concise review: modeling central nervous system diseases using induced pluripotent ... ·...

Pluripotent Stem Cells

Concise Review: Modeling Central Nervous SystemDiseases Using Induced Pluripotent Stem Cells

XIANMIN ZENG,a,b JOSHUA G. HUNSBERGER,c ANTON SIMEONOV,d NASIR MALIK,c YING PEI,b

MAHENDRA RAOc,e

Key Words. Central nervous system disease x iPSC x Cell-based screening x Genetic engineering

ABSTRACT

Induced pluripotent stem cells (iPSCs) offer an opportunity to delve into the mechanisms underlyingdevelopment while also affording the potential to take advantage of a number of naturally occurringmutations that contribute to either disease susceptibility or resistance. Just as with any new field,several models of screening are being explored, and innovators are working on the most efficientmethods to overcome the inherent limitations of primary cell screens using iPSCs. In the present re-view, we provide a background regarding why iPSCs represent a paradigm shift for central nervoussystem (CNS) disease modeling. We describe the efforts in the field to develop more biologically rel-evant CNS disease models, which should provide screening assays useful for the pharmaceutical in-dustry. We also provide some examples of successful uses for iPSC-based screens and suggest thatadditional development could revolutionize the field of drug discovery. The development and imple-mentation of these advanced iPSC-based screens will create amore efficient disease-specific processunderpinned by the biological mechanism in a patient- and disease-specific manner rather than bytrial-and-error. Moreover, with careful and strategic planning, shared resources can be developed thatwill enable exponential advances in the field. Thiswill undoubtedly lead tomore sensitive and accuratescreens forearlydiagnosisandallowthe identificationofpatient-specific therapies, thus,paving thewayto personalized medicine. STEM CELLS TRANSLATIONAL MEDICINE 2014;3:1418–1428

INTRODUCTION

The development of the central nervous system(CNS) occurs over a prolonged period. It starts fromearly embryonic development when the epiblastsegregates to form the neuroectoderm. The neuro-ectoderm undergoes additional development toform the CNS and the peripheral nervous system(PNS). The neural tube, destined to form the CNS,undergoesclassicmorphogeneticmovements.Ros-trocaudal and dorsoventral specification gives riseto the forebrain (rostrally) and the spinal cord (cau-dally). The PNS is generated from the placodes inthe skin and the neural crest stem cells. These cellsmigrate to give rise to sensory, enteric, and sympa-thetic neurons. They also support glia and a host ofnon-neural derivatives, including melanocytes andcraniofascial mesenchyme [1–3].

Neural stem cells (NSCs) are the earliest borncells. They are often termed “neuroepithelialstem cells.” They first give rise to radial glial cells,whichprovide the scaffoldonwhich subsequentlyborn neurons migrate to reach appropriate posi-tions in the developing brain. Oligodendrocyteprecursors are born next, followed by astrocytes,which are born at or around the time of birth,followedbymaturation of oligodendrocytes. Dur-ing the first decade of life, oligodendrocytes

myelinate the developing axons. During this pe-riod, the final number of neurons becomes spec-ified, connections are pruned, and the adultstructures are established. Although synapticconnections are dynamic, the total number ofneurons remains relatively static throughout life.Astrocytes, in general, do not proliferate, unlessthey are responding to injury. Oligodendrocyteprecursors persist in the adult brain and replenishthemyelination of new connections that neuronsmight make. Overall, stem cells do not play a ma-jor role in repairing the adult brain. Estimates ofNSC numbers suggest that they represent a verysmall fraction of the total number of cells presentin the brain. Therefore, repair in the brain is be-lieved to be induced by a glial response, whichincludes scarring and associated synaptic reorga-nization to achieve functional restoration, witholigodendrocytes, perhaps, myelinating the newconnections [4].

Many of these processes have been modeledusing animal models or in vitro culture systems.However, certain processes have clearly beendifficult to model. For example, modeling the ep-ithelial tomesenchymal transition that generatesneural crest or modeling the neural tube foldingdefects that underlie several developmental ab-normalities, such as spina bifida, are difficult to

aXCell Science Inc., Novato,California, USA; bBuckInstitute for Research onAging, Novato, California,USA; cLaboratory of Stem CellBiology, NIH Center forRegenerative Medicine,Bethesda, Maryland, USA;dNational Center forAdvancing TranslationalSciences, NIH, Bethesda,Maryland, USA; eNew YorkStem Cell Foundation, NewYork, New York, USA

Correspondence: Xianmin Zeng,Ph.D., Buck Institute for Researchon Aging, 8001 RedwoodBoulevard, Novato, California94945, USA. Telephone: 415-209-2211; E-Mail: [email protected]

ReceivedMay 13, 2014; acceptedfor publication September 19,2014; first published online inSCTM EXPRESS November 3,2014.

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http://dx.doi.org/10.5966/sctm.2014-0102

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PLURIPOTENT STEM CELLS

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achieve with current in vitro technology. More success has beenachieved by characterizing the properties of individual cells,modeling cell-to-cell interactions, and modeling some complexcellular interactions in a dish. Some of these successes includeneuron-astrocyte interactions, in vitro models of the blood-brain barrier, synaptogenesis, and simplified models of learningand memory [5–8].

