Induced pluripotent stem cells for modeling of pediatric neurological disorders.

www.biotechnology-journal.com

Page - 1 -

Biotechnology Journal

Accepted Article

Review

Induced pluripotent stem cells for modeling of pediatric neurological disorders Jiho Jang1, Zhejiu Quan2, Yunjin J. Yum2, Hyo Sook Song2, Seonyeol Paek2, Hoon-Chul Kang2 1

Department of Physiology, 2Division of Pediatric Neurology, Department of Pediatrics,

Severance Children’s Hospital, Epilepsy Research Institute, Yonsei Stem Cell Research Institute, Yonsei University College of Medicine, Seoul, Korea

Key Words: Induced pluripotent stem cells, Pediatric neurological disorders, Disease modeling, Drug screening, Cell-based therapy Abbreviations: ALD, X-linked adrenoleukodystrophy; AMN, adrenomyeloneuropathy; CCALD, childhood cerebral linked adrenoleukodystrophy; CDKL5, cyclin-dependent kinase-like 5; CFTR, cystic fibrosis transmembrane conductor receptor; CNS, central nervous system; CRISPR, clustered regularly interspaced short palindromic repeat; Dppa2, developmental pluripotency associated 2; DSB, double-stranded break; ESCs, embryonic stem cells; Esrrb, estrogen related receptor beta; FMR1, fragile X mental retardation 1; FMRP, fragile X mental retardation protein; HDR, homology-directed repair; HR, homologous recombination; hTERT, human telomerase reverse transcriptase; iN, induced

neuronal cells; iNSC, induced neural stem cells; iPSCs, induced pluripotent stem cells; NHEJ, nonhomologous end joining; Mbd3, methyl-binding protein 3; MeCP2, methyl CpGbinding protein; SMA, Spinal muscular atrophy; SMEI, severe myoclonic epilepsy in infancy; SV40LT, simian vacuolating virus 40 large T; TALEs, transcription activator-like

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/biot.201400010. Submitted: Revised: Accepted:

09-Jan-2014 29-Mar-2014 15-May-2014

This article is protected by copyright. All rights reserved.


Page - 2 -


effectors; TALENs, transcription activator-like effector nucleases; TGF, transforming

Accepted Article

growth factor; VLCFA, very-long chain fatty acids; ZFN, zinc finger nuclease Corresponding author: Dr. Hoon-Chul Kang, Division of Pediatric Neurology, Department of Pediatrics, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea E-mail: [email protected]


Page - 3 -


Accepted Article

ABSTRACT Understanding the pathophysiological mechanisms of neurological disorders and the

advancement in novel cell-based therapies has previously been inconceivable due to lack of suitable and applicable disease models. Although animal models showed promising results, they possess potential risks of malfunctions in reprogramming that should be avoided at all costs before these results can fully be exploited with translation to the clinic. Human embryonic stem cells (ESCs), are also unsuitable candidates for disease modeling because of ethical and technical challenges. Induced pluripotent stem cells (iPSCs), on the other hand, closely resemble the key features of human ESCs including self-renewal and pluripotent potentials. With this advanced iPSC technology, various neurological disorders can now be extensively studied. In this paper, we provide an overview of various gene delivery systems

for generating iPSCs, the current state of modeling early-onset neurological disorders and the ultimate application of these in-vitro models in cell therapy through the correction of diseasespecific mutations.


Page - 4 -


Accepted Article

1 Introduction The neurodegenerative disorders of childhood are a diverse group of rare diseases

that pose a considerable burden in the child, the family, and the community. This downhill track includes a progressive loss of speech, hearing, and vision often accompanied by seizures, feeding difficulties, and loss of intellect. Whilst previous studies were, to some extent, successful at delaying the progress of these disorders, various disease mechanisms remain unknown. Early studies of human brain and its neuronal function had been done using postmortem tissues that were not always well preserved and often represented the end-stage of the disease [1]. The following researches performed their experiments on animal disease models as an alternative to postmortem tissues, yet they, too, failed to recapitulate the complexity of human pathology. Thus, the lack of applicable and feasible disease models has impeded the progress of understanding the underlying mechanisms of neurological disorders. In 2007, Takahashi and Yamanaka shed light on this problem by generating

pluripotent cells from human fibroblasts through the retroviral-mediated induction of four pluripotency genes, including OCT4, SOX2, KLF4, and c-MYC [2]. Thomson’s follow-up research generated iPSCs using different set of four transcription factors (OCT4, SOX2, NANOG and LIN28) [3]. This alternative source of customized pluripotent cells closely resembles the properties of human embryonic stem cells (ESCs), including the ability to selfrenew and to differentiate into cells of all three germ layers. Even though iPSCs may not accurately reproduce the epigenetic status of ESCs [4], they have substantial advantages over ESCs such as circumvention of moral and ethical dilemmas. In addition, this is a notable feat because iPSCs differentiated in vitro can now replace human tissue and be utilized for modeling and investigating pathophysiology of early-onset pediatric neurological disorders, and ultimately developing drug treatment and patient-specific cell therapies. In this article, we outline the recent advancements in iPSC technology and

thoroughly review its application in cutting-edge researches on pediatric neurological disorders. Additionally, some concerns and challenges regarding modeling and therapeutic cell-based therapy of pediatric neurological diseases are addressed.


Page - 5 -


Accepted Article

2 iPSC technology 2. 1 Generating iPSCs What are some criteria to consider when generating clinically relevant iPSC lines? Of

all, the induction of iPSCs free of transgenic sequences in their genomes is the utmost feature to be acknowledged. The most commonly used method is the use of viral transduction of defined factors to somatic cells. Lentiviral-based systems, for example, are reasonably efficient and reproducible which can reprogram somatic cells - known as the hardest to reprogram of all cell types – by forcing the integration of the reprogramming factors. Unfortunately, viral-based disease models still bear risks of insertional mutagenesis, reactivation of reprogramming factors, oncogene reactivation, immunogenicity, and uncontrollable silencing of transgenes, hence are inadequate for human clinical applications. Because the long-lasting expression of reprogramming factors is necessary for successful reprogramming, any insertion of transgenes into patient’s chromosomes will cause a risk of random insertional mutagenesis in the host genome sequence. If arbitrary insertion site is related to the gene or the promoter of the gene linked to pathogenesis, it is hard to analyze the disease using iPSC-derived disease models. Another major issue with viral systems is reactivation and residual expression of the integrated reprogramming factors, which can impede differentiation into target cells or bias understanding of pathophysiology. In the aspect of safety of reprogramming, various different approaches have been developed instead of integrating vectors. In order to generate clinically approachable iPSCs, the following gene delivery

strategies have been used (Table 1): plasmids [5], the Cre/loxP system [6], piggyBac vectors [7], and minicircle vectors [8]. Recent studies have successfully generated transgene-free and genetically immaculate iPSCs using protein transduction [9], non-integrating viral vectors such as Sendai virus [10], episomal vectors [11], transfection of modified mRNA transcripts

