtodifferenttypesof ventral progenitors and, hence, to distinct subtypes of neurons, from hypothalamicand striatal projection neu-ronstocorticalandstriatalinterneurons[25,32,33], remi-niscentofthe timedependenceofSHHsignalling invivo

[34].Moreover,theidentityofESC-derivedventral telen-cephalon progenitors can be furtherrefined through the manipulationofanteriorandposteriorregionalpatterning

Progenitor amplificaon

VI V IV

III/II

Human neurogenesis Maturaon

VI

V IV III/II

Mouse neurogenesis Maturaon

Shh ming Ventral telencephalon Early Late POA MGE LGE Corcal interneurons Striatal interneurons ? Telencephalic precursors Fgf8 Notch Dorsal telencephalon Bcl6

Corcal pyramidal neurons Caudal (occipital) Rostral (frontal) Corcal progenitors _Astrocytes

Deep layers Upper layers

Default

Noggin

Dual Smad inhibion

TGFβ Wnt BMP FGF/IGF

Wnt inhibion Pluripotent stem cells

Default

(A)

(B)

E9 E11 E15 E18

GW5 GW8 GW12 GW30 Wnt inhibion Cyclopamine Defa ult Shh TRENDS in Neurosciences

Figure1.Invitropluripotentstemcell(PSC)-derivedcorticogenesisrecapitulatesinvivodevelopmentalmilestones.(A)PSCculturedunderminimalconditionsorinthe presenceofWnt/transforminggrowthfactor(TGF)-b/bonemorphogeneticprotein(BMP)morphogeninhibitors,undergodifferentiationtowardsforebrainortelencephalic identity.Asinvivo,furtherspecificationintoventraltelencephalicprogenitorsandneuronsrequiresactivationoftheSonicHedgehog(Shh)pathway.Differenttypesof ventralprogenitor[preopticarea(POA),medialganglioniceminence(MGE),andlateralganglioniceminence(LGE)]aregenerated,includingcorticalinterneurons, dependingonthetimingandconcentrationofShh.Conversely,inabsenceorlowlevelsofShhsignalling,PSCwillmostlydifferentiateintoacollectionofprogenitorsof dorsaltelencephalonorcorticalidentity.Subsequentgenerationofcorticalpyramidalneurons,underthebalancedinfluenceofNotchandpro-neurogenicfactors,suchasB cellCLL/lymphoma6(BCL6),followsatemporalpatterning,withdeeplayerneuronsbeinggeneratedearlierthanupperlayerneurons,eventuallyfollowedbyaswitchto astrocyteproduction,similartoinvivo.(B)Humancorticogenesisandneuronalmaturationfollowamoreprotractedtime-coursethantheirmousecounterparts,whichis alsoobservedinvitro.Abbreviations:E,embryonicday;FGF,fibroblastgrowthfactor;GW,gestationalweeks;IGF,insulin-likegrowthfactor.

cues,suchasfibroblastgrowthfactor8and15(FGF8and FGF15),andactivin,togeneratespecificsubtypesof inter-neurons[25,35].

Collectively, these data demonstrate that ESC and iPSC, whether of human or mouse origin, can generate eitherventralordorsaltelencephalicprogenitors, depend-ing on the levels of SHH signalling. Eventually, these progenitors produce specific types of neurons, whereby dorsalcellsgenerateessentiallyglutamatergicpyramidal neurons, whereas ventral cells generate mostly GABA-ergic neurons. Interestingly, human and mouse PSC do not differinthisrespect,in linewith resultsobtained in humanexvivocultureswhere,asintherodent,theventral telencephalonappearstobethesiteoforiginofmost,ifnot all,corticalinterneurons[36,37].

Corticalneurogenesis:ataleoftransitions

The mammalianneocortex isorganisedintosix different layers, each of which comprises a collection of neurons displayingspecificpatternsofgeneexpressionand connec-tivity[2,38–40](Figure1B).Whichlayeraneuronsettles in, is tightly linked to its birthdate, with deeper layer neuronsbeinggeneratedearlierthanupperlayerneurons. Thisprocessoftemporalpatterningiscentraltothe gen-erationoflayer-specifictypesofcorticalneurons. In vivo andinvitro studieshaveshownthatitinvolves the pro-gressive restriction of progenitor competence, which is controlledbybothintrinsic,cell-autonomousprogrammes andbyenvironmentalinfluences[2,38–40].