However, progress in modeling CNS diseases has been ham-pered by two important facts. First, acquiring human neural cellsis difficult, because to accomplish this would require invasive sur-gery. Second, obtaining sufficient numbers of neurons, which donot proliferate from adult or cadaveric samples, has been limitedfor most applications. Given that neurons are postmitotic andrarely form tumors (with the possible exception of retinoblasto-mas and a subclass of medulloblastomas), this severely limits thesheer quantity of neurons available for investigation, even fromtumor samples. Furthermore, glial tumors, which are relativelymore abundant, appear to be irreversibly altered when propa-gated under standard culture conditions [9]. Much of our workon neuronal biology has used neuroblastomas from the PNSand pheochromocytomas of chromaffin cells, which are a non-neural endocrine derivative of the neural crest. In addition tothe limitation of obtaining human neural cells in sufficient num-bers, researchers have faced the uncomfortable realization thatcomplex behavior simply cannot be modeled well in other spe-cies. Even more disconcerting is the species to species variabilityand the realization that even when the final output of a modelseemed identical, different species can have variable responsesto the exact same insult, leading to developing cures for one an-imal species (e.g., a mouse) that do not translate into cure forhumans. Such has been the case with the knockout of SOD1 inmice to model familial amyotrophic lateral sclerosis, or the lossof c-ret signaling inmodeling enteric disease. Thus, different spe-cies could respond to the same insult differently, and screening inanimals could lead to a cure for the animal but not work forhumans. Studies in mice have also led to the realization thatthe same gene knockouts can have a different phenotypic effect,depending on the genetic background of the rodent. Althoughthis can be addressed by examining the phenotype in different ro-dent strains, it presents the challengeof how to assess the geneticbackground inhumancells. For instance, howmanydifferentphe-notypic cell lines should be examined to conclude that the resultscan be generalized to treat human patients with diverse geneticphenotypes? This secondhurdle couldbeovercomewith compar-ative data on patient cells to identify the patient characteristicsthat could be used to form subgroups and quantify the allelic var-iability and other attributes that will enhance the utility of theproposed therapy for a specified group of patients. However, thisrequires developing panels of lines that have been difficult to cul-ture thus far.

DO IPSC-DERIVED CELLS RESOLVE SOME OF THESE ISSUES?

iPSCs as a source of differentiated cells offer numerous possibil-ities and resolve some of the constraints with current non-iPSCbased models. iPSCs are reprogrammed stem cells that are akinto embryonic stem cells (ESCs), with the advantage of being ableto be generated from any individual at any point. This offers theadvantage of modeling diseases across a lifetime in a patient-specificmanner [10, 11]. Just aswith their ESC counterparts, iPSCscanbemaintained indefinitely, thusprovidinga limitless supply of

well-characterized cells of a defined allelic phenotype. Equally im-portant, these cells can be further differentiated into any cell type(or cocultures consisting of multiple cell types) and assessed toidentify aberrant processes that can be targeted for the amelio-ration of diseases (Fig. 1). This offers the advantage of examiningthe effect of a perturbation on isogenic-differentiated cell popu-lations. This was not feasible when wewere limited to examiningcell lines or even primary cells from rodents, other species, oreven humans. Likewise, minimizing the confounding effect of al-lelic variability, as well as harnessing allelic variability, to under-stand the effect of modulators on primary gene disorders canbe achieved using iPSCs from both diseased individuals and theirclosely related normal relatives [12, 13].

Engineering iPSCs offers another unique advantage. iPSCs canbe engineered usingmultiplemethods to investigate howgeneticalterations modulate physiological and disease processes. Theseengineered tools can be further applied in disease-pertinent cel-lular lineages and in developing isogenic and reporter cell lines[14]. Another advantage is the ability to derive iPSCs from differ-ent cellular sources. iPSCs can be reprogrammed from patient-derived cell sources, including blood [15–18], skin fibroblasts[19, 20], hair follicles [21], dental tissue [22], urine [23], and evenpostmortem tissue [24–26]. Another unique consideration is thatpatient cellular sources canbe collected across a lifetime to exam-ine the effects of the environment and age on the differentiationability over time.

These advantages allow for investigations that are now nolonger limited by tissue availability and access to isogenic con-trols. Additionally, iPSCs can be differentiated into various celltypes of the CNS, including neurons, astrocytes, and oligodendro-cytes [27–29]. Each of these neural cell types has been suggestedor used for drug discovery and toxicity tests [29–31]. Given thatmany protocols that generate these CNS cell types have beenreported [32–34], this also provides theopportunity for investiga-tion of multiple cell types to elucidate differences between cellautonomous and cell intrinsic effects. It is also relatively straight-forward to share samples and obtain independent validations,given the unlimited nature of the cell. Collectively, these advan-tages, illustrated in Figure 2, generate optimism that the drug dis-covery process can become more efficient and productive.

Although iPSCs offers unique advantages to screening in theCNS field, it is important to remember that iPSCs are certainly nota universal panacea and, certainly, iPSCs will not offer a solutionmany diseases (Table 1) [35]. For instance, iPSC linesmight not beable to be derived from some disorders that affect DNA repair,aging disorders, and disease that affects protein pathology, suchas prion protein disease. Genomic imprinting, inwhich epigeneticmodifications in the DNA (e.g., DNAmethylation or histonemod-ification) can influence gene expression without altering thegenomic sequence, could also prove difficult to model using aniPSC-based approach. In addition, disorders of the mitochondrialgenome could be difficult to address. Likewise, even after the suc-cessful generation of iPSC lines, an unmet need still exists to con-struct biologically relevant three-dimensional (3D) models thatcan integrate multiple cell types. Similarly, achieving appropriatebiological maturity to manifest late-stage disease phenotypesin a cell-based model to interrogate neurological diseases needsfurther development, although some early success has beenreported [36]. Also, recapitulating in vivo interactions in a dishthat have enabled us to model highly heterogeneous diseasescould prove challenging. In addition, in vivo models in which

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human cells are placed in a relevant animal model could be diffi-cult to generate, because we still have many limitations with de-veloping humanized immune systems in rodents and viableimmunosuppression models for xenotransplants. Finally, somecommon challenges in the reprogramming field need to be con-sideredwhenusing iPSC-basedmodels. These include the hetero-geneity of the reprogramming methods and efficiency, becausethey could lead to alterations in the iPSCs, which would make re-producibility difficult.

