Modeling Neurodevelopmental Disorders Using Human Pluripotent Stem Cells

Modeling Neurodevelopmental Disorders Using HumanPluripotent Stem Cells

Michael Telias & Dalit Ben-Yosef

# Springer Science+Business Media New York 2014

Abstract Neurodevelopmental disorders (NDs) are impair-ments that affect the development and growth of the brainand the central nervous system during embryonic and earlypostnatal life. Genetically manipulated animals have contrib-uted greatly to the advancement of ND research, but many ofthem differ considerably from the human phenotype. Cellularin vitro models are also valuable, but the availability of humanneuronal cells is limited and their lifespan in culture is short.Human pluripotent stem cells (hPSCs), including embryonicstem cells and induced pluripotent stem cells, comprise apowerful tool for studying developmentally regulated dis-eases, including NDs. We reviewed all recent studies in whichhPSCs were used as in vitro models for diseases and syn-dromes characterized by impairment of neurogenesis or syn-aptogenesis leading to intellectual disability and delayedneurodevelopment. We analyzed their methodology and re-sults, focusing on the data obtained following in vitro neuraldifferentiation and gene expression and profiling of the de-rived neurons. Electrophysiological recording of action poten-tials, synaptic currents and response to neurotransmitters ispivotal for validation of the neuronal fate as well as forassessing phenotypic dysfunctions linked to the disease inquestion. We therefore focused on the studies which includedelectrophysiological recordings on the in vitro-derived neu-rons. Finally, we addressed specific issues that are critical forthe advancement of this area of research, specifically in pro-viding a reliable human pre-clinical research model and drugscreening platform.

Keywords Human pluripotent stem cells .

Neurodevelopmental disorders . Diseasemodels . Neuraldifferentiation . Electrophysiology

Introduction

Neurodevelopmental disorders (NDs) are impairments thataffect the development and growth of the central nervoussystem (CNS) during embryonic and early postnatal life[1, 2]. There are several currently available research modelsfor studying NDs. Genetically manipulated animals have beenused as a common research model for studying human geneticdiseases, and they have contributed greatly to the advance-ment of ND research. Many of these models, however, oftendiffer considerably from that of the human phenotype due tointra-species variations, as evidenced by anatomical, develop-mental and even some biochemical differences [3].Additionally, the evolutionary distance between humans andthese animals (e.g., mice, frogs, fish, flies) limits the use ofthese models for genotype-phenotype studies, especially inareas such as the neocortex and the hippocampus. Moreover,there are several naturally occurring mutations in humanswhich have no counterparts in other animals (e.g., the CGGexpansion mutation in fragile X syndrome and trisomy ofchromosome 21 in Down syndrome), and several NDs whichare caused by mutations that remain to be identified [4]. Forthese cases, a cellular in vitro paradigmmay be another optionto model human disorders. One such approach is the genera-tion of postmortem human neural primary cultures [5–7], butthis methodology is limited due to the specific range of tissuesfrom which cells can be obtained and the short lifespan ofthese primary cultures. One way of overcoming these limita-tions is to use human embryonic stem cells (hESCs). Sincetheir first establishment in 1998 [8], hESCs have revolution-ized our understanding of cell biology, opening a whole new

M. Telias :D. Ben-Yosef (*)The Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis MaternityHospital, Tel-Aviv SouraskyMedical Center, Department of Cell andDevelopmental Biology, Sackler Faculty of Medicine, Tel-AvivUniversity, Tel-Aviv, Israele-mail: [email protected]

Stem Cell Rev and RepDOI 10.1007/s12015-014-9507-2

spectrum of possible therapeutic applications through regen-erative medicine. In the area of basic scientific research,hESCs have advanced our knowledge in countless fields, frommolecular biology to tissue engineering. Furthermore, re-search on hESCs led to the establishment of human inducedpluripotent stem cells (hiPSCs) [9], in which adult cells can bereprogrammed back to the pluripotent stage. Both hESCs andhiPSCs have been proven to recapitulate development in vitroat the molecular and cellular levels [10–12]. For these reasons,human pluripotent stem cells (hPSCs) are powerful tools inthe field of disease modeling. In the present review, we willfocus on their use in studying the pathophysiology of NDs.

Neurodevelopmental Disorders – Definition and Etiology

An ND is a disease or condition affecting the normal devel-opment of the CNS, resulting in learning and memory disabil-ities and/or motor or sensory dysfunction, and/or cognitiveimpairments [1, 2, 13–15]. At the cellular level, an ND dem-onstrates abnormal neurogenesis and/or synaptogenesis [16].The CNS starts to develop in the third week of embryogenesisfollowing embryo implantation in the uterus when theneuroectoderm folds to form the neural tube [17–19].During the early stages of neurogenesis, neural precursor cellsand neuroblasts proliferate, differentiate and migrate. It hasbeen established that neurogenesis takes place mostly duringembryonic life while synaptogenesis occurs mostly during theearly postnatal period [19]. Spatial and temporal regulation ofthese processes determines the identity of neurons and thebasic properties of initial synaptic contacts, and is also crucialfor the development of glial cells [20, 21]. During adulthood,progenitor neural stem cells in the brain can differentiate intonew neurons (i.e., adult neurogenesis), although this process islimited to specific areas of the brain and activated mainlyunder stress conditions. Adult neurogenesis is responsiblefor ongoing maintenance of the dentate gyrus of the hippo-campus as well as replenishment of the olfactory bulb withnew neuroblasts [22, 23]. Similarly, while synaptogenesistakes place mostly in the mature fetus by the end of pregnancyand especially during early postnatal life, continuing throughthe first years of infancy [24], synapses are continually beingre-shaped and re-tuned during life in a process known assynaptic plasticity [25]. NDs often present with a decreasedmass of neurons as a result of affected neurogenesis duringembryonic life, and/or with a decreased ability for synapticformation as a result of a faulty synaptogenic process duringearly post-natal life. However, it is possible that some NDscan further affect the brain during adulthood by impairingadult neurogenesis and synaptic plasticity.

NDs can be divided into two main groups based on theiretiology: genetic (or endogenously caused) and environmental(or exogenously caused) [26]. Environmental NDs are caused

by exogenous factors, such as chemical, mechanical, immu-nological and microbiological agents [27–31]. Examples ofexogenously caused NDs are fetal alcoholic syndrome inwhich the intake of alcohol by the mother during pregnancyresults in aberrant neurogenesis/synaptogenesis [32], and ce-rebral palsy in which hypoxia of the developing brain causesneuronal cell death [33]. In this review, we will focus ongenetic (endogenously caused) NDs that have been recentlymodeled using hPSCs [34–36]. This group of NDs includesmonogenic or multifactorial disorders, inherited or spontane-ous de novo gene mutations and chromosomal aberrations,with known or unknown gene targets [37, 38]. We summarizethe most prominent studies that used hESCs, pre-implantationgenetic diagnosis (PGD) hESCs and hiPSCs, in order tomodel endogenous NDs which affect the development of theCNS during neurogenesis and/or synaptogenesis. A total of 44manuscripts are examined for which we analyzed the type ofhPSC used as well as the differentiation method and mainresults, focusing on those studies which included neural andneuronal differentiation and, when available, electrophysio-logical recordings (see Table 1). Twenty-eight out of those 44papers reported on neural differentiation of diseased hPSCs,and only 7 of them performed electrophysiological recordingson the in vitro-derived neurons.

