review article neural conversion and patterning of human...

Review ArticleNeural Conversion and Patterning of Human Pluripotent StemCells A Developmental Perspective

Alexandra Zirra1 Sarah Wiethoff123 and Rickie Patani1345

1Department of Molecular Neuroscience UCL Institute of Neurology Queen Square London WC1N 3BG UK2Center for Neurology and Hertie Institute for Clinical Brain Research Eberhard-Karls-University 72076 Tubingen Germany3National Hospital for Neurology and Neurosurgery UCL Institute of Neurology 33 Queen Square London WC1N 3BG UK4Department of Clinical Neurosciences University of Cambridge Cambridge CB2 0QQ UK5Euan MacDonald Centre for MND University of Edinburgh Edinburgh EH16 4SB UK

Correspondence should be addressed to Rickie Patani rickiepataniuclacuk

Received 30 October 2015 Accepted 24 January 2016

Academic Editor Jason Weick

Copyright copy 2016 Alexandra Zirra et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Since the reprogramming of adult human terminally differentiated somatic cells into induced pluripotent stem cells (hiPSCs)became a reality in 2007 only eight years have passed Yet over this relatively short period myriad experiments have revolutionizedprevious stem cell dogmataThe tremendous promise of hiPSC technology for regenerativemedicine has fuelled rising expectationsfrom both the public and scientific communities alike In order to effectively harness hiPSCs to uncover fundamental mechanismsof disease it is imperative to first understand the developmental neurobiology underpinning their lineage restriction choices inorder to predictablymanipulate cell fate to desired derivatives Significant progress in developmental biology provides an invaluableresource for rationalising directed differentiation of hiPSCs to cellular derivatives of the nervous system In this paper we begin byreviewing core developmental concepts underlying neural induction in order to provide context for how such insights have guidedreductionist in vitro models of neural conversion from hiPSCs We then discuss early factors relevant in neural patterning againdrawing upon crucial knowledge gained from developmental neurobiological studies We conclude by discussing open questionsrelating to these concepts and how their resolution might serve to strengthen the promise of pluripotent stem cells in regenerativemedicine

1 The Developmental Origins of the NervousSystem An Overview

The process of neurodevelopment is spatiotemporally reg-ulated and necessitates sequential progressive restrictionsin cell fate Although some interspecies differences in bothcytoarchitecture and molecular machinery do exist betweenmouse and man rodent models have illuminated key under-lying mechanisms of lineage restriction to a variety of celltypes These insights have provided invaluable guidance forthe predictablemanipulation of human pluripotent stem cells(hPSCs) into myriad cell fates From the point of fertilisationof the secondary oocyte cells commence asymmetric divisionand sequentially give rise to the 2- 4- and then 8-cell stageblastomere which subsequently develops into the blastocyst(Figure 1) Oct34 serves to maintain pluripotency in theinner cellmass (ICM) of the blastocyst Although interspecies

differences in cell-type specific factors exist ultimately andfollowing implantation and gastrulation 3 distinct germlayers emerge endoderm (which forms the lining of internalorgans) mesoderm (which gives rise to bone muscle andvasculature) and ectoderm (from which results the nervoussystem and skin) Figures 1 and 2(a) describe developmen-tal processes involved in specification of the 3 germ lay-ers During gastrulation this 3-layered structure undergoesprogressive and stereotyped morphological transformationsThe mesoderm and endoderm invaginate inwards and theectoderm forms an epithelial sheetwhich ensheathes a centralcavity The region of the ectoderm surrounding the neuralplate becomes epidermis (Figure 2(a)) An important aspectof embryogenesis is the assignment of developmental axesldquoAnterior-posteriorrdquo can be used to refer to the proximal-distal axis which is based on proximity to the future placenta(in the early blastocyst the proximal pole is represented by

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 8291260 14 pageshttpdxdoiorg10115520168291260

2 Stem Cells International

Proximalrostral (anterior)

Distalcaudal (posterior)

Dorsal(posterior)

Oocyte Blastomere

2-cell stage 4-cell stage 8-cell stage 16-cell stage

Ventral(anterior)

Inner cell massOct34

TrophectodermCdx2

Early blastocyst

Late blastocyst

EpiblastOct34 + Nanog

Oct34 + GATA4Endoderm

Ectoplacental cone

Extraembryonic ectoderm

Embryonicendoderm

Extraembryonicendoderm

Cdx2

AVE (anterior visceralendoderm)

DVE (distal visceralendoderm)

Mesoderm(primitive streak)

MousehumanE55E7ndashE12


MousehumanE6-E7E13ndashE17

Gastrulation

Figure 1 Developmental stages of mouse embryo First row (left to right) from the secondary oocyte the blastomere develops (2-cell 4-cell 8-cell and 16-cell stages) to give rise to the early blastocyst formed of trophectoderm (cells that express Cdx2) and inner cell mass cells(that express Oct34) Later the inner cell mass gives rise to the epiblast (cells that express Oct34 and Nanog) and endoderm (expressingOct34 and GATA4) Second row (right to left) in the late mouse blastocyst Cdx2 positive cells give rise to the extraembryonic ectoderm andectoplacental cone At the same time the endoderm divides into an embryonic endoderm and an extraembryonic endodermThe epiblast andthe extraembryonic ectoderm form a cavity lined by embryonic endoderm From the embryonic endoderm the distal visceral endoderm isformed (DVE) Third row (left to right) the DVE migrates proximally and will be known as the anterior visceral endoderm (AVE) The finalimage (third row right) shows the development of the primitive streak (mesodermal cells) at the opposite (posterior) pole from the AVE NBThere are 2 different types of endoderm called extraembryonic and embryonic these differ in their potency and give rise to distinct cellularderivatives All timelines are given for mouse and human embryonic development

the ectoplacental cone as depicted in Figure 1) Later theproximal-distal axis will become the future rostrocaudal axisin vertebrates However the term ldquoanterior-posterior axisrdquocan also sometimes refer to the dorsoventral axis in the adultstate a distinction that is primarily based on position of theabdomen (ventral) as opposed to the backspinal column(dorsal) Therefore for ease of reference this review will usethe terms rostrocaudal (ldquoR-Crdquo) and dorsoventral (ldquoD-Vrdquo)axes

Three principal events characterise early neurodevel-opment First the process of neural induction specifies aregion of the embryonic ectoderm to form the neural plate(Figure 2(a) [1]) Second a process termedneurulation occursthrough serial morphological transformations to give rise tothe neural tube (Figure 2(b) [2]) This process consequentlyimparts further histological architecture to the developingneuraxis Third the neural tube is divided into functionallyand spatially distinct regions by a programme of inductive

interactions called neural patterning (Figure 2(c) [3]) Inhumans neurulation occurs at 21 days after conception anddepends on a precise sequence of changes in the three-dimensional shape of individual cells including changesin cell-cell adhesion Specific gene expression profiles arecontrolled by neuraxial position and local extrinsic mor-phogenetic instruction Gastrulation leads to the formationof the notochord a distinct cylinder of mesodermal cellsextending along the midline Ectoderm lies adjacent to thenotochord from which it receives inductive signals to formneuroectoderm Neuroepithelium of the neural plate thenundergoes complex morphogenetic movements involvingcell division morphological changes and migration to per-mit neural tube formation Following neural tube closurethe dorsomedial borders of the neural folds become neuralcrest derivatives Cell movements at this stage are critical inproducing different neuraxial regions For example in theventral midline of the neural tube cells become a specialized

Stem Cells International 3

AVEAVE

NodeNeuralinductionNeuroectoderm


MousehumanE75E16-E17

(a)

Neural crest

Neural crest

Neural tube

MousehumanNeurulation

E75ndashE9E16ndashE20(b)

Forebrain

Midbrain

Hindbrain

Spinal cord

Neural patterningMousehuman

E75ndashE10E16ndashE24

Rostral

Caudal

(c)

Figure 2 Neural induction neurulation and neural patterning overview (a) Neural induction neuroectoderm (neural plate) differentiationhappens under the influence of theAVEThemesodermal cells startmigrating in all directions and envelop the embryo between the endodermand the ectoderm At the distal pole of the embryo the node develops to further act as the ldquotrunk organiserrdquo (b) Neurulation from the neuralplate cells start to proliferate and invaginate in order to form the neural tube and neural crest which derives from the dorsomedial bordersof the neural folds (c) Neural patterning cells from the neural tube start to differentiate into precursors for forebrain midbrain hindbrainand spinal cord according to a rostrocaudal axis All timelines are given for mouse and human embryonic development

region called the floor plate (Figure 4(d)) A large variety ofdistinct neuronal subtypes are generated during mammalianneurodevelopment This diversity is an absolute prerequisitefor the establishment of functional neuronal circuits