[12], and chemicals [13]. Attempts at reprogramming with proteins have been made, yet the efficiency of production is extremely low (~0.001). Current researches focus on the use of Adenoviruses and Sendai viruses due to their

ability to avoid exogenous DNA integration into the host genome. The results, however, have been unsuccessful most of the time. A modified mRNA-based strategy is currently being explored to produce transgene-free iPSCs. Other methods dealing with small molecules such as 5-aza-2′deoxycytidine [14], forskolin [15], valporic acid [16], vitamin C [17], butyrate [18], RNA-145 inhibitor and TGF beta ligands [19], Rapamycin [20], and lithium [21] have also


Page - 6 -


been reported to improve the efficiency of iPSCs derivation. Furthermore, P53 siRNA,

Accepted Article

human telomerase reverse transcriptase (hTERT) and Simian Vacuolating Virus 40 large T (SV40LT) Antigen successfully accelerated the reprogramming kinetics [22, 23]. Estrogen

related receptor beta (Esrrb), Utf1, Lin28, and developmental pluripotency associated 2

(Dppa2) further enriched the study by generating fully reprogrammed iPSCs in the absence of the Yamanaka factors with single-cell level identification of reprogramming events [24]. The typical yields of iPSCs generation by methods mentioned above ranged from 0.01%–5%, depending on the cell type and reprogramming system. Rais et al. recently jumped on the board and reported about the reprogramming efficiency of the methyl-binding protein 3 (Mbd3) deletion that reached up to nearly 100% within few days, suggesting that iPSCs reprogramming renders a deterministic process [25].. 2.2 Correcting disease-specific mutations To date, gene targeting by homologous recombination (HR) conventionally enable in

the generation of knockout/knock-in transgenic mouse models using mouse ES cells thereby improving various genetic studies. However, the efficiency of gene targeting in human ESCs still remains very low [26], as compared to that is routinely performed in mouse ES cells. Because they grow poorly as single cells and selection of targeted clones is of rare, compared with mouse ES cells. Patient-specific iPSCs are huge resources for investigating diverse human diseases for disease modeling and for cell therapy in the future. Currently, several new technologies including zinc fingers (ZFs), transcription

activator-like effectors (TALEs), and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system have drastically increased the targeting efficiency and thus are widely adopted for the purpose of correcting disease-specific mutations [27-29]. These new technologies have been widely used in the targeted correction of disease-specific mutations, as well as gene knock-in studies. A sequence-specific gene targeting making use of the cell’s HR and changing patient’s permanent mutation to a normal status is absolutely needed for full application of these iPSCs such as correction of disease phenotypes and cell-replacement therapy. Especially, gene correction could produce malady-free autologous cells with therapeutic potentials. This technology will accelerate the development of the personalized treatment of various diseases. ZFs and TALEs are DNA-binding proteins that combine with Fok1 nucleases and recognize specific DNA sequences, known as ZFNs and transcription activator-like effector


Page - 7 -


nucleases (TALENs) [30]. While Fok1 was capable of breaking DNA double-strands only,

Accepted Article

Cas9 nuclease in the bacterial CRISPR-Cas9 system targets and cleaves specific DNA sequences entirely when guide RNAs bind to the particular DNA sequence. ZFN and TALEN systems cooperate with two hemi-nuclease domains and provoke double-stranded break (DSB) at the target site. The resulting DSB is repaired by cellular repair mechanisms including error-prone non-homologous end joining (NHEJ), gene repair by HR, or homology-

directed repair (HDR) facilitated by an exogenous DNA donor. NHEJ causes arbitrary

insertions/deletions at the incision site. Long, double-stranded DNA donors that induced variation in the genome (selection markers) are necessary for HR. Utilizing these toolboxes, several groups have made great advancement in altering disease phenotypes in patientderived hiPSCs, which encourages a viewpoint of this expertise in the future. Jaenisch et al. successfully demonstrated disease-phenotype correction by using zinc finger nuclease (ZFN) in human iPSCs derived from the monogenic blood disease sickle-cell anemia, followed by correction of α-synuclin in Parkinson’s disease iPSCs using ZFN-mediated genome editing

[31, 32]. Correcting pathogenic mutations of disease-specific iPSCs suggests the encouraging perspective of this technology and provides a powerful tool for treating many genetic disorders. Recent report on CRISPR-Cas9 system addressed its connection to an organoid

culture technology in patients of cystic fibrosis transmembrane conductor receptor (CFTR) and demonstrated a recovery of CFTR function in corrected organoids, suggesting a potential strategy for future cell-based therapies [33]. Though these tools have a relatively high efficiency compared to that of conventional

HR, they possess certain risks of potential off-target effects and genetic toxicity. It is essential to ensure that the gene editing procedures do not introduce any unexpected mutation. Currently, bringing gene targeting into the clinical setting heavily depends on whether or not a sequence-specific nuclease can be designed to target the designated area. Future research should focus on examining and minimizing off-target effects of these nucleases before it can be translated into clinic. Therefore, to this end, more efficient as well as safer gene correction strategies may be required for the generation of mutation-free human iPSCs before the therapeutic application in the future.