Surprisingly,bothmouseandhumanPSC-derived cor-ticogenesis recapitulate robustly temporal patterning in vitro:neuronsthatexpressmolecularmarkersandproject to targets specific ofupper layer neurons are generated consistentlylaterthanneuronswithadeeplayeridentity

[11,18,24,41,42].Fromclonalcellanalyses,itwas further-more shownthat mouseESC-derived neural progenitors areinitiallymultipotentandcanchangeandrestricttheir competence over time[11], similarly to what was previ-ouslydemonstratedusingexvivoculturesofearlycortical progenitors [43]. However, whereas in vitro systems of corticogenesisdisplayremarkablesimilaritieswithinvivo developmentalprocesses,itstilldiffersinsignificantways from in vivo corticogenesis, depending on culture condi-tions.Indeed,whereasinvivodeepandupperlayer neu-ronseachrepresentapproximatelyhalfofthecortex, ESC-derivedpyramidalneuronsarestronglyskewedtowardsa deep layer identity following monoadherent culture in minimaldifferentiationconditionsintheabsenceofadded morphogens [11,18]. Importantly, thiswas alsothe case whennativemousecorticalprogenitorsweregrownexvivo atclonaldensities[43].Conversely,ahigherproportionof upper layer neuronsappearstobegenerated when ESC arefirstdifferentiatedathighdensityand/or supplemen-tedwithextrinsiccues,suchasretinoicacid[27]orascell aggregates [24,44]. Although direct comparison between various studiesis notalways straightforward becauseof different markersbeing analysed,thesefindings suggest thatextrinsiccuesthatmaybemissinginminimalculture systems, are required for the proper generation of the upperlayerneurons[45].Consistentwiththishypothesis, whereashumanPSC-derivedcorticalprogenitorscultured

in minimal conditions generate only a few upper layer neuronsevenafterprolongedperiodsinvitro,they gener-ate manyupper layer neuronsfollowing transplantation intothe mouse newborn cortex[18]. Thesedata suggest thatcorticalprogenitorscompetenttogenerateneuronsof allsixlayerscanbegeneratedfollowingminimalinvitro conditionsfrom ESC,butthat cuespresentinthemouse newbornbrainarenecessarytoenhanceupperlayer neu-ronproductionfromthesecells.Suchcuescouldbeeither producedbycorticalprogenitorsandneuronsthemselves, orderivedfromextrinsicsources,suchasmeninges, cere-brospinal fluid, or the vascular niche [45]. PSC-derived corticogenesis may providea useful platform toidentify thesecuesandtheirmechanismsofaction.Specifically,it will be interesting to determinewhether andhow these cuesacttochangethecompetenceofcorticalprogenitorsto generate upper layer neurons, and if they act on the specificationor amplificationofspecifictypes of progeni-tors,suchasintermediateprogenitorsorouterradialglial cells,asexplainedfurtherbelow.

In parallelwith temporalpatterning, akeytransition forthegenerationofcorticalneuronsisneurogenesisitself, whereby cortical progenitors differentiate into cortical neurons,eitherdirectlyorindirectlyfollowingbrief ampli-ficationthroughintermediateprogenitors[46,47].Therate andtiming of neuronal differentiation from cortical pro-genitorsisdynamicallyregulatedbyvariousintrinsicand extrinsiccues,includingNotchandproneuralfactors,such asneurogenins,whichcollectivelycontrolthenumberand fate of cortical neurons [45,48–50]. ESC-derived cortical neurogenesiscloselyrecapitulatestheseprocesses[11,24], andhas beenused togain mechanistic insightsintothe factors involved in this process (Figure 1A). The mouse ESCcell-basedmodelofcorticogenesiswasusedtoscreen for novel transcription factors involved in neurogenesis, leadingto theidentification ofa novelpotent pro-neuro-genicgene,BcellCLL/lymphoma6(BCL6)[41].Whereas BCL6 was uncovered in an ESC-based screen, it was subsequentlyfoundtobeexpressedincorticalprogenitors invivo,duringthetransitionfromcorticalprogenitorsto pyramidal neurons. Furthermore, the analysis of BCL6 knockoutmiceledtotheconclusionthatBCL6isrequired for proper cortical neurogenesis in vivo [41]. Combined studiesonthe ESC systemandinvivo cortexconverged todemonstratethatBCL6actsthroughdirectandstable epigeneticrepressionoftheNotchtargetHes5promoter, thereby enabling the neurogenic transition to proceed irreversibly. This study illustrates how the use of ESC-based modelsofcorticogenesis canlead tonovelinsights intotheinvivomechanismsofcorticalneurongeneration. Corticalarealidentity:intrinsicinsightsfrom