In summary the consecutive steps of neurodevelopmentinclude neural induction from embryonic ectoderm pat-terning along rostrocaudal (R-C) and dorsoventral (D-V)axes (allowing regionally determined functional heterogene-ity) and subsequently terminal differentiation into diversepostmitotic neuronal subtypes [2] Such insights from devel-opmental neurobiology provide a conceptual framework forthe directed differentiation of hPSCs and allow experimentalinterrogation of the molecular ldquologicrdquo of neuronal subtypediversification [4] Taken together with the understandingthat region andor subtype specific degeneration of neuronsunderpin the majority of neurodegenerative diseases thesefacts provide a compelling rationale to predictably manip-ulate the cell fate of hPSCs in order to generate clinicallyrelevant populations of region specific neurons and glia forfurther study [5]

2 Neural Induction

The first mechanistic insights into neural induction originatefrom seminal experiments by Spemann and Mangold in theearly part of the twentieth century In these studies dorsalmesodermwas transplanted into the ventral embryo and gen-erated a secondary host-derived neural tube The graft itselfwas found to contribute to secondary mesodermal structuresincluding the notochord while the neural tissue was host-derived The ability of the dorsal blastopore lip to reprogramsurrounding tissues when transplanted ectopically justifiesits designation as ldquoorganiser tissuerdquo Equivalent organiserregions in other vertebrates were subsequently discoveredby the elegant work of Waddington in the 1930s includingldquoHensenrsquos noderdquo in birds andmammals (Figure 2(a)) Organ-iser tissuersquos capacity to precipitate ectopic neural inductioninterspecies suggests evolutionary conservation of underlyingmechanisms The notion of inductive signals orchestratingthe process of neural induction has become widely accepted

Accumulating evidence suggests a spatiotemporal interde-pendence of several signalling pathways in neural induc-tion which somewhat challenges the concept of organisertissue The molecular pathways underlying neural inductionremained elusive until the 1990s when Xenopus studies firstreported that transient dissociation of gastrula-stage animalcaps into single cells resulted in neural fate acquisition andthat misexpression of a dominant-negative Activin receptorsince being discovered to inhibit multiple transforminggrowth factor (TGF120573-) related factors ectopically generatedneural tissue at the expense of mesoderm specificationThesestudies suggest that neural induction may occur through aldquode-repressionrdquo strategy (ie the removal of an inhibitorysignal) Figure 3 depicts the relevant pathways in this process

21TheRole of TGF-120573 Signalling SuperfamilyMembers inNeu-ral Induction Themolecular machinery of TGF-120573 signallingis relatively well understood ligand binding causes receptordimerization and initiates a signal transduction pathway andactivates a family of cytoplasmic proteins the Smads byphosphorylation Eight Smad proteins are encoded in thehuman genome although only five of these (Smad 1 Smad2 Smad 3 Smad 5 and Smad 8) act as substrates for the TGFreceptor family these are commonly referred to as ldquoreceptor-regulated Smadsrdquo or just ldquoRSmadsrdquo Broadly the TGF-120573signalling superfamily encompasses both the ActivinNodaland bone morphogenetic protein (BMP) signalling pathways[6] The substrates for BMP signalling are Smads 1 5 and8 while the ActivinNodal receptors activate Smads 2 and3 Co-Smad (Smad 4) functions as a common partner forall RSmads whereas Smad 6 and Smad 7 are inhibitorySmadSmad 4 complexes translocate to the nucleus andactivate gene expression

211 BMPAntagonism In the early 1990sNoggin Follistatinand Chordin were identified as genes encoding proteinswith neuralizing activity that were expressed in organisertissue These proteins are inhibitors of BMP signalling witha particular bias towards antagonising BMP4 an inhibitor


+

+

+

WNT NODAL


BMP4

DVE

(a)

Dickkopf


Cerberus1LeftyCaudal

RostralBMP4

FGF8

DVE

WNT NODAL

(b)

DickkopfVentral Dorsal


Cerberus1Lefty

BMP4

FGF8

AVE

WNT

NODAL

(c)

Ventral Dorsal

Neuroectoderm


Caudal

Rostral

(d)

Figure 3Molecular pathways in neural induction (a)The epiblast (depicted in pink) expresses NodalThe epiblast throughNodal stimulates(pink arrow) the expression of BMP4 (depicted in blue) in the extraembryonic ectoderm (blue cells) The extraembryonic ectoderm by theaction of BMP4 stimulates (blue arrow) the WNT (depicted in pink) pathway in the epiblast that in turn further activates (pink arrow)Nodal expression Thus there is a positive feedback loop between Nodal BMP and WNT Colour scheme arrows corresponds to therelated tissuemorphogen (b) The DVE (depicted in red) expresses Cerberus1 and Lefty (also depicted in red) to inhibit Nodal expressiontherefore downregulating Nodal in its proximity It also expresses Dickkopf (depicted in red) a protein that inhibits WNT3 signals closeto the DVE Downregulating Nodal and WNT also inhibits BMP4 expression close to the DVE Thus there is a gradient of Nodal WNTand BMP with a high expression rostrally and low expression caudally FGF8 (pink and blue) expressed both in the epiblast (pink) andextraembryonic ectoderm (blue) also inhibits BMP4 contributing to the gradient Colour scheme arrows corresponds to the secretedinhibitory moleculestissue source (DVE) They show the consequence of the negative feedback that creates the morphogen gradients in theR-C axis (c)TheDVEmigrates into the AVE and the gradients are thus remodelled with lowNodalWNT and BMP expression ventrally andhigh dorsally (d) Due to these gradients the neuroectoderm is formed at the ventral pole of the epiblast Colour scheme arrows correspondsto the secreted inhibitory moleculestissue source (AVE) They show the consequence of the negative feedback that creates the morphogengradients in the D-V axis All timelines are given for mouse and human embryonic development

of neural fate BMP4 is expressed widely at the onset ofgastrulation (Figure 3(a)) but is subsequently downregulatedin the neural plate following the emergence of the organiserregion (Figure 2(b)) Blockade of BMP signalling leads to anexpanded neural plate in whole embryos while mice withnull mutations in BMP antagonists (such as Noggin andChordin) show a significantly reduced brain size [1] Thewider roles of BMP pathway in embryo development arecomprehensively reviewed elsewhere [7]

These facts taken together allow a simple molecularpathway for neural induction to be considered the extraem-bryonic ectoderm produces BMPs to promote epidermaldifferentiation while neural inducing regions (organisertissues) antagonize BMPs to permit neural induction (Figures3(a)ndash3(d)) This can be achieved by blocking BMP mRNAat the pregastrula stage by Fibroblast Growth Factor (FGF)Alternatively the BMP protein can be antagonised at the gas-trula stage by aforementioned factors secreted from organiserregions Against this background the ldquodefault modelrdquo of neu-ral induction was formulated hypothesizing that gastrula-stage ectodermal cells have an autonomous predilection to

differentiate into neural tissue and that this process is inhib-ited by BMPs In contrast to this model subsequent studieshave demonstrated that organiser tissueBMP antagonismcan be dispensable for neural induction suggesting that addi-tional mechanismssignalling pathways merit considerationin this review given their potential significance in informingstrategies for neural conversion of hPSCs [1 8 9]

212 ActivinNodal Antagonism A significant majority ofstudies have focused on the role of BMP inhibition inneural induction during vertebrate development Howeverthe importance of other members of the TGF-120573 superfamilyincluding Nodal is also well established [10] Nodal acts asan inhibitor of neural induction [11] while Nodal knockoutembryos show increased neuroectoderm specification [12] Arole forNodal inhibition in neural induction frommouse andhuman embryonic stem cells (ESCs) is well established bothalone [13ndash15] and combinatorially with BMP antagonism[16] Nodal is expressed throughout the epiblast (Figure 3(a))and inhibitors of this pathway have been identified in theDVEAVE [17] which play crucial regulatory roles both in


Forebrain

Midbrain

Hindbrain

Spinal cord

Rostral

Caudal

MousehumanE75ndashE10E16ndashE24

(a)


(b)