Human Pluripotent Stem Cells

The first hESC line was established through the isolation ofthe inner cell mass (ICM) of a human blastocyst and cultureunder pluripotency-supporting conditions [8]. Since then,hundreds of hESC lines were established in different labsusing different techniques for derivation and culture [39–48].A critical property of hESCs is their capacity for unlimitedself-renewal, remaining pluripotent in culture for long periodsof time [49, 50]. Furthermore, they can be induced to differ-entiate into all cell types in the body by recapitulating themolecular and cellular mechanisms characterizing in vivodevelopment [51–55]. For this reason, hESCs can be used ashuman in vitro developmental models, with two main advan-tages over its alternatives: (1) human embryos/fetuses areethically restricted for research, and studying human embryosduring development in the uterus is obviously not an option;(2) animal developmental models (both in vitro and in vivo)do not always resemble the human process due to evolution-ary divergence, and they frequently require genetic manipula-tions which can affect the expression of additional unknowngenes and their molecular pathway.

Developmental disorders can be investigated using hESCsby taking advantage of clinically oriented PGD, duringin vitro fertilization treatments [40, 50, 56–60]. PGD is offeredto patients who are carriers of severe genetic disorders toensure the birth of a healthy baby. Diagnosis of the embryo

Stem Cell Rev and Rep

Table 1 List of reports on modeling neurodevelopmental disorders using human pluripotent stem cells

Disorder (Gene) Reference Model Neuraldifferentiation

Electrophysiologicalrecordings

ASD (multiple genes) Lin et al., PLoS One [102]a hiPSCs Yes No

Lin et al., PLoS One [103]a hiPSCs Yes No

DeRosa et al., Neurosci. Lett. [106] hiPSCs Yes No

Zeng et al., PLoS One [107] hESCs, hiPSCs Yes No

Timothy syndrome (CACNA1C) Pasca et al., Nat. Med. [111] hiPSCs Yes Yes

Krey et al., Nat. Neurosci. [113] hiPSCs Yes No

Fragile X syndrome (FMR1 full mutation) Eiges et al., Cell Stem Cell [130] PGD-hESCs No –

Turetsky et al., Hum. Reprod., 2008 PGD-hESCs No –

Urbach et al., Cell Stem Cell [131] PGD-hESCs, hiPSCs No –

Sheridan et al., PLoS One [132] hiPSCs Yes No

Bar-Nur et al., J. Mol. Cell Biol. [133] hiPSCs Yes No

Telias et al., Dev. Biol. [80] PGD-hESCs Yes Yes

Alisch et al., BMC Med. Genet. [135] hiPSCs No –

Fragile X-associated tremor/ataxiasyndrome (FMR1 premutation)

Liu et al., Hum. Mol. Genet. [138] hiPSCs Yes Yes

Rett syndrome (MeCP2) Hotta et al., Nat. Methods [146] hiPSCs No –

Muotri et al., Nature [147] hiPSCs Yes No

Marchetto et al., Cell [148] hiPSCs Yes Yes

Di Stefano et al., Stem Cells [151] hiPSCs No –

Pomp et al., Cell Stem Cell [152] hiPSCs No –

Cheung et al., Hum. Mol. Genet. [153] hiPSCs Yes No

Ananiev et al., PLoS One [154] hiPSCs Yes No

Kim et al., PNAS [155, 217] hiPSCs Yes No

Larimore et al., PLoS One [157] hiPSCs Yes No

Rett syndrome (CDKL5) Amenduni et al., Eur. J. Hum. Gen. [158] hiPSCs Yes No

Ricciardi et al., Nat. Cell Biol. [159] hiPSCs Yes No

Down syndrome (trisomy 21) Biancotti et al., Stem Cells [172] hESCs No –

Mou et al., Stem Cell Res. Ther. [173] hiPSCs No –

Chou et al., PNAS [174] hiPSCs No –

Maclean et al., PNAS [175] hiPSCs No –

Li et al., Cell Stem Cell [176] hiPSCs No –

Lu et al., Exp. Cell. Res., 2012 hiPSCs Yes No

Briggs et al., Stem Cells [178] hiPSCs Yes No

Weick et al., PNAS [179] hiPSCs Yes Yes

Jiang J et al., Nature [180] hiPSCs Yes No

Down syndrome associated Alzheimer’s disease(trisomy 21)

Shi et al., Sci. Transl. Med. [181] hiPSCs Yes Yes

Prader-Willi & Angelman (15q11.2-q13; UBE3A) Chamberlain et al., PNAS [186, 188] hiPSCs Yes Yes

Yang et al., J. Biol. Chem. [187] hiPSCs Yes No

Lesch-Nyhan syndrome (HPRT1) Urbach et al., Stem Cells [197] hESCs No –

Park et al., Cell [198]b hiPSCs No –

Khan et al., Mol. Ther. [199] hESCs, hiPSCs No –

Mastrangelo et al., PNAS [200] hESCs, hiPSCs No –

Mekhoubad et al., Cell Stem Cell [201] hiPSCs Yes No

Type II/III Gaucher’s disease (GBA) Panicker et al., PNAS [206] hiPSCs Yes No

Tiscornia et al., Hum. Mol. Genet. [207] hiPSCs Yes No

List of peer-reviewed papers published on the use of human pluripotent stem cells as models for neurodevelopmental disorders. The name of the disease,its affected gene, and the specific model used are summarized. Data also include whether or not cells were subjected to neural differentiation andelectrophysiological recordings

ASD – Autism Spectrum Disorders; FXTAS – fragile X-associated tremor/ataxia syndrome; hiPSCs – human induced pluripotent stem cells; hESCs –human embryonic stem cells; PGD – pre-implantation genetic diagnosisa Studies were performed on hiPSC lines derived from healthy donors and schizophrenia patientsb This study included the derivation of additional hiPSC lines for Down syndrome and Gaucher’s disease Type III


is performed on single cells that had been biopsied frompreimplantation embryos. Monogenic inherited diseases areusually analyzed by single-cell polymerase chain reaction(PCR), and chromosomal aberrations are assessed by fluores-cence in-situ hybridization (FISH) or chromosome microarrayanalysis (CMA) [61]. Embryos diagnosed by PGD as havinginherited the normal allele are transferred to the uterus forpregnancy, while those diagnosed as having inherited theaffected allele can be donated for research, following theprovision of a written consent by the parents, and in accor-dance to the stipulations of an Ethics Committee. These af-fected embryos are grown to the blastocyst stage, and hESClines carrying natural mutations for specific genetic diseasescan be derived [40]. Derivation of diseased hESCs is limitedby the availability of PGD treatments and the frequency of thespecific mutations in a given population [62–67]. However,when successful, this approach provides researchers with aunique platform to establish a human disease-in-a-dish modelin which there is no need for genetic manipulation. In addition,cell and tissue differentiation can be fate-directed, includingthe possibility of studying mid-step precursor cells with norestriction of biological material. For diseases in which thespecific mutation is unknown and/or those that result from denovo mutations for which PGD is not an option, geneticmanipulation of wild-type hESCs can be another option forgenerating a human in vitro model [68–71]. A more efficientalternative has been developed by reprogramming fully dif-ferentiated cells (such as fibroblasts) obtained from affectedpatients into ES-like cells known as hiPSCs [9, 72]. Since thedifferentiated cells subjected to reprogramming are taken di-rectly from affected patients, hiPSCs can be used to modelthose diseases in which the specific genetic cause is unknown[10]. Taken together, hESCs and hiPSCs offer a valuablehuman in vitro model which can recapitulate the developmentof the CNS, and thus provide the tools for elucidating theetiology and pathophysiology of NDs.