Overall, however, iPSCs clearly represent a radical advance inour capability of developing many simple models. These modelsare already proving useful for a field that has been severely lim-ited by access to human cells. Nevertheless, significant challengesto expand the scope and utility of iPSCs remain, and these repre-sent technical challenges for investigators to overcome.

SUCCESSES IN USING IPSC-BASED MODELS

Neurotoxic Developmental Assays and Survival Assays

Current toxicology studiesdonoteffectivelypredicthowadiversepopulation will react to drugs, pollutants, and other environmen-tal chemicals. Moreover, these studies are expensive and time-consuming andoften require a large number of animals for testing.Consequently, only a small number of chemicals have been eval-uated using these methods. Current tests also provide littleinformation on the modes and mechanisms of action, whichare critical for understanding interspecies differences in toxicity.

Furthermore, little to no information is available for assessing thevariability in human susceptibility. In vitromechanistic tests using

human cells can provide rapid evaluations of a large number of

chemicals, greatly reducing the need for live-animal use and

might provide results potentiallymore relevant to human biology

and human exposures. A fundamental understanding of the cel-

lular responses to toxicants, combined with knowledge of tissue

dosimetry in cell systems and in exposed human populations, will

provide a suite of tools to permitmore accurate predictions of the

conditions under which humans can be expected to show path-

way perturbations from toxicant exposure.In collaboration with the National Institute of Environmental

Health Sciences, we undertook a demonstration screen of 80compounds (mostly developmental neurotoxicants) to demon-strate the feasibility of such an iPSC-based approach. We ob-tained cells in sufficient numbers and purity to rapidly evaluatethe drug response using a 96-well format. Because the cells canbe maintained in culture for prolonged periods, both viabilityassays and high content screening (HCS) can be performed. Theeffect of a single compound could be examined using cell viabilityassays (MTT- and/or ATP-based assays) on isogenic iPSC and iPSC-derived homogeneous neural populations (NSCs, neurons, andastrocytes). The 80 compounds used in our study produced spe-cific cellular responses according to cell type and species, a criticalstep in validating this approach and developing it further toscreenmore compounds and for optimization for higher well for-mats to enable 384- and 1,536-based neurotoxicity assays.

Figure1. Human iPSC-derivedneural cells. Schematic summaryofdifferent typesofneural cells that canbederived fromhumanESC/iPSC lines.The applications of iPSC-derived neural cells are listed in the square box on the right. Abbreviations: ESC, embryonic stem cell; iPSC, inducedpluripotent stem cell; NSC, neural stem cell; OLIGO, oligodendrocyte; PNS, peripheral nervous system; Tox, toxicity.

1420 Modeling CNS Diseases Using iPSCs

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Dissecting the contributions of enhancers andmodulators onallelic diversity can now be ascertained. Well-characterized iPSClines can be differentiated using identical differentiation proto-cols in human cell lines. Such efforts, thus far, have only beenpos-sible in rodents and have required a tremendous investment indeveloping inbred lines and diversity panels. We now havea model system in which rodent and human cell lines can berun in parallel.

The same strategy can be used to identify the mechanism oftoxicity and the differential specificity of cellular responses to thesame toxic agent. Our laboratories have optimized methods forfeeder-free culture of pluripotent stem cells (PSCs) and PSC-derived NSCs to facilitate automated screening [37, 38].With thisoptimized differentiation platform, we screened a collection ofFood and Drug Administration (FDA)-approved drugs (∼1,000compounds) to identify compounds that have differential toxicityto PSCs and their neural derivatives [37]. Using comparativescreening of PSCs and PSC-derived homogenous NSCs, we wereable to identify compounds that had differential toxicity toboth cell populations. “Hits” obtained in the primary screenwere then retested, and a small subset was assayed for dose-responsiveness. One confirmed dose-responsive compound,amiodarone, was further tested for toxicity in postmitotic neu-rons. We found amiodarone to be toxic to NSCs but not to post-mitotic neurons. Using time-lapsed imaging, we were able toshow that the cells died extremely rapidlywithin less than 4hoursand did so with little change in gene expression. Thus, the mech-anismof deathwas likely osmotic changes resulting fromblockingof a channel transporter [37].

Such rapid analysis with off-the-shelf human neural cellsoffers an unprecedented opportunity to understand neurotoxic-ity [39–41] in the developing nervous system. In addition, by

combining this with HCS, one can rapidly dissect out the mecha-nism of action of the compound.

Modeling Monogenic CNS Disorders With iPSCs

Modeling hereditary CNS disorders represents a unique advan-tage in which screens can be developed that takes advantage ofthe known genes that have been implicated in that particulardisease and correlate it with a function that can be dissectedout in vitro. By combining data from familial mutant lines withisogenic controls, in which the same gene is knocked out in a de-fined andwell-characterized line, one can dissect out the role ofthe gene at high resolution. Testing the hypothesis in lines gen-erated from patients who do not carry the mutation but displaythe same phenotype or introducing additional mutations will al-low one to develop models of how genes interact. Such modelswere possible, although difficult, in mice but are now readilydeveloped in human iPSCs because of the availability of linesand the advances in gene engineering technologies (Figs. 3,4). We provide five examples (below) in which single genescan be corrected, knocked out, and engineered to develophigh-throughput screens for drug discovery or to interrogatedisease mechanisms (Table 2).