Mousehuman

DorsalVentral

SHH WNT

BMP4

E75ndashE10moreE16ndashE24more

NODAL BMP4 RA FGF8

PAX6 OTX2

OTX2EN1PAX2

EN1PAX2 GBX2 FGF8

Mousehuman

Floor plate

Roof plate

V3

SHH

BMP4 WNT

Ventral

Dorsal

V0

V1

V2MN


(d)(c)

minus

minus

Figure 4 Neural patterning (a) Rostrocaudal gradients of Nodal BMP4 RA (retinoic acid) and FGF8 important in rostrocaudal patterning(b) The interplay between different factors encoding forebrain (PAX6 and OTX2) midbrain (PAX6 OTX2 and EN1PAX2) and hindbrain(EN1PAX2 GBX2 and FGF8) The forebrain-midbrain barrier is defined by the mutually exclusive expression of PAX6 (forebrain) andEN1PAX2 (midbrain) while the midbrain-hindbrain boundary by OTX2 (midbrain) and GBX2 (hindbrain) OTX2 and GBX2 are regulatedby FGF8 expression (c) Dorsoventral patterning with dorsal gradients for BMP4 and WNT and with a ventral gradient of SHH (Sonichedgehog) (d) Transverse section through the neural tube depicting various neurons specified by the gradient of SHH from the floor plateand the BMP4 and WNT from the roof plate V0ndash3 interneurons and MN motor neurons

neural induction and in repositioning morphogen gradientsbetween the R-C and D-V axes (Figures 3(b) and 3(c))Against this background we and others have utilised Nodalantagonism alone to achieve neural specification from hPSCsin suspension culture [14ndash16 18] although the most widelyadopted approach to neural conversion fromhPSCs is termeddual-Smad inhibition and utilises both Nodal and BMP4antagonists in combination [16]

22 Other Factors Implicated in Neural Induction

221 Fibroblast Growth Factors (FGFs) FGFs are a diversecollection of secreted diffusible glycoproteins that act bybinding with differential affinity to four classes of extracellu-lar receptor (FGFR 1ndash4) The precise role of FGF signallingin neural induction remains controversial but studies col-lectively suggest an early function to promote competence

for neural conversion and later functions in transcriptionalantagonism of BMP Another important member of theFGF family FGF8 is expressed in the mouse embryo inthe extraembryonic ectoderm and the epiblast before andduring gastrulation (Figures 3(b) and 3(c)) FGF8 activatescalcineurin which dephosphorylates Smad 15 the maincomponents of the BMP4 pathway [19] Thus FGF8 caninhibit BMP4 signalling leading to neural induction Thisfinding further supports the complexity of neural inductionand somewhat challenges the previous ldquodefaultrdquo modelHuman PSC biology has also contributed to understand-ing the relevance of FGF in neural induction with somestudies demonstrating that FGF withdrawal or antagonism(together with Nodal and BMP4 antagonism) facilitatesneural conversion [20ndash22] and others suggesting that FGFhas neural inducing capacity [23ndash26] These seemingly con-tradictory findings can be at least partially reconciled through


recognition that different culture conditions were employedin each of these studies (eg monolayer versus suspensionculture different programmes of coadministered extrinsicsignals) which may alter the influence of FGF on neuralinduction in a context-dependent fashion

222 WNT Signalling WNTs are secreted glycoproteinsresponsible for establishment of the dorsoventral axis of theembryo a direct consequence of which is the acquisition ofneural identity Administration of mRNA encoding WNTs(or their effectors) into the animal hemisphere of one-cellembryos by injection generates ectopic neural tissue WNTsignalling is itself activated by BMP4 and implicated in aNodal positive feedback loop [27] (Figure 3(a)) The AVEsecretes Dickkopf a WNT pathway antagonist contributinginitially to the R-C and later the D-V Nodal gradient(Figures 3(b) and 3(c)) However WNT3 activation doesnot impair neural induction in mouse embryos [28] mESCs[29] and hiPSCs [30] An extra layer of complexity is addedby the different ways in which WNT can act throughoutdevelopment the canonical 120573-catenin pathway (to promoteproliferation) or the noncanonical JNK pathway (to promoteneuronal differentiation) in an FGF2-dependentmanner [31]

These findings collectively suggest that neuroectodermspecification is likelymore complex than the ldquodefaultrdquo (BMP4inhibition) or ldquoorganiserrdquo (combined BMP4 WNT3 andNodal inhibition) models might suggest The effects of eachrelevant signalling pathway are temporally regulated anddetermined by developmental context justifying their sys-tematic investigation (both individually and combinatorially)in the neural conversion of hPSCs [26]

3 Neural Patterning An Overview

Once specified the neuroectoderm is subsequently regional-ized along the R-C axis of the embryonic body (Figures 2(c)and 4(a)) Organiser regions can be divided into those thatare involved in generating rostral versus caudal structuresin the neuraxis [32] More specifically following gastrulationthe head organiser tissue lies under the prechordal neuralplate (anterior neurectoderm) whereas tail organiser tissuebecomes notochord and somites and lies beneath the epi-chordal neural plate (posterior neurectoderm) Interestinglythere is evidence that during neural induction in mESCsWNTandFGF signalling promote neuromesodermal precur-sors a population of cells that gives rise to spinal cord neuronsand paraxial mesoderm [29] Signals that inhibit BMPs (egNoggin) and WNTs (eg Dickkopf) stimulate productionof the prechordal plate insights which have again guidedontogeny recapitulating hPSC differentiation protocols [33]

The precise timing and mechanisms of neuraxial pat-terning remain unresolved A popular model is that neuralinduction initially specifies rostral precursors upon whichcaudalising signals subsequently respecify positional identityin a progressive and stereotyped manner to establish sub-divisions of the posterior neuraxis Some of the signallingpathways implicated in neural induction also appear to playkey roles in early R-C and D-V patterning at later stages [10]they establish a matrix of positional cues (Figures 4(a) and

4(c)) which in turn influence precursor cell fate specificationthrough graded concentrations of morphogenetic signalsIn broad terms the anterior neuroectoderm generates theforebrain and the posterior neuroectoderm gives rise to themidbrain hindbrain and spinal cord [32]TheD-V signallingpathways have more pertinent roles in generating neuralcell-type diversity within each of the aforementioned R-Csubdivisions (Figure 4(c)) It is noteworthy that othermecha-nisms such as local signals between developing neurons alsocontribute to the full ensemble of neuronal subtypes Figure 4summarizes some of the relevant concepts here which areexplained in further detail below

31 Early Patterning in the R-C Axis Evidence from animalstudies suggests that spatially and functionally distinct cellpopulations organise development of head and trunk struc-tures [32] The head organiser tissue is located in the AVEand the trunk organiser in the node and anterior primitivestreak (Figure 2(a)) A wealth of evidence implicates BMPantagonism in forebrain development (Figure 4(a)) Indeedneural conversion strategies utilising BMP antagonism inhPSCs generally report forebrain precursor specification [1623 34 35]

Studies using a range of approaches have shown that AVEis necessary for normal forebrain development with Nodalsignalling being critical in this process [1] Collectively thesestudies suggest that partial reduction of Nodal signallingprimarily affects specification of the prechordal mesendo-derm which is necessary for antagonising caudalising signalsand thus perturbs forebrain development Therefore Nodalsignalling is necessary for proper R-C patterning of theneuroectoderm (Figure 4(a)) Smad 2 and Smad 3 are requi-site intracellular effectors of Nodal signals Previous reportsimplicate Smad 23 in neural development in mice forexample Smad 2+minus and Smad 3minusminusmutant embryos exhibit aminiaturized head-like structure [36] In zebrafish injectionof mRNAs encoding dominant-negative Smad 23 mutantsalso results in a smaller head [37] However the preciseroles of Smad 23 in neural induction and neuroectodermalpatterning remain incompletely understood Against thisbackground and consistent with these findings we and othershave demonstrated that small molecule inhibition of Smad23 imposes caudal regional identity on hPSC-derived neuralprecursors [15 26]

A FGF signalling gradient operates along the R-C axis toinduce the expression of paralogous Hox genes in the neuraltube Hox genes located at one end of the cluster (31015840 end) areexpressed more rostrally in response to low levels of FGFconversely genes at the opposite end (51015840 end) are expressedcaudally in response to high levels of FGF (Figure 4(a))Different Hox genes are consequently expressed at brachial(Hox4ndashHox8) thoracic (Hox8-Hox9) and lumbar (Hox10ndashHox13) levels of the neural tube [38] The mechanisms bywhich a Hox-based transcriptional network choreographsthese processes are now being systematically resolved [39]These graded FGF signals regulate the primary Hox geneexpression pattern before further superimposed cues refinesubset-specific Hox expression Rostrally retinoic acid (RA)


regulates Hox expression at cervicalbrachial levels in part byantagonising the FGF gradient (Figure 4(a)) More caudallyGdf11 (also a member of the Tgf-120573 superfamily) plays animportant role in Hox8ndashHox10 gene expression at thoracicand lumbar neural tube regions [40]

