Induced Pluripotent Stem Cells Chapter 8

187G. Steinhoff (ed.), Regenerative Medicine: From Protocol to Patient,DOI 10.1007/978-90-481-9075-1_8, © Springer Science+Business Media B.V . 2011Abstract Nearly 50 years have passed since the concept of nuclear r eprogramming was proposed for the first time. Since then, several approaches have been developed to convert somatic cells to a pluripotent state. Direct reprogramming with defined factors, is the newest of these approaches. This method requires just a few genes, and it also has a great reproducibility. Applying this method to humans seems to open the door to cell transplantation therapy without immune rejection, drugd iscovery, and elucidation of the pathogenesis of intractable diseases. However, this concept still faces some issues which must be overcome before application due to a shortage of experience. This chapter introduces an overview of direct reprogram-ming as well as a special focus on its potentials and challenges.8.1 I ntroductionPluripotent stem cells such as embryonic stem (ES) cells may be a hopeful resource for regenerative medicine to repair degenerative or damaged tissues. ES cells have the potential to differentiate into all cell types in the body including germ cells. Not only pluripotency but also their growth properties are substantially superior. In conventional conditions, ES cells can grow infinitely while maintaining K. Takahashi and S. Yamanaka ( )Center for iPS cell Research and Application, Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japane-mail: takahash@cira.kyoto-u.ac.jpS. YamanakaDepartment of Stem Cell Biology, Institute for Frontier Medical Sciences,Kyoto University, Kyoto 606-8507, JapanandYamanaka iPS Cell Special Project, Japan Science and Technology Agency,Kawaguchi 332-0012, JapanandGladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USAChapter 8Induced Pluripotent Stem CellsKazutoshi Takahashi and Shinya Yamanaka188K. Takahashi and S. Yamanaka an undifferentiated state. Combined with the techniques of gene targeting and transgenes, these characters have allowed the generation of genetically modified animals (Robertson et al. 1986; Thomas and Capecchi 1987; Doetschman et al. 1987). To this end, the mechanisms or causes of many diseases have been eluci-dated. Reports on the establishment of human ES cells brought great expectations to regenerative medicine of various diseases (Thomson et al. 1998). In the last decade, scientists have established appropriate culture conditions, differentiation protocols and guidelines for users.On the other hand, ES cells face two big issues that need to be overcome before successful medical application can be achieved. One is immune rejection caused by the mismatching of human leukocyte antigen (HLA) haplotypes between ES cells and patient. Another is the usage of human embryos. Although the response to e thical issues may vary depending on regions and civilizations, it should be ear-nestly addressed in any case.One of the solutions to overcome these issues is to reprogram a patient’s own somatic cells directly to pluripotent stem cells. The reprogramming of the somatic nucleus was firstly demonstrated by Sir John Gurdon in 1958 (Gurdon et al. 1958). He developed cloned frogs by injecting the nucleus of a tadpole somatic cell into an egg. This report suggested that frog eggs have some reprogramming factor(s) that could initialize somatic state back to totipotency. In 1997, the group of Sir Ian Wilmut and Dr. Keith Campbell with their famous sheep, Dolly, showed that not only flexible species such as amphibians but also mammals have the reprogram-ming factor(s) in their eggs by using nuclear transfer technology (Wilmut et al. 1997). In the last year of the twentieth century, Dr. Takashi Tada and his colleagues showed that the nucleus of somatic cell also could be reset to the pluripotent state by electrical-fusion with mouse ES cells (Tada et al. 2001). All these findings and following associated reports suggested that ES cells as well as eggs have such reprogramming factor (s). At a later date, the same phenomenon was confirmed in human cells (Cowan et al. 2005).What is the reprogramming