transplantationexperiments

Inadditiontolayer-specificidentity,neuronsfrom differ-entcortical areas alsodevelopselectivepatterns ofgene expression and connectivity. The patterning of cortical areas is a complex process resulting from the interplay betweenfactorsintrinsictothecortex,aswellasextrinsic factorsfromoutsidethebrain[22,51].Surprisingly,invivo transplantation experiments revealed that mouse ESC-derived cortical neurons seem to acquire mainly limbic

andvisual(occipital)identities[11].Following transplan-tation,ESC-derivedcorticalneuronssendaxonstospecific visualandlimbictargets,withapatternofprojectionthat is strikingly similar to grafted embryonicvisual cortical tissue[11,52].Importantly,theseresultswereallobtained withgraftswithin thefrontalcortex,suggestingthatthe highlyselectivepatternofprojectionswasnotduetoinvivo respecification of the grafted neurons. Confirming this hypothesis, examination of the molecular identity of ESC-derivedcorticalprogenitorsandneuronsbefore graft-ingrevealedthatmostofthemexpressedtypicalmarkers of the occipital cortex, in particular chicken ovalbumin upstream promoter transcription factors (Coup-TF) I and II [11]. By contrast, the areal fate of ESC-derived corticalprogenitorsinvitrocouldbemodifiedbythe addi-tionofextrinsiccuesknowntoinducefrontalcorticalfates invivo,suchasFGF8[24,53].

Corticogenesis from human ESC in similar minimal conditions also results in many Coup-TFI/II-expressing progenitors that display visual and limbic-like patterns ofaxonalprojectionsshortlyaftertransplantationintothe frontalcortexofneonatalmice[18].However,unlikeinthe mouse,when the grafts were left forlonger periods, the transplantedcellstendedtolosemarkersofoccipital iden-tityandtheiraxonalprojectionscorrespondedtoawider rangeofarealidentities[18].Theseobservationsare con-sistentwithamodelwherebyspecificpatternsofvisualor limbic areal identity may be acquired during in vitro minimaldifferentiationfromhumanESC,asinthemouse, butthat, followinggrafting, asubstantial fraction ofthe cellscanbe specified toother areal identitiesover time, perhaps in relation to their relatively earlier stage of maturationatthe timeofgraftingand, therefore,higher

susceptibilitytoextrinsiccuesfromthefrontalcortex.This isreminiscentofdataobtainedwithrodentnativecortical cells,whichshowdifferentpatternsofarealfate specifica-tiondependingontheirdegreeofcommitmentand matu-rationatthetimeofgrafting[54].Theseissuesneedtobe dissectedfurther,butthedataavailablesofarsuggestthe existence ofa conserved programme ofspecificationinto visual and limbic areal identity from PSC in minimal conditions,whichmightperhapscorrespondtoanancient ‘primitive’patternofarealspecification.

Cytoarchitectureinadish:howfarcanwego3D? Thecortexismuchmorethanmerelyasumofitspartsand, ifonewishestomodelthisinvitro,its3Dcytoarchitecture shouldalsoberecapitulated.Wenowknowthatatleastpart oftheintricateorganisationofmanyorganscanalsoemerge invitro:astrikingexamplewasprovidedthroughthe au-tonomousformationofafullyformedopticcupandderived retinalstructurefromESCaggregates[55].For corticogen-esis,thisgoalisfarfrombeingcompletelyachievedand,in principle,would bemorechallenging; however,somekey aspects of the patterned organisation of the developing cortex can be recreated in vitro (Figure 2). Even in 2D systems,neural progenitorsexhibitspatialpatterns, with cellsoftenorganisedas rosettesdisplayingapicobasal po-larityandincludingfeaturestypicallyfoundinthe neuroe-pitheliumandventricularzone,suchasinterkineticnuclear migration[11,19,27].WhenmouseorhumanESCare cul-turedasbowlsofcellsand differentiatedintocortical-like progenitors,thisleadstoamorerobustpolarisedcellular organisation[24],withprogenitorsoccupyingdeeperlayers ofthebowls,andneuronsaccumulatingattheirperiphery, following anorganisationhighlyreminiscent ofanascent