In vitro Neural Differentiation and Characterizationof the Derived Neurons

Several research models have been established to study em-bryonic neurogenesis in humans using neural precursor cellsor different neuronal cells extracted from aborted fetuses[73–75]. However, this approach is limited because it employscells and tissues that have a short lifespan in culture and werederived at very different developmental stages, making com-parisons among obtained samples problematic. From thispoint of view, in vitro neural differentiation of hESCs andhiPSCs is currently the best option for modeling humanembryonic neurogenesis in health and disease [76–78].Undifferentiated hESCs and hiPSCs can be synchronouslydifferentiated into neurons. Furthermore, the process of

in vitro differentiation can potentially recapitulate the wholespan of in vivo development, allowing the study of the effectof the disease not only on the neuronal endpoint, but duringthe different stages of neurogenesis as well (see Fig. 1)[79–81]. Moreover, this approach provides a steady ongoingsource of human neurons that can then be analyzed at alllevels, from gene expression and morphological assessmentto electrophysiological functionality. Neurons that have beendifferentiated in a dish can generate neural networks, includ-ing the formation of active synapses that can be pharmacolog-ically tested [12, 82]. Specific neurons can be obtained inorder to develop therapeutic strategies for specific diseases,such as dopaminergic neurons for Parkinson’s disease[83–86], or motor neurons for amyotrophic lateral sclerosis(ALS) [87–89]. Furthermore, in vitro neural differentiation ofhESCs and hiPSCs can also be used to study gliogenesis anddemyelination disorders, such as multiple sclerosis [90, 91].

A critical factor in performing in vitro neural differentiationis the assessment of the specific neuronal identity of thederived cells. It is commonly accepted that the expression ofcertain proteins is correlated with specific types of neuronsand with their specific maturation stage. Of the 44 papersreviewed here, 28 reported in vitro neuronal differentiation.We summarized the different sets of biological markers usedby them to identify and classify the generated neurons (seeTable 2). As can be seen in the table, the most commonpositive markers for identifying neurons in vitro are the cyto-skeletal proteins associated with their axons and/or dendrites,such as TUJ1 and MAP2. However, these markers cannotindicate whether the neuron in question is in its final post-mitotic stage or still proliferating, nor it can demonstrate itsspecific neuronal subtype. The expression of transcriptionfactors (TFs), such as NeuN and NeuroD1, can indicate ifthe analyzed neurons are post-mitotic or have not yet reachedthat stage. The presence or absence of neurotransmitters,synaptic proteins and ion channels can all be useful to identifyspecific types of neurons. The presence or absence of glialmarkers (such as GFAP or O4) is important for evaluating thepurity of the neuronal population in culture, and the fraction ofglial cells comprising the whole population. Furthermore, thespecific neuron subtype (cortical, forebrain, anterior, dorsal)can be determined by the expression of specific subsets of TFs.

Modeling Neurodevelopmental Disorders with HumanPluripotent Stem Cells

Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are a collective terminol-ogy for NDs characterized by impaired social interactionand communication as well as stereotypic repetitive behavior[92, 93]. ASDs include four separate disorders: autism,


Asperger’s disorder, childhood disintegrative disorder (CDD)and otherwise not specified pervasive developmental disorder(PDD) [94, 95]. ASDs are complex disorders, and extensiveresearch has thus far revealed little about their genetic regula-tion [96–99]. Furthermore, several other diseases and syn-dromes have been linked symptomatically to ASDs, includ-ing, among others, Rett syndrome, fragile X syndrome andTimothy syndrome, in which is possible to observe “autistic-like traits”, including the avoidance of eye contact and socialreclusion. The epidemiology of ASDs is also very complicat-ed and varied, but it is widely accepted that they affect 60 to 70infants among 10,000 live births, making them the mostprevalent group of ND disorders [100]. Collectively, ASDsare more common in males than females (incidence ratio of 4to 1) [101]. The main reason for the poor understanding ofsuch a widespread condition is the lack of a proper humanresearch model.

In 2011 and 2012, two consecutive studies used hiPSCs foridentifying gene candidates for the etiology of ASDs [102,103]. However, those reports did not include hiPSCs derivedfrom autistic patients but rather from schizophrenia patients,based on already published data showing a link betweenautism and schizophrenia. Using RNA sequencing analysis,they uncovered a series of mRNAs and long non-codingRNAs that significantly changed their expressions during theprocess of neural differentiation. Of those, the synaptic regu-lators neurexin and neuroligin have already been revealed asbeing ASD candidates [104, 105]. The identified ASD andschizophrenia candidate genes were shown in those studies tobe expressed in an allele-biased manner. In another study,ASD hiPSCs were established from peripheral blood mono-nuclear cells [106]. Two different ASD hiPSC lines were usedto produce GABAergic neurons in vitro, and they showed noabnormal phenotype other than the presence of immature cells

Fig. 1 Schematic representationof in-vitro neural differentiationprocess as we previouslypublished [80]. The principalsteps of in-vitro neuraldifferentiation are shown incorrelation to in vivo embryonicneurogenesis. The whole processof in vitro neural differentiationcan be divided into early and latestage. During the early stage,primitive neural tube-like cellsand progenitors develop in vitro,along with many other non-specific neural cell types. Incontrast, the late stage ischaracterized by synchronousgeneration of neuroblasts andsubsequent neurons. NIM –Neural Induction Medium; NDM– Neuronal DifferentiationMedium


Tab

le2

Identificationof

neuronsderivedfrom

pluripotentstem

cells

Disorder

Reference

Neuronalidentity

Positiv

emarkers

Negativemarkers

Cytoskeleton

Transcriptio

nfactors

Neurotransm

itters,Synaptic

proteins

&ionchannels

Autism

&ASD

Lin

etal.,PL

oSOne

[102]

N/A

Expressionprofile

Expressionprofile

Lin

etal.,PL

oSOne

[103]

DeR

osaetal.,Neurosci.