factor? People have hypothesized that factors playing important roles in ES cell identities, such as differentiation potentials and tumor-like growth properties, play a crucial role in the induction of pluripotency in somatic cells. The concept that the genes expressed specifically in stem cells p rovided multipotency to the cells has been established and called stemness (Ramalho-Santos et al. 2002). Therefore, the genes expressed predominantly in eggs and/or ES cells, and some non-ES cell specific genes which are important for the character of pluripotent cells can be candidates of reprogramming factor(s).The importance of pluripotent cell-associated genes has been proposed in s everal studies (Boiani and Schöler 2005; Niwa 2007).Oct3/4 (also known as Pou5f1) one of the most famous ES cell-specific genes, is an octamer sequence binding transcription factor (Okamoto et al. 1990; Schöler et al. 1990). Deletion of Oct3/4 gene caused the loss of pluripotency in both ES cells and early embryos (Nichols et al. 1998). Oct3/4-null embryos die at around the implantation stage. The inner cell mass (ICM) of Oct3/4-deficient embryo can no longer outgrow. Detailed analyses have shown that Oct3/4 prevents the d ifferentiation8 Induced Pluripotent Stem Cells189 into trophectoderm by suppressing the function of Cdx2 which plays essential role in trophectoderm development (Niwa et al. 2005). On the other hand, only a 1.5-fold increase of Oct3/4 expression in ES cells is sufficient to trigger differentiation into either mesoderm or primitive endoderm (Niwa et al. 2000). These data indicated that Oct3/4 is one of the most important regulators to prevent differentiation into specific lineages and maintain ES cells in a pluripotent state.The other important player is Sox2. The expression of Sox2 is restricted in nerve tissue and pluripotent cells including germ cells (Kamachi et al. 2000). Structural and biochemical analyses demonstrated that Sox2 binds directly to Oct3/4 and acts as a regulator of ES cell-specific gene expression such as Utf1, Fgf4 and Lefty1 (Ambrosetti et al. 1997; Reményi et al. 2003; Nishimoto et al. 1999). Homozygous deletion of Sox2 gene leads to embryo death immediate after implantation with the lack of epiblast formation (Avilion et al. 2003). Blastocysts carrying a null mutation of Sox2 gene looked normal, but the ICM could not expand in vitro whereas tropho-blasts and primitive endoderm cells can continue to proliferate. In addition, Sox2-deficient ES cells cannot maintain an undifferentiated state in conventional conditions (Masui et al. 2007). Taken together, Sox2 is crucial for the maintenance of pluripotent cells in both ES cells and early embryos.However, the functions of Oct3/4 and Sox2 are not sufficient to explain the molecular mechanisms underlying pluripotent stem cell identity. The presence of other co-factors has been speculated. Many exhaustive analyses were performed in the early 2000s, and databases of gene expression profiles were advanced rapidly such as expressed sequence tags (ESTs) and microarray technology (Kawai et al. 2001). To this end, hundreds of stemness-relating genes were found as candidates to define the stem cell phenotype (Tokuzawa et al. 2003; Takahashi et al. 2003). An in silico analysis and functional screening identified a novel stemness gene, designated Nanog at the same time (Mitsui et al. 2003). Nanog is a transcription factor which contains a paired-like homeobox. The expression pattern of Nanog in early development is more restricted than that of Oct3/4 or Sox2. Embryos carrying a null-mutation of Nanog gene die during the post-implantation period lacking epiblasts (Mitsui et al. 2003). Outgrowth of Nanog-deficient ICM is also defective, although primitive endoderm-like cells can proliferate. On the other hand, ES cells carrying homozygous deletion of the Nanog gene can proliferate in vitro although morphologies and gene expression patterns were changed to primitive endoderm-like state (Mitsui et al. 2003). In addition, forced expression of the Nanog gene allows cells to maintain pluripotency even without leukemia inhibitory factor (LIF) which is an essential component for self-renewal