Arch Pharm Res Vol 35, No 2, 245-260, 2012DOI 10.1007/s12272-012-0205-9

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Induced Pluripotent Stem Cell Research: A Revolutionary Approach to Face the Challenges in Drug Screening

Minjung Song*, Saswati Paul*, Hyejin Lim, Ahmed Abdal Dayem, and Ssang-Goo ChoDepartment of Animal Biotechnology, Animal Resources Research Center, and SMART-IABS, Konkuk University, Seoul143-701, Korea

(Received October 12, 2011/Revised November 8, 2011/Accepted November 10, 2011)

Discovery of induced pluripotent stem (iPS) cells in 2006 provided a new path for cell trans-plantation and drug screening. The iPS cells are stem cells derived from somatic cells thathave been genetically reprogrammed into a pluripotent state. Similar to embryonic stem (ES)cells, iPS cells are capable of differentiating into three germ layers, eliminating some of thehurdles in ES cell technology. Further progress and advances in iPS cell technology, from viralto non-viral systems and from integrating to non-integrating approaches of foreign genes intothe host genome, have enhanced the existing technology, making it more feasible for clinicalapplications. In particular, advances in iPS cell technology should enable autologous trans-plantation and more efficient drug discovery. Cell transplantation may lead to improved treat-ments for various diseases, including neurological, endocrine, and hepatic diseases. In studieson drug discovery, iPS cells generated from patient-derived somatic cells could be differenti-ated into specific cells expressing specific phenotypes, which could then be used as diseasemodels. Thus, iPS cells can be helpful in understanding the mechanisms of disease progres-sion and in cell-based efficient drug screening. Here, we summarize the history and progressof iPS cell technology, provide support for the growing interest in iPS cell applications withemphasis on practical uses in cell-based drug screening, and discuss some challenges faced inthe use of this technology. Key words: Induced pluripotent stem (iPS) cells, Magnet-based nanofection, Cell transplan-tation, Disease modeling, Drug discovery

INTRODUCTION

Human world is developing and at the same time,there has been a continuous evolution of diseases anddisease agents. Therefore, it is necessary to developmethods to cure and combat the most daunting dis-eases. One significant step in this direction has beenthe discovery of stem cells, followed by the suc-cessful isolation of human embryonic stem (ES) cells.More recently, the induced pluripotent stem (iPS) celltechnology revolutionized the world of scientific re-search (Maherali and Hochedlinger, 2008). Develop-

ment of iPS cells through reprogramming of somaticcells was a landmark discovery in the field of stemcells, since it made it possible to generate cell linesfrom adult tissues and most importantly, from pati-ent-derived tissues, which have the potential for usein cell-based drug discovery, as well as in cell therapy(Amabile and Meissner, 2009).

Stem cells are classified into several groups, depend-ing on their differentiating ability and developmentalstage at which they are obtained (Lodi et al., 2011).Adult stem cells are multipotent cells isolated fromadult or fetal tissues; these cells have the ability torenew themselves and differentiate into the special-ized cell type of the originating tissue (Weissman,2000). They are mainly used for replacing damagedand injured tissue, and to date, they have been deriv-ed from the brain, bone marrow, spinal cord, skin,liver, retina, and many other tissues (Pittenger et al.,

*These authors contributed equally as a first author.Correspondence to: Ssang-Goo Cho, Department of Animal Bio-technology and Animal Resources Research Center, KonkukUniversity, Seoul 143-701, KoreaTel: 82-2-450-4207, Fax: 82-2-450-4207E-mail: ssangoo@konkuk.ac.kr

REVIEW

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1999). However, it is difficult to identify, isolate, andculture the adult stem cells due to their rareness inadult tissues.

ES cells, on the other hand, are derived from theembryo and are capable of forming stable cell lines,which retain the pluripotency to differentiate intocells from all three germ layers (ectoderm, mesoderm,and endoderm) (Thomson et al., 1998; Cowan et al.,2004). Due to the extensive developmental potential ofES cells, scientists can manipulate ES cells in vitro toform various cell types (Keller, 2005). Although manystudies have reported on ES cells in animal models,applications of the findings in humans are still notpredictive and need further extensive studies. The useof human ES cells can overcome many hurdles thatexist in conducting studies using animal models, butit also introduces two important issues. A disadvan-tage of using ES cells is the destruction of embryos,something that has raised moral and ethical issuesamongst the public. In addition, an ES cell transplantcarries a risk of immunogenic reactions, since ES cellsfrom a random embryo donor are likely to face immunerejection after transplantation.

Generation of iPS cells from somatic cells by retro-virus-mediated transduction of four transcriptionfactors (Takahashi and Yamanaka, 2006) provided arevolutionary platform for stem cell use in cell-baseddrug screening and disease therapy. The iPS cells aresimilar to ES cells in their morphology, gene expres-sion, epigenetic status of pluripotent cell-specific genes,and pluripotency (i.e. differentiating into all threegerm layers) (Takahashi et al., 2007; Yamanaka, 2009).Importantly, iPS cells provide an alternative to theuse of human embryos, overcoming ethical issues. Inaddition, iPS cell technology allows the use of patient-specific somatic cells to generate therapeutic iPS cells,thus overcoming the potential for immune rejection.The recent advances made with iPS cells hold poten-tial for creating patient-specific disease model iPS celllines that can be used for disease mechanism studiesand drug discovery. In this review, we describe thehistory and progress of iPS cells as well as discuss thecurrent and future applications of iPS cells in diseasemodeling and drug discovery. Furthermore, stem celltherapy through cell transplantation is also discussed.