Complex and organised Simple and reduconist

2D modelling 3D modelling (A) (B) Radial glia Intermediate progenitors Upper layer neurons Basal surface Apical surface Outer radial glia Co r c a l p la te IS V Z VZ O S VZ a diate ors Upper lay neuro a TRENDS in Neurosciences Deep layer neurons

Figure2.Differentmodelsofinvitrocorticogenesisfrom2Dto3D.(A)2Dmodelsaremorereductionistbutneverthelessrecapitulatekeyaspectsofcorticogenesisandcan beusedtostudyvariousaspectsofcorticalneurongenerationandfunction,includingfollowinginvitroscreensorinvivotransplantation.3Dmodelscanrecapitulateina strikinglyfaithfulwaytheinvivoorganisationofcorticalprogenitorsandneurons,therebyprovidinguniquetoolstostudyspatialpatterningandcytoarchitecture formation.(B)Schematicsoftherelationsbetweenthevariouscellularplayersofcorticogenesisfoundinvivo.Abbreviations:OSVZ/ISVZ,outer/innersubventricularzone.

corticalprimordium,includingtheventricularzoneandthe cortical plate.Acrucialchallengeof3Dmodels istokeep themforlongperiodsofculturewhilepreservingaccessto gas and nutrients. This important aspect was recently improvedtoallowneuralandcorticaldifferentiationtogo on for several months and to examine later aspects of development,includingtheemergenceofspecificdomains ofprogenitorsandneurons[53,56].Moststrikingly,usinga long-termcortical3Dmodel,thefinalpositionoftheneurons withinacorticalplate-likestructurewasfoundtodependon theirneuronalbirthdate,whereneuronsbornearlierwere foundindeeperpositionsthanlater-bornones,thus reca-pitulating the fundamental inside-out pattern of cortical neurogenesis[53].Itwillbefascinatingtodetermine wheth-erthisspatiallayer-likearrangementofneuronsis estab-lishedinasimilarwayasinvivo(i.e.,byrelyingonactive neuronalmigrationalongtheradialscaffoldandoncomplex guidancebyextrinsiccues,suchasReelin).

Altogether,thesedataconstituteafirstproofof princi-plethatacortical-likecytoarchitecturecanalsoemergein vitro.Similartotheobservationthatthetemporal pattern-ingleadingtosequentialgenerationofcorticalneuronsis encoded in neural progenitors themselves [11,43], it is remarkable that morphogenesis and compartmentalisa-tion also emerge as self-organising properties [57]. This constitutes a promising system to decipher some of the underlying mechanismsofcorticalpatterning,andopens newroadstooptimisefurtherinvitrocorticogenesisandto modelmorecloselyinvivocorticalneuronalnetworks(Box 1).

PSC-derivedcorticogenesisandhuman evolution:time istheessence

Thebrainand,moststrikingly,theneocortex,have under-gonearapidandconsiderableincreaseinrelativesizeand complexityduringthepastfewmillionsofyearsofhominid (human and great apes) evolution [1]. This has led to enlargement of the surface and thickness of the cortex, associated with an increased number and diversity of cortical neurons. Thus, manyof the species-specific fea-tures of the human cortex are thought to be linked to differencesinthegeneration,specification,and differenti-ation of cortical neurons [1,58,59], which may directly impactonthetotal numberanddiversityofcortical neu-rons[1,60–62].

Althoughcorticalneurogenesisappearstobewell con-servedamongmammals,severaldivergentfeatureshave alsobeenidentified,whicharethoughttobemainlylinked to the properties of cortical progenitors [1,59,63–65]

(Figure 1B). Neuroepithelial(NE) cellsarethe firsttype toemergeduringearlydevelopment,andarecharacterised by symmetricproliferativedivisionsthatamplify the ini-tialpoolofcorticalprogenitors.Thisamplificationprocess mayhaveadirectimpactonthefinalnumberofneurons

[61]andisexpandedinspeciesdisplayingbrainsoflarger size,suchasprimates(takingweeksinsteadof2–3daysin mice)[61,66].NEprogenitorsthenconvertintoradialglial (RG) progenitors, which constitute the major subtype of neurogenic cortical progenitors [46,48,67–69]. RG cells undergo multiple rounds of asymmetric cell divisions, therebyenablingthegenerationofdiversetypesofneurons

whilemaintainingapoolofprogenitors,thus,followinga stemcell-likebehaviour[70,71].Inprimates,theprocessof neuronal production is considerably protracted in time, takingseveralmonthsinsteadofseveraldaysinthemouse

[66].