of mouse ES cells in serum- containing medium (Chambers et al. 2003; Mitsui et al. 2003). Therefore, these data suggested that Nanog acts as a switch between the pluripotent state and primi-tive endoderm.ChIP on chip analyses, which combine chromatin immunoprecipitation, microarray, and a transcriptome, revealed global target genes of Oct3/4, Sox2 and Nanog in ES cells (Chew et al. 2005; Loh et al. 2006; Boyer et al. 2005). More than 300 genes were categorized as common targets of these three transcription factors, including both expressed (Oct3/4, Sox2, Nanog, Stat3, Zic3) and not expressed190K. Takahashi and S. Yamanaka genes (Hoxb1, Pax6, Lhx5, Myf5) in ES cells. These data suggested that a circuit consisting of Oct3/4, Sox2 and Nanog promotes the expression of genes supporting the self-renewal of ES cells, suppresses those genes required for differentiation into three germ layers such as homeobox genes, and thus maintains pluripotency.On the other hand, several studies reported that some oncogenes also played important roles in ES cell identity (Chambers and Smith 2004; Cheng et al. 1998). The most famous oncogene related to self-renewal of mouse ES cells is Stat3. L IF-mediated Stat3 activation is essential and sufficient for the maintenance of pluripotency (Niwa et al. 1998). One of its downstream targets in LIF/Stat3 signal-ing pathway is c-Myc (Cartwright et al. 2005). An overexpression of c-Myc in mouse ES cells allows LIF-independent self-renewal. On the other hand, another well known oncogenic pathway, Ras/MAPK, negatively regulates the maintenance of pluripotency. Inhibition of the Ras/MAPK pathway by a small molecule or gene targeting blocks differentiation (Cheng et al. 1998; Burdon et al. 1999). It is not always true that these factors are expressed specifically in pluripotent cells. ES cells display tumor-like properties with regard to their growth including anchorage independence, infinite expansion and tumorigenicity. As a result, it is no wonder that tumor-related genes join the network of pluripotency.In 2006, direct reprogramming of mouse somatic cells was accomplished by introducing the combination of just four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc (Takahashi and Yamanaka 2006). These reprogrammed cells artificially induced by the defined factors were named induced Pluripotent Stem (iPS) cells. In 1 year, human iPS cells were also generated from adult dermal fibroblasts (Fig. 8.1) (Takahashi et al. 2007; Yu et al. 2007). Since the first announcement of these findings, this field has rapidly expanded and developed in various directions.In this chapter, I will introduce the expected potential and possible problems of iPS cells based on the latest findings.Fig. 8.1iPS cells derived from adult human dermal fibroblasts191 8 Induced Pluripotent Stem Cells8.2 D erivation8.2.1 R eprogramming Factors and SubstitutesMouse iPS cells were initially established by forced-expression of Oct3/4, Sox2, Klf4 and c-Myc in mouse embryonic fibroblasts (MEFs) or tail-tip fibroblasts (TTFs) from adult mice (Takahashi and Yamanaka 2006). Some of these factors can be replaced with related genes. For example, Klf2 or Klf5 can mimic Klf4 func-tions, and Sox1, Sox3, Sox15, Sox17 and Sox18 are also able to substitute for Sox2 (Nakagawa et al. 2008). Estrogen-related receptor, beta (Esrrb), can contribute to direct reprogramming with Oct3/4 and Sox2 (Feng et al. 2009). In addition, treat-ment with kenpaullone along with transduction of Oct3/4 and Sox2 also can p roduce ES-like colonies from MEFs (Lyssiotis et al. 2009).All of the Myc family genes in mammals, c-Myc, N-Myc and L-Myc, dramati-cally enhance the efficiency of iPS cell generation (Nakagawa et al. 2008). Although Myc is dispensable for direct reprogramming, the number of iPS cell colonies gener-ally diminishes to approximately 150 without Myc (Nakagawa et al. 2008; Wernig et al. 2008b). The effects of Myc on iPS cell generation can be mimicked by the activation of the canonical Wnt pathway (Marson et al. 2008). Myc proteins are substrates of Glycogen synthase kinase-3 (GSK3) which is activated by Wnt signals (Cohen et al. 2001). Phosphorylated Myc proteins