Page - 8 -


Accepted Article

2.3 Bringing the Bench to Bedside Despite the controversies on the immunogenicity of iPSCs [34-36], some clinical

studies such as the ones conducted by the Takahashi group are being performed. Takahashi’s lab conducts transplantation of iPSCs-derived retinal pigment epithelium into the subretinal space of six severe age-related macular degeneration patients. Age-related macular degeneration is characterized by progressive deterioration of the retinal pigment epithelium (RPE) resulting in loss of vision due to leakage caused by neovascularization [37]. This year, the same group has reported that hiPSC-derived RPE cell sheets that are optimized to meet clinical requirements including quality, quantity, consistency, and safety have been successfully generated. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. This pilot study has motivated many researchers to try the transplantation of autologous iPSCs-derived retinal pigment epithelium cell sheets for age-related macular degeneration in human patients. A similar clinical trial related to macular generation using human ESCs was published last year which showed no hyperproliferation, abnormal growth, or immune-mediated transplant rejection after the transplant [38]. Functional advancements after transplantation of human ESC-/iPSC-derived neurons

in animal models have been acknowledged in many preclinical studies [39]. However a thorough exploration regarding differentiation, maturation, and integration of the grafted cells into the endogenous neural circuitry should be considered to significantly promote human ESCs/iPSC-based cell replacement therapy into clinical application. Moreover, finding proper biomarkers for cell sorting of the desired cells, reducing the risk of tumorigenesis in

vivo for suicide genes, and optimizing cell dosage for transplantation are critical for designing human trials. Finally, the therapeutic adequacy in vivo and the precise mechanisms of the functional recovery should be evaluated and elucidated in detail. 3 Ideal, human-like iPSC-derived disease modeling in vitro Neurological disorders exclusively dealing with pediatrics are distinguished by

impaired neuronal functions during early development and degenerative neuronal structure. Conventional therapies of these pediatric neurological disorders can alleviate certain symptoms, yet fail to provide the complete treatment. Previous studies on determining pathogenesis of various pediatric neurological disorders have utilized non-central nervous system (CNS) tissues and transgenic animal models. Because of ethical and procedural issues,


Page - 9 -


live CNS tissue samples could not be collected and this impeded the progress in

Accepted Article

understanding of pediatric neuro-pathological deficits that develop during the course of disease. Of course, animal studies alone were insufficient in unraveling the complexities of the human brain, either. With the advancement in iPSC technology, however, cells differentiated in vitro from patient-derived iPSCs can be used instead of human tissues for these purposes. Using this in vitro patient model, one can analyze brain disorders even during the progression of the disease. Overall, In vitro approaches in generating patient iPSCderived neural lineages have largely changed the paradigm in studying neurological disorders. In addition, human iPSC–based disease modeling can identify disease-associated

phenotypes when they are differentiated into the designated cell type. In order to effectively model brain diseases, one must consider a cell type, develop differentiation protocols to

generate the target cells from iPSCs, and identify disease-related cellular phenotypes.

Currently, human iPSC-based neurodegenerative disease models are being utilized worldwide to investigate the molecular basis of disease and potential therapeutic approaches (Figure 1). Specifically, iPSC technology aids in the study and treatment of early-onset pediatric neurodegenerative diseases such as Rett syndrome, Down syndrome, Angelman syndrome and Prader–Willi syndrome, Friedreich Ataxia, Spinal muscular atrophy (SMA), Fragile X Syndrome X-linked adrenoleukodystrophy (ALD), and SCN1A gene related epilepsies. Therefore, a breakthrough discovery of cellular reprogramming from human fibroblasts into iPSCs has enlightened the world of regenerative medicine. 4 iPSC for early-onset neurological disorders 4.1 Rett syndrome Rett syndrome is a progressive neurological disorder caused by mutations in the X-

linked gene encoding methyl CpG-binding protein (MeCP2), that almost exclusively affects females [40]. Rett syndrome symptoms are easily confused with those of autism since they share similar signs such as no verbal skills and repetitive stereotyped hand movements [41]. Human model of Rett syndrome by generating iPSCs from fibroblast of Rett syndrome patients enabled researchers to observe Rett syndrome specific neuronal phenotype due to neuronal maturation defects [42]. The initial neuronal differentiation of patient-derived Rett syndrome iPSCs was identical to that of wild type iPSCs and human ESCs. During late neuronal maturation, however, they had a reduced number of dendritic spines and synapses,


Page - 10 -


smaller soma size, altered calcium signaling, and elevated L1 retrotransposon mobility when

Accepted Article

compared to unaffected controls [43]. When these mutated iPSCs were treated with IGF1, neurons had increased number of synapse in some colonies, which ultimately stimulated glutamatergic Rett syndrome neurons above normal levels. Given a lower dosage of gentamicin, neurons derived from Rett syndrome iPSCs expressed increased level of fulllength MeCP2 and were able to rescue glutaminergic synapses, just like when they were

treated with IGF1 [44]. The current research is focused on the activity of MeCP2, which is known to

epigenetically regulate the target gene. L1 retrotransposon activity is critical during brain development because it controls gene expression and neuronal function [45]. Mutori et al. have shown an increased level of neuronal transcription and retrotransposition in Rett syndrome animal models in the absence of MeCP2. In accordance with the animal model, neuronal progenitor cells from Rett syndrome iPSCs also revealed that L1 retrotransposition can be controlled in a tissue-specific manner and MeCP2 mutation can influence the frequency of neuronal L1 retrotransposition [46]. Recent studies have investigated on the mosaic expression of Rett syndrome patient’s

MeCP2 and found that it is caused by X-chromosome inactivation in MeCP2 gene. Cheung et

al. generated patient-derived iPSCs that managed to remain inactive in a nonrandom pattern. They also generated a pair of isogenic Rett syndrome-human iPSC lines exhibiting either the wild type or mutant MeCP2 expression pattern upon differentiation into neurons. Their

phenotypic analysis of mutant iPSCs-derived neurons indicated soma size reduction when compared to isogenic control Rett syndrome-human iPSCs-derived neurons from the same Rett syndrome patient [42]. Further studies are expected to focus on the pathogenesis of Rett syndrome and more importantly, the specific role of MeCP2 in human neurons. Unlike the familiar Rett syndrome mentioned above, an atypical form of Rett

syndrome is caused by a mutation in the gene encoding cyclin-dependent kinase-like 5

(CDKL5). CDKL5 mutations have been found in both females with an early onset seizure variant of Rett syndrome and in males with X-linked epileptic encephalopathy [47]. Amenduni et al. demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome

inactivation and their clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control [48].