Remarkably, human ESC-derived corticogenesis pre-sents temporal specificities that are reminiscent of hu-man-specific features of cortical development [18,24,26,

27,44].Directcomparisonbetweenmouseandhuman

cor-ticogenesisfromPSC,usingexactlythesameculture con-ditions, revealed that the time line for corticogenesis is considerably extended in the human versus the mouse

[18].HumanESC-corticalderivedprogenitorsstartto gen-eratepostmitoticneuronsafteramuchlongerperiod(i.e., approximately4weeksinsteadof6–8daysinthemouse) andthiswascorrelatedwiththeappearanceofmolecular markersofRGcells. Similartoinvivo,the generationof distincttypesofcorticalneuronsisalsomuchprotractedin time:whereasmouseESCcorticogenesistakes2–3weeksto becompleted,ittakes10–15weeksforhumanESC[11].The underlyingmechanismsthatenabletheprimateembryonic braintogenerate neurons fora prolongedperiodoftime remainlargelyunknown[72],buttheymightbelinkedto species-specific properties intrinsic to RG cells, such as differentialcellcyclecontrolortuningofself-renewalversus terminal differentiation [73]. In addition, the emergence and/or amplification of different species-specific types of progenitorsmightbeanothermechanismthatcontributes tocorticalneurondiversityandtoevolutionarychangesin corticalneurogenesis.Theseprogenitorsincludethe recent-lydescribedouterRG(oRG)cells[64,65,74–76].oRGcells sharemanyfeatureswithRGcells,includingthepotential forself-renewal,buttheylackanyapicalprojectionreaching the ventricularsurface. Most strikingly, whereas human oRGcellscangenerateneuronsdirectly,theirprogenytend toundergomultipleroundsofdivisions,thusprovidingan additionalmechanismforincreasedneuronal outputand corticalexpansion.ThedetectionofoRG-likecellsinvitro fromhumanPSCwasreportedin2D[27]and3Dsystems

[53,56].Importantlythesecells werenotfoundinsimilar

differentiationparadigmsfrommousePSC[56],providing evidenceofspeciesspecificity,althoughperhapsthemost importantfeatureofoRGcells(i.e.,theircapacityto gener-atemanyneuronsthroughtransientamplification)stillhas tobedeterminedininvitroconditions.

Another important aspect that appears to be species specific is neuronal maturation: human cortical neurons display more prolonged patterns of morphological and electrophysiological maturation, as well as synaptogen-esis, which might underliesome of the relative neoteny thatcharacteriseshumanbrainmaturation[77,78]. Simi-larly,theinvitromaturationofthehumancortical excit-atoryneuronsfollowsaslowerpace comparedwith their mousecounterparts. Invitro-derived humanneurons ex-hibitimmatureprofilesofgeneexpressionandexcitability for several weeks, and extended periods of culture are needed to observe mature patterns of action potentials and signs of significant synaptic activity [18,27,44]. A similarlyprotractedpatternisobservedforcortical inter-neurons:GABA-ergicneuronspresentanimmatureprofile ofactivityevenafter8–9weeksincultureandonlystartto

displaymorematurefunctionalpropertiestypicallyafter 15–30weeks[28].Thisslowprocesscanbeacceleratedby co-culture with murine astroglial or embryonic cortical cultures, but only to a partial extent [28,30]. Besides and most strikingly, in vivo transplantation of human PSC-derived excitatory and inhibitory cells into mouse modelshasshownthat thehumancellsseemtofollowa species-specificprogrammefordelayedneuronal matura-tion and synaptogenesis [18,20,28,30]. Indeed, whereas differentiated human pyramidal neurons extend axons towardsspecificcorticaltargetsfrom4–6weeks post-trans-plantation,thefull extentof axonalgrowthfrom human corticaltransplantedneuronswasonlyreached6months aftertransplantationin themouseneonatalcortex. Den-dritic maturation also progressed at a slow pace even within themousebrain,where neuronspresenteda ma-ture complex dendrite arborisation pattern, dendritic spines,andfunctionalsynapticactivityonly9monthsafter transplantation[18].Similarly,thepatternsofmorphology andfunctionality of xenotransplanted human interneur-ons are not yet fully reached even 6 months post-trans-plantation[28,30].