by GSK3 are rapidly degraded by proteasomes. These data suggest that Myc acts as a booster of direct reprogramming.8.2.2 S election of Reprogrammed CellsGenerally, mice carrying a reporter system of fluorescent proteins and/or drug resistance genes driven by the promoters of pluripotent cell-associated genes are used for the establishment of mouse iPS cells. First, iPS cells are generated from MEFs or TTFs carrying b geo, which is a beta-galactosidase and neomycin resis-tance fusion gene knocked into the Fbx15 locus (Takahashi and Yamanaka 2006). Thereafter, other reporter systems have been designed for this purpose. Nanog or Oct3/4 can also work as selection markers of reprogramming (Okita et al. 2007; Wernig et al. 2007; Maherali et al. 2007). For example, green fluorescence protein (GFP) and puromycin resistance gene controlled by Nanog or Oct3/4 promoter are widely used in the field. The other indicator of reprogramming is the silencing of the retrovirus promoter (Nakagawa et al. 2008). The long terminal repeat (LTR) of mouse molony-leukemia virus (MMLV) can act as a strong promoter. In contrast, the activity of MMLV LTR is effectively silenced in pluripotent cells such as ES cells and iPS cells. Although some researchers have tried to elucidate this phenom-enon, the underlying mechanisms still remain unclear (Wolf and Goff 2007, 2009). Nevertheless, when a retrovirus encoding fluorescent protein is transduced along192K. Takahashi and S. Yamanaka with the reprogramming factors into somatic cells, the disappearance of the f luorescence suggests that reprogramming has been completed.These reporters are employed because it is quite hard to distinguish r eprogrammed cells from the non-reprogrammed cells just by morphology in mice. In contrast, there is little unrest for human cells. Human iPS cells form distinctive flat, tightly packed and clear edged colonies like human ES cells, whereas non-reprogrammed cells show granular morphologies and tend to form rough colonies (Takahashi et al. 2007; Lowry et al. 2008).8.2.3 E pigeneticsSome reports have shown that treatment with histone deacetylase (HDAC) inhibi-tors, such as Trichostatin A (TSA), valproic acid (VPA), DNA methyltransferase (DNMT) inhibitor, and 5-Aza-2¢-deoxycytidine can improve the frequency of iPS cell establishment (Huangfu et al. 2008a; b; Mikkelsen et al. 2008). Inhibition of histone methyltransferase G9a by treatment with a small molecule, BIX-01294, is also effective. These data suggest that epigenetic modifications are closely linked to nuclear reprogramming (Shi et al. 2008).8.2.4 O ther ElementsIn addition to the above, some other factors which can improve the reprogramming efficiency were identified. The transduction of microRNAs such as miR-291-3p, miR-294, miR-295 elevates the number of iPS cell colonies by about 10-fold (Judson et al. 2009). In addition, RNA-related protein LIN28 enhances the efficiency of human iPS cells generation (Yu et al. 2007; Liao et al. 2008). The inhibition of both Mitogen-Activated Protein Kinase (MEK) and GSK3, which is generally called 2i in serum-free culture, increases the reprogramming efficiency (Silva et al. 2008). The overexpression of Spalt-like 4 (Sall4) or the suppression of transformation related protein 53 (Trp53) increase the reprogramming efficiency both in mouse and human (Tsubooka et al. 2009; Hong et al. 2009; Kawamura et al. 2009; Li et al. 2009; Marión et al. 2009; Utikal et al. 2009; Banito et al. 2009). In the case of human cells, telomerase reverse transcriptase (TERT) and Simian virus 40 large T (SV40LT) antigen that promote the immortalization of human primary fibroblasts, can enhance the reprogramming efficiency (Park et al. 2008b; Mali et al. 2008).8.2.5 C ell SourcesIn mouse, MEFs and TTFs are commonly used as sources of iPS cells in many reports. Various other cell types were demonstrated to be sources of iPS cells, such193 8 Induced Pluripotent Stem Cellsas neural progenitors, adrenal glands, keratinocytes, muscular cells, intestinal e pithelium cells, mesenchymal stem cells and hematopoietic cells can be (Aoi et al. 2008; Wernig et al. 2008a; Silva et al. 2008; Kim et al. 2008; Eminli et al. 2008, 