HISTORY AND PROGRESS OF iPS CELLTECHNOLOGY

Previous work in research ranging from nucleartransfer and cloning to the establishment of immortalpluripotent cell lines influenced and led to the presentstatus of iPS cell technology. In mammals, embryonic

development is characterized by a gradual restrictionin the developmental potential of the cells that con-stitute the embryo. The zygote and blastomeres of theearly morula stage are totipotent and further divide toform the blastocyst (Fig. 1A). The inner cell mass ofthe blastocyst is pluripotent and self-renewing, whilethe outer cells of the embryo develop into the pla-centa. Adult stem cells, derived from various adulttissues, are multipotent and also capable of self-re-newal (Thomson et al., 1998; Cowan et al., 2004; Keller,2005).

To understand the developmental potential ofnuclei, a cloning technique was established by trans-planting isolated nuclei into enucleated oocytes(Briggs and King, 1952; King and Briggs, 1955). Thiswas later followed by the generation of a cloned frog(Gurdon, 1962) and a sheep (Wilmut et al., 1997),which further supported the finding that fully spe-cialized cells remain genetically totipotent (Fig. 1A).In 1964, researchers established the pluripotent terato-carcinoma cell line (Kleinsmith and Pierce, 1964),which replicated and grew in culture as stem cellscalled embryonal carcinoma (EC) cells. Although ECcells provide a model system for the study of cellularcommitment and differentiation, they are tumor cellsand typically aneuploid. These disadvantages associ-ated with the use of EC cells led to the establishmentof ES cells. In 1981, Evan and Martin first derivedpluripotent ES cells from a blastocyst stage mouseembryo (Evans and Kaufman, 1981; Martin, 1981).The discovery of lineage-associated transcription fac-tors, which help maintain cellular identity duringdevelopment, contributed to the discovery of iPS cells.These transcription factors were first shown in fibro-blast cell lines transduced with retroviral vectors(Davis et al., 1987), and subsequently, transcriptionfactors responsible for expression of cell-type specificgenes that are important for pluripotency were ob-served (Xie et al., 2004; Laiosa et al., 2006; Zhou et al.,2008; Vierbuchen at al., 2010). In the study to identifytranscriptional regulators responsible for reprogram-ming somatic cells into pluripotent cells, Takahashiand Yamanaka first successfully produced iPS cellsfrom mouse fibroblasts using four retrovirally trans-duced transcription factors, c-Myc, Oct3/4, Sox2, andKlf4 (Takahashi and Yamanaka, 2006) (Fig. 1B). HumaniPS cells were derived in 2007 by the transduction ofeither the same set of transcription factors (c-Myc,Oct3/4, Sox2, and Klf4) or another set of transcriptionfactors (Oct3/4, Sox2, Nanog, and Lin28) into humansomatic cells (Takahashi et al., 2007; Yu et al., 2007;Nakagawa et al., 2008; Park et al., 2008b).

Initially, iPS cells were derived from somatic cells

iPS Cell Research: Application in Drug Screening 247

by retroviral (Takahashi and Yamanaka, 2006) orlentiviral (Blelloch et al., 2007; Yu et al., 2007;Brambrink et al., 2008; Stadtfeld et al., 2008a) trans-duction of transcription factors (Table I). However,such a method of establishing iPS cells with carcino-genic c-Myc and retrovirus may induce tumor forma-tion and affect the efficiency of somatic cells to pro-duce iPS cells (Okita et al., 2007), thus hindering theapplication of iPS technology in disease therapy. Toavoid genetic modification and to improve the effici-ency of iPS cell generation and differentiation, iPS cellproduction technology was advanced by techniquesthat avoided stable integration of foreign geneticmaterial into the host genome (Table I). Stadtfeld et

al. (Stadtfeld et al., 2008b) for the first time used anadenovirus carrying Oct4, Sox2, c-Myc, and Klf4 togenerate iPS cells from mouse hepatocytes, but thismethod did not receive much clinical application dueto low efficiency. Vector integration-free mouse iPScells have been derived from embryonic fibroblasts byrepeated plasmid transfections (Okita et al., 2008),however, the practical approaches of this method islimited by the associated low reprogramming fre-quency. Human iPS cells have also been derived usingnon-integrating episomal vectors (Yu et al., 2009) andvectors based on Sendai virus (Fusaki et al., 2009).Further research, using the PiggyBac transposon inboth mouse and human fibroblast cells, has led to the

Fig. 1. (A) Stage-by-stage hierarchy of stem cell development. During development, the differentiation potency decreasesand the specialization increases at each stage. Reprogramming potential was first confirmed in animal cloning throughsomatic cell nuclear transfer (SCNT) and then at the cellular level by generation of iPS cells without the use of oocytes. (B)Generation of iPS cells from somatic cells using various approaches, including non-viral approaches (synthetic mRNA/miRNA, proteins, and small molecules), viral approaches (retrovirus, lentivirus, adenovirus, and Sendai virus), vectors(episomal vectors), transposon (PiggyBac transposon), transient transfection and magnet-based nanofection.