Modeling Familial Dysautonomia With iPSCs

Familial dysautonomia (FD) or hereditary sensory and autonomicneuropathy III (Riley-Daysyndrome)affects theautonomicnervoussystem and is due to a point mutation in the gene for IkB kinasecomplex-associated protein (IKBKAP). This point mutation causesa tissue-specific splicing deficit in peripheral neurons, reducedlevels of IKAP protein, and altered cell motility. Investigators de-rived iPSC lines from patients with FD, a fatal autosomal recessive

Figure 2. iPSCs offer a paradigm shift in central nervous system (CNS) disease modeling. iPSCs can be reprogrammed from a variety of tissuesources and differentiated into many different cell types that would be useful for modeling disease aspects in the CNS. For instance, differen-tiation into midbrain dopaminergic neurons would provide a cell type applicable for modeling Parkinson’s disease. These disease models andneurodevelopmental processes can be developed into screening assays to interrogate further for drug screening and gene profiling studies.These screeningapplications canbeaided furtherby thedevelopmentof iPSCpanels of controls, patient-specific lines, isogenic lines, engineeredlines, and reporter lines. Abbreviations: iPSC, induced pluripotent stem cell; NSC, neural stem cell.



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disease, tomodel this disease in a tissue- and patient-specificman-ner that was further developed into assays to probe for candidatedrugs [42].

The investigators were able to differentiate these FD-iPSClines into all three germ layers and confirm the low levels of nor-mal IKBKAP transcript in FD-iPSC derived neural crest precursors.In patients with FD, autonomic and sensory neurons have beenlost; however, the exact mechanisms remain elusive, and, cur-rently, no animal models are available to investigate FD diseasepathology. These FD-iPSC models identified deficits in IKBKAPsplicing and showed a reduced ability of FD-iPSC derived neuralcrest precursors to undergo neuronal differentiation and de-creased migration in FD-iPSCs compared with control iPSC-derived neural crest precursors using the wound healing assay[42]. In turn, these models identified a candidate drug, kinetin,a plant hormone that promotes cell division. Acute treatmentwith this plant hormone was able to reduce the mutant IKBKAPsplice formand increase normal IKBKAP levels. Chronic treatmentincreased the rate of neurogenesis and peripheral neuronmarkers but did not have significant effects on FD-iPSC neuralcrest precursor cell migration.

In addition to interrogating disease mechanisms and devel-oping disease- and cell type-specific assays for novel drug dis-covery for the treatment of FD, progress has been made indifferentiating neural crest stem cells into a specific type of sen-sory neuron, nociceptors. Chambers et al. have succeeded indirecting differentiation from human PSCs to nociceptors usinga cocktail of small molecules [43]. This has opened the door forinvestigating the transduction of pain mechanisms in a clinicallyrelevant cell type.

Modeling Rett Syndrome With iPSCs

Rett syndrome (RTT) is a neurodevelopmental disorder due toa mutation in the X-linked gene encoding methyl-CpG-bindingprotein 2 [44]. Marchetto et al. recently developed a humanmodel of RTT using an iPSC-based approach [45]. They generatediPSCs from fibroblasts taken from patients with RTT and controls.They then differentiated these iPSCs into neurons and foundmany disease characteristics. These included RTT-iPSC-derivedneuronswith fewer dendritic spines, fewer synapses, a decreasedsoma size, altered calcium signaling, and electrophysiologicaldefects compared with control iPSC-derived neurons. Thesedisease-specific characteristics were then used to test candidatedrugs that would restore these deficits and altered responses to-ward the control levels. They found that insulin-like growth factor1 increased the glutamatergic synapse number in treated RTT-derived neurons. Future studies should validate these diseasespecific deficits using high-throughput screens to identify themost robust models to be used for novel drug discovery.

Modeling Parkinson’s Disease With iPSCs

Parkinson’s disease (PD) is a neurodegenerative disorder primar-ily targeting dopaminergic neurons in which a specific brain re-gion, the substantia nigra, is affected. Modeling this diseaserequires, first, generating iPSC-derived dopaminergic neuronsand, second, challenging thesecellswith known toxins alreadyde-veloped in human and animal models to elucidate the mecha-nisms underlying the demise of these dopaminergic neurons. Ina recent study, Peng et al. reported a screening platform withassays suitable for automated readout using primary dopaminer-gic neurons derived from PSCs and ran a small screen ofcompounds for neuroprotective effects in two toxic models(1-methyl-4-phenylpyridinium [MPP+]- and rotenone-inducedtoxicity) [46]. The investigators chose only known compoundsthat have been reported for neuroprotection in human immor-talized cell lines, rodent primary cells, or in vivo in rodent cells. Apositive feature of this strategy is that these compounds havebeen suggested to act via different protective mechanisms,making validating the mechanism of action easier. Of the 44compounds screened, the investigators found only 18were neu-roprotective in human primary dopaminergic neurons, althoughall were reportedly protective in rodent or cell line models.Equally important, the compounds identified as effective weremostly those that have been used in human trials. These resultsclearly show that screens with primary cells derived from PSCsoffer advantages compared with screens run in immortalizedcell lines and those tested on rodent cells. Although it is difficultto run high-throughput screens with iPSCs, the investigatorsshowed the assays can beused to study themechanismof actionand possible synergic effects of combinations of drugs. In addi-tion, these assays allow investigators to evaluate the long-termeffects of exposure to a particular treatment or drug, becausethe cells can be maintained in culture for prolonged periods.