2009). In addition, terminally differentiated cells such as mature B cells, T cells and pancreatic b-cells can also be reprogrammed into iPS cells (Hong et al. 2009; Hanna et al. 2008; Stadtfeld et al. 2008a).In human, fibroblasts derived from a fetus, neonatal foreskin and adult dermis are widely used (Takahashi et al. 2007; Yu et al. 2007). Human iPS cells were also generated from keratinocytes, mesenchymal stroma cells and hematopoietic cells (Aasen et al. 2008; Loh et al. 2009). The efficiency of iPS cell generation from these cell types thus seems to be similar, although direct comparisons among them still need to be carried out.8.3 P ropertiesThe characteristics of mouse iPS cells are almost completely equivalent to those of mouse ES cells. The morphology of both mouse ES cells and iPS cells is identical. Those two cells types can grow in serum-containing medium in the presence of leukemia inhibitory factor (LIF) and/or feeder cells (Okita et al. 2007). They can also be maintained in serum-free medium supplemented 2i (Silva et al. 2008).The expression levels of pluripotent cell marker genes such as ERas, Rex1 and Esg1are indistinguishable between ES cells and iPS cells. Microarray analyses have also shown the global gene expression patterns of mouse iPS cells to be very similar to those of mouse ES cells (Okita et al. 2007; Wernig et al. 2007; Maherali et al. 2007). In addition, iPS cells also have the potential to undergo homologous recombination that is a useful property of ES cells in biology (Hanna et al. 2007).Not only the transcripts but the epigenetic status of iPS cells and ES cells, including DNA methylation and histone modification, are quite similar (Meissner et al. 2008). CpG methylation in the promoter regions of pluripotent cell marker genes such as Oct3/4, Rex1and Nanog are highly unmethylated in iPS cells, whereas those of MEFs or TTFs were steadily methylated. Previous reports d emonstrated that both the 4th (K4) and 27th (K27) lysine residues of histone H3 are methylated around the locus of differentiation-associated genes such as Gata4, Pax6 and Msx2 in mouse ES cells (Bernstein et al. 2006). Generally, K4 is methy-lated in the regions transcriptionally activated. In contrast, methylation of K27 reflects silencing in the vicinity. These bivalent patterns of histone methylation may imply that ES cells are always ready and waiting to initiate differentiation. In addi-tion, mouse iPS cells also show bivalent patterns of histone methylation in differ-entiation marker genes as similar to mouse ES cells (Maherali et al. 2007). Therefore, in the broad view of both external and inner aspects, it seems that iPS and ES cells have a striking resemblance. However, detailed analyses suggest that194K. Takahashi and S. Yamanaka the epigenetic patterns are not completely identical between ES cells and iPS cells (Mikkelsen et al. 2008). On the other hand, differences in these properties can be found among iPS clones.These similarities and differences are also found in human cells. In particular, the patterns of global DNA methylation differ between human iPS cells and human ES cells (Chin et al. 2009). On the other hand, various differences such as gene expres-sion patterns, statuses of X-chromosome inactivation and differentiation capacities have been reported even among human ES clones (International Stem Cell Initiative 2007). One of the conceivable reasons for this is the variety of genetic backgrounds among humans, unlike the situation regarding experimental animals. In fact, ES clones derived from different blastocysts provided by the same couple have been shown to have a strong resemblance to each other (Chen et al. 2009).8.4 D ifferentiation Capacity and Their PrecursorsMany protocols for in vitro differentiation of mouse ES cells have been estab-lished. Most of them can be applied to iPS cells. The traditional method with embryoid body formation allows iPS cells to differentiate into all three germ layers of endoderm, mesoderm and ectoderm (Takahashi and Yamanaka 2006; Wernig et al. 2007; Maherali et al. 2007). In vitro differentiations of iPS cells into specific cell types such as cardiac cells, bloods, adipocytes