Lett.[106]

GABAergic

TUJ1,M

AP2

,NCAM

NeuN,S

IX3,HOXB4

VGAT,

GAD67,S

ynapsin

OTX2

Zengetal.,PL

oSOne

[107]

N/A

TUJ1,E

xpression

profile

N/A

N/A

Expressionprofile

Tim

othy

syndrome

Pascaetal.,Nat.M

ed.[111]

Mixed

populatio

nsMAP2

,NCAM

DCX

VGLUT1,VGLUT2,DRD2,

GAD65,G

AD67,V

GAT

N/A

Cortical

N/A

FOXP1

,ETV1,SA

TB2,

CTIP2,CUX1,RELN

N/A

N/A

Kreyetal.,Nat.N

eurosci.[113]

N/A

MAP2

N/A

Synapsin

N/A

Fragile

Xsyndrome

Sheridan

etal.,PLoS

One

[132]

N/A

TUJ1

N/A

N/A

GFA

P

Bar-N

uretal.,J.Mol.

CellB

iol.[133]

N/A

NCAM

N/A

N/A

N/A

Teliasetal.,Dev.B

iol.[80]

Glutamatergica

TUJ1,M

AP2

,TAU

NeuN

Synaptophysin,S

ynaptotagm

inGFA

P

FragileX-associated

trem

or/ataxiasyndrome

Liu

etal.,Hum

.Mol.G

enet.[138]

N/A

TUJ1,M

AP2

N/A

Synapsin,V

GLUT1,PS

D95,

GPHN,V

GAT

N/A

Rettsyndrom

eMuotrietal.,

Nature[147]

N/A

TUJ1

N/A

N/A

N/A

Marchetto

etal.,Cell[148]

Mixed

TUJ1,M

AP2

N/A

GABA,V

GLUT1,Synapsin

N/A

Cheungetal.,Hum

.Mol.

Genet.[153]

N/A

TUJ1,M

AP2

N/A

N/A

N/A

Ananiev

etal.,PLoS

One

[154]

Postm

itotic

TUJ1

N/A

N/A

N/A

Kim

etal.,PNAS[155,217]

N/A

TUJ1

N/A

SCN1A

,SCN1B

GFA

P

Larim

oreetal.,PLoS

One

[157]

N/A

TUJ1

NeuroD1

TUJ1,N

euroD1

GFA

P

Amendunietal.,Eur.J.H

um.G

en.

[158]

Mixed

populatio

nsTUJ1,M

AP2

TBR1

VGLUT1,VGLUT2,TBR1,

GAD65/67

GFA

P

Ricciardi

etal.,Nat.C

ellB

iol.

[159]

Forebrain

TUJ1,M

AP2

EMX1,TBR1,CTIP2,CUX1,

FOXG1,CAMKII

VGLUT1,VGAT,TH,G

AD65,

PSD

95,S

ynaptophysin

N/A

Dow

nsyndrome

Luetal.,Exp.C

ell.Res.,2012

N/A

TUJ1

N/A

N/A

N/A

Briggsetal.,Stem

Cells[178]

Anterior

TUJ1,M

AP2

OTX1,OTX2,HOXB4

N/A

GFA

P,S1

00B,G

LAST

,NFIA

Weick

etal.,PN

AS[179]

Dorsaltelencephalic

TUJ1

PAX6,LHX2,TBR1,

FOXG1,OTX2,

Expressionprofile

N/A

NKX2.1,GBX2,HOXB4

Forebrain

TUJ1

N/A

Synapsin,G

ABA,V

GAT

N/A

JiangJetal.,Nature[180]b

N/A

N/A

N/A

Dow

nsyndromeassociated

Alzheim

er’sdisease

Shietal.,

Sci.T

ransl.Med.[181]

Cortical

TUJ1,M

AP2

,TAU

TBR1,CUX1,BRN2,

SATB2,CTIP2

PSD95,Synaptophysin,H

omer,

Munc13

S100B


still expressing doublecortin and lack of the OTX2 marker. Adifferent approach used to establish hPSC-based models forASDs is to manipulate candidate genes and observe the im-pact of such manipulation on neural differentiation capabilityand neuronal fate [107]. When neurexin-1 was knocked-downin hESC- and hiPSC-differentiated cells, the expression of 33neuron-specific and cell-adhesion genes was altered, includ-ing 4 genes that were already demonstrated as ASDs candi-date genes. More importantly, several synaptotagmins hadreduced expression levels as well as the glial marker GFAP.The additional finding that neurexin-1 down-regulation im-paired astrocyte differentiation can suggest a novel role forglial development in the pathology of ASDs. None of thesestudies performed electrophysiological recordings of ASD-derived neurons.

Timothy Syndrome

Timothy syndrome (TS) is a rare autosomal dominant disordercharacterized by the unique comorbidity of long Q-T syn-drome and syndactyly [108]. Eighty percent of TS patientswho survive long enough for evaluation are diagnosed ashaving ASDs [109]. TS is caused by a missense mutation inthe CACNA1C gene, which encodes the calcium channelCav1.2 α-subunit [110]. The first study that used TS-hiPSCsfound several abnormalities during in vitro neurogenesis [111,112]. Neural differentiation resulted in heterogeneous popula-tions of excitatory and inhibitory neurons, including a highproportion of immature doublecortin cells and an excess num-ber of tyrosine hydroxylase-positive dopaminergic neurons.Current clamp recordings of TS neurons revealed wider actionpotentials, implying an underlying mis-regulation in ion-channels activity. In a follow-up study, dendrites in TS-hiPSCs-derived neurons retracted upon stimulation, as op-posed to controls in which stimulation increased dendritelength [113].

Fragile X Syndrome

Fragile X syndrome (FXS) is the most common form ofinherited cognitive impairment [114]. It is one of the leadingcauses of autism, with 20–25 % of FXS patients being diag-nosed as autistic and 5 % of all autistic patients being diag-nosed as having FXS [115–117]. FXS also includes epilepsy(20–25 % of cases), hyperextensible joints, macroorchidismand a stereotypic face. It is an X-linked monogenic diseasecaused by inactivation of the FMR1 gene and absence of itsencoded protein FMRP [118]. The FMR1 gene contains aCGG repeats region located upstream to its promoter [119].In healthy individuals, this region includes up to 55 suchrepeats. However, for reasons yet unknown, the CGG repeatregion tends to undergo expansion during gametogenesis and,when it extends to over 200 CGG repeats, FMR1 becomesT

able2

(contin

ued)

Disorder

Reference

Neuronalidentity

Positiv

emarkers

Negativemarkers

Cytoskeleton

Transcriptio

nfactors

Neurotransm

itters,Synaptic

proteins

&ionchannels

Prader-W

illi&

Angelman

Yangetal.,J.Biol.Chem.[187]

N/A

TUJ1,M

AP2

N/A

N/A

GFA

P

Chamberlainetal.,PNAS

[186,188]

N/A

TUJ1

N/A

N/A

S100B

Lesch-N

yhan

syndrome

Mekhoubad

etal.,CellS

tem

Cell[210]

N/A

TUJ1,M

AP2

N/A

N/A

N/A

Type

II/IIIGaucher’s

disease

Panicker

etal.,PNAS[206]

N/A

TUJ1,M

AP2

N/A

GABA,D

βH,T

HGFA

P,O4

Tiscorniaetal.,Hum

.Mol.