Modeling Huntington’s Disease With iPSCs

Huntington’s disease (HD) is a neurodegenerative disease caused byCAG expansion that leads to a host of symptoms, including chorea,cognitive decline, and neuropsychiatric abnormalities. ResearchershavegeneratedHD-iPSCsandcorrectedHD-iPSCsusinghomologousrecombination [47]. That report showed that the correction per-sisted in iPSC-derived DARPP-32 positive neurons both in vitro

Table 1. Limitations of iPSC-based models

Limitations of iPSC-based models

Reproducibility from line to line

Heterogeneity of reprogramming and efficiency; also differentiation intospecific cell lineages from iPSCs are still challenging; for example,differentiation into placodal cells, cranial crest, and enteric glia arelimited and cannot make cerebellar granule cells

Inability to grow in 1,534-well plates

Purity of some populations

Unable to generate disease-specific iPSC lines of sufficient maturity (bothdegree of maturation and long period of maturation); for example,diseases of old age

Absence of cross-species data and references or standards

Current limited number of iPSC reporter lines

Difficulty introducing genes in postmitotic cells

Lack of a good myelination model

Genomic imprinting (epigenetic mechanisms); for example, fragile X

Diseases of DNA repair in which the iPSC process might not occur or theline is unstable; for example, polyglutamine Q diseases and trisomies

Mitochondrial diseases with mutations in the mitochondrial genome; forexample, Leber hereditary optic neuropathy, dystonia, andLeigh’s disease

Recapitulating in vivo interactions and complex modeling of genetic andenvironmental interactions over time; for example, neural tube closure,blood-brain barrier, cell migration, synaptic physiology, retinalphysiology, circuit defects, epilepsy, and neuropsychiatric disorders areall difficult to model using iPSC-based in vitro approaches

Abbreviation: iPSC, induced pluripotent stem cell.



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and invivo.Additionally, the correctedHD-iPSCs reversed theabnor-malities in thecommonHDsignalingpathways (e.g., cadherin,BDNF,and caspase activation) and disease-specific phenotypic abnormali-ties. A relevant disease model with identical genetic backgrounds(isogenic controls) is a critical step to advancing new therapiesand will pave the way for personalized medicine.

Modeling Spinal Muscular Atrophy Using iPSCs

Spinal muscular atrophy (SMA) is an autosomal recessive diseasethat has a defect in the SMN1 gene that leads to a loss of motorneurons. Because the SMN1 gene is mutated in SMA-affectedindividuals, correction of this deletion, occurring at exon 7, orother point mutations could provide a unique model systemfor investigating the SMA disease mechanisms using an iPSC-basedmodel. A recent studygenerated iPSCs fromskin fibroblastsfrom patients with SMA and genetically corrected these iPSCs[48]. The motor neurons differentiated from uncorrected SMA-iPSCs showed a disease-specific phenotype that was lost in themotorneuronsderived fromthe corrected SMA-iPSCs.Moreover,in amousemodel of SMA, transplantation of these correctedmo-tor neurons derived from SMA-iPSCs extended the life span andreduced the disease burden in the mice. If similar studies can bedone in humans, this would suggest a clear therapeutic interven-tion for using corrected motor neurons derived from SMA-iPSCs.

Summary

As is clear fromtheseexamples, thegeneral approachhasbeento (a)develop anassayusing generic iPSC lines that arewell characterized,(b) determine whether a phenotype can be obtained in vitro using

patient-specific lines, and (c) assay compoundsusing standardviabil-ity, toxicity, survival, or functional assays as a readout and couple

themwithHCS tounderstand themechanismof action. This processcould provide a unique advantage for orphan and rare diseases for

which tissue samples have often been rate limiting. With iPSC tech-nology, this is no longer true. Standardized lines and the ability toengineer isogenic controls offer an opportunity for new investiga-

tors to enter this field. Several elegant results have been obtainedby simply performing a drug-repurposing screen using already ap-

proved FDA compounds as a screening library. Such a screen atNational Center for Advancing Translational Sciences led to the

identification of d-tocopherol and cyclodextrin as potential treat-ment of Niemann-Pick type C and identified six new classes of com-

pounds for the treatment of Gaucher’s disease [49, 50].Hasson et al. at the NIH were able to use siRNA libraries to

identify key interacting proteins that might be important in the

pathology of PD [51]. We believe iPSC-based screening of rareCNS diseases might be able to identify common mechanisms of

action that contribute to multiple rare diseases that, collectively,are predicted to affect millions of Americans each year. Another

Figure 3. Lineage-specific reporter line generation by zinc-finger nuclease (ZFN)-mediated gene targeting. The process for creating knock-in in-duced pluripotent stem cell (iPSC) lines by ZFN technology is illustrated. ZFNmRNAs and donor vector are inserted into the iPSC via nucleofection,and iPSCsthatwere successfully integratedwereselected forviadrugselection.Anexampleofadonorvectorwitha reporterandaselectionmarkerstrategy is shown. The surviving clones (∼10–50 clones picked) are expanded for 2-4 weeks, with genomic DNA used to screen and confirmfor mutations. The targeted iPSC lines are then assayed for pluripotency and genomic stability and other characterization desired. Abbreviations:2A, 2A peptide; d, day; gDNA, genomic DNA; GFAP, Glial fibrillary acidic protein; LA, left homologous recombination arm; Neo, Neomycin resistantgene; PGK, phosphoglycerate kinase promoter; Puro, puromycin resistant gene; RA, right homologous recombination arm.