32 Patterning in the D-V Axis The D-V arrangement ofneuraxial anatomy is closely correlated to functional organ-isation This anatomical polarity is clearly evident in thespinal cord where motor neurons reside in the ventral hornsand sensory neurons are positioned in dorsal root gangliaIn the rostral neuraxis structures such as the basal ganglia(including the substantia nigra) are ventrally located whilethe cerebral cortex is dorsally positioned R-C and D-Vpatterning is carefully integrated in a highly stereotypedmanner Broadly ventral regional specification requires acti-vation of both the Nodal and Sonic hedgehog pathways withantagonism of BMP signalling Over and beyond its rolein R-C patterning RA is required for intermediate zonespecification within the D-V axis Likewise FGF also playsimportant roles in ventral domain specification The majorcontributors to D-V axis formation are BMPs and WNTsdorsally and Sonic hedgehog ventrally [41] Distinct neuronalsubtypes are generated through interaction of opposingD-V morphogenetic gradients which form a matrix ofldquocoordinatesrdquo that combinatorially encode discrete precursordomains in a stereotyped D-V array [2 3] In the neural tubethis developmental strategy underlies motor neurogenesisand ventral interneurogenesis (Figure 4(d)) Ventral neuralpatterning results from morphogens originating from thefloor plate and the notochord In the early 1990s differentlabs cloned vertebrate homologues of the Drosophila genehedgehog which encode secreted signalling proteins Sonichedgehog (SHH) transpired as the ventrally secreted mor-phogen conferring D-V neural tube polarity (Figure 4(c))It is now well established through a variety of gain- andloss-of-function studies in different species that SHH playscrucial and indispensible roles in specifying ventral cell typesthroughout the neuroectoderm [41] SHH is first expressedin the notochord and later the floor plate likely secondary toauto induction (Figure 4(d)) Its function is concentration-dependent and its major effector mechanism is repression ofGLI3 transcription factor Spinal motor neuron generationfor example depends on two temporally distinct phasesof SHH signalling an early period where it ventralizesneural plate precursors and a late period where it promotesdifferentiation of these precursors into motor neurons atwhich point there is a concentration-dependent specificationof ventral precursors into motor neurons or interneurons(Figure 4(d))

How is positional identity imposed on precursor cellsSeveral studies have implicated a group of factors pre-dominantly the homeodomain (HD) and basic helix-loop-helix (bHLH) transcription factors as crucial regulators hereThese are expressed in strictly organised arrays along the D-V axis of the neural tube Individual proteins are designatedas classes I or II by their response to SHH signallingClass I proteins are repressed by SHH thus defining theirventral limit of expression while class II protein expression

is induced by SHH and defines dorsal expression boundariesSpecifically in the context of spinal cord development suchcross-repressive interactions allow the establishment of fivedistinct ventral precursor domains which in turn permit thespecification of distinct neuronal subtypes Gain- and loss-of-function experiments have further supported this putativemechanism across different species where ectopic expressionof HD proteins predictably changed the regional allocation ofindividual neuronal subtypes within the neural tube [38 42]A similar cross-repressive interaction between protein classesI and II also underlies the developmental ldquologicrdquo of ventralspinal neurogenesis The most ventral aspects of neuralpatterning (ie floor plate) requireNodal signalling and FGFhas also been broadly implicated in ventral patterning withinthe neuraxis [41]

SHH signalling does not appear to contribute to pattern-ing in the dorsal neural tube However BMPs have similarand complementary roles in dorsal patterning of the neuraltube and telencephalon (Figure 4(c)) These serve as theprimary dorsal morphogenetic cues by establishing a highto low concentration from dorsal to ventral positions In asimilar fashion to SHH in the ventral neural tube this BMPgradient enables distinct precursor domains to be definedthus permitting the generation of diverse dorsal neuronalsubtypes [43]

4 Directed Differentiation of hPSCs

These aforementioned developmental studies provide a con-ceptual framework to rationalise both neural inductionstrategies and bespoke programmes of morphogenetic cuesfor the directed differentiation of hPSCs to clinically relevantand region specific neurons (summarized in Figure 5 andTable 1)

41 Forebrain ldquoDefaultrdquo neural conversion from hPSCs toforebrain neuronal subtypes has been demonstrated in avariety of systems including chemically defined suspensionculture not requiring extrinsic signals as well as in anadherent culture method [16 44 45] These studies began in2007 with the discovery that a selective Rho-associated kinase(ROCK) inhibitor permits survival of dissociated hPSCsthus allowing systematic manipulations to cell fate afterdissociation [44] A year later the same lab again employedserum-free embryoid body-like (SFEB) culture but this timeto recapitulate cell intrinsic and temporally regulated corticallaminar determination in vitro [45] These and subsequentstudies have confirmed cortical layer specific expression ofdifferent markers including Reelin in layer 1 (Cajal-Retziusneurons) TBR1 andCTIP2 in deep layers and SATB2 BRN2and CUX1 in superficial cortical layers [46] Such defaultdorsal telencephalic differentiation strategies tend to give riseto predominantly glutamatergic but also some GABAergicneurons [47]

Prior to terminal differentiation if specified dorsal telen-cephalic precursors are exposed to SHH andor a WNTantagonist they are ventralised to generate subpallial deriva-tives (ie of the lateral and medial ganglionic eminencesLGE and MGE respectively) Upon terminal differentiation


Table 1

Cell type Study Culture method Programme of developmental cues for neuralconversion and patterning Duration (days)

Cortical precursors Watanabe et al 2007 [44]Serum-free

embryoid body-like(SFEB)

BMP antagonist (BMPRIA-Fc)ActivinNodal antagonist (LeftyA)Wnt antagonist (Dkk1)

35

Cortical neurons Eiraku et al 2008 [45] SFEB derivativeBMP antagonist (BMPRIA-Fc)ActivinNodal antagonist (LeftyA)Wnt antagonist (Dkk1)

45ndash60

Cortical neurons Chambers et al 2009 [16] Monolayer BMP antagonist (NOGGIN)ActivinNodal antagonist (SB431542) 19

Cortical neurons andMGELGE neurons Li et al 2009 [47] Suspension

None for cortical (endogenous Wnt)For MGE and LGE derivativesWnt antagonist (Dkk1)Sonic hedgehog (SHH)

30ndash35

Cortical neurons Shi et al 2012 [46] Monolayer BMP antagonist (NOGGIN)ActivinNodal antagonist (SB431542) 80ndash100

Midbraindopaminergic neurons Kriks et al 2011 [50] Monolayer

BMP antagonist (NOGGIN or LDN)ActivinNodal antagonist (SB431542)Sonic hedgehog (SHH and purmorphamine)Fibroblast Growth Factor 8b (FGF8b)Wnt agonist (CHIR99021)

80

Midbraindopaminergic neurons Kirkeby et al 2012 [48] Embryoid body

BMP antagonist (NOGGIN)ActivinNodal antagonist (SB431542)Wnt agonist (CT99021)Sonic hedgehog (SHH-C24II)

35

Midbraindopaminergic neurons Jaeger et al 2011 [52] Monolayer

BMP antagonist (NOGGIN)ActivinNodal antagonist (SB431542)FGFERK antagonist (PD0325901)Fibroblast Growth Factor 8b (FGF8b)Sonic hedgehog (SHH)

30ndash35

Cerebellar neurons Erceg et al 2012 [59] Embryoid body

Fibroblast Growth Factors (FGF8 FGF4 andFGF2)Retinoic acid (RA)Wnt agonists (Wnt1 Wnt3a)BMPs (BMP4 BMP6 BMP7 and GDF7)Sonic hedgehog (SHH)

35

Cerebellar neurons Muguruma et al 2015 [57] SFEBq

ActivinNodal antagonist (SB431542)Fibroblast Growth Factors (FGF2 FGF19)InsulinStromal cell-derived factor 1 (SDF-1)(co-culture with mouse granule cells togenerate Purkinje cells)

35ndash135

Cerebellar neurons Wang et al 2015 [58] Embryoid body

Fibroblast growth factor (FGF2)InsulinSonic hedgehog antagonist (cyclopamine)(coculture with rat organotypic cerebellar sliceto generate Purkinje cells)

20ndash65

Spinal cord motorneurons Li et al 2005 [63] Monolayer

Retinoic acid (RA)Sonic hedgehog (SHH)Fibroblast Growth Factor (FGF2)

21ndash35

Spinal cord motorneurons Patani et al 2011 [18] Suspension

ActivinNodal antagonist (SB431542)Sonic hedgehog (purmorphamine)Fibroblast Growth Factor (FGF2)