Genet.[207]

Dopam

inergic

TUJ1,N

FNeuN

TH,S

ynapsin

N/A

The

fulllisto

fpositiv

eandnegativ

emarkersused

toidentifyneurons(asdefinedby

theauthors)forthosestudiesin

which

human

pluripotentstem

cells

weredifferentiatedin

vitrointo

neuronsin

the

contextof

modelingneurodevelopmentaldisorders(Table

1).Po

sitiv

emarkers

werefurtherdividedinto

threecategories:cytoskeletal

markers,transcriptionFactors,andNeurotransm

itters,Synaptic

Proteins

andIonChannels

N/A

–notavailable

aBased

onpatch-clam

precordings

ofneuronsexposedto

glutam

ateatphysiologicalconcentratio

nsbNeurald

ifferentiatio

nwas

performed

only

upto

theneuralprecursorstage


hypermethylated and consequently inactivated, causing FXS.Hence, the FMR1 mutation is called an expansion or ‘dynam-ic’ mutation [120]. However, in spite of the presence of thefull expansion (>200 CGG), silencing of FMR1 expressiondoes not take place until at least week 12–14 of gestation[121]. Furthermore, there is no evidence that the hypermethy-lation event occurs simultaneously at every cell or tissue. Inunaffected individuals, FMR1 is expressed ubiquitously at thebeginning of development, and its expression becomes pro-gressively restricted to the CNS, neural crest and testis [122,123]. FMRP has been shown to act as an mRNA-bindingprotein which regulates the translation of several hundredsof targeted mRNAs by preventing their interaction with ribo-somes [124, 125]. FMRP granules are found in dendriticspines and synaptic terminals, suggesting that FMRP is in-volved in the molecular fine tuning of synaptic activity at alocal level [126]. The most prolific model for FXS has longbeen the fmr1−/− (knockout) mouse [127]. FXS models havemore recently been established in other species as well, amongthem zebrafish [128] and drosophila [129]. Although thosemodels are valuable for studying the molecular function ofFMRP, their main disadvantage is the fact that, unlike thehuman fetus, FMR1 is inactivated at every developmentalstage. In 2007, our lab published the first study of a geneticalbeit disorder using an FX-hESC line derived from affectedblastocysts following PGD [130]. Albeit the presence of 200–1,000 CGG repeats, FMR1was unmethylated in FX-hESCs atthe undifferentiated pluripotent stage, and they activelyexpressed FMRP, similar to non-mutated controls. FMR1was hypermethylated and FMRP was absent only upon dif-ferentiation. hiPSC lines were subsequently generated fromfibroblasts of FXS individuals [131]. Despite successfulreprogramming of FXS somatic cells, FMR1 remained inac-tive and FMRP expression was absent, even at the undiffer-entiated stage, resembling fmr1−/− models. These datahighlighted critical differences between hESCs and hiPSCsinmodeling developmentally regulated diseases, such as FXS.In vitro neural differentiation of FX-hiPSCs demonstratedabnormal neurodevelopment, including a significant loweryield of Tuj1+ cells and a larger yield of GFAP+ cells com-pared to controls [132]. Additionally, these neuronal cellsdisplayed fewer and shorter neurites. In another study, treat-ment of FX-hiPSCs and their neuronal derivatives with thedemethylation agent 5-azacytidine was able to reactivateFMR1 and restore FMRP [133]. However, 5-azacytidine is ageneral de-methylation drug and is not specific to FMR1, thusits effects on the expression of other genes cannot be exclud-ed. We recently reported the abnormal neurogenesis of threefull mutation male FX-hESC lines in comparison to normalhESC lines [80]. We were able, for the first time, to createmature neuronal networks following in vitro differentiation ofFX-hESCs by recapitulating the different steps of embryonicneurodevelopment (see Fig. 2). The ability to generate human

FXS neurons from pluripotent hESCs enabled us to dissect thephenotype of this disease from a developmental point of view,an approach that is extremely difficult to implement in othermodels in which FMR1 is inactivated in all stages of devel-opment. Abnormal neurogenesis was observed in both thetiming of development and in the morphology of the differentcellular structures, as well as in the aberrant expression ofseveral neural markers [80]. Surprisingly, we found abnormalgene expression already at early stages of neural differentia-tion, a previously unknown in vitro phenotype of FXS. Inaddition, similar to hiPSCs [132] and human neural precursorcells [134], more glial/less mature neuronal cells (GFAP+)were created compared to control lines. Notably, this was thefirst study to produce electrophysiological data on humanFXS neurons derived from hPSCs [80]. We found that al-though FXS neurons fire action potentials similar to controlcounterparts, they do so in reduced frequency and amplitude.In vitro-derived human FXS neurons developed poor sponta-neous synaptic activity and did not respond to glutamate.The most recently published study using FX-hiPSCsfocused on the molecular mechanism of FMR1 inactivation:it demonstrated that FMR1 methylation is restricted tothe FMR1 locus and does not extend to other genomiclocations [135]. That study did not, however, include anyneurodevelopmental assays.

Individuals with an FMR1 containing 55–200 CGG repeatsare defined as pre-mutation carriers, and their incidence is 1 in100 in the overall population [136]. Pre-mutation carriers donot exhibit cognitive impairments, but they may suffer fromFX-associated tremor/ataxia syndrome (FXTAS), a neurode-generative disorder that appears in later life [137]. FemaleFMR1-premutation human fibroblasts were used to createFXTAS hiPSCs [138]. In vitro neural differentiation of pre-mutation neurons showed shorter neurites and reduced expres-sion of post-synaptic density protein 95 (PSD-95). Moreimportantly, spontaneous calcium transients were increasedin affected neurons, which can explain the decrease inPSD-95, although the molecular cause behind such alteredspontaneous oscillations remains unclear.

Rett Syndrome

Rett syndrome (RTT) is a monogenic X-linked disease exclu-sively affecting females. RTT males die between pre-term to2 years of age due to severe encephalopathy [139]. At least90 % of cases are caused by a mutation in the MeCP2 genelocated on the X chromosome [140], although mutations inCDKL5 and FOXG1 have also been described as being asso-ciated with RTT [141]. More than 95 % of RTT cases arecaused by de novo mutations and are not inherited by off-spring [142]. A striking feature of RTT females is their appar-ent normal development until they reach the age of 6–18 months [139]. Since RTT females are genetically MeCP2


mosaics (due to X inactivation), they present a wide diversityand severity of symptoms and pathologies. The neuropathol-ogy of RTT includes autistic-like behavior, loss of speech,seizures, lack of eye contact, sensory problems, ataxia, micro-cephaly, chorea, hypotonia/dystonia and gait disturbances[143]. It is currently regarded as an ASD, since autism has a100 % penetrance in RTT. On the other hand, RTT patientscomprise around 5 % of all autistic patients [144, 145]. It isonly recently that several research teams have generated RTThuman models using pluripotent stem cells [76].