Page - 11 -


Accepted Article

4.2 Fragile X Syndrome Fragile X syndrome is a single-gene disorder associated with learning problems,

autism, and anxiety. The disorder itself is associated with the expansion of the CGG trinucleotide repeat that affects the proximal regulatory region of fragile X mental retardation 1 (FMR1) gene on the X chromosome [49]. As a result, decreased or absent level of fragile X mental retardation protein (FMRP) is observed [50]. Although FMR1 silencing and FMR1 CGG repeat expansion may possibly be related to each other, current knowledge on epigenetic modifications at the FMR1 locus and the results of the loss of function in FMRP on human neurodevelopmental process remain poor. In ESC lines generated from human embryos carrying the fragile X mutation in their

gene, the FMR1 gene is expressed in undifferentiated cells but undergoes a transcriptional silencing when differentiated [51]. Disease specific iPSCs, however, show that the FMR1 gene remained inactive and carried the same DNA methylation and histone modifications, indicating inactive heterochromatin [52]. Thus, fragile X syndrome-derived iPSCs were believed to be unsuitable to depict the effects of FMR1 silencing during neuronal differentiation. However, Sheridan et al. showed that reprogrammed cell lines from a mosaic patient

having both normal and pre-mutation length CGG repeats resulted in a genetically matching

iPSC clones differing in FMR1 promoter CpG methylation and FMRP expression [53]. Using this Fragile X syndrome patient-derived iPSCs model, they detected an atypical neuronal differentiation, correlating with epigenetic modification of the FMR1 gene and a loss of FMRP expression. These findings provide an evidence for the roles of FMRP in early neurodevelopment prior to synaptogenesis and show potential for modeling Fragile X syndrome with iPSCs technology. 4.3 Down Syndrome Down syndrome is a genetic disorder caused by trisomy of chromosome 21 and is the

most frequent genetic cause of mental retardation [54]. Other than its characteristic dysmorphology of the facial and physical features, Down syndrome is associated with increased risks of leukemia, immune system defects, and an early Alzheimer-like dementia [55]. Individuals with Down syndrome have a very high risk of developing Alzheimer’s disease because the extra copy contains Alzheimer’s disease-associated gene that encodes


Page - 12 -


amyloid precursor protein [56]. In order to study the underlying mechanisms, human neural

Accepted Article

progenitor cell lines derived from Down syndrome patients are being used [57]. But understanding and investigating the Down syndrome mechanisms are still challenges because of the genetic complexity and individual variability of Down syndrome phenotypes. In the current study, iPSCs generated from Down syndrome patients are used as an

effective cell models for the study of the Down syndrome mechanisms as well as for drug

screening. Shi et al. successfully generated cortical neurons from Down syndrome derived iPSCs and showed that these neurons develop the two main characteristic pathological hallmarks of Alzheimer’s disease in a short amount of time, including coagulation of amyloid peptides due to impairment in amyloid precursor protein and neurofibrillary tangles of hyperphosphorylated tau protein [56]. These two characteristics were developed in both Down syndrome iPSCs-derived and ESCs-derived cortical neurons, suggesting that these pathologies are reproducible in cortical neurons derived from two different sources. Moreover, it is clear that their formation was not at all influenced by the cellular reprogramming strategy used to derive iPSCs from adult fibroblasts [58]. The expansion of pathological amyloid aggregates in in-vitro human cortical neuron

model could be blocked by a drug called γ-secretase. It is the same drug that is being tested in clinical trials for treating Alzheimer’s disease. This absolutely amazing work of Shi et al. suggests that Down syndrome derived iPSCs will be a useful tool for drug screening for potential candidates and developing new disease intervention strategies for treating Alzheimer’s disease. 4.4 Angelman Syndrome and Prader–Willi syndrome Angelman

syndrome

(AS)

and

Prader–Willi

syndrome

(PWS)

are

neurodevelopmental disorders associated with genomic imprinting. The common cause of AS comes from a mega base maternal deletion in chromosomal region 15q11-13, resulting in negative UBE3A expression [59]. In PWS, seven genes on chromosome 15 (q 11–13) are either deleted or unexpressed on the paternal chromosome [60]. Even though iPSCs demonstrate a priceless approach in modeling human disease, their practicality can be limited in AS and PWS by disturbed genomic imprinting marks during the nuclear reprogramming of somatic cells to pluripotent stem cells. Nonetheless, Chamberlain et al. established AS and PWS derived iPSCs that show no evidence of DNA methylation imprint erasure at the cisacting PSW imprinting center [61]. Moreover, they unraveled that the imprinting of UBE3A


Page - 13 -


in normal brain was generated during neuronal differentiation of AS-derived iPSCs, with the

Accepted Article

repression of paternal or maternal UBE3A allele accompanying up-regulation of the UBE3A antisense transcript. These results indicate that iPSCs may be a useful and promising tool to study both AS and PWS regarding developmental timing and mechanism of UBE3A repression in human neurons [62]. 4.5 Spinal Muscular Atrophy Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by a

genetic defect in the SMN1 gene, resulting in selective degradation of motor neurons [63]. Diminished abundance of the SMN protein leads to death of neuronal cells in the anterior horn of the spinal cord and, subsequently, system-wide muscle atrophy. Because of its unique anatomy and physiological properties, researchers were unable to study SMA extensively [64]. With the iPSCs technology, however, SMA became the first neurological disease to be modeled using human iPSCs and vast amount of studies in attempt to reveal pathogenesis are currently being performed [65]. Ebert et al. reported about generating iPSCs from skin fibroblast samples taken from

a child with SMA [66]. These patient-derived iPSCs, while maintaining the disease genotype, expanded firmly in culture and differentiated into motor neurons expressing certain defects compared to those derived from the child’s unaffected mother. In addition, they analyzed whether or not SMN-inducing compounds could elevate SMN levels in the SMA-iPSCs in vitro, and concluded that SMA-derived iPSCs treated with either valporate or tobramycin

showed a significant increase in SMN levels compared to untreated iPSCs. Further studies include Chang et al. reporting about the establishment of five iPSC

lines from the fibroblasts of a type 1 SMA patient [65]. They cultured SMA-derived iPSCs and differentiated them into neurons, which exhibited a reduced capacity to form motor neurons and an aberrant neurite outgrowth. Ectopic SMN expression in these iPSC lines restored normal motor neuron differentiation and rescued the phenotype of delayed neurite outgrowth. Comprehensively, these results suggest that the observed abnormalities are indeed caused by SMN deficiency and not by iPSCs clonal variability.