21ndash35

Spinal cord motorneurons Calder et al 2015 [67] Monolayer

ActivinNodal antagonist (SB431542)BMP antagonist (LDN193189)Retinoic acid (RA)

35ndash40


hiPSCepiblast equivalents

Neural induction

BMP inhibitiondual-Smad inhibition(BMP4 NODAL inhibition)

Neuroectoderm

Directeddifferentiation

Adherent culture(monolayercoculture)

orsuspension culture

Forebrain no external

Midbrain

Hindbrain

Spinal cordWNT FGF and RA

WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens

Figure 5 Methods for directed differentiation hPSCs can be directed to undergo neural conversion by applying developmental principlesof inhibiting BMP4 andor Nodal From neuroectoderm differentiation of different neuraxial regions can be achieved by recapitulatingdevelopmental morphogenetic instruction forebrain (default) midbrain (WNT FGF8 activation) hindbrain (WNT FGF8 and others RA)and spinal cord (WNT FGF8 and others RA) These gradients are shown on the right of the figure for the rostrocaudal axis Anotherimportant morphogenetic cue used in directed differentiation is SHH for its ventralising effect within the D-V axis All timelines are givenfor mouse and human embryonic development

these ventralised telencephalic cultures give rise to GABAer-gic projection neurons and interneurons Clinically relevantcell types originate from the LGE (eg medium spiny projec-tion GABAergic neurons which are relevant to Huntingtonrsquosdisease and dystonia) and the MGE (eg basal forebraincholinergic neurons relevant to Alzheimerrsquos disease) Furthersophistication can be added to the aforementioned directeddifferentiation strategies by carefully regulating SHH andWNT pathways (which orchestrate dorsoventral positionalidentity in this context) For example a low concentration ofSHH alone permits the specification of both LGE and MGEderivatives whereas if a WNT antagonist is added to SHHthe more ventral MGE (ie NKX21 expressing) neurons arepreferentially specified at the expense of LGE (ie GSX2DLX MEIS2 and ISLET1 expressing) neurons Some elegantand ontogeny recapitulating strategies have been defined forthe generation of authentic DARPP32 expressing mediumspiny projection neurons [33 47]

42 Midbrain Differentiating hPSCs into midbraindopaminergic neurons has maintained great enthusiasmlikely owing to their potential to understand and treatParkinsonrsquos disease Although dopaminergic neurons existthroughout the nervous system there is a region-specificfunctional heterogeneity that has been experimentallydemonstrated by performing anisotopic implantationexperiments [48] Midbrain dopaminergic neurons aredevelopmentally partitioned to three distinct nuclei (i) thesubstantia nigra pars compacta (A9 group) which is primar-ily affected in Parkinsonrsquos disease (ii) the ventral tegmentalarea (A10 group) and (iii) the retrorubral field (A8 group)Noting that hPSC-derived neural precursors have a defaultrostral (forebrain) and dorsal (cortical) identity morphogen-guided positional respecification or patterning to theventral mesencephalon is necessary for the differentiationof authentic midbrain dopaminergic neurons Feeder-freeand feeder-dependent differentiation approaches have bothbeen employed to generate midbrain dopaminergic neurons

from hPSCs Feeder-dependent differentiation strategieshave utilisedmouse stromal cell lines (eg PA6) which eventhough relatively easy to establish carry the main disadvan-tage of being chemically undefined and animal-derived Fromdevelopmental in vivo studies we are guided by the insightthat FGF8 signalling leads to a cross-repressive interactionbetween Otx2 and Gbx2 defining the midbrain-hindbrainboundary (MHB Figure 4(b)) and imparting rostrocaudalpositional identity to precursors of the MHB [49] Otx2and Gbx2 control patterning in this region by regulating theexpression of two morphogenetic cues WNT1 in midbrainand FGF8 in the hindbrain Furthermore in combinationwith Otx2 expression cross-repressive mechanisms betweenPax6 and En1Pax2 define boundaries of regional fateallocation to either forebrain or midbrain (Figure 4(b))

Against this background initial approaches to midbraindifferentiation were based on FGF8 for R-C patterning tothe region of the midbrain and SHH for ventralization intodopaminergic neurons although the yields were low (approx30) using such strategies Furthermore subsequent studieshave raised the possibility that PA6 and SHHFGF8-basedapproaches alone are not sufficient to generate authenticmidbrain dopaminergic neurons [50 51] The field thenunderwent a period of reevaluation where protocols thatrecapitulated ventral mesencephalic development with morefidelity and precision were developed During this timeearlier protocols were systematically refined and supersededby studies using WNT agonists [48 50] most notablyfrom the Studer Lab who established an efficient midbrainfloor plate differentiation strategy through which dopamin-ergic neurons were efficiently specified Crucially this studydemonstrated functional engraftment and recovery in micerats and nonhuman primates with Parkinsonrsquos disease [50]Contemporaneous studies showed that by using canonicalWNT agonists at different concentrations and for defineddurations the generation of diverse regionally specifiedprogenitors from fore- to hindbrain is possible Interestinglythe generated midbrain dopaminergic neurons but not


their telencephalic counterparts could reverse structural andfunctional deficits in animal models of Parkinsonrsquos diseaseThis subtype specificity highlights the unparalleled potentialof directed differentiation of hPSCs in regenerative medicine[48] A further notable study in this arena used transientblockade of FGF signalling to refine midbrain positionalidentity and yield authentic dopaminergic neurons withhigh efficiency [52] Although it can be argued that theselater studies yield more authentic midbrain dopaminergicneurons because they utilised developmentally rationalisedcues it should be noted that the GSK3120573 inhibitors suchas CHIR99021 used here for WNT pathway activation dohave off target effects (ie they regulate pathways other thanWNT) [53] Additionally it is noteworthy that more specificWNT pathway activators (eg WNT3a) do not reproduciblygenerate midbrain dopaminergic neurons with the sameefficiency as the GSK3120573 inhibitor CHIR99021 [50 54] Infuture studies the absolute requirement forGSK3120573 inhibitionand the identification of additional key regulatory pathwayswould be of great importance to establish

43 Hindbrain and Cerebellum Broadly evolutionary path-ways appear to be more conserved in caudal (primitive)regions of the CNS such as the hindbrain The hindbraincan be divided into rostral and caudal portions which areseparated by rhombomere 4 (r4) Neurons derived fromrostral regions project to and innervate myriad brain regionswhereas the caudal portion located in the myelencephalongives rise mainly to descending spinal projections Thebrain innervating central serotonergic neurons originatingfrom r2-3 of the rostral raphe contribute to higher orderbrain functions and are implicated in a range of psychi-atric disorders By using EGF and FGF2 in the mainte-nance media so-called ldquolong-term self-renewing rosette-typerdquo hPSC-derived neural precursors can be expandedwhich exhibit a ventral anterior hindbrain-like expressionprofile after prolonged culture [55] These precursors prefer-entially generated GABAergic neurons some of which wereserotonergic neurons This finding likely reflects positionalrespecification of the default forebrain identity secondaryto protracted culture in FGF2 which is known to havecaudalising properties Very recently a protocol for directeddifferentiation of hPSCs to functionally validated hindbrainserotonergic neurons through activation of the WNT andSHH pathways was reported [56]

There are few reports of cerebellar differentiation withdemonstration of electrophysiologically mature and func-tional Purkinje- and granule-cell specification [57 58]A recent study generated MATH1-positive cerebellar-likegranule cells from iPSCs using a complex programme ofsequentially administeredmorphogens including FGF8 RAFGF4 FGF2 WNT1a WNT3a BMP4 GDF7 BMP7 BMP6SHH BDNF Jagged1 and NT3 [59] More recently anontogeny recapitulating strategy for cerebellar neurogenesisachieved efficient directed differentiation of hPSCs usingthree morphogens only [57] Here hPSC-derived embryoidbodies were first positionally specified to the midbrain-hindbrain boundary and subsequently directed to cerebellarplate neuroepithelium (CPNE) CPNE in turn gave rise

to functionally mature Purkinje- and granule cells DCN-neurons and various interneurons in specific coculture set-tings by sequentially administering FGF2 FGF19 and SDF1A contemporaneous study used insulin FGF2 and an antag-onist of SHH signalling (cyclopamine) again necessitatingcoculture with rat cerebellar slices to reinforce the validityof this approach for directed differentiation to cerebellarneurons [58] Both of these recent studies relied to somedegree on coculture with isotopic organotypic slicesrodentcerebellar derivatives Future studies in this area should focuson overcoming reliance on coculture with rodent or humancerebellar slice cultures by identifying the requisite extrinsicsignals for specifying cerebellar derivatives at each stage oftheir lineage restriction