The first study in which RTT hiPSCs were established[146] demonstrated their ability to differentiate into all threegerm layers, including the neuroectoderm layer. Although theexpression of the neural markers nestin and β-III-tubulin wasobserved in these cells, there were no resultant insights intoin vitro human RTT neurons. The ensuing reports on theestablishment of RTT-hiPSCs were published in 2010 [147,148]. Those RTT hiPSCs were used to confirm that MeCP2-mutated human neural precursor cells possess increased L1transcription and retrotransposition, as had previously beenobserved in rodent models of RTT. Extensive analysis of theseRTT neurons unveiled abnormal morphology, includingsmaller soma size and reduced spine density, as well as keyfunctional features, such as decreased glutamatergic synapto-genesis, decreased frequency of calcium oscillations and de-creased frequency of post-synaptic currents. Furthermore,these RTT neurons were used to show that insulin growthfactor 1 (IGF1) could induce an increase in glutamatergicsynapse numbers, thereby partially rescuing the RTT neuronalphenotype. Importantly, the described impaired phenotypeswere observed across four different RTT hiPSC lines that hadbeen established from four patients, each with a differentMeCP2 mutation [149, 150]. Other studies have reported theestablishment of MeCP2-mutated RTT hiPSCs, but neuraldifferentiation was either not attempted [151, 152], or it re-sulted in neuronal-like cells with reduced soma size [153, 154]

and reduced numbers of β-III-tubulin-expressing cells [155],partially confirming the results obtained by the Muotri labo-ratory [147, 148]. iPSCs were also established from MeCP2-deficient mice [156], and the neurophysiological alterationsdescribed in their in vitro-differentiated neurons were similarto those described in the hiPSCs. A recent study performed onRTT neurons derived from both hiPSCs and mutant mice[157] showed significantly reduced transcription of β-III-tubulin and neuroD1 in RTT neurons. More importantly, thisstudy uncovered a reduced expression of pallidin, a memberof the multi-subunit protein complex BLOC-1, which is re-quired for normal biogenesis of specialized organelles of theendosomal-lysosomal system, a previously unknown mecha-nism that could be partially responsible for the pathology ofRTT.

In 2011, one study reported on the establishment ofCDKL5-mutant hiPSCs from a RTT female patient as wellas from an X-linked epileptic encephalopathy male patient[158]. These mutant hiPSCs could shed light on the functionof CDKL5 in NDs in general, as well as on the role of Xchromosome inactivation in the pathology of RTT. However,when these hiPSCs lines were differentiated into glutamater-gic and GABAergic neurons, no significant differences wereobserved between them or between these two lines and controllines. In 2012, a second study elucidated the role of CDKL5using hiPSCs [159]. Interestingly, control and CDKL5-mutanthiPSC lines were derived from the same RTT patient byselecting clones based on their X-chromosome inactivationstatus. Neural differentiation of these hiPSCs revealedthat CDKL5 expression is required mainly at the latestages of neuronal maturation and that it is highly enrichedat dendritic spines. Although a similar rate of neuronal matu-ration and development was observed in control and mutantcells, the lack of CDKL5 led to a significantly decreasednumber of synaptic contacts as well as to abnormal spinemorphology [159].

Fig. 2 Neurons in vitrodifferentiated from fragile Xsyndrome human embryonic stemcells (a). A single highlyarborized neuron differentiatedin vitro from the male FX-hESCline Lis_FX6, positively stainedfor the neuronal cytoskeletonmarker MAP2 (red). (b) Anintricate compact neuronalnetwork differentiated fromanother male FX-hESC line,SZFX6, positively stained forMAP2 (red) and the synapticprotein synaptotagmin (greenpunctae; overlay in yellow)


Down Syndrome

Down syndrome (DS) is the most common form of intellec-tual disability, with the affected individuals having an averageIQ of 50 [160–163]. It is caused by trisomy of chromosome21, which is the most common chromosomal abnormality,affecting 14.47 per 10,000 live births in the USA [164].Trisomy 21 is not inherited but rather a de novo chromosomalaberration, resulting from meiotic nondisjunction, with 88 %of cases arising from nondisjunction in the maternal gamete.In addition to cognitive impairment, speech delay and delayedmotor skills, DS individuals also exhibit symptoms of non-nervous tissue pathologies, such as congenital heart disease, ahigher incidence of acute lymphoblastic and acute myeloge-nous leukemia, dysfunction of the thyroid gland, increasedrisk of Hirschsprung’s disease and infertility. Due to medicaladvancements in surgery and physical therapy, the median ageat death of individuals with DS has risen in the US from25 years in 1983 to 49 years in 1997 [165]. This increase inlife expectancy had an unexpected consequence: the DS sub-jects who survived to reach older ages (40–60 years) had anincreased incidence of early-onset Alzheimer’s disease[166–168]. This could be attributable to the fact that theamyloid precursor protein (APP), a key marker in AD etiolo-gy, is located at chromosome 21. Hence, neurodevelopmentalstudies of DS can shed light not only on the pathophysiologyof DS itself, but on Alzheimer’s disease as well.

The first pluripotent model for DS was established usingmESCs following microcell-mediated chromosomal transfer[169]. Using this technique, microcells containing single orsmall numbers of chromosomes are fused with whole cells,after which hybrids are selected [170, 171]. The first humanDS ESC line was derived following preimplantation geneticscreening (PGS) for aneuploidy embryos [172]. These hESClines were successfully differentiated in vitro by generatingembryoid bodies and in vivo into teratomas, but no significantimpaired phenotype was observed. In 2012, four parallelstudies were conducted on hiPSC lines established from DSsubjects, but none of them included neural differentiation ofthe cells. Instead, these DS hiPSCs were used to investigatewhether their pluripotency is affected by the extra chromo-some 21 [173] in order to better understand the hematopoieticdefects in DS [174, 175] and to test a novel method to removethe extra chromosome 21 by a combination of gene targetingand negative selection [176]. In 2013, the first two studieson in vitro neural differentiation of DS hiPSCs werepublished with contradictory results. One of them re-ported differences between DS and control cells already atthe neural progenitor stage [177], accompanied by significant-ly increased expression of APP and reduced expression ofMeCP2 in DS. Furthermore, neuronal differentiation resultedin significantly lower efficiency for DS cultures to generateneurons. In contrast, the other study found that DS hiPSCs

differentiated similarly to control cells during the early stagesof neurodevelopment in terms of neural potential, timing,proliferation and neurite extension [178]. However, at laterstages of neural differentiation that also allows the initiation ofgliogenesis, DS neurospheres produced a 2-fold higher pro-portion of glia than control cultures concomitantly with areduced number of developing neurons. Interestingly, a simi-lar glia/neuron bias has been reported in FXS (see above). Itwas also reported that although DS neurons are normallyproduced from hiPSCs, they are more sensitive to oxidativestress-induced apoptosis than non-DS neurons. Another study[179] revealed that DS does not affect differentiation ofhiPSCs into neural progenitors or the production of earlycortical neurons. Whole-cell patch-clamp recordings con-firmed that DS neurons at this stage are functionally equiva-lent to control cells in terms of firing of action potentials, butthey display a significantly reduced incidence and frequencyof spontaneous post-synaptic currents and a reduced numberof synapsin punctae on their neurites. Trisomy of chromosome21 did not, however, alter the production of GABAergic andglutamatergic neurons following cortical differentiation, andthe relative rate of these populations was similar between theDS and control lines. The most recent study using DS hiPSCscontributed to greater understanding of the role of the XISTgene in the epigenetic regulation of DS neural rosettes bydemonstrating that DS hiPSCs produced neural rosettes witha 4 day delay and that XIST induction corrected this aberrantphenotype [180].