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benefit is that by identifying these mechanisms of action, itcould facilitate the development of new therapeutic agents thatmight have wider uses and thus could attract more commercialattention.

Modeling Polygenic CNS Disorders With iPSCs

Although monogenic disorders represent the most obviousscreening choice for iPSC-based research, the ability to obtainlarge panels of lines and develop complex culture assays suggestthat iPSC technology could have someutility in our understandingof polygenic diseases and other complex disorders. This conceptinvolves investigating disease-resistant and -susceptible mecha-nisms in disease-relevant cell types that will enable the discoveryof novel therapies. Several investigators have shown promisingresults, and in the section below we provide some specific exam-ples (Table 2).

Modeling Alzheimer’s Disease With iPSCs

In a recent study, iPSCs were generated from patients with famil-ial and sporadic Alzheimer’s disease (AD) andwere further differ-entiated into neurons and astrocytes [52]. One hypothesis of thecontributing factors to the pathophysiology of AD is the accumu-lation of oligomeric forms of amyloid-b peptide (Ab). In the ADiPSC-derived neurons and astrocytes, an accumulation of thisAb oligomer was found, leading to endoplasmic reticulum (ER)and oxidative stress. Docosahexaenoic acid (DHA) treatment,anomega-3 fatty acid abundant in theCNSandamain constituentof the neuron’s plasmamembrane, reduced the ER and oxidativestress response in AD-iPSC-derived neurons. Moreover, DHA isdecreased in AD brains [53, 54], and decreased DHA serum con-tent correlateswith impaired cognitive ability [55–59]. Therefore,

AD-iPSC-derived cells were able to model a particular aspect ofthe disease. This model can be further interrogated using drugscreening to identify potential compounds to reduce ER and ox-idative stress.

In addition to investigating the disease mechanisms of ADthat go awry, another modeling approach is to investigate themechanisms underlying healthy controls. An extreme exampleof this is the development of iPSCmodels from centenarians. Thiswas done and compared with iPSC-derived neurons from thosewith familial Alzheimer’s disease and familial Parkinson’s disease[60]. Notably, disease-specific pathologic features were absent inthe centenarian iPSC-derived neuronal cultures.

Modeling Schizophrenia With iPSCs

Schizophrenia (SCZD) is a neurodevelopmental disorder charac-terized by hallucinations, delusions, and cognitive deficits. It alsohas a high degree of heritability, with some estimates as high as80% [61]. Given that SCZD is highly heritable, we anticipate thatgenerating panels of iPSC lines according to subgrouping by geneexpressionorepigenetic patternswill lead to identifying interven-tions that are effective for the specific subgroups. This will enableclinicians to truly practice personalized medicine in a complexheterogeneous disease such as SCZD.

Modeling cellular characteristics of SCZD using iPSCs thatwere differentiated into neurons has been achieved [62]. Bren-nand et al. reprogrammed fibroblasts to generate iPSC lines thatwere then differentiated into neurons (mostly glutamatergic,∼30% GABAergic and ∼10% dopaminergic) for both controls(n = 6) and SCZD (n = 4). Although this was only a small sample size,their study does present some interesting findings. First, SCZDneurons demonstrated phenotypic differences compared with

Figure 4. Genetic engineering of induced pluripotent stem cells (iPSCs) by zinc-finger nuclease (ZFN)-mediated gene targeting. The process forcreating knock-out iPSC lines by ZFN technology is illustrated. ZFN mRNAs are inserted into the iPSCs via nucleofection, and iPSCs that are suc-cessfully integrated are screened via polymerase chain reaction and sequencing. The selected clones are expanded for 2-4 weeks, and genomicDNA is used to confirm for heterozygote or homozygote. More than one round of nucleofection and screening can be required to generatehomozygotes. The targeted iPSC lines are thenassayed for pluripotency andgenomic stability andother characterizationdesired. Abbreviations:d, day; gDNA, genomic DNA.



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controls (e.g., decreased neurite number, decreased neuronalconnectivity, and decreased synaptic protein), and, at the sametime, showed similar electrophysiological responses comparedwith the control neurons. Second, gene expression differenceswere also apparent where cAMP and WNT signaling pathwaygenes were altered in SCZD neurons. Third, treatment with anantipsychotic drug (loxapine) ameliorated the changes in geneexpression (e.g., NRG1, GRIK1, ADCY8, PRKCA, WNT7A, TCF4).Finally,NGR1geneexpressionwaselevated inSCZDneurons com-paredwith the controls andwas specific for the relevant cell type,becausenodifferenceswere foundbetween theSCZDandcontrolfibroblasts.

It is also feasible now to generate integration-free SCZDiPSC lines from skin biopsies [63]. This advent permits thegeneration of more consistent iPSC lines that are not plaguedwith the potential of foreign DNA integration, which can dis-rupt the host’s genome and lead to unintended consequences(e.g., reactivation of oncogenes). Moreover, iPSCs can also begenerated using human postmortem tissue (e.g., dura materand scalp) to make use of postmortem human samples fromlarge cohorts of well-characterized subjects stored in brainbanks.

OTHER INNOVATIVE STRATEGIES USING IPSC-BASED SCREENS

This section provides additional examples for other innovativestrategies using iPSC-based screens. These innovative strategiesincludemodeling neurotrauma,modeling CNS infections, screen-ing prospectively to personalize therapy, complementing adverseevent screening during clinical trials, developing diversity panels,conducting genome-wide association study (GWAS) screens, anddeveloping shared resources. Collectively, these innovative strat-egies can be used to create more advanced iPSC-based screensthat are more sensitive and effective predictors for disease sus-ceptibility and treatment.