and retinal epitheliums has also been achieved (Schenke-Layland et al. 2008; Narazaki et al. 2008; Tashiro et al. 2009; Senju et al. 2009). The gold standard to test the pluripotency is the genera-tion of chimeric mice and the subsequent germ-line transmission. Similar to ES cells, mouse iPS cells are able to contribute to the development of chimeric mice including germ cells (Okita et al. 2007; Wernig et al. 2007; Maherali et al. 2007). The successful rate of germ-line transmission for iPS cells is no less than that for ES cells. In addition, mouse iPS cells have hurdled the stricter challenge of tetra-ploid complementation (Kang et al. 2009; Zhao et al. 2009; Boland et al. 2009). The tetraploid embryos generated by the fusion of two eggs can contribute only to the placenta instead of the pup body. When iPS cells are injected into a tetraploid blastocyst, the entire body of the pup should be derived from the injected iPS cells. The pups only from iPS cells are then successfully born. These results suggest that mouse iPS cells have already reached the pluripotency of ES cells.Most of the protocols for human ES cell differentiation are also applicable to human iPS cells. Random differentiation of human iPS cells can be achieved by embryoid body formation in vitro or teratoma experiments in vivo (Takahashi et al. 2007; Yu et al. 2007). Directed differentiation into specific cell lineages has also succeeded, such as differentiation into retinal cells, vascular cells, pancreatic i nsulin-producing cells, hepatocyte-like cells, functional cardiomyocytes and neuronal cells (Osakada et al. 2009; Viczian et al. 2009; Taura et al. 2009; Zhang et al. 2009a, b; Song et al. 2009; Gai et al. 2009; Karumbayaram et al. 2009; Chambers et al. 2009).195 8 Induced Pluripotent Stem Cells8.5 P otential Applications for TherapiesOne of the potentially useful applications of iPS cell technology is that the source of regenerative medicine using a patient’s own pluripotent stem cells (Yamanaka 2009). These order-made iPS cells would thus make it possible to achieve autolo-gous transplantation. The first study for therapeutic application of iPS cells was reported with the mouse model of sickle-cell anemia, a blood disease caused by a defect in the b-globin gene (Hanna et al. 2007). They established iPS cells from the diseased mouse, and then repaired their genetic defects by homologous recombina-tion. The injection of hematopoietic progenitors derived from genetically-corrected iPS cells could cure the diseased donor mouse. Another report showed the injection of iPS-derived cells directly into the liver of irradiated hemophilia A mice to improve their phenotypes (Xu et al. 2009). These are examples of ideal models for regenerative medicine. In human case, the idea of iPS cell therapy was also brought up by Raya and colleagues (Raya et al. 2009). At first, they corrected genes by introducing a lentiviral vector encoding FANCA or FANCD2in keratinocytes derived from Fonconi anemia patients. iPS cells derived from the patients with genetic correction can be differentiated into the hematopoietic lineage.Moreover, dopaminergic neurons differentiated from iPS cells were able to improve the behavior of a Parkinson’s disease model rat through transplantation into the brain (Wernig et al. 2008c). On the other hand, however, they found that residual undifferentiated iPS cells included in the cells for transplantation caused a teratoma. Undifferentiated cells and the subsequent tumor formation after trans-plantation remain to be a major complication of pluripotent cell therapies, not only with iPS cells, but also with ES cells.The depletion of SSEA-1 positive cells, which means undifferentiated cells, from the cultures by fluorescence-activated cell sorter (FACS) can reduce the risk of teratoma formation after cell transplantation (Wernig et al. 2008a). This issue was analyzed with a different point of view in the work of Miura and colleagues (Miura et al. 2009). They differentiated 36 mouse iPS cell lines derived from embryonic or adult cells as well as ES cells into neural progenitor cells using the sphere culture method (Miura et al. 2009). They found that