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generation of iPS cells without vector integration (Kajiet al., 2009; Woltjen et al., 2009).

In 2008, Bosnali and Edenhofer (Bosnali andEdenhofer, 2008) generated a transducible version oftranscription factors, OCT4 and SOX2, and used avector system to fuse it with a TAT sequence. The re-combinant OCT4 and SOX2 proteins displayed DNA-binding properties, exhibited cellular entry, and main-tained pluripotency in mouse stem cells. Moreover, in2009, DNA-free iPS cells were generated from human

(Kim et al., 2009) and mouse (Zhou et al., 2009) fibro-blasts by direct delivery of four reprogramming factors(OCT4, SOX2, KLF4, and c-MYC). These DNA-free iPScells were similar to ES cells, in terms of morphology,proliferation, and gene expression. They were capableof differentiating into three germ layers and led toteratoma formation, but the efficiency was significant-ly lower than virus-based methods (Kim et al., 2009).The addition of small molecules was successfully ableto increase the reprogramming efficiency (Huangfu et

Table I. Methods for iPS cell generationMethod Advantage Disadvantage References

Retrovirus Self-silencing eliminates need fortimed factor withdrawal

Genomic integrationIncreased tumor incidenceInfection limited to dividing cells

(Takahashi and Yamanaka, 2006;Takahashi et al., 2007; Yu et al.,2007; Nakagawa et al., 2008;Park et al., 2008b)

Lentivirus Infects both dividing and nondi-viding cells

Polycistronic vector availableTet-inducible expression availableTransgene-free through CRE/loxP

excision

Genomic integrationLack of silencing in pluripotent state

(Blelloch et al., 2007; Okita et al.,2007; Brambrink et al., 2008;Stadtfeld et al., 2008a)

Adenovirus Low frequency of genomic integra-tion

Repeated infection requiredDelayed kinetics of reprogrammingSome generation of tetraploid cells

(Stadtfeld et al., 2008b)

Episomal vectors Low frequency of genomic integra-tion

Excision may be inefficient and la-borious

(Yu et al., 2009)

Sendai vectors No viral componentsNo genomic integration

Inefficiency (Fusaki et al., 2009)

Transienttransfection

No viral componentsLow frequency of genomic integra-

tionTechnically simple procedure

Multiple applications required Lower levels of expression Delayed kinetics of reprogrammingIntegration provides selective advan-

tage and necessitates clone screen-ing

(Okita et al., 2008)

PiggyBactransposon

Precise deletion possible Extremely low efficiency (Kaji et al., 2009; Woltjen et al.,2009)

Minicircle DNA No viral componentsFree of bacterial DNAHigh expression

Inefficiency (Jia et al., 2010; Narsinh et al.,2011)

Sythetic mRNAmiRNA

No viral componentsNo genomic integrationHigh efficiency

Multiple applications required (Lin et al., 2008; Li et al., 2011;Yang et al., 2011)

Proteintransduction

Direct delivery of transcriptionfactors avoids complications ofnucleic-acid-based delivery

No viral components

Multiple applications requiredSome proteins difficult to purify

(Bosnali and Edenhofer, 2008;Kim et al., 2009; Zhou et al.,2009)

Small molecules No viral componentsTransient controllable activityTechnically easy to work with

Issue of toxicity versus efficacyUndefined/nonspecific effects

(Huangfu et al., 2008; Chen etal., 2011; Wang et al., 2011)

Nanofection No viral componentsTransient controllable activityTechnically easy to work with

Less efficient than viral infection (Gersting et al., 2004; Lee et al.,2008; Lee et al., 2011)

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al., 2008). Interestingly, Lin et al. (2008) reported thatreprogramming of human skin cells can be achievedusing only micro RNAs (miRNAs), specifically by ex-ogenous expression of the miR-302 cluster. More re-cently, miR-93 and its family members were found toenhance iPS cell generation in mouse, demonstratingthat iPS cell induction efficiency can be greatly en-hanced by modulating miRNA levels in cells (Li et al.,2011). The role of miRNAs involved the regulation ofmultiple signaling networks and has been reported tohave a similar role in iPS cell generation (Yang et al.,2011). The iPS cells can also be established by variousgrowth factors and chemical agents. For instance,lithium, a drug used to treat mood disorders, greatlyenhanced the efficiency of iPS cell generation inmouse embryonic fibroblasts and human umbilicalvein endothelial cells by enhancing the transcriptionalactivity of Nanog (Wang et al., 2011). Another set ofcompounds, including rapamycin and curcumin, werefound to enhance the efficiency of somatic cell repro-gramming to iPS cells (Chen et al., 2011). In a recentreport, iPS cells were generated from adult humanadipose tissues using non-viral mini-circle DNA vectorscontaining the reprogramming genes, Oct4, Sox2,Nanog, and Lin28 (Jia et al., 2010; Narsinh et al.,2011). In the experiment, they used plasmid vector toyield a mini-circle vector separated from the parentalplasmid vector containing the bacterial elements. Anadvantage of this technique is that iPS cells are de-rived with no viral sequence or c-Myc oncogene, over-coming possible safety concerns. In a slightly differentapproach, magnet-based nanofection was shown to bean efficient method of transfection in various cell types,including ES cells (Gersting et al., 2004; Lee et al.,2008), which could avoid the harmful genome-inte-grating effect of viral DNAs. On the basis of this study,Lee et al. (2011) successfully performed the nanofec-tion method in mouse embryonic fibroblast (MEF)cells and could generate iPS cells with the ES-like pro-perties of self-renewal and pluripotency. In the study,a magnetic-nanoparticle coated with a biodegradablecationic polymer was employed to transfect the iPSfactors into mouse fibroblasts to generate iPS cellswithout using a viral system. The reprogrammingefficiency of this method for generating iPS cells wascomparatively higher than other non-viral systems.