Overall,thesedataindicatethatthefundamental prin-ciplesthatcontrolthetimingofgenerationandmaturation ofcorticalneuronsappeartobespeciesspecificanddonot dependmuchontheinvivocontext.Instead,theypointto cortex-intrinsicmechanismsthatcontroltheclock of cor-ticogenesis,forwhichPSC-basedmodelsmayprovide at-tractive experimental set-ups to dissect the underlying mechanisms.

Itwaspreviouslyproposedthatsuchprotracted neuro-nalmaturationprovidesabasisfortheneotenythatmay characterisehumancorticalneurondevelopment(i.e.,the retention of juvenile traits even at an older age) [77]. Althoughlargelyspeculative,thispossibilityisattractive enoughtoexplorefurther,forinstanceby examiningthe consequences of the transplantation and integration of ‘juvenile’ human neurons into the mouse cortex at the circuitandbehavioural levels.

Modellingpathologicalcorticaldevelopmentand degeneration

TheadventofiPSCtechnology[7]offersinprinciplemany novel opportunities to model brain diseases, including thosethatstrikethedevelopingcortex[8,79].Atthispoint, mostcanprobablybelearned from modellingmonogenic disorderswith high penetrance[80],forwhich gain-and loss-of-function paradigms could confirm the role of the affectedgene.However, there arefewexamplessofarof studiesthat haverelied oniPSC-derived corticalcells of definedidentitytomodelcorticalneurodevelopmental dis-eases. Among these, one striking example is Timothy syndrome, a monogenic disorder caused by a mutation in an L-type voltage-gated calcium channel, which is stronglyassociatedwithdevelopmentaldelayandautism. Examination of cortical cells from iPSC derived from patients with Timothy syndrome revealed several inter-estingphenotypes.Theseincluded,asexpected,defectsin calcium signalling andneuronal activity,but also some-whatmoresurprisinglydefectsinthegenerationofspecific typesofneurons(i.e.,callosalprojectionneurons),aswell

as defects in dendrite remodelling[81,82].These pheno-types wererecapitulated ina mousemodel presentinga similar mutation, thus confirming the validity of the in vitro cellular human model. Other examples of autistic syndromes are Phelan–McDermid syndrome caused by 22q13 deletion or Rett syndromecaused by methyl CpG bindingprotein2(Mecp2)mutations. The22q13deletion syndrome was modelled in a similar way with patient-derivediPSC,whichrevealedsynapticdeficitsthatcould be largelyrescued by re-expressionof SH3 andmultiple ankyrin repeat domains 3 (Shank3), one of the genes involved in the deletion syndrome [83]. To study Rett syndrome,theauthorsusedisogenicESClinesgenerated through homologousrecombination,which appearstobe most promising to cope with the inherent variability of iPSClinesderivedfromdifferentindividuals[79,84].This approachrevealedseveraldefectsinthestudiedneurons, including a neuron-specific global alteration of gene ex-pression that could underlie many of the cellular and functionaldefectsobservedinRettsyndrome-affected neu-rons.Earlydefectsofcorticaldevelopmentthatareatthe origin of most severe forms of brain malformations and dysfunctionconstituteothercandidatediseasestobe mod-elledbyinvitrocorticogenesis.Forinstance,iPSCfroma patientwithmicrocephalyaffectedbyaCDK5regulatory subunitassociatedprotein2(CDK5RAP2)mutation[56], have enabled researchers to recapitulate some of the defectspreviouslydescribedinmousemodels[85,86]. Cor-ticogenesis from iPSC was alsoapplied to study Down’s syndrome,amajorcause ofmental retardationand neu-rodegeneration,revealingearlysynapticdefectsinaffected neurons [87],as wellas amyloid peptide overproduction

[88].