As noted above, a striking feature of DS is early onset AD[181]. DS hiPSCs were successfully differentiated into gluta-matergic cortical neurons. Electrophysiological analysis dem-onstrated that DS neurons showed normal action potentialsand miniature excitatory post-synaptic currents. Nevertheless,these neurons produced higher levels of the Aβ40 and Aβ42peptides, displaying amyloid plaques and abnormal localiza-tion of hyperphosphorylated tau protein, all hallmarks of thepathophysiology of AD.

Prader-Willi & Angelman Syndromes

Prader-Willi and Angelman syndromes (PWS and AS, respec-tively) are complex genetic disorders that primarily affect thenervous system. These are rare imprinting diseases: PWSoccurs in about 1 in every 15,000–25,000 live births and ASaffects 1 in 12,000–20,000 people. PWS and AS usuallyreferred to as ‘sister disorders’, since they are caused by thesame set of genes located on the q arm of chromosome 15[182, 183]. While the maternal allele is imprinted and thepaternal allele is mutated in PWS, the opposite occurs in AS(i.e., paternal imprinted and maternal mutated). PWS isdue to the absence of paternally expressed imprintedgenes at 15q11.2-q13 through paternal deletion of this region(65–75 % of individuals), maternal uniparental disomy 15


(20–30 %), or an imprinting defect (1–3 %) [182]. In contrast,AS is attributable to the reduced expression of the maternallyinherited ubiquitin-protein ligase E3A gene (UBE3A) onchromosome 15 [184]. Although each of the two syndromesshows very unique symptoms, they do share several features,most importantly developmental delay, cognitive disability,and speech impairment [185, 186].

The first report on PWS hiPSCs was published in 2010[187]. Several clones of hiPSCs were derived from the samepatient and compared to two control lines of hESCs. PWShiPSCs were successfully differentiated into Tuj1+ andMAP2+ cells as well as astrocyte-like cells, without any sig-nificant difference from control lines in the parameters thathad been examined. In another study, hiPSCs were obtainedfrom both PWS and AS [188]. The characteristic imprintingpattern was confirmed in all diseased lines, but only AShiPSCs were subjected to in vitro differentiation into func-tional neurons. That study demonstrated that the AS hiPSCs-derived neurons were not different from the control neurons,even in their electrophysiological properties, exhibiting firingof action potentials as well as AMPA-mediated excitatorypost-synaptic currents.

Lesch-Nyhan Syndrome

Lesch-Nyhan syndrome (LNS) is characterized by neurolog-ical and behavioral abnormalities. It is an extremely rarerecessive X-linked disease, affecting 1 in 380,000 livebirths [189–191]. LNS is caused by mutations in theHPRT1 gene, which encodes the hypoxanthine-guaninephosphoribosyltransferase (HGPRT). This enzyme plays acritical role in the generation of purine nucleotides, andHGPRT insufficiency causes the accumulation of high levelsof uric acid in the blood. During fetal life, uric acid is clearedby the placenta, but the symptoms of LNS begin to emerge atbirth, and they include kidney dysfunction, testicular atrophyand gout, as well as mental retardation, poor muscle controland self-injuring behavior [192, 193]. While the non-neurological symptoms of LNS can be treated with drugs,there is no current effective treatment for the neurologicaldamage. Moreover, its molecular properties are poorly under-stood [194–196].

In the first attempt to model LNS using hPSCs, the authorsmutated the HPRT1 gene in ‘wild-type’ hESCs using homol-ogous recombination [197]. These cells recapitulated some ofthe characteristics of LNS by showing a higher rate of uricacid accumulation than the control cells. Their neural differ-entiation was not examined in that study. LNS hiPSCs werefirst established in 2008 [198], but no information on theirneural potential was provided. Both hESCs and hiPSCs werelater used to test whether gene therapy could be a suitabletreatment for LNS by correcting mutations in the HPRT1 geneusing an adeno-associated virus vector [199]. In vitro human

models for LNS were also established by shRNA-mediatedgene knockdown of HPRT1 in both hESCs and hiPSCs [200].Several important insights regarding the molecular mecha-nisms downstream to the HPRT1 mutations were achieved,but neural differentiation was not performed. The only pub-lished study that included in vitro neural differentiation ofpatient-derived LNS hiPSCs revealed a significantly reducednumber of Tuj1+ cells as well as decreased neurite lengths[201]. In addition, there was no improvement in neuralproduction and neurite morphology when those cells wereco-cultured with glial cells.

Type II/III Neuropathic Gaucher’s Disease

Gaucher’s disease (GD) is the most common of the lysosomalstorage diseases [202–204]. It is an autosomal recessive diseaseresulting from mutations in the lysosomal glucocerebrosidasegene (GBA). Type I GD is the most common form of GD andhas no neurological effects whatsoever. In contrast, type II andIII neuropathic GD are rare manifestations of the disease,whereby different mutations in the same gene cause eithermental retardation (in type II GD), or dementia (in typeIII GD) [205].

Due to the rarity of type II and III GD, there has beenlimited research into these neurological manifestations. Onlytwo studies have been published in which hPSCs were used tostudy type II/III GD. In the first study, hiPSC lines weregenerated from all three types of GD [206]. These cells weresuccessfully differentiated into monocytes and macrophages,and their metabolic impairment was reverted by treatmentwith recombinant glucocerebrosidase. Neuronal differentiationof these cells was also successful, and no impairments in theneurogenesis or synaptogenesis were observed, although therewere very low levels of glucocerebrosidase enzymatic activityand accumulation of glucosylsphingolipids. In the secondstudy [207], although hiPSCs were derived only for type IIGD, the results were almost identical: neuronal differentiationwas successful, producing neurons which did not show anyphenotypic difference from their control counterparts, butexpressing much lower levels of the GBA-encoded enzyme.