As the aforementioned history and progress in iPScell research indicates, the reprogramming of somaticcells into pluripotent cells has opened a new avenuefor generating patient- and disease-specific pluripotentstem cells. Although further work is needed to in-crease the efficiency of reprogramming and to overcomethe carcinogenic effects, application of iPS cell technol-

ogy for the treatment of human disease will help toenhance our understanding of disease mechanisms;further, this technology may have applications in drugscreening, toxicity screening, and regenerative medi-cine.

THERAPEUTIC APPLICATIONS OF iPSCELLS

The use of iPS cell technology in clinical applicationsis gaining a lot of attention because it provides apotential avenue for novel therapeutic approaches anda great scope for customized cell transplantation ther-apy, disease-modeling, and drug-discovery. Therapeuticapplications mainly involve cell transplantation anddrug discovery. In this section, recent cases of celltransplantation are reviewed and the application ofiPS cells in disease modeling/drug discovery is dis-cussed.

TransplantationThe iPS cells have three advantages over ES cells

starting with bypassing ethical concerns regarding theuse of human embryos (Green, 2002; George andGomez-Lobo, 2005). In addition, this technology enablesautologous transplant, which is considered as the “goldstandard” method. With allogeneic ES cell transplan-tation, patients must receive immunosuppressive ther-apy for at least 4 months and need to be monitored forsigns of rejection for 15 years (Charron et al., 2009;Boyd and Fairchild, 2010). Shortly after iPS cells wereintroduced, several cases of transplantation using iPScell technology were reported. Fig. 2A demonstratesthe general transplantation procedures for iPS cells.The therapeutic effect of iPS cells was first describedin a Parkinson’s disease (PD) model by Wernig et al.(Wernig et al., 2008). Dopaminergic neuronal death inthe midbrain is known as the main cause of PD.Wernig et al. successfully reprogrammed fibroblastsinto iPS cells and differentiated them into neural pre-cursor cells before finally differentiating them into do-paminergic neurons. When these functional neuronswere transplanted into a rat model of PD, normalizeddopamine activity was reported and alleviation of PDsymptoms was observed. iPS cell therapy was alsoused to treat an inherited genetic disease, hemophiliaA (Xu et al., 2009). Hemophilia A is caused by amutation in factor VIII (FVIII), which is involved inblood clotting; thus, hemophilia A patients suffer frominternal or external bleeding. In the study, iPS cells,reprogrammed from mouse fibroblasts, were differ-entiated into endothelial progenitor cells (EPC) andendothelial cells, and FVIII protein release from these

250 M. Song et al.

cells was measured. Following transplantation of FVIII-producing EPC/endothelial cells, mice with hemophiliaA survived from a death-inducing bleeding assay,demonstrating pathologic phenotype correction (Xu etal., 2009). iPS cells have also been differentiated intofunctional cardiac myocytes (Mauritz et al., 2008), inan attempt to treat cardiac infarcts. Additionally, iPScells generated from type I diabetes patients weredifferentiated into insulin-producing pancreatic pro-genitor cells (Maehr et al., 2009). Two years later,another group published similar results, in which iPScells were successfully differentiated into insulin-pro-ducing cells and injected into a mouse model of type Idiabetes (Bar-Nur et al., 2011). Insulin generation inthese cells was confirmed through glucose challengeexperiments. Another case of functional recoverythrough iPS cell transplantation was reported with aspinal cord injury model (Tsuji et al., 2010). In thisreport, iPS cells produced neurospheres that differ-entiated into three neural lineages, functional neurons,astrocytes, and oligodendrocytes, in vitro. The iPScell-derived neurospheres were then transplanted intothe brain of a spinal cord injury mouse model. After 9days, functional recovery was confirmed on the basis

of re-myelination and axonal regrowth.Finally and most importantly, there is a repairing

chance of disease-causing mutations by iPS cell trans-plantation, which was unsuccessfully attempted severaltimes using adult stem cells (Fig. 2A) (Stadtfeld andHochedlinger, 2010). Mutation repair can be employedfor diseases caused by genetic disorders. After fibro-blast cells are reprogrammed to iPS cells, disease-causing mutations are corrected with the traditionalgene therapy techniques of insertion, alteration, orremoval of genes. Briefly, iPS cell therapeutic ap-proaches follow four steps: (1) iPS cell generation frommutant donor somatic cells, (2) genetic correction ofmutant genes with gene therapy, (3) in vitro differ-entiation of corrected iPS cells into mature cells, and(4) transplantation. To date, two such studies havebeen reported (Hanna et al., 2007; Raya et al., 2009).Hanna et al. applied this method to the humanizedsickle cell anemia mouse model (Hanna et al., 2007).First, iPS cells generated from fibroblasts were usedto correct the human sickle hemoglobin allele by gene-specific targeting. These corrected iPS cells were thendifferentiated into hematopoietic stem cells in vitro,followed by transplantation. Eight weeks later, the