44 Spinal Cord The generation of functional spinal cordderivatives includingmotor neurons has been achieved fromhPSCs through a variety of approaches using insights fromdevelopmental biology [15 60ndash62]These strategies employedeither simultaneous or sequential administration of caudal-ising (eg RA) and ventralising (eg SHH) morphogensprior to terminal differentiation These studies confirmedthe expression of specific motor neuron fate determin-ing factors including HB9 specific enzymestransportersincluding choline acetyltransferase (ChAT) and the vesicularacetylcholine neurotransmitter transporter (vAChT) andalso coculture with myotubes to demonstrate the formationof physiologically relevant neuromuscular junctions [18 6063] Electrophysiological studies confirm that hPSC-derivedmotor neurons acquire appropriate functional properties[60] Motor neuron precursors have importantly been shownto survive and integrate in rodent embryonic spinal cord[64 65] and to project axons forming physiological synapses

Treating cultures with RA typically results in a cervicalor brachial positional identity [18 65] More caudal (lumbar)motor neuron fates can also be achieved in the absenceof RA signalling likely in response to FGF2 indeed wehave reported a retinoid independent strategy for motorneurogenesis from hPSCs that yields a lumbar spinal subtypeidentity and favours medial motor columnar specification[18] This retinoid-mediated diversification of motor neuronsubtypes was further supported by a parallel study usingmouse embryonic stem cells [65] A recent study employedcombined retinoic acid andWNTagonism to generate cranialmotor neurons from hPSCs [66] Yet another subsequentstudy reported the derivation of motor neurons under RAtreated but SHH free conditions uncovering importantinsights into humanmotor neurodevelopmental biology [67]

45 Neural Crest Neural crest cells are highly migratoryand give rise to myriad differentiated cell types including(i) sensory and autonomic neurons and Schwann cells (ii)chromaffin cells in the adrenal medulla (iii) melanocytesand (iv) cranial skeletal and connective tissue componentsThe fate of the neural crest cells is largely determined bywhere they migrate tosettle From an hPSC perspectivestriking phenotypic consequences have been demonstratedbased on plating density and this provides a strategy to


generate neural crest derivatives A high plating densityfavours PAX6 expressing central nervous system precursorswhile low plating density specified neural crest-like differen-tiation [16] Using variations of this approach stage-specificisolationdifferentiation of hPSC-derived neural crest cellshas been achieved using a combination of in vitro expansiondirected differentiation via extrinsic signals and cell sortingFor example serum-free conditionswith subsequent bespokeprogrammes of extrinsic cues can permit specification ofSchwann cells autonomic or sensory neurons while serumbased approaches tend to favour mesenchymal derivativesincluding adipocytes osteocytes chondrocytes and smoothmuscle Functional validation has been demonstrated bytransplantation of hPSC-derived neural crest cells into a chickembryo where they exhibit preserved neural crest identity inthe context of survival migration and differentiation [68]

5 Concluding Remarks

Theunrivalled complexity of themammalian central nervoussystem is enabled by a series of progressive and sequentialevents during embryogenesis The degree of interconnect-edness within the central neuraxis is somewhat surpris-ing given its impressively precise organisation into discreteregions Evolutionary conservation of developmental pro-cesses underlying the organisation of such discrete neuralregions becomes increasingly less applicable to more rostral(ie evolutionarily ldquonewerrdquo) components like the forebrainThe hPSC platform is emerging as an important reductionistin vitro system to interrogate aspects of human developmentwhich have remained experimentally inaccessible until now

Current approaches towards such directed differentiationof hPSCs often fail to capture the dynamic and overlappingnature of neurodevelopmental processes For instance neuralinduction and patterning are often conceptualised as mecha-nistically distinct processes However a bias towards differentregional fates will likely be determined by the neural con-version paradigm employed Similarly current differentiationstrategies do not yet fully acknowledge or exploit the ability toinfluence cell (subtype) fate decisions postmitotically whichhas been reported [69ndash71] As such the fieldrsquos approach todirected differentiation to individual cellular subtypes couldpotentially benefit from being more closely aligned to eachrespective stage of neurodevelopment leading to bespokeconditions for each stage of lineage restriction (ie neuralconversion patterning and terminal differentiation)

Developmental principles are a crucial resource for defin-ing ontogeny recapitulating directed differentiation protocolsfor hPSCs (Figure 5) In addition to the wealth of knowl-edge that already exists from rodent developmental biologythere is an increasing number of publicly available humanbrain region-specific and transcriptome-wide datasets fromstudies using a diverse range of tissue from fetal throughto adult stages [72ndash74] In addition to highlighting thematurational status of hPSC-derived neurons [75] suchdevelopmentalstage-specific data sets could now serve asa gold standard for validating directed differentiation pro-tocols to region-specific cell types Indeed these datasetsshould eventually contribute to experimental design when

a relatively unexplored region of the nervous system is beinginvestigated using hPSCs The utilisation of human brain-derived data bypasses potential issues of evolutionary diver-gence betweenmouse andman especially in themore rostral(evolutionarily newer) regions of the neuraxis Couplinginsights gained from these invaluable resources together withhigh throughput platforms for protocol discovery would bea future avenue for improving the robustness of currentdirected differentiation strategies [66]

Finally the hPSC field stands to benefit from defin-ing multiple directed differentiation protocols that employclosely aligned methods for neural conversion and similarprotocol durations This may then permit more meaningfulcomparison between region-specific neurons without thepotentially confounding issue of differential cellular matu-ration Indeed such an approach was recently utilised toshow region-specific phenotypes using iPSCs derived frompatients with Alzheimerrsquos disease and motor neuron disease[76] Taken together such standardizations in directed dif-ferentiation of hPSCs may help to drive the identificationof robust strategies to specify enriched populations of allclinically relevant region-specific subpopulations of humanneurons for further study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Alexandra Zirra and Sarah Wiethoff performed literatureresearch contributed to writing of the paper and constructedfigures Rickie Patani directed the analysis and wrote thepaper

Acknowledgments

Sarah Wiethoff is supported by a BRT-Studentship (BrainResearch Trust) Rickie Patani is a Wellcome Trust Interme-diate Clinical Fellow (101149Z13Z) and an Anne RowlingFellow in Regenerative Neurology

References

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[2] C G Becker and R Diez Del Corral ldquoNeural development andregeneration itrsquos all in your spinal cordrdquo Development vol 142no 5 pp 811ndash816 2015

[3] A Kicheva and J Briscoe ldquoDevelopmental pattern formation inphasesrdquo Trends in Cell Biology vol 25 no 10 pp 579ndash591 2015

[4] L A Williams B N Davis-Dusenbery and K C EgganldquoSnapShot directed differentiation of pluripotent stem cellsrdquoCell vol 149 no 5 pp 1174ndash1174e1 2012


[5] S S W Han L A Williams and K C Eggan ldquoConstructingand deconstructing stem cell models of neurological diseaserdquoNeuron vol 70 no 4 pp 626ndash644 2011

[6] J Massague ldquoTGF120573 signalling in contextrdquo Nature ReviewsMolecular Cell Biology vol 13 no 10 pp 616ndash630 2012

[7] E Bier and E M De Robertis ldquoEMBRYO DEVELOPMENTBMP gradients a paradigm for morphogen-mediated develop-mental patterningrdquo Science vol 348 no 6242 2015

[8] E M Pera H Acosta N Gouignard M Climent and I ArregildquoActive signals gradient formation and regional specificity inneural inductionrdquo Experimental Cell Research vol 321 no 1 pp25ndash31 2014

[9] M Z Ozair C Kintner and A H Brivanlou ldquoNeural inductionand early patterning in vertebratesrdquo Wiley InterdisciplinaryReviews Developmental Biology vol 2 no 4 pp 479ndash498 2013

[10] A J Levine and A H Brivanlou ldquoProposal of a model ofmammalian neural inductionrdquoDevelopmental Biology vol 308no 2 pp 247ndash256 2007

[11] L Vallier D Reynolds and R A Pedersen ldquoNodal inhibitsdifferentiation of human embryonic stem cells along the neu-roectodermal default pathwayrdquoDevelopmental Biology vol 275no 2 pp 403ndash421 2004

[12] A Camus A Perea-Gomez A Moreau and J CollignonldquoAbsence of Nodal signaling promotes precocious neural dif-ferentiation in the mouse embryordquo Developmental Biology vol295 no 2 pp 743ndash755 2006

[13] K Watanabe D Kamiya A Nishiyama et al ldquoDirected dif-ferentiation of telencephalic precursors from embryonic stemcellsrdquo Nature Neuroscience vol 8 no 3 pp 288ndash296 2005