Page - 14 -


Accepted Article

4.6 Friedreich Ataxia Friedreich's ataxia is an inheritable, autosomal recessive disease characterized by

degeneration in cardiomyopathy as well as neurons. It is due to an amplified intronic GAA repeats in the FXN gene located on chromosome 9. This particular GAA repeat expansion reduces frataxin levels. Frataxin is an iron-binding protein responsible for forming iron– sulfur clusters. One crucial outcome of frataxin deficiency is an overload of irons in mitochondria, which can damage many proteins [67]. The exact role of frataxin in normal physiology, however, remains unknown. Ku et al. was the first to establish Friedreich’s ataxia patient-derived iPSCs with

transcription factors[68]. In their research, FXN gene repression was maintained in the iPSCs and GAA•TTC repeated uniquely in FXN in the iPSCs, exhibiting repeat instability similar to that of patient families. They also showed that the mismatch repair enzyme MSH2, which

plays a role in repeat instability in other triplet repeat diseases, was highly expressed in pluripotent cells, and the silencing of MSH2 impeded repeat expansion, providing a possible molecular explanation for repeat expansion in Friedreich’s ataxia. iPSC lines were also generated from skin fibroblasts of two Friedreich's ataxia

patients by Liu et al [69]. When placed in in vitro setting, the disease-specific iPSCs gave rise to two primarily affected cell types of FA; peripheral neurons and cardiomyocytes. They also have shown that securely inserting a functional human BAC containing the intact FXN gene into iPSCs results in the expression of frataxin protein in differentiated neurons. Taken together, these Friedreich’s ataxia iPSC lines are powerful tools to study the

cellular pathology of Friedreich’s ataxia. Also, with the correction of the mutated gene, FAderived iPSCs could yield in useful information on immunocompatible cells for transplantation therapy. 4.7 X-Linked Adrenoleukodystrophy X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in ABCD1 gene

that results in the accumulation of very-long chain fatty acids (VLCFA), primary concentrated in the nervous system, the adrenal cortex, and the Leydig cells of the testis [70]. ABCD1 gene located on the X chromosome encodes a peroxisomal ATP-binding cassette transporter, ABCD1, which plays an important role in the entry of long chain fatty acids into peroxisomes for degradation [71]. Two major types include childhood cerebral ALD


Page - 15 -


(CCALD) and adrenomyeloneuropathy (AMN). CCALD (onset at 3-10 years of age) is

Accepted Article

known to have the most severe symptoms with a rapidly progressive inflammatory demyelination and death within a few years after onset of symptoms. AMN, on the other hand, is a milder adult form that shows slow progression and symptoms that are specifically limited to the spinal cord and peripheral nerves [70]. There are, however, no obvious correlations between ABCD1 gene mutations and the clinical phenotypes [72]. To investigate further, Jang & Kang et al. were the first to generate iPSCs of both

CCALD and AMN, and differentiate them into oligodendrocytes and neurons. These iPSCsbased cell models of X-ALD show remarkable increase in VLCFA levels after differentiating into oligodendrocytes. This novel finding is congruous to the previous finding that microsomal VLCFA elongation activity is high during myelin development in mice around 20 days after birth. Jang et al. also detected increased VLCFA level in CCALD oligodendrocytes than in AMN oligodendrocytes, possibly explaining the observations made in the postmortem brains [73]. Subsequent drug screening results demonstrated effectiveness of 4-PBA and lovastatin in reducing the level of VLCFA in oligodendrocyte derived from CCALD iPSCs via up-regulating ABCD2 gene [74]. Overall, X-ALD iPSC model recapitulates the key

events of disease development and allows to find novel approaches for drug therapy [73]. 4.8 SCN1A gene related epilepsies Patient-derived iPSCs have also been suitable for modeling and understanding

SCN1A gene related epilepsies. Because of the early onset of the disorder, SCN1A gene related epilepsies are a highly suitable candidate for the investigation by using iPSCs [75-77]. These specific epilepsies can be caused by mutations in the SCN1A gene that normally encodes pore-forming ߙ-subunit of the voltage-gated sodium channel [78]. Interestingly, a mutated SCN1A gene results in either a gain or loss of function in the sodium channel, and the outcomes of each are still under the investigation [79, 80]. Depends on the type of seizure or prognosis, epilepsies linked to SCN1A mutations can further be categorized from a milder form, a generalized epilepsy with febrile seizures to a more serious form, known as Dravet syndrome, that involves severe myoclonic epilepsy in infancy (SMEI) [78]. Genetic tests for SCN1A gene mutations are available; yet, poor correlation between the genotypic and the phenotypic properties poses difficulties to reveal the pathophysiologic mechanisms of the disease.


Page - 16 -


Patient-derived iPSCs facilitate the research to move forward in generating more

Accepted Article

human relevant models that aid researchers to accurately study SCN1A gene related epilepsies. However, the results of the studies have been inconsistent in their findings. Higurashi et al.

have reported the generation of iPSCs derived from a Dravet syndrome patient with a truncation in the fourth homologous domain of the Nav1.1 [75]. Overall, they showed a functional decline in Dravet neurons, particularly in the GABAergic subtype, which supports the previous findings using specific murine disease models where inhibition of a loss-offunction in GABAergic neuron appears to be the main driver in epileptogenesis. In contrast to Higurashi et al, Liu et al. derived forebrain-like pyramidal

(glutaminergic)- and bipolar (GABAergic)-shaped neurons from two Dravet syndrome subjects respectively with a splice donor site mutation and a nonsense mutation and three human controls with iPSCs reprogramming from fibroblasts [76]. In this experiment, Dravet syndrome patient-derived neurons showed an increase in sodium currents in both pyramidaland bipolar-shaped neurons. Consistent with this result, both types of patient-derived neurons showed a spontaneous bursting and other evidence of hyperexcitability. Jiao et al. recently established iPSCs from two patients; one from severe SMEI and

another from a patient with mild febrile seizures [77]. Contrary to previous reports, both patients had missense mutations of SCN1A together. By electrophysiological analysis using differentiated glutamatergic neurons, they could observe a hyperexcitable state of enlarged and persistent sodium channel activation. The hyperexcitability of the neurons derived from SMEI patient was far more serious than that of mild febrile seizure patient, which is consistent with the severity of the symptoms of each type. They also confirmed that the treatment with phenytoin, a conventional antiepileptic drug, alleviates the hyeprexcitability of the neurons. Similarly, induced neurons were directly converted from patient fibroblasts, which also showed a delayed inactivation of sodium channels. The failure to define the consistent results of pathophysiologic studies may be due to

many factors including differences in phenotype, genotype and neuronal types. Therefore, in the future, more detailed studies are required to reveal the definite pathophysiologic mechanisms of SCN1A gene related epilepsies. 5 Conclusion Reprogrammed iPSCs are central in modeling and studying early-onset neurological diseases that was once thought to be inconceivable. It not only offers an affordable and


Page - 17 -


applicable approach to investigate the pathophysiological mechanisms of pediatric

Accepted Article

neurological disorders, but also provides an insight into future cell-based therapy. When dealing with iPSC-derived models, it is essential to consider the inherent variation among patient samples, including genetic and epigenetic diversities in the phenotypic analysis of these disease models. Despite its potentials, iPSCs-based clinical trials cannot be performed unless the limitations are fully resolved. For instance, one must examine the nature of all transplanted cells to assure the absence of undifferentiated cells that may form teratomas. Any abnormalities concerning tumor formations or malfunctions of epigenetic memory

should also be closely monitored. There are many more steps before iPSCs can fully be applied to treat pediatric neurological disorders. Taken together, various reprogramming technologies should further be developed to aid the process of establishing stable and relevant cell models that can potentially be applied in the clinic.