Conclusions and Open Questions

Human Pluripotent Stem Cells: “Modeling Disease in a Dish”

hESCs, PGD-hESCs and hiPSCs are valuable for investigat-ing human pathologies since they offer ample naturallyinherited impaired human cells in culture. The availability ofhESC and hiPSC lines from genetically affected embryos andadult patients facilitates basic research on developmentallyregulated mechanisms that are responsible for the develop-ment of specific diseases. The advantages and disadvantages


of the in vitro approach are derived mainly from the compar-ison of hPSC-based models to the available animal modelsthat have been in use for a longer period of time. The gener-ation of animal models for human diseases, such as the trans-genic mouse, includes the use of highly interbred animals withminimal ‘genetic background noise’. In fact, each mousecould be considered an identical copy of the other. This allowsfor a specific genotype to be pinpointed to a specific pheno-type, and the molecular pathway between them can subse-quently be uncovered. However, this approach has its limita-tions when modeling human NDs since the murine homolo-gous gene used to recapitulate the human disease is not alwaysidentical, and, unlike humans, there are no different levels ofpenetrance and incidence of comorbidity in transgenic ani-mals. Asmodels for human diseases, hPSCs offer a solution tocircumvent some of these problems. PGD hESC and hiPSClines are derived from different patients with varied geneticbackgrounds, and therefore resemble the general variability inthe human population, thus providing a platform to studydifferent molecular phenotypes of the disease within theirnatural variability. However, if phenotypic variance representsa significant challenge, then isogenic lines can be establishedby using unaffected siblings or parents [154, 179, 208, 209].Another option is to manipulate ‘wild-type’ hPSCs to createthe desired mutation or to fix the mutation in a sub-clone ofaffected lines to serve as isogenic controls [199, 200].

Diseased hESCs and hiPSCs can be exceptionally usefulfor the development, screening and evaluation of drugs. Mucheffort is currently being invested in the development of newdrugs by trying to understand the pathology of the targeteddisease. This drug screening disease-oriented approach re-quires model systems that are based on human cells ratherthan on animal cells, which are frequently unsuitable.Obviously, the best model system would be specific humancell types that manifest the clinical phenotype. However, thecurrently available cellular models in humans are limited bythe lack of relevant and validated cell types. Most are subop-timal since they are based on the use of abnormal cancer celllines or on primary cell cultures. Diseased hESCs and hiPSCs,however, may be exceptionally useful for unraveling devel-opmentally controlled mechanisms associated with humangenetic disorders as well as for large-scale drug screening,and this may lead to the identification of new drug targets. Forexample, IGF1 has been proposed as a potential drug to treatRTT and ASDs following the developmental analysis of RTThiPSCs [148], and 5-azacytidine for reactivation of FMR1 inFXS hiPSCs [133].

Analysis of Neuronal Fate and Functionality

It is crucial to be able to differentiate the hPSCs into neuronsfor the establishment of reliable in vitro models of humanNDs. There are many different protocols available for in vitro

generation of committed neural stem cells and neurons[210–215]. A critical point to address in any study is whetherthe selected protocol recapitulates the different stages ofneurogenesis that are known or suspected to be affected bythe disease in question. The use of protocols that bypass someof these stages can be rapid and effective in producing largepopulations of homogenous mature neurons, but it can alsooverlook molecular and cellular phenotypes that might beimportant in elucidating the mechanisms underlying the dis-ease. On the other hand, prolonged protocols aimed at reca-pitulating several stages of the neurogenesis process can leadto the emergence of in vitro artifacts that are difficult todistinguish from the true pathology. The specific protocolfor neural in vitro differentiation should, therefore, be chosenand/or adapted to the specific needs of the planned research.For example, since the accumulation of uric acid in LNS isprevented by the placenta during fetal life, the symptoms ofthe disorder will therefore arise only after birth when mostneurons are already formed. In this case, a differentiationmethodology which bypasses the mid-step progenitors, suchas genetically induced neuronal differentiation, would be use-ful [216]. In contrast, when investigating a disease such asFXS, the importance of assessing the pathology at every stepof neurodevelopment cannot be underestimated. Since themechanism and timing of FMR1 silencing in humans is stilla matter of debate, it is necessary to implement differentiationprotocols that can recapitulate every possible stage ofneurogenesis [80]. Furthermore, it is important to establishwhether the generation of glial lineages along the neuraldifferentiation process could be an insignificant byproduct, adetrimental phenomenon, or an opportunity for research. Forexample, a bias towards in vitro generation of astrocytes hasbeen observed in both FXS and DS, unveiling a very specificin vitro phenotype that can be used to elucidate the molecularmechanisms behind these disorders [80, 178].

As outlined in Table 2, revealing the specific identity of thein vitro neuronal cells is critical for the research of NDs.However, the myriad of positive and negative markers incurrent use are adopted from previous animal-based neurobi-ological works in which these markers are only a means forthe validation of neuronal identity in cells isolated from aspecific known anatomical compartment. For example, gluta-matergic neurons are present in the cortex of the brain as wellas in the sensory afferents of the dorsal root ganglion, and bothtypes express NMDA receptors even though they have verydifferent functions and properties. In contrast, neurons obtain-ed in vitro from hPSCs cannot be related to an anatomicalcompartment or subtype of neurons based solely on the ex-pression of a few markers. There is a pressing need to over-come this limitation by developing reliable methods for pro-filing neurons obtained in vitro at the genetic and epigeneticlevel, and for comparing them to data obtained from animalmodels, especially those from primates. Electrophysiological


recordings of action potentials, synaptic currents and responseto neurotransmitters are pivotal to the validation of the neuro-nal fate as well as for assessing phenotypic dysfunctionslinked to the disease in question [217]. Nevertheless, as canbe seen from Table 1, hPSCs have been mostly analyzed bybiochemical andmolecular approaches, and only a few studiesadditionally examined their electrophysiological functionality.Those latter studies produced important conclusions regardingthe pathophysiology of the studied ND. For example, electro-physiological data from neurons derived from FX hESCs andRTT hiPSCs suggest that the cognitive disabilities observed inthese NDs are mainly glutamatergic post-synaptic impair-ments [80, 148]. In contrast, recordings from TS hiPSCs-derived neurons suggest a pre-synaptic mechanism [111].Considering that FXS, RTT and TS are comorbid with autismand ASDs, elucidating the role of the post- vs. the pre-synapticmechanisms is key to the understanding of the pathology ofNDs and the eventual development of new treatments, hencethe importance of performing electrophysiological recordingswhen modeling NDs with hPSCs.

Final Remarks

As the field of human in vitro disease modeling evolves,pluripotent stem cells show the greatest promise of providinga suitable platform due to the many advantages they carry.Their application to the understanding of the molecular path-ophysiology of NDs can be pivotal in both scientific advance-ment and therapeutic strategies. More work is needed, such asthe derivation of additional hPSC lines carrying the molecularhallmarks of NDs, followed by thorough protocols for neuraldifferentiation. Detailed molecular and electrophysiologicalcharacterization of these in vitro-derived neurons is funda-mental for reliable phenotypic recapitulation as well as foruncovering the molecular mechanisms responsible for thedisease.

Acknowledgments We thank Prof. Menahem Segal from the Depart-ment of Neurobiology at Weizmann Institute of Science, and Dr. MiraMalcov and Dr. Yael Kalma from the Wolfe PGD-Stem Cell Lab atTel-Aviv Sourasky Medical Center, for critical reading of the manu-script. Esther Eshkol is thanked for editorial assistance. This study wassupported by NNE-Teva scholarship (M. Telias) and Tel-Aviv MedicalCenter (D. Ben-Yosef).

Conflict of interest The authors declare no conflict of interest.

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Modeling Neurodevelopmental Disorders Using Human Pluripotent Stem Cells

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