[14] J R Smith L Vallier G Lupo M Alexander W A Harrisand R A Pedersen ldquoInhibition of ActivinNodal signalingpromotes specification of human embryonic stem cells intoneuroectodermrdquoDevelopmental Biology vol 313 no 1 pp 107ndash117 2008

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[17] M Yamamoto Y Saijoh A Perea-Gomez et al ldquoNodal antago-nists regulate formation of the anteroposterior axis of themouseembryordquo Nature vol 428 no 6981 pp 387ndash392 2004

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[20] LVallierMAlexander andRA Pedersen ldquoActivinNodal andFGF pathways cooperate to maintain pluripotency of humanembryonic stem cellsrdquo Journal of Cell Science vol 118 no 19pp 4495ndash4509 2005

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[39] H Jung J Lacombe E O Mazzoni et al ldquoGlobal control ofmotor neuron topography mediated by the repressive actionsof a single hox generdquo Neuron vol 67 no 5 pp 781ndash796 2010

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[42] J S Dasen and T M Jessell ldquoHox networks and the originsof motor neuron diversityrdquo in Current Topics in DevelopmentalBiology vol 88 chapter 6 pp 169ndash200 Elsevier New York NYUSA 2009

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[46] Y Shi P Kirwan J Smith H P C Robinson and F J LiveseyldquoHuman cerebral cortex development from pluripotent stemcells to functional excitatory synapsesrdquo Nature Neurosciencevol 15 no 3 pp 477ndash486 2012

[47] X-J Li X Zhang M A Johnson Z-B Wang T LaVauteand S-C Zhang ldquoCoordination of sonic hedgehog and Wntsignaling determines ventral and dorsal telencephalic neurontypes fromhuman embryonic stem cellsrdquoDevelopment vol 136no 23 pp 4055ndash4063 2009

[48] A Kirkeby S Grealish D A Wolf et al ldquoGeneration ofregionally specified neural progenitors and functional neuronsfrom human embryonic stem cells under defined conditionsrdquoCell Reports vol 1 no 6 pp 703ndash714 2012

[49] NA Sunmonu K Li QGuo and J YH Li ldquoGbx2 and Fgf8 aresequentially required for formation of the midbrain-hindbraincompartment boundaryrdquo Development vol 138 no 4 pp 725ndash734 2011

[50] S Kriks J-W Shim J Piao et al ldquoDopamine neurons derivedfrom human ES cells efficiently engraft in animal models ofParkinsonrsquos diseaserdquoNature vol 480 no 7378 pp 547ndash551 2011

[51] M Denham L HThompson J Leung A Pebay A Bjorklundand M Dottori ldquoGli1 is an inducing factor in generating floorplate progenitor cells from human embryonic stem cellsrdquo StemCells vol 28 no 10 pp 1805ndash1815 2010

[52] I Jaeger C Arber J R Risner-Janiczek et al ldquoTemporallycontrolled modulation of FGFERK signaling directs midbraindopaminergic neural progenitor fate in mouse and humanpluripotent stem cellsrdquo Development vol 138 no 20 pp 4363ndash4374 2011

[53] E-M Hur and F-Q Zhou ldquoGSK3 signalling in neural develop-mentrdquo Nature Reviews Neuroscience vol 11 no 8 pp 539ndash5512010

[54] E R Andersson C Salto J C Villaescusa et al ldquoWnt5a coop-erates with canonical Wnts to generate midbrain dopaminergicneurons in vivo and in stem cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 110 no7 pp E602ndashE610 2013

[55] P Koch T Opitz J A Steinbeck J Ladewig and O BrustleldquoA rosette-type self-renewing human ES cell-derived neuralstem cell with potential for in vitro instruction and synapticintegrationrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 106 no 9 pp 3225ndash3230 2009

[56] J Lu X Zhong H Liu et al ldquoGeneration of serotonin neuronsfrom human pluripotent stem cellsrdquo Nature Biotechnology vol34 pp 89ndash94 2016

[57] KMuguruma A Nishiyama H Kawakami K Hashimoto andY Sasai ldquoSelf-organization of polarized cerebellar tissue in 3Dculture of human pluripotent stem cellsrdquo Cell Reports vol 10no 4 pp 537ndash550 2015

[58] S Wang B Wang N Pan et al ldquoDifferentiation of humaninduced pluripotent stem cells to mature functional Purkinjeneuronsrdquo Scientific Reports vol 5 article 9232 2015

[59] S Erceg D Lukovic V Moreno-Manzano M Stojkovic and SS Bhattacharya ldquoDerivation of cerebellar neurons from humanpluripotent stem cellsrdquo Current Protocols in Stem Cell Biologychapter 1 unit 1H 5 2012

[60] J S Toma B C Shettar P H Chipman et al ldquoMotoneuronsderived from induced pluripotent stem cells develop maturephenotypes typical of endogenous spinal motoneuronsrdquo TheJournal of Neuroscience vol 35 no 3 pp 1291ndash1306 2015

[61] X-J Li B-Y Hu S A Jones et al ldquoDirected differentiationof ventral spinal progenitors and motor neurons from humanembryonic stem cells by small moleculesrdquo STEM CELLS vol26 no 4 pp 886ndash893 2008

[62] M W Amoroso G F Croft D J Williams et al ldquoAcceleratedhigh-yield generation of limb-innervating motor neurons fromhuman stem cellsrdquoThe Journal of Neuroscience vol 33 no 2 pp574ndash586 2013

[63] X-J Li Z-W Du E D Zarnowska et al ldquoSpecificationof motoneurons from human embryonic stem cellsrdquo NatureBiotechnology vol 23 no 2 pp 215ndash221 2005

[64] H Wichterle I Lieberam J A Porter and T M JessellldquoDirected differentiation of embryonic stem cells into motorneuronsrdquo Cell vol 110 no 3 pp 385ndash397 2002

[65] M Peljto J S Dasen E O Mazzoni T M Jessell and HWichterle ldquoFunctional diversity of ESC-derived motor neuronsubtypes revealed through intraspinal transplantationrdquo CellStem Cell vol 7 no 3 pp 355ndash366 2010

[66] Y Maury J Come R A Piskorowski et al ldquoCombinatorialanalysis of developmental cues efficiently converts humanpluripotent stem cells into multiple neuronal subtypesrdquo NatureBiotechnology vol 33 no 1 pp 89ndash96 2015

[67] E L Calder J Tchieu J A Steinbeck et al ldquoRetinoic acid-mediated regulation of GLI3 enables efficient motoneuronderivation from human ESCs in the absence of extrinsic SHHactivationrdquo The Journal of Neuroscience vol 35 no 33 pp11462ndash11481 2015

[68] G Lee H Kim Y Elkabetz et al ldquoIsolation and directeddifferentiation of neural crest stem cells derived from humanembryonic stem cellsrdquo Nature Biotechnology vol 25 no 12 pp1468ndash1475 2007

[69] T M Jessell ldquoNeuronal specification in the spinal cord induc-tive signals and transcriptional codesrdquoNature Reviews Geneticsvol 1 no 1 pp 20ndash29 2000

[70] J Livet M Sigrist S Stroebel et al ldquoETS gene Pea3 controlsthe central position and terminal arborization of specific motorneuron poolsrdquo Neuron vol 35 no 5 pp 877ndash892 2002


[71] C M William Y Tanabe and T M Jessell ldquoRegulation ofmotor neuron subtype identity by repressor activity of Mnxclass homeodomain proteinsrdquo Development vol 130 no 8 pp1523ndash1536 2003

[72] J A Miller S-L Ding S M Sunkin et al ldquoTranscriptionallandscape of the prenatal human brainrdquo Nature vol 508 no7495 pp 199ndash206 2014

[73] H J Kang Y I Kawasawa F Cheng et al ldquoSpatio-temporaltranscriptome of the human brainrdquo Nature vol 478 no 7370pp 483ndash489 2011

[74] A Ramasamy D Trabzuni S Guelfi et al ldquoGenetic variabilityin the regulation of gene expression in ten regions of the humanbrainrdquo Nature Neuroscience vol 17 no 10 pp 1418ndash1428 2014

[75] R Patani P A LewisD Trabzuni et al ldquoInvestigating the utilityof human embryonic stem cell-derived neurons to model age-ing and neurodegenerative disease using whole-genome geneexpression and splicing analysisrdquo Journal ofNeurochemistry vol122 no 4 pp 738ndash751 2012