Acknowledgment: This work was supported by National Research Foundation grant funded by the Korea Government (MEST, 2010-0020353) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A2A2A01014108). The authors thank Dong-Su Jang for his excellent support with medical illustration.

Disclosure The authors declare no financial or commercial conflict of interest.

6 References 1.

2. 3. 4.

5. 6.

Marchetto, M.C., et al., Induced pluripotent stem cells (iPSCs) and neurological disease modeling: progress and promises. Hum Mol Genet, 2011. 20(R2): p. R109-15. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20. Kim, K., et al., Epigenetic memory in induced pluripotent stem cells. Nature, 2010. 467(7313): p. 285-90. Okita, K., et al., Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322(5903): p. 949-53. Sommer, C.A., et al., Induced pluripotent stem cell generation using a single


8. 9.

10. 11.

12. 13.

14. 15. 16.

17.

18. 19.

20.

21.

22.

23.

24.

25.

26.

27. 28.


lentiviral stem cell cassette. Stem Cells, 2009. 27(3): p. 543-9. Woltjen, K., et al., piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 2009. 458(7239): p. 766-70. Jia, F., et al., A nonviral minicircle vector for deriving human iPS cells. Nat Methods, 2010. 7(3): p. 197-9. Kim, D., et al., Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell, 2009. 4(6): p. 472-6. Ban, H., et al., Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci U S A, 2011. 108(34): p. 14234-9. Okita, K., et al., A more efficient method to generate integration-free human iPS cells. Nat Methods, 2011. 8(5): p. 409-12. Warren, L., et al., Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010. 7(5): p. 618-30. Hou, P., et al., Pluripotent stem cells induced from mouse somatic cells by smallmolecule compounds. Science, 2013. 341(6146): p. 651-4. Mikkelsen, T.S., et al., Dissecting direct reprogramming through integrative genomic analysis. Nature, 2008. 454(7200): p. 49-55. Hanna, J., et al., Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci U S A, 2010. 107(20): p. 9222-7. Huangfu, D., et al., Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol, 2008. 26(11): p. 1269-75. Esteban, M.A., et al., Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 2010. 6(1): p. 71-9. Mali, P., et al., Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotencyassociated genes. Stem Cells, 2010. 28(4): p. 713-20. Moriguchi, H., et al., Generation of human induced pluripotent stem cells from liver progenitor cells by only small molecules. Hepatology, 2010. 52(3): p. 1169; author reply 1169-70. Chen, T., et al., Rapamycin and other longevity-promoting compounds enhance the generation of mouse induced pluripotent stem cells. Aging Cell, 2011. 10(5): p. 90811. Wang, Q., et al., Lithium, an anti-psychotic drug, greatly enhances the generation of induced pluripotent stem cells. Cell Res, 2011. 21(10): p. 1424-35. Zhao, Y., et al., Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell, 2008. 3(5): p. 475-9. Mathew, R., et al., Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. FASEB J, 2010. 24(8): p. 2702-15. Buganim, Y., et al., Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell, 2012. 150(6): p. 1209-22. Rais, Y., et al., Deterministic direct reprogramming of somatic cells to pluripotency. Nature, 2013. 502(7469): p. 65-70. Thomson, J.A., et al., Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7. Boch, J., et al., Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 2009. 326(5959): p. 1509-12. Sanjana, N.E., et al., A transcription activator-like effector toolbox for genome

Accepted Article

7.

Page - 18 -


30.

31.

32. 33. 34.

35. 36.

37. 38. 39.

40. 41.

42. 43.

44.

45. 46.

47.

48. 49. 50.


engineering. Nat Protoc, 2012. 7(1): p. 171-92. Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6. Merkle, F.T. and K. Eggan, Modeling human disease with pluripotent stem cells: from genome association to function. Cell Stem Cell, 2013. 12(6): p. 656-68. Hanna, J., et al., Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 2007. 318(5858): p. 1920-3. Soldner, F., et al., Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell, 2011. 146(2): p. 318-31. Schwank, G., et al., Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients. Cell Stem Cell, 2013. 13(6): p. 653-8. Zhao, T., et al., Immunogenicity of induced pluripotent stem cells. Nature, 2011. 474(7350): p. 212-5. Emborg, M.E., et al., Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain. Cell Rep, 2013. 3(3): p. 646-50. Araki, R., et al., Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature, 2013. 494(7435): p. 100-4. Homma, K., et al., Developing rods transplanted into the degenerating retina of Crxknockout mice exhibit neural activity similar to native photoreceptors. Stem Cells, 2013. 31(6): p. 1149-59. Schwartz, S.D., et al., Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet, 2012. 379(9817): p. 713-20. Nicholas, C.R., et al., Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell, 2013. 12(5): p. 573-86. Amir, R.E., et al., Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet, 1999. 23(2): p. 185-8. Hagberg, B., et al., A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett's syndrome: report of 35 cases. Ann Neurol, 1983. 14(4): p. 471-9. Cheung, A.Y., et al., Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum Mol Genet, 2011. 20(11): p. 2103-15. Kim, K.Y., E. Hysolli, and I.H. Park, Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome. Proc Natl Acad Sci U S A, 2011. 108(34): p. 14169-74. Marchetto, M.C., et al., A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell, 2010. 143(4): p. 527-39. Singer, T., et al., LINE-1 retrotransposons: mediators of somatic variation in neuronal genomes? Trends Neurosci, 2010. 33(8): p. 345-54. Muotri, A.R., et al., L1 retrotransposition in neurons is modulated by MeCP2. Nature, 2010. 468(7322): p. 443-6. Evans, P.D., et al., Microcephalin, a gene regulating brain size, continues to evolve adaptively in humans. Science, 2005. 309(5741): p. 1717-20. Amenduni, M., et al., iPS cells to model CDKL5-related disorders. Eur J Hum Genet, 2011. 19(12): p. 1246-55. Warren, S.T. and C.T. Ashley, Jr., Triplet repeat expansion mutations: the example of fragile X syndrome. Annu Rev Neurosci, 1995. 18: p. 77-99. Garber, K.B., J. Visootsak, and S.T. Warren, Fragile X syndrome. Eur J Hum Genet, 2008. 16(6): p. 666-72.