Fig. 2. (A) Generation of iPS cells from patient-derived somatic cells, using reprogramming factors to cause differentiationinto various human cell types. These cells could be re-transplanted into the same patient for treatment of various diseases.(B) Possible procedures for using of iPS cells in drug screening: differentiation of iPS cells into various human cell types (forexample, hepatocytes, neurons, cardiomyocytes or pancreatic cells) that could be exploited in the drug screening process tofind potent compounds by using patient-derived iPS cells, testing toxicity of compounds using an animal model (preclinicaldevelopment), using these compounds in human therapy after passing a screening process that includes three phases ofclinical trials.

iPS Cell Research: Application in Drug Screening 251

corrected iPS cells had reconstituted the hematopoieticsystem of sickle mice and restored hemoglobin func-tions. Additionally, Raya et al. generated iPS cellsfrom a patient with Fanconi’s anemia (FA), a commonbone marrow failure syndrome that results from 13gene mutations in the FA pathway (Raya et al., 2009).

The iPS cells derived from the FA patient were genet-ically corrected by reprogramming and differentiatedinto hematopoietic progenitor cells. In vitro, thecorrected iPS cells showed functional FA pathway re-establishment.

In addition to cell transplantation, another applica-

Fig. 2. Continued

252 M. Song et al.

tion of iPS cells is in the development of a cell-basedvaccine for human immunodeficiency virus (HIV)/AIDS (Yoshizaki et al., 2011). The authors of this studyintroduced HIV-gp160 into iPS cells and pre-treatedthe HIV-gp160-expressing iPS cells with irradiation.Interestingly, the HIV-gp160-expressing iPS cells re-leased pre-inflammatory cytokines upon irradiation.After the cells were inoculated into a xenogenic modelmouse, the cellular immune response was induced,suggesting a promising application for iPS cells incell-based vaccine production.

Application of iPS cells for drug discoveryTo launch a novel drug into the market, a phar-

maceutical company goes through the following steps:drug discovery (evaluation of the efficacy and toxicityof the novel compound at the cellular level), pre-clinical development with animal models, and phase I,II, and III clinical trials (Paul et al., 2010). Dependingon the success rate of treating the disease in a diseasemodel, the drugs may progress into clinical trials inhumans. However, current drug discovery procedureshave many limitations. First, disease-specific cells arenot available for many diseases, especially many de-generative diseases, including Alzheimer's disease(AD), PD and amyotrophic lateral sclerosis (ALS), inwhich it is difficult to obtain cells or tissues withpathological phenotypes. Moreover, it is difficult toprepare relevant cells for diseases occurring in sporadicforms and those affected by complicated unknowngenetic aspects, such as autism spectrum disordersand type I diabetes. The current iPS technology mayresolve these issues by using its “disease modeling”potential (Fig. 2B). When iPS cells derived from apatient's fibroblasts are differentiated, mature cellsexpress disease phenotypes and show disease progres-sion. Therefore, disease modeling aids in both the un-derstanding of disease mechanisms and the discoveryof novel therapeutic compounds (Paul et al., 2010).

Another limitation in current drug discovery is thatwhile drugs may show a significant activity in cell cul-ture and animal models during the discovery steps,they might not show any activity during clinical trials(DiBernardo and Cudkowicz, 2006). These clinicalfailures may result from differences in disease mecha-nisms and developmental in addition to physiologicalaspects between humans and animals. Moreover, toxiceffects of compounds that are specific to humans maynot be detected in cell lines and animal models. Thesediscrepancies can be resolved with iPS cell technology,which reprograms a patient’s fibroblasts and differ-entiates them into affected cells. In addition, iPS celltechnology opens the door for personalized medicine.

In many cases, different patients respond differentlyto the same drug. If drugs can be prescreened withpatient-derived cells before injection, drug efficacy canbe optimized and adverse effects could be reduced.

Drug discovery procedures with iPS cellsTo apply iPS cells in drug discovery, scientists need

to overcome several hurdles, shown in Fig. 2B. First,iPS cells need to be successfully generated from non-pluripotent patient cells, mainly through fibroblastreprogramming. Second, these cells need to differ-entiate into the three germ layers and commit to ma-ture cells that express the disease phenotype, which iscalled “disease modeling”. Third, novel compoundsapplied to these disease-specific cells are screened forefficacy (e.g., drugs that reverse disease phenotypes ata cellular level) and toxicity. After successful screen-ing of particular drugs in iPS cells, preclinical devel-opment with animal models and phase I, II, and IIIclinical trials are followed for final drug launching.