Thesefindingsillustratethat,whenappliedtoneurons of defined identity, the iPSC modelling technology can yieldvaluableinsightintopathogenicmechanismsof com-plexneurodevelopmentaldiseases.Inaddition,more com-plexbraindevelopmentaldisorders,suchasschizophrenia

[89]orfragileXsyndrome[90],althoughmorechallenging, couldalsobenefitfrommodellingusinginvitro corticogen-esis. Finally,itcouldalsobeappliedtodegenerative dis-eases that primarily affect cortical neurons, including Alzheimer’sdiseaseandvariousotherformsofdementia. However,inthiscase,itwillbeimportanttofindoutwhich critical aspects of these diseases can be recapitulated faithfullyinvitro,giventhefactthattheyprimarilyaffect the adult or agingnervous system.Indeed,so far iPSC-derivedor directly reprogrammedneuronsfrompatients with familial [amyloid precursor protein (APP) duplica-tions,presenilin1or2(PSEN1orPSEN2)mutations]and sporadicAlzheimer’sdisease,werefoundtomimica limit-edsetofthepathologicalfeaturesencounteredindiseased brain tissue, including altered APP processing and Tau phosphorylation [87,88,91–94]. By contrast, major hall-marks of thedisease, such as Tauneurofibrillary tangle formation,neuronaldysfunctionanddeath,havenotbeen reported so far. In this respect, it will be interesting to explorefurtherartificialwaystoaccelerateneuronal mat-uration,orevenaginginvitro[95]ortrytomodel neuro-degeneration following xenotransplantation in the aging mousebrain.

Concludingremarksandperspectives

ThemergeofPSCtechnologyanddevelopmental neurobi-ologyrevealsunexpectedopportunitiestostudythe forma-tion and maintenance of the cerebral cortex (Box 1). However, toholdits promises, PSC-basedmodelling will havetocontributesignificantlytouncovernovelfeaturesof normal and pathological mechanisms of corticogenesis. One areaof interestin thiscontext is touse ESC-based systemsforunbiasedscreens,inlinewiththediscoveryof BCL6 as anovel cortical pro-neurogenic factor. Another exciting focus will betoinvestigate the mechanisms un-derlyingthespecies-specificfeaturesofcorticogenesis,by exploringexperimentallythegeneticlinksbetween devel-opment andevolution of the human brain.Finally,PSC modelsmaybringnovelinsightsintothepathophysiology ofhumandiseasesthatarenotrecapitulatedasseverelyor clearlyinmousemodels,suchascorticalmalformationsand alterationsofhighercognitivefunctions.However, whatev-erPSCmayrevealwillhavetobeconfrontedwithinvivo data,nomatterhowchallengingthismaybe.Indeed, mod-els, whetherin art or science, can merelyrepresent,but cannotreplace,theoriginalcomplexityoflife.

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Box1.Outstandingquestions:What’snext,inandoutof

thedish?

Howmuchcanmodelsof2Dand3Dcorticogenesisrecapitulate

accuratelythefullrepertoireofcorticalneuronalidentities?

Canwedesignandimprove thetoolsofPSCdifferentiationto

generatehighlyhomogenouspopulationsofneuronsofasingle,

physiologicallyrelevant,identity?

Howmuchcan3Dmodelsrecapitulatetheprocessesofcortical

neurogenesis, including polarised cell movements and

asym-metricdivisions?

Howmuchcan3Dmodelsrecapitulatethelayeredorganisationof

theneocortex?

To what extent are 3D models capable of reproducing the

emergenceofdistinctcorticalarealidentities?

What are the patterns of connectivity of cortical neurons

generatedin3Dmodels?Canwegenerateacorticalcolumnin

vitro?Doneuronsdisplaygenuinelayer-specificpatternsofinput

andoutput?

How much specificity and accuracy of connectivity can be

achieved followingtransplantationofPSC-derived cortical

neu-rons in thebrain? Canshort- and long-range patterns lead to

physiological circuits? If so, how much can lead to cortical

functionrestoration?

Arethecorticogenesismodelsrobustenoughtonotonlyconfirm,

butalsopredictmechanismsofdevelopmentordisease,which

couldbefurtherexploredinvivo?

CanPSC-derivedmodelsofcorticogenesisbeusedtomodelthe

eventsthatoccuronlyintheadultorevenagingbrain,including

mostaspectsofneurodegeneration?

CanhumanPSCmodelsbeimprovedtoallowcost-effectiveand

robusthigh-throughputgeneticorchemicalscreens?

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