Accepted Article

29.

Page - 19 -


52. 53. 54.

55.

56.

57.

58. 59.

60.

61. 62.

63. 64.

65. 66.

67. 68.

69.

70.

71. 72.


Eiges, R., et al., Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell, 2007. 1(5): p. 568-77. Urbach, A., et al., Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells. Cell Stem Cell, 2010. 6(5): p. 407-11. Sheridan, S.D., et al., Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome. PLoS One, 2011. 6(10): p. e26203. Antonarakis, S.E. and C.J. Epstein, The challenge of Down syndrome. Trends Mol Med, 2006. 12(10): p. 473-9. Antonarakis, S.E., et al., Chromosome 21 and down syndrome: from genomics to pathophysiology. Nat Rev Genet, 2004. 5(10): p. 725-38. Shi, Y., et al., A human stem cell model of early Alzheimer's disease pathology in Down syndrome. Sci Transl Med, 2012. 4(124): p. 124ra29. Esposito, G., et al., Genomic and functional profiling of human Down syndrome neural progenitors implicates S100B and aquaporin 4 in cell injury. Hum Mol Genet, 2008. 17(3): p. 440-57. Park, I.H., et al., Disease-specific induced pluripotent stem cells. Cell, 2008. 134(5): p. 877-86. Williams, C.A., D.J. Driscoll, and A.I. Dagli, Clinical and genetic aspects of Angelman syndrome. Genet Med, 2010. 12(7): p. 385-95. Bittel, D.C. and M.G. Butler, Prader-Willi syndrome: clinical genetics, cytogenetics and molecular biology. Expert Rev Mol Med, 2005. 7(14): p. 1-20. Chamberlain, S.J., et al., Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes. Proc Natl Acad Sci U S A, 2010. 107(41): p. 17668-73. Yang, J., et al., Induced pluripotent stem cells can be used to model the genomic imprinting disorder Prader-Willi syndrome. J Biol Chem, 2010. 285(51): p. 40303-11. Prior, T.W., Perspectives and diagnostic considerations in spinal muscular atrophy. Genet Med, 2010. 12(3): p. 145-52. Lorson, C.L., H. Rindt, and M. Shababi, Spinal muscular atrophy: mechanisms and therapeutic strategies. Hum Mol Genet, 2010. 19(R1): p. R111-8. Chang, T., et al., Brief report: phenotypic rescue of induced pluripotent stem cellderived motoneurons of a spinal muscular atrophy patient. Stem Cells, 2011. 29(12): p. 2090-3. Ebert, A.D., et al., Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature, 2009. 457(7227): p. 277-80. Pandolfo, M., Friedreich ataxia. Arch Neurol, 2008. 65(10): p. 1296-303. Ku, S., et al., Friedreich's ataxia induced pluripotent stem cells model intergenerational GAATTC triplet repeat instability. Cell Stem Cell, 2010. 7(5): p. 631-7. Liu, J., et al., Generation of induced pluripotent stem cell lines from Friedreich ataxia patients. Stem Cell Rev, 2011. 7(3): p. 703-13. Moser, H.W., A. Mahmood, and G.V. Raymond, X-linked adrenoleukodystrophy. Nat Clin Pract Neurol, 2007. 3(3): p. 140-51. Mosser, J., et al., Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature, 1993. 361(6414): p. 726-30. Kemp, S., et al., ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations. Hum Mutat, 2001. 18(6): p. 499-515.

Accepted Article

51.

Page - 20 -


73.

75. 76. 77.

78. 79.

80.

81. 82.

83. 84. 85. 86.

87.

88.

89.

90. 91.

92.

93.


Jang, J., et al., Induced pluripotent stem cell models from X-linked adrenoleukodystrophy patients. Ann Neurol, 2011. 70(3): p. 402-9. Singh, I., K. Pahan, and M. Khan, Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X- adrenoleukodystrophy. FEBS Lett, 1998. 426(3): p. 342-6. Higurashi, N., et al., A human Dravet syndrome model from patient induced pluripotent stem cells. Mol Brain, 2013. 6: p. 19. Liu, Y., et al., Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism. Ann Neurol, 2013. 74(1): p. 128-39. Jiao, J., et al., Modeling Dravet syndrome using induced pluripotent stem cells (iPSCs) and directly converted neurons. Hum Mol Genet, 2013. 22(21): p. 4241-52. Stafstrom, C.E., Severe epilepsy syndromes of early childhood: the link between genetics and pathophysiology with a focus on SCN1A mutations. J Child Neurol, 2009. 24(8 Suppl): p. 15S-23S. Stafstrom, C.E., Persistent sodium current and its role in epilepsy. Epilepsy Curr, 2007. 7(1): p. 15-22. Yu, F.H., et al., Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci, 2006. 9(9): p. 1142-9. Lowry, W.E., et al., Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A, 2008. 105(8): p. 2883-8. Moore, J.C., Generation of human-induced pluripotent stem cells by lentiviral transduction. Methods Mol Biol, 2013. 997: p. 35-43. Maherali, N., et al., A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell, 2008. 3(3): p. 340-5. Somers, A., et al., Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells, 2010. 28(10): p. 1728-40. Stadtfeld, M., K. Brennand, and K. Hochedlinger, Reprogramming of pancreatic beta cells into induced pluripotent stem cells. Curr Biol, 2008. 18(12): p. 890-4. Zhou, H., et al., Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 2009. 4(5): p. 381-4. Si-Tayeb, K., et al., Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors. BMC Dev Biol, 2010. 10: p. 81. Hu, K., et al., Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood, 2011. 117(14): p. e109-19. Narsinh, K.H., et al., Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc, 2011. 6(1): p. 78-88. Kumano, K., et al., Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples. Blood, 2012. 119(26): p. 6234-42. Churko, J., P. Burridge, and J. Wu, Generation of Human iPSCs from Human Peripheral Blood Mononuclear Cells Using Non-integrative Sendai Virus in Chemically Defined Conditions, in Cellular Cardiomyoplasty, R.L. Kao, Editor. 2013, Humana Press. p. 81-88. Zou, J., et al., Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell, 2009. 5(1): p. 97-110. Miyoshi, N., et al., Reprogramming of Mouse and Human Cells to Pluripotency Using Mature MicroRNAs. Cell Stem Cell, 2011. 8(6): p. 633-638.