[76] K Imaizumi T Sone K Ibata et al ldquoControlling the regionalidentity of hPSC-derived neurons to uncover neuronal sub-type specificity of neurological disease phenotypesrdquo Stem CellReports vol 5 no 6 pp 1010ndash1022 2015

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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Proximalrostral (anterior)

Distalcaudal (posterior)

Dorsal(posterior)

Oocyte Blastomere

2-cell stage 4-cell stage 8-cell stage 16-cell stage

Ventral(anterior)

Inner cell massOct34

TrophectodermCdx2

Early blastocyst

Late blastocyst

EpiblastOct34 + Nanog

Oct34 + GATA4Endoderm

Ectoplacental cone

Extraembryonic ectoderm

Embryonicendoderm

Extraembryonicendoderm

Cdx2

AVE (anterior visceralendoderm)

DVE (distal visceralendoderm)

Mesoderm(primitive streak)



MousehumanE6-E7E13ndashE17

Gastrulation

Figure 1 Developmental stages of mouse embryo First row (left to right) from the secondary oocyte the blastomere develops (2-cell 4-cell 8-cell and 16-cell stages) to give rise to the early blastocyst formed of trophectoderm (cells that express Cdx2) and inner cell mass cells(that express Oct34) Later the inner cell mass gives rise to the epiblast (cells that express Oct34 and Nanog) and endoderm (expressingOct34 and GATA4) Second row (right to left) in the late mouse blastocyst Cdx2 positive cells give rise to the extraembryonic ectoderm andectoplacental cone At the same time the endoderm divides into an embryonic endoderm and an extraembryonic endodermThe epiblast andthe extraembryonic ectoderm form a cavity lined by embryonic endoderm From the embryonic endoderm the distal visceral endoderm isformed (DVE) Third row (left to right) the DVE migrates proximally and will be known as the anterior visceral endoderm (AVE) The finalimage (third row right) shows the development of the primitive streak (mesodermal cells) at the opposite (posterior) pole from the AVE NBThere are 2 different types of endoderm called extraembryonic and embryonic these differ in their potency and give rise to distinct cellularderivatives All timelines are given for mouse and human embryonic development

the ectoplacental cone as depicted in Figure 1) Later theproximal-distal axis will become the future rostrocaudal axisin vertebrates However the term ldquoanterior-posterior axisrdquocan also sometimes refer to the dorsoventral axis in the adultstate a distinction that is primarily based on position of theabdomen (ventral) as opposed to the backspinal column(dorsal) Therefore for ease of reference this review will usethe terms rostrocaudal (ldquoR-Crdquo) and dorsoventral (ldquoD-Vrdquo)axes

Three principal events characterise early neurodevel-opment First the process of neural induction specifies aregion of the embryonic ectoderm to form the neural plate(Figure 2(a) [1]) Second a process termedneurulation occursthrough serial morphological transformations to give rise tothe neural tube (Figure 2(b) [2]) This process consequentlyimparts further histological architecture to the developingneuraxis Third the neural tube is divided into functionallyand spatially distinct regions by a programme of inductive

interactions called neural patterning (Figure 2(c) [3]) Inhumans neurulation occurs at 21 days after conception anddepends on a precise sequence of changes in the three-dimensional shape of individual cells including changesin cell-cell adhesion Specific gene expression profiles arecontrolled by neuraxial position and local extrinsic mor-phogenetic instruction Gastrulation leads to the formationof the notochord a distinct cylinder of mesodermal cellsextending along the midline Ectoderm lies adjacent to thenotochord from which it receives inductive signals to formneuroectoderm Neuroepithelium of the neural plate thenundergoes complex morphogenetic movements involvingcell division morphological changes and migration to per-mit neural tube formation Following neural tube closurethe dorsomedial borders of the neural folds become neuralcrest derivatives Cell movements at this stage are critical inproducing different neuraxial regions For example in theventral midline of the neural tube cells become a specialized


AVEAVE




(a)

Neural crest

Neural crest

Neural tube



Forebrain

Midbrain

Hindbrain

Spinal cord



Rostral

Caudal

(c)




2 Neural Induction






+

+

+

WNT NODAL


BMP4

DVE

(a)

Dickkopf



RostralBMP4

FGF8

DVE

WNT NODAL

(b)



Cerberus1Lefty

BMP4

FGF8

AVE

WNT

NODAL

(c)

Ventral Dorsal

Neuroectoderm


Caudal

Rostral

(d)







Forebrain

Midbrain

Hindbrain

Spinal cord

Rostral

Caudal


(a)


(b)

Mousehuman

DorsalVentral

SHH WNT

BMP4


NODAL BMP4 RA FGF8

PAX6 OTX2

OTX2EN1PAX2

EN1PAX2 GBX2 FGF8

Mousehuman

Floor plate

Roof plate

V3

SHH

BMP4 WNT

Ventral

Dorsal

V0

V1

V2MN


(d)(c)

minus

minus




























Table 1





35


45ndash60




30ndash35




80



35



30ndash35



35



35ndash135



20ndash65



21ndash35



21ndash35



35ndash40



Neural induction


Neuroectoderm





Midbrain

Hindbrain


WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens


























Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AVEAVE




(a)

Neural crest

Neural crest

Neural tube



Forebrain

Midbrain

Hindbrain

Spinal cord



Rostral

Caudal

(c)




2 Neural Induction






+

+

+

WNT NODAL


BMP4

DVE

(a)

Dickkopf



RostralBMP4

FGF8

DVE

WNT NODAL

(b)



Cerberus1Lefty

BMP4

FGF8

AVE

WNT

NODAL

(c)

Ventral Dorsal

Neuroectoderm


Caudal

Rostral

(d)







Forebrain

Midbrain

Hindbrain

Spinal cord

Rostral

Caudal


(a)


(b)

Mousehuman

DorsalVentral

SHH WNT

BMP4


NODAL BMP4 RA FGF8

PAX6 OTX2

OTX2EN1PAX2

EN1PAX2 GBX2 FGF8

Mousehuman

Floor plate

Roof plate

V3

SHH

BMP4 WNT

Ventral

Dorsal

V0

V1

V2MN


(d)(c)

minus

minus




























Table 1





35


45ndash60




30ndash35




80



35



30ndash35



35



35ndash135



20ndash65



21ndash35



21ndash35



35ndash40



Neural induction


Neuroectoderm





Midbrain

Hindbrain


WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens


























Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


+

+

+

WNT NODAL


BMP4

DVE

(a)

Dickkopf



RostralBMP4

FGF8

DVE

WNT NODAL

(b)



Cerberus1Lefty

BMP4

FGF8

AVE

WNT

NODAL

(c)

Ventral Dorsal

Neuroectoderm


Caudal

Rostral

(d)







Forebrain

Midbrain

Hindbrain

Spinal cord

Rostral

Caudal


(a)


(b)

Mousehuman

DorsalVentral

SHH WNT

BMP4


NODAL BMP4 RA FGF8

PAX6 OTX2

OTX2EN1PAX2

EN1PAX2 GBX2 FGF8

Mousehuman

Floor plate

Roof plate

V3

SHH

BMP4 WNT

Ventral

Dorsal

V0

V1

V2MN


(d)(c)

minus

minus




























Table 1





35


45ndash60




30ndash35




80



35



30ndash35



35



35ndash135



20ndash65



21ndash35



21ndash35



35ndash40



Neural induction


Neuroectoderm





Midbrain

Hindbrain


WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens


























Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Forebrain

Midbrain

Hindbrain

Spinal cord

Rostral

Caudal


(a)


(b)

Mousehuman

DorsalVentral

SHH WNT

BMP4


NODAL BMP4 RA FGF8

PAX6 OTX2

OTX2EN1PAX2

EN1PAX2 GBX2 FGF8

Mousehuman

Floor plate

Roof plate

V3

SHH

BMP4 WNT

Ventral

Dorsal

V0

V1

V2MN


(d)(c)

minus

minus




























Table 1





35


45ndash60




30ndash35




80



35



30ndash35



35



35ndash135



20ndash65



21ndash35



21ndash35



35ndash40



Neural induction


Neuroectoderm





Midbrain

Hindbrain


WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens


























Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Table 1





35


45ndash60




30ndash35




80



35



30ndash35



35



35ndash135



20ndash65



21ndash35



21ndash35



35ndash40



Neural induction


Neuroectoderm





Midbrain

Hindbrain


WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens


























Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Neural induction


Neuroectoderm





Midbrain

Hindbrain


WNT FGF and RA

Ventral Dorsal

Rostral

Caudal

WNTRA

SHH

FGF8

WNT (Chir) and FGF8

morphogens


























Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Acknowledgments


References
























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article neural conversion and patterning of human...

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