residual undifferenti-ated cells existed in less than 0.5% of ES cells or MEF-derived iPS cell lines. In contrast, TTF-derived iPS cell lines reproducibly produced a significantly higher ratio of undifferentiated cells. Interestingly, such differences showed no clear rela-tionship with the presence or absence of Myc transgenes.8.6 C onclusion and PerspectiveHuman iPS cells have another potential utility as a tool for drug discovery or under-standing the pathogenesis of diseases. Over the last couple of years, iPS cells were established from somatic cells of patients with refractory diseases such as Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Down s yndrome and196K. Takahashi and S. Yamanaka Parkinson’s disease, milial dysautonomia, type I diabetes, beta-thalassemia and Fanconi anemia (Park et al. 2008a; Maehr et al. 2009; Ye et al. 2009; Soldner et al. 2009). Heretofore, it was difficult to analyze the pathogenesis of specific diseases in vitro because large amount of the patient’s cells are needed. iPS cells established from the patients may overcome the issue with their indefinite self-renewal ability. Combining the proliferation of undifferentiated iPS cells and the differentiation of specific cell types can yield an enormous amount of cells carrying the causal factors of diseases as associated with genetic mutations can be obtained, and used for large-scale screening or analyses.If pathological conditions can be recapitulated in vitro using iPS cells from the patients, they could be used for screening therapeutically-effective molecules, and/ or for the evaluation of medicine side-effects for each individual patient. For example, Long-QT syndromes (LQTS) are congenital or acquired types of diseases with delayed repolarization and subsequent depolarization of the heart. This can lead to a risk of ventricular fibrillation and unexpected death. A mutation in one of several disease-causing genes can bring on LQTS, and the effective medication is different among each type of LQTS. An epinephrine-loading test seems to be effec-tive to determine if a patient has LQTS. However, this test may carry a certain degree of risk. Functional cardiomyocytes derived from patient’s iPS cells can be used for the in vitro diagnosis of LQTS, thus reducing the burden and risk for the patient.However, recapitulation pathogenesis with disease-specific iPS cells does not always work well. Dimos et al. established iPS cells derived from an ALS patient (Dimos et al. 2008). They successfully differentiated ALS-iPS cells into both glial cells and motor neurons. However, although the glial cells of ALS patient produce toxins and result in destroying motor neurons, they failed to show the phenomenon in vitro with iPS cells. In contrast, Ebert and colleagues generated iPS cells carrying spinal muscular atrophy (SMA) which is a neurological disorder developed during infancy and causes death. Motor neurons derived from SMA patient’s iPS cells showed a defect in the maturation of motor neurons (Ebert et al. 2009). Another paper also reported the success of reproducing a pathological condition. They choose Familial dysautonomia (FD) as a model of pathogenesis, which shows peripheral neuropathy induced by a reduction in the expression of the IKBKAP gene. They developed neural crest precursor cells from FD-iPS cells. As result, they observed a defect of cell migration and spontaneous differentiation into TuJ1 or ASCL1 positive neurons. In addition, they treated the cells with kinetin which could correct the expression of IKBKAP in mutant cells, and it markedly improved the phenotypes required for cell migration. Therefore, although not in all cases, these data indicate that the recapitulation of the pathogenesis and subsequent tests for drug effects are feasible.First iPS cells were generated by transduction with four reprogramming factors with retroviral vectors (Takahashi and Yamanaka 2006). This method has proven to be the most effective. However, viral integration into the genome should be avoided for clinical applications, because of destruction or unexpected activation of adjacent genes, which may result in tumor formation. Many attempts have been。