CURRENT iPS CELL APPLICATIONS INDRUG DISCOVERY

Table II shows the current status of iPS cell appli-cation in drug screening for various diseases (Park etal., 2008a; Kiskinis and Eggan, 2010; Stadtfeld andHochedlinger, 2010). In neurological, immune, mus-cular, genetic, hematological and hepatic diseasemodels, iPS cells were successfully generated withvarious reprogramming methods. Moreover, generat-ing mature cells with disease phenotypes was onlysuccessful in neurologic, genetic, hematological, andhepatic diseases. A motor neuron study on ALS, aneurological disease that results from destroyed spinalcord motor neurons and leads to progressive paralysisand death, was successfully conducted (Dimos et al.,2008). Because most motor neurons in ALS patientsdie, it has been difficult for scientists to study the dis-ease’s progression and to screen therapeutic drugs forefficacy. Dimos et al. produced iPS cells from fibro-blasts of ALS patients and differentiated them intomotor neurons; the iPS cells were then used to screennovel compounds and to study their interactions withother cells as well as their susceptibility to environ-mental conditions (Dimos et al., 2008). Long-QT syn-drome (LQTS) and LEOPARD syndrome (LS) providegood examples of genetic diseases that are treatablewith iPS cell therapy (Carvajal-Vergara et al., 2010;Moretti et al., 2010). Moretti et al. derived iPS cellsfrom patients with type 1 LQTS, differentiated the iPScells into cardiomyocytes, and recapitulated nearly allmajor aspects of the LQTS phenotype (Moretti et al.,

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Table II. Current progress of disease-specific iPS cells for drug screeningDisease Differentiated cell types Drug screening References

Neurological diseaseAmyotrophic lateral sclerosis (ALS) Motor neurons No (Dimos et al., 2008)Spinal muscular atrophy (SMA) Mature motor neurons VPA, (Ebert et al., 2009)

Astrocyte TobramycinParkinson disease (PD) Dopaminergic neurons No (Park et al., 2008a; Soldner et al., 2009;

Hargus et al., 2010; Swistowski et al., 2010)

Huntington disease (HD) Striatal neurons No (Park et al., 2008a; Zhang et al., 2010)Rett syndrome (RTT) Neuronal progenitor

cellsIGF1, Gentamycin (Hotta et al., 2009; Marchetto et al.,

2010)Prader-Willi syndrome (PWS) Neurons No (Yang et al., 2010)Shwachman-Bodian-Diamond syndrome(SBDS)

ND No (Mali et al., 2008; Park et al., 2008a; Somers et al., 2010)

Alzheimer’s disease (AD) Neurons γ-secretase inhibitorsand modulators(Compound W)

(Yagi et al., 2011)

Schizophrenia Neurons Loxapine (Brennand et al., 2011)Immune system diseaseADA-SCID (ADA) ND No (Park et al., 2008a)Endocrinology/Metabolism diseaseGaucher disease type III (GD) ND No (Park et al., 2008a)Juvenile diabetes mellitus (JDM) ND No (Park et al., 2008a)Lesch-Nyhan syndrome (LNSc) ND No (Park et al., 2008a; Hotta et al., 2009)Type 1 diabetes Pancreatic beta cells No (Maehr et al., 2009; Si-Tayeb et al., 2010)Muscular system diseaseDuchenne muscular dystrophy (DMD) ND No (Park et al., 2008a)Becker muscular dystrophy (BMD) ND No (Park et al., 2008a)Genetic diseaseDown syndrome (DS) ND No (Park et al., 2008a)LEOPARD syndrome (LS) Cardiomyocytes No (Carvajal-Vergara et al., 2010)Familial dysautonomia (FD) Central NS Kinetin (Lee et al., 2009)

Peripheral NSHematopoietic cellsEndothelial cellsEndodermal cells

Friedreich’s ataxia (FRDA) ND No (Ku et al., 2010)Long-QT syndrome type 1 (LQTS1) Cardiomyocytes No (Moretti et al., 2010)Long-QT syndrome type 2 (LQTS2) Cardiomyocytes Nifedipine,

Ranolazine(Itzhaki et al., 2011)

PinacidilHematological diseaseMyeloproliferative disorders (MPD) Hematopoietic cells No (Ye et al., 2009b)Fanconi anemia (FA) ND No (Raya et al., 2009)b-Thalassemia Hematopoietic cells No (Ye et al., 2009a)Hepatic diseaseAlpha 1-antitrypsin deficiency (A1ATD) Hepatocytes No (Rashid et al., 2010; Somers et al., 2010)Glycogen storage disease type 1a (GSD1a) Hepatocytes No (Rashid et al., 2010)Glycogen storage disease type 1b (GSD1b) Hepatocytes No (Ghodsizadeh et al., 2010)Familial hypercholesterolemia (FH) Hepatocytes No (Ghodsizadeh et al., 2010)Crigler–Najjar syndrome (CND) Hepatocytes No (Ghodsizadeh et al., 2010; Somers et al.,

2010)Tyrosinemia type 1 (TYR1) Hepatocytes No (Ghodsizadeh et al., 2010; Somers et al.,

2010)HER1 Hepatocytes No (Ghodsizadeh et al., 2010)

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2010). LS, which arises from two genetic mutations,T468M and Y279C, is a difficult genetic disease tostudy because its only animal models are Drosophilaand zebrafish. Recent studies have demonstrated thesuccessful differentiation of iPS cells into cardiomyo-cytes with hypertrophic features that are phenotypi-cally associated with LS (Carvajal-Vergara et al.,2010). For the study of inherited hepatic disease,dermal fibroblasts were obtained from patients withα1-antitrypsin deficiency (A1ATD), familial hyperchole-sterolemia (FH), and glycogen storage disease type 1a(GSD1a), and iPS cells were generated (Rashid et al.,2010). These iPS cells were differentiated into endo-derm cells, hepatic progenitors as well as maturehepatocytes. The hepatocytes expressed key pathologi-cal features of the disease from which the fibroblastsoriginated.