Modeling Neurotrauma With iPSCs

To improve the treatment of patients and thereby decrease theassociated mortality, morbidity, and cost, several in vivo modelsof CNS injury have been developed and characterized during thepast twodecades. To complement theability of these in vivomod-els to reproduce the sequelae of human CNS injury, in vitro mod-els of neuronal injury have also been developed [64]. Despite theinherent simplifications of these in vitro systems,many aspects of

Table 2. Successful modeling of CNS disorders with iPSC-based approaches

Disease Progress References

Monogenic CNS disorders

iPSCs were derived from patients with FD. Mis-splicing of IKBKAP was identified by geneexpression analysis in FD-iPSC-derived lineages. Low levels of IKBKAP were reported inpatient-specific neural crest precursors. An FD-iPSC assay was used to evaluate candidatedrugs.

Lee et al. [42], 2009

FD Developed efficient protocol for deriving human nociceptors from iPSCs using smallmolecules.

Chambers et al. [43], 2012

RTT RTT-iPSCs were generated and differentiated into neurons that displayed disease-specificcharacteristics (fewer synapses, reducedspinedensity) that could thenbedeveloped intoanassay to screen candidate drugs.

Marchetto et al. [45], 2010

PD Human iPSC-derived dopaminergic neurons were used to evaluate neuroprotectivecandidates from 2 common neurotoxicity models (MPP and rotenone) and identified 16promising candidates.

Peng et al. [46], 2013

HD HD-iPSCs were corrected using homologous recombination and further differentiated intoDARPP32-positive neurons that reversed common disease-specific abnormalities (e.g.,cadherin, BDNF, and caspase activation).

An et al. [47], 2012

SMA SMA-iPSCs were generated and genetically corrected (SMN1 gene correction) and furtherdifferentiated into motor neurons, where the disease-specific phenotype was absentcompared with uncorrected SMA-iPSC-derived motor neurons. Using a mouse model ofSMA, these corrected motor neurons were transplanted and extended the lifespan andreduced the disease burden in mice.

Corti et al. [48], 2012

Polygenic CNS disorders

AD Patient-derived iPSCs were derived from patients with familial and sporadic AD and furtherdifferentiated into neurons and astrocytes. The AD iPSC model mimicked disease aspectssuch as accumulation of Ab oligomer. iPSCs derived from centenarians were comparedagainst iPSC-derived from AD, where specific disease pathologies were absent incentenarian iPSC-derived neuronal cultures.

Kondo et al. [52], 2013;Yagi et al. [60], 2012

SCZD Patient-derived iPSCs reprogrammed from fibroblastsweredifferentiated intoneurons thatdemonstrated phenotypic differences compared with controls (e.g., decreased neuritenumber). Geneexpression identifiedaltered cAMPandWNTsignalingpathways. Treatmentwith an antipsychotic medication ameliorated changes in gene expression (e.g., NRG1,GRIK1, ADCY8, PRKCA, WNT7A, TCF4). NRG1 gene expression was elevated in iPSC-derivedneurons but not in patient fibroblasts, supporting the importance of investigating relevantcell types.

Brennand et al. [62], 2011;Chiang et al. [63], 2011

Abbreviations: AD, Alzheimer’s disease; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; DARPP32, dopamine, cAMP-regulatedphosphoprotein of 32 kDa; FD, familial dysautonomia; HD, Huntington’s disease; iPSCs, induced pluripotent stem cells; PD, Parkinson’s disease; RTT,Rett syndrome; SCZD, schizophrenia; SMA, spinal muscular atrophy.



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the post-traumatic sequelae, including primary, secondary, andtertiary responses to blast injuries, can be faithfully reproducedin cultured cells. The changes that have been monitored includeultrastructural changes, ionic derangements, alterations in elec-trophysiology, free radical generation, and alterations in geneexpression.

Additional development of these models has been limitedowing to the lack of availability of human cells and the abilitytogrowcells forprolongedperiods.However, iPSCs andotherplu-ripotent cells allow one to overcome this limitation, and manyinvestigators have now begun to transfer their rodent in vitromodels to in vivo models. Advances in 3D culture and the abilityto develop myelination models has further improved the fidelityof the models. These models have also been extended to investi-gate radiation damage.

Modeling Infections in the CNS

Lafaille et al. engineered iPSCs from childrenwith inborn errors oftoll-like receptor 3 (TLR3) immunity,whoareprone toherpes sim-plex virus 1 (HSV-1) encephalitis (HSE), andused these cells to pin-point the underlying causes of the disease [65]. They illustratedthat HSE involves nonhematopoietic CNS resident cells and thatiPSC-derived neurons were far more susceptible than UNC93B-deficient patients and controls. These iPSC lines were differenti-ated into purified populations of NSCs, neurons, astrocytes, andoligodendrocytes. More importantly, the team showed that theinduction of interferon-b (IFN-b) and/or IFN-l1 in response tostimulation by the double-stranded RNA analog polyinosinic-polycytidylic acid [poly(I:C)] was dependent on TLR3 and UNC93Bin all cells tested. However, the induction of IFN-b and IFN-l1 inresponse to HSV-1 infection was impaired selectively in UNC93B-deficient neurons and oligodendrocytes. These cells were alsomuch more susceptible to HSV-1 infection than were the controlcells, although the UNC93B-deficient NSCs and astrocytes werenot. TLR3-deficient neurons were also susceptible to HSV-1 infec-tion. The rescue of UNC93B- and TLR3-deficient cells with the cor-responding wild-type allele showed that the genetic defect wasthe cause of the poly(I:C) and HSV-1 phenotypes. The viral infec-tion phenotype was rescued further by treatment with exoge-nous IFN-a or IFN-b (IFN-a/b), but not IFN-l1. Thus, impairedTLR3- and UNC93B-dependent IFN-a/b intrinsic immunity toHSV-1 in the CNS, in the neurons and oligodendrocytes in partic-ular, might underlie the pathogenesis of HSE in children withTLR3-pathway deficiencies.