Accepted Article

74.

Page - 21 -


Page - 22 -


Accepted Article

Legends

Figure 1. Patient specific-iPSCs are established by introducing Yamanaka’s factors (Oct4, Klf4, Sox2, and c-Myc: OKSM) or other factors into somatic cells (biopsy skin or blood) from children with neurological diseases using various delivery system (virus, Sendai virus, mRNA, episomal, chemicals and proteins). Induced neural stem cells (iNSCs) are also produced by infecting induction transcription factors to patient-derived skin or blood utilizing

the virus system or chemicals. The iPSCs or the iNSCs can be differentiated into target cells that are relevant for the study of neurological disorders. Neural progenitors from iPSCs or iNSCs give rise to glial cells (oligodendrocytes and astrocytes) and to subtype neurons. Mutations in iPSCs (or iNSCs) could have been corrected via genome editing technologies by

using the zinc finger nucleases (ZFNs), the transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPER-Cas9)

system before patient-specific iPSCs are developed as cell therapy. Distinct neuronal and glial cell types differentiated from iPSCs or iNSCs can be employed to investigating disease pathophysiology (disease modeling), to screening effective drug candidates among chemical libraries and to testing toxicity and efficacy of the newly identified compounds. As a recent novel approach, the advanced studies of direct conversion and in vitro self-organization of complex organ buds using 3D stem cell culture might be open a new avenue for underlying pathological mechanisms and finding novel approaches to drug therapy.


Page - 23 -


Accepted Article

Table 1. Various methods used to generate induced pluripotent stem cells

Delivery System Retroviral transduction

Target cell Efficiency Genome type (%) integration fibroblast, ~0.001–1 yes neuronal, keratinocyte, blood cells, adipose, and liver cells Lentiviral fibroblast ~0.1–1.1 yes transduction and keratinocyte Inducible fibroblast, lentiviral melanocytes, transduction beta-cells, blood cells, and keratinocyte loxPfibroblast flanked lentiviral

Advantages reasonable efficiency

incomplete proviral silencing and insertional mutagenesis

[2, 81]

transduces dividing and non-dividing cells

incomplete proviral silencing and insertional mutagenesis requirement for transactivator expression

[3, 82]

labor-intensive screening of excised lines, and loxP sites retained in the genome labor-intensive screening of excised lines slow and inefficiency

[84]

~0.1–2

yes

controlled expression of transgenes

~0.1–1

no

transgenefree

Transposon

fibroblast

~0.1

no

transgenefree

Adenoviral

fibroblast and liver cells fibroblast

~0.001

no

integrationfree

~0.001

no

integrationfree

fibroblast, dental pulp cells mononuclear bone marrow and

~0.3-0.4

no

high induction of efficiency from adult somatic cells after only one

Plasmid

Episomal vectors

Disadvantages References

very low efficiency, rare genomic integration, and requirement for multiple rounds of transfection rare genomic integration event and need to check genomic integration

[83]

[7]

[85], [86] [5, 87]

[11, 88]


Page - 24 -

Accepted Article

cord blood cells


transfection, transgenefree nonviral, transgenefree, a single vector

Minicircle DNA

adult human adipose stem cells

~0.005

no

Sendai virus

fibroblast, T cells

~1

no

free of viral gene integration

Protein

fibroblast

~0.001

no

direct delivery of transcription factors and free of gene materials

Modified mRNA

fibroblast

~1–4.4

no

MicroRNA (miR-200c, miR-302s, miR-369s)

adipose stromal cells and dermal fibroblast

~0.1

no

bypasses innate antiviral response, faster reprogramming kinetics, controllable and high efficiency no exogenous transcription factors and integration-free

Chemicals

fibroblast

~0.2

no

less risk of mutating the genes and free of gene materials

requirement for sorting of transfected cells, low efficiency difficulty in removing cells of replicating virus in cytoplasm, technically complex

[89]

[10, 90] [91]

very low efficiency, short half-life, and requirement for large quantities of pure proteins and multiple applications of protein requirement for multiple rounds of transfection, technically complex

[9, 92]

lower efficiency than other commonly used methods, laborintensive unknown side effects and toxicity of chemicals

[93]

[12]

[13]

Accepted Article


Page - 25 -


Hoon-Chul Kang is a professor in the department of pediatric neurology at Yonsei University College of Medicine. Dr. Kang received his M.D. from Yonsei University in 1992 and his Ph.D. in 2006. After completing his clinical fellowship in the division of Pediatric Neurology at Severance Hospital, he joined the Molecular Neurology lab at McLean Hospital in Boston MA as a research fellow, concentrating on reprogrammable stem cell research. Dr. Kang was the first to establish iPSCs derived from adrenoleukodystrophy and he currently focuses on generating iPSCs from early-onset neurodegenerative diseases including SCN1A-related epilepsy.

Induced pluripotent stem cells for modeling neurological disorders.

Modeling human neurological disorders with induced pluripotent stem cells.

Generating CNS organoids from human induced pluripotent stem cells for modeling neurological disorders.

Modeling Genomic Imprinting Disorders Using Induced Pluripotent Stem Cells.

Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling.

Patient-specific induced pluripotent stem cells in neurological disease modeling: the importance of nonhuman primate models.

Modeling neurodevelopmental disorders using human pluripotent stem cells.

Modeling of Menkes disease via human induced pluripotent stem cells.

Biomedical and clinical promises of human pluripotent stem cells for neurological disorders.

Utility of Induced Pluripotent Stem Cells for the Study and Treatment of Genetic Diseases: Focus on Childhood Neurological Disorders.

Modeling pulmonary alveolar proteinosis with induced pluripotent stem cells.

Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells.

Modeling retinal dystrophies using patient-derived induced pluripotent stem cells.

Concise Review: Cardiac Disease Modeling Using Induced Pluripotent Stem Cells.

Modeling familial cancer with induced pluripotent stem cells.

Modeling Cancer with Pluripotent Stem Cells.

Stem cells on the brain: modeling neurodevelopmental and neurodegenerative diseases using human induced pluripotent stem cells.

Human induced pluripotent stem cell for modeling cardiovascular diseases.

Induced pluripotent stem cells for regenerative medicine.

Pluripotent stem cells in neuropsychiatric disorders.

Modeling neurological diseases with induced pluripotent cells reprogrammed from immortalized lymphoblastoid cell lines.

Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy.

In Vitro Modeling of Alcohol-Induced Liver Injury Using Human-Induced Pluripotent Stem Cells.

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.