Six individual studies have reached the final stagesof novel compound screening in disease-specific cells.Each study provides much information to its respec-tive field. In a study of a neurological disease, motorneurons were generated from a spinal muscularatrophy (SMA) patient (Ebert et al., 2009). SMA is aneurodegenerative disease in which deficiency of sur-vival motor neuron (SMN) protein leads to the loss oflower motor neurons. The researchers obtained skinfibroblasts from SMA patients and generated iPS cellsfrom the SMA fibroblasts. The SMA-derived iPS cellswere expanded and differentiated into motor neuronswith a SMN deficit. Using the SMA neurons to screenvarious drugs, the study found that treatment withvalproic acid (VPA) and tobramycin led to an increasein the level of SMN protein. The following year, iPScells were derived from the fibroblasts of a patientwith Rett syndrome (RTT) (Marchetto et al., 2010).RTT is a progressive neurological disease and is consi-dered as part of the autism spectrum disorders (ASD).This neuronal disease is caused by a mutation in theMECP2 gene resulting in fewer synapses, reducedspine density, and smaller soma size. Marchetto et al.differentiated iPS cells into RTT-neurons, which wereused in drug screening, and showed that IGF1 andgentamycin could rescue synaptic defects (Marchettoet al., 2010). In a recent study, iPS cells have shownpromise for the treatment of AD. AD is an autosomaldominant disease mutated in presenilin 1 and pre-senilin 2, and it is known as the most common form ofdementia, afflicting 35 million people worldwide (Yagiet al., 2011). At present, there is no effective treatmentfor delaying its onset or attenuating its symptoms.Yagi et al. generated iPS cells from fibroblasts of ADpatients and differentiated these cells into neuronsthat had increased amyloid β42 secretion, resulting

from mutant presenilin. When the neurons weretreated with various novel compounds, Compound W(γ-secretase inhibitors and modulators) decreasedamyloid β42 secretion, indicating its potential as atherapeutic drug. In addition, iPS cell technology hasbeen applied to another neurological disorder, schizo-phrenia (SCZD), which is a mental disorder character-ized by disintegration of thought processes and dimin-ished emotional responsiveness (Brennand et al.,2011). Existing studies have demonstrated abnormalhistology in the prefrontal cortex and hippocampus inaddition to unusual neuropharmacological activities,but the underlying disease mechanism is still un-known. Brennand et al. used iPS cell technology togenerate neurons for studying the underlying mecha-nism of SCZD, which revealed a differential gene ex-pression pattern in the cAMP and Wnt signaling path-ways, as well as a decrease in neuronal connectivity(Brennand et al., 2011). When loxapine was applied,improved neuronal connectivity was observed. In thegenetic disease field, Lee et al. (2009) produced famil-ial dysautonomia (FD)-associated iPS cells from patient-derived fibroblasts. FD is a rare autosomal syndromecaused from a point mutation in the IKBKAP gene. InFD patients, peripheral neurons, such as sensory andautonomic neurons, are depleted. Systemic analysis ofdisease progression and functional validation of can-didate drugs have been limited by the lack of FD tis-sues and the absence of a disease mouse model. UsingiPS cell technology, neuronal precursor cells weregenerated as a disease model used for drug screening.Continuous kinetin treatment showed partial restora-tion of disease phenotypes. In 2011, iPS cells fromanother genetic disease, type 2 LQTS, were generatedfrom patient-derived fibroblasts (Itzhaki et al., 2011).LQTS patients have an abnormal ion channel functionthat leads to sudden cardiac death. The underlyingdisease mechanism has not been extensively studieddue to an absence of human cardiomyocytes. In thestudy discussed here, iPS cells differentiate into car-diomyocytes which are considered a model for patient-specific LQTS and disease phenotypes were ameli-orated upon treatment with pharmacological agents(nifedipine, pinacidil, and ranolazine).

Clinical trials require tremendous cost (e.g., $1,778million/drug), and many clinical applications end upin failure (Rubin, 2008). Considering all the previousstudies and examples, we conclude that iPS cells arevery promising for drug development by providing ahigh quality and low-cost drug screening system thathelps to overcome current drug discovery limitationsin addition to providing potential for personalizeddrug therapy.

iPS Cell Research: Application in Drug Screening 255

PROBLEMS AND CHALLENGES FACEDBY iPS CELL RESEARCH

The successful reprogramming of somatic cells intoiPS cells has ushered in a new era of application of SCsin disease research, drug screening, and regenerativemedicine. With further progress in iPS cell research,iPS cell technology may provide powerful new alterna-tives to animal models, representing a significantbreakthrough that can be used to validate clinical andtherapeutic applications of drugs in specific groups ofdisease patients. However, many questions remainunanswered about the safety of iPS cells in diseasetherapy and their accuracy in drug screening. The mostconcerning obstacles for using iPS cells in clinicaltherapy and drug screening are: (1) heterogeneity ofiPS cell culture and difficulty in distinguishing thepatient-specific therapeutic population from the massof differentiated cells, (2) the potential of residual iPScells to form teratomas in culture, and (3) immuno-genicity of the therapeutically viable iPS cells.

iPS cells are found to have epigenetic memory,which favors differentiation along lineages related tothe somatic tissue origins and restricts alternative cellfates (Kim et al., 2011). This property should beconsidered in order to efficiently differentiate the iPScells into the desired cell types for cell therapy anddrug screening.