Screening Prospectively to Personalize Therapy

Cancer biologists have suggested that tumor recurrence likelyoccurs because a rare tumor population escapes standard ther-apy. If the population was isolated and approved drugs weretested, perhaps a tailored optimized cocktail of drugs could berapidly developed to administer to a patient inwhom the therapyhas failed. This strategy has shown some success and is a novelapproach to screening with immediate benefits.

iPSC-based therapy to treat gliomas is gaining speed throughtheuseof engineering iPSC-derivedNSCs as vehicles todeliver an-ticancer gene therapy [66]. Other investigators have argued thatthe process of iPSC generation might mimic the changes that oc-cur in cancer and that studying the process and drugs that affectthe process could provide clues to cancer therapy.

Adverse Event Screening During Drug Trials

An important issue for the pharmaceutical industry has been therisk of anunexpected rare adverse event thatwill bemissed as thedrug proceeds through the standard clinical evaluation phase, ne-cessitating a late recall. Drugs have beenwithdrawn despite theirefficacy in a large population because of this risk to a rare popu-lation. iPSCs offer a solution to this problem. Patients withadverse events canbe identified, and their iPSCs canbegeneratedand used to extensivelymap the drug response and compare it tothat of patients who had a beneficial effect. This sort of testingcould allow one to salvage drugs as biomarkers and could be de-veloped further to identify patients who should not be adminis-tered such a drug. Likewise, in most clinical trials, some patientswill have a treatment response and otherswill not. Thus, compar-ing responders to nonresponders could provide novel insightsand help stratify patients for therapy. No fundamental technicalissue exists in performing such screens and the size of the screensis not large; thus, it is simply a matter of time and a decision toimplement.

Developing Diversity Panels

One can imagine developing panels of lines that are genetically di-verse and testing them routinely with any new approved drug toreduce the risk of unexpected events when a drug is widely used.Suchapaneldoesnot exist andwould requirea public-privatepart-nership.However, it is certainly technically feasible, andwebelieveits benefits will far outweigh the cost associated with such an en-deavor. Efforts to develop these lines are being initiated (Fig. 4),and, with careful attention to ensuring adequate diversity, wemight soon have a panel of iPSC lines for such a purpose.

GWAS Type Screens

iPSCs have also offered a solution to the allelic variability issue illus-trated in pharmacogenomics [67]. The problem is simply thathumans are outbred, which means the allelic variability reflectedin gene expression and variation in gene levels is in the same rangeas across species. This has been the basis of the reluctance in usingprimary cells, becausewedonot knowhownormal the responseofany given line will be. With iPSCs, we can generate a large numberof lines and return to the same allelic phenotype because of the in-trinsic lack of senescence in the cells. Thus,we can achievemany ofthe advantages of an immortalized cell line without its disadvan-tages. Efforts along these lines have already begun. Disease-specific panels in which sporadic and familial cases from patientswith the same disease are obtained using a standardized methodwill allow one to test hypotheses and drugs in a panel to identifyunique and uniform responses and dissect stochastic differencesfrom biologically relevant ones. Panels for PD, HD, and cardiac hy-pertrophy have been developed. More ambitious proposals havesuggested that a similar panel-based approach can be successfullyimplemented for polygenic disorders. These proposals would in-clude even larger polygenic disease panels in which screening iscombined with whole genome, exome, and epigenome sequenc-ing. This is an ambitious idea but, if successful, could completelychange how we perform screens.

Developing Shared Resources

Too often resources are not developed to allow the field to ad-vance rapidly.Wesuggest that thedevelopmentof several shared



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resources will enable exponential advances in the field. Theseshared resources include (a) access to disease panels of iPSCsand their differentiated/engineered products, (b) access to com-mon controls, (c) access to common patient data for selectingappropriate samples, and (d) sophisticated bioinformatic toolsthat can integrate patient data and cell assay data in a cloud-based format for global use. These resources, if developed andimplemented strategically, will allow preventative medicine tobe fully exploited such that disease-specific, cell-based screenscan be personalized for each patient to, not only predict suscep-tibility to disease, but also to develop innovative therapies to curethat disease.

CONCLUSION

iPSC-based CNS disease modeling holds tremendous potential.The cells can be used in multiple ways. No doubt exists that addi-tional screens such as those related tomyelination, novel screens

related to modeling the blood-brain barrier or neural folding, orentirely new modes of therapy will be developed. Our sense isthat the current successes are only the tip of the iceberg. As inves-tigators become more familiar with the system and the costs ofsuch assays continue to decline, one will see even more innova-tion in this area of toxicology research.

AUTHOR CONTRIBUTIONS

X.Z. andM.R.: conception and design, financial support, adminis-trative support, collection and/or assembly of data, manuscriptwriting, final approval of manuscript; J.G.H.: conception and de-sign, manuscript writing; A.S., N.M., and Y.P.: manuscript writing.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

X.Z. has uncompensated ownership interest in XCell Science Inc.

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