Genomic instability has been observed in humaniPS cells (Laurent et al., 2011), making it necessary togenomically monitor iPS cells prior to clinical applica-tion. It was recently observed that reprogramming ofhuman iPS cells led to an increase in mutations,which may be due to the pre-existing mutation andmutations arising after reprogramming (Gore et al.,2011). In 22 human iPS cell lines reprogrammed byfive different methods, protein-coding point mutationswere reported and some mutations were found to benew mutations linked to reprogramming. Moreover,using a high-resolution single nucleotide polymorphismarray, Hussein et al. (2011) compared copy numbervariations (CNVs, structural variations in the DNA ofa genome, resulting in cells having an abnormalnumber of copies of one or more sections of the DNA)of different passages of human iPS cells with theirfibroblast cell origins and with human ES cells. Theyreported a higher level of CNVs associated with repro-gramming and also found that during moderate lengthculture, however, iPS cells undergo a selection process,leading to a decreased mutation load equivalent tothat seen in ES cells.

Chromosomal aberrations and increased expressionof cell cycle-related genes, which may lead to tumor

formation and restriction of differentiation ability,were observed in reprogrammed human iPS cells(Mayshar et al., 2010). Variations in the propensity ofteratoma formation were also reported for iPS cells(Miura et al., 2009). These findings were significantlyrelated to the tissue of origin, which must be properlyevaluated before iPS cell-based therapy is used. Im-munogenicity of iPS cells is another factor for consi-deration before further clinical application of iPS celltechnology occurs (Zhao et al., 2011).

In drug screening and cell therapy, iPS cells havemany advantages over ES cells; however, because theconsistency of pluripotency and the tendency of thesecells to develop into the cell types desired for thera-peutic application are still under investigation, thereare many challenges involved in the application of iPScell technology. The first major concern is to identifythe best reprogramming method to achieve high iPScell generation efficiency and less genetic alteration inthe generated iPS cell lines. Another challenge isovercoming the heterogeneity of iPS cells in culture,which makes it important to optimize the protocols forderivation of target cell types without inducing largeundifferentiated cell (which may lead to tumor forma-tion). Heterogeneity of iPS cells in culture may alsodistort the high-throughput screening of compounds,as homogeneity of cells is essential for drug screening(Ellis and Bhatia, 2011). The application of iPS cells tostudy diseases such as AD and PD, which take a longtime for the first onset of disease symptoms, posesanother challenge. A possible alternative could be theapplication of external stress to speed up the onset ofdisease in culture (Saha and Jaenisch, 2009). Finally,for drug screening, cell-to-cell interactions are theimportant criteria to consider, other than the cell’sautonomous program. In this regard, the use of tissueengineering to generate a desired disease-relevantphenotype in iPS cells may provide a more in-depthunderstanding of particular drug's effects (Saha andJaenisch, 2009).

CONCLUSION

Recent advances in iPS cell technology has urgedscientists to study diseases in vitro, looking for diseasephenotypes, applying micro-environmental stresses,and testing new drugs. Although the reprogrammingprocess for efficient generation of iPS cells faces manychallenges, many groundbreaking approaches havehelped to overcome some of the challenges. iPS celltechnology provides a revolutionary method for drugdiscovery, which bridges genetics, cell biology, andphysiology. As we have discussed, progress in iPS cell

256 M. Song et al.

technology has improved the potential for using stemcells in drug discovery, as well as in cell transplanta-tion. By transplanting iPS cell-derived autologouscells, functional improvement was observed in manydiseases, including PD, hemophilia A and sickle cellanemia, without serious immune rejection issues. Inthe drug discovery field, patient-derived iPS cells couldbe differentiated into disease-specific mature cells fordisease modeling. Various drug candidates were appliedto the iPS cell-derived disease model cells and theeffects of particular drugs were scrutinized for patho-logical phenotypes in neurological and genetic diseases.Some issues regarding iPS cell applications remain tobe resolved, but this technology can be a valuable toolin treating diseases. Broader applications of iPS cellsto medicine and therapy are expected in the near futureand will result from advances made in developingsafer and more efficient methodology in iPS cell tech-nology.

ACKNOWLEDGEMENTS

This work was supported by a grant from the KoreaHealth 21 R&D Project, Ministry for Health, Welfareand Family Affairs, Republic of Korea (A08-4065 andA10-1712) and by National Research Foundation(NRF) grant funded by the Korea government (MEST)(No. 2010-0020348). We acknowledge a graduate fello-wship provided by the Ministry of Education andHuman Resources Development through the BrainKorea 21 project, Republic of Korea.

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