iPSCs are somatic cell-derived stem cells similar to ESCs in terms of self-renewal and pluripotency. iPSCs were first established by Takahashi and Yamanaka in 2006 [14] (Fig. 31.1). They introduced Oct3/4, Sox2, Klf4, and c–Myc using retrovirus vector into mouse embryonic fibroblasts and selected using the Fbx15 reporter system, which is specifically expressed in mouse ESCs and early embryos, but is dispensable for the maintenance of pluripotency and mouse development. Resulting cells were similar to mouse ESCs in many points such as an expression of stem cell-specific genes and surface glycoproteins, chromatin methylation patterns, and differentiation potency into three germ layers in vivo and in vitro. However, these cells were not able to contribute to adult chimeras. To improve the quality as pluripotent cells, fully reprogrammed cells were selected by the expression of Nanog, which is required for maintaining pluripotency, and germ-line chimeras were obtained as a result [16]. In the next step, human iPSCs were established by using the same set [17] or another set of transcription factors (OCT3/4, SOX2, NANOG, LIN28) [18]. These human iPSCs also fulfilled the same criteria as mouse iPSCs and ESCs, except for confirmation of chimera formation and germ-line transmission, which is obviously ethically unacceptable.

After the generation of iPSCs from fibroblasts [17], iPSCs have been generated from various types of cells to demonstrate a proof of principle that all somatic cells can be reprogrammed into pluripotency: murine liver and stomach cells [19], pancreatic β-cells [20], terminally differentiated lymphocytes [21], cord blood cells [22], third molar [23], keratinocytes of plucked hair [24], and neural stem cells (NS cells) [25, 26]. iPSCs were generated also from cochlear epithelial cell-derived otospheres of neonatal mice [27]. Although otospheres resemble multipotent NS cells in their overall gene expression pattern and epigenetic status [28], the reprogramming efficacy of NS cells by four factors was approximately 10 times higher than that of otospheres [25, 27]. Differences in demethylated patterns of NS cell-specific Sox2 enhancers may partly cause the difference in reprogramming efficacy.

More recently, it was reported that less factors are required to reprogram somatic cells into pluripotency. iPSCs were generated from mouse and human fibroblasts without Myc [29] and from keratinocyte and mesenchymal stem cells by three factors: OCT3/4, SOX2, and KLF4 except c–MYC [30, 31]. Additionally, iPSCs were generated from human NS cells by introduction of OCT3/4 and KLF4, or only OCT3/4, suggesting that endogenous expression of the reprogramming factors can complement exogenous factors [32].

The genetic mutation in a genome of a patient is taken over to iPSCs, when somatic cells of the patient are reprogrammed to iPSCs. These cells, so-called disease-specific iPSCs, provide opportunities to develop novel strategies to elucidate the pathological mechanisms of disease, together with physiological mechanisms by comparing with wild-type cells. Differentiated cells from disease-specific iPSCs into the target or precursor cells serve as in vitro disease models, that is, recapitulation of the disease phenotype at cellular level, and are used to reveal molecular mechanisms of the disease. These differentiated cells are also useful to develop new treatments. Soon after the establishment of human iPSCs [17], mutant iPSCs were successfully generated from several diseases including amyotrophic lateral sclerosis (ALS) [33], muscular dystrophy, Parkinson disease, and Huntington disease [34], followed by the research for cell survival and function of spinal muscular atrophy patient iPSC-derived motor neurons [35]. Since then, disease-specific iPSCs have already been generated to establish disease models, from neurological disorders [36–46], cardiovascular diseases [47–51], metabolic abnormalities [36, 52, 53], and hematological insufficiency [54–56].

The major advantage of the use of disease-specific iPSCs owes much to the ability to generate a specific tissue/cell type from the cells obtained from another tissue. For example, the pathogenetic mechanism in Alzheimer’s disease (AD) comes from chronic accumulation of abnormal amyloid-β peptide (Aβ) oligomers in the brain tissue. However, brain biopsy for the diagnosis or research is not easy, and, before the time of diagnosis of AD, neural tissue has been extensively modified due to the chronic accumulation of Aβ. By using several lines of iPSCs derived from AD patient’s skin fibroblasts (AD-iPSC), Kondo et al. [42] induced AD-iPSC-derived neurons. They successfully detected stress responses as an early cellular phenotype and showed that docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD-iPSC-derived neurons from a subset of patients. This not only clarified the early mechanism of the disease, but also may help select patients who are responsive to DHA treatment. iPSCs derived from skin fibroblasts of dilated cardiomyopathy (DCM) patients [50] also provided a good opportunity to perform cellular study, because cardiac tissues from DCM patients are difficult to obtain and do not survive in long-term culture.

Disease- or patient-specific iPSCs are used for drug screenings in vitro to identify candidate chemical compounds for the therapeutic purpose [39, 40, 42, 50, 57] or to test toxicity of drugs [48, 51]. For example, a histone acetyltransferase inhibitor called anacardic acid was identified that rescued the phenotype of abnormal motor neuron differentiated from ALS-iPSCs [39].

Mutations in mitochondrial DNA (mtDNA) are implicated in numerous human diseases. Artificial modification of mtDNA would be one possible strategy to treat diseases due to mutations in mtDNA. However, the modification of mtDNA is difficult because mtDNA is encapsulated in the mitochondrial outer and inner membrane, and multiple copies of mtDNA exist in each cytoplasm. Mitochondria in each somatic cell are maintained during the process of reprogramming for the generation of iPSCs. Thus, resulting iPSCs that carry the mutant mtDNA serve as an in vitro model for these diseases. Disease-specific iPSCs have been established from those patients including mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) [58, 59] and Pearson marrow-pancreas syndrome [60].

As genomic DNA of iPSCs (and ESCs) has an accessible chromatin state, genomic modification is relatively easier compared to other somatic cells. Thus, recent precision gene modification techniques such as the helper-dependent adenoviral vector [61], zinc-finger nucleases (ZFN) [62], transcription activator-like effector nucleases (TALEN) [63], and CRISPR-Cas9 system [64, 65] are suitable to be applied to modify a genome of pluripotent stem cells. For example, ZFN was used to introduce XIST (the intrinsic X chromosome-inactivating gene) into a trisomic chromosome 21 in Down’s syndrome patient-derived iPSCs, followed by successful trisomy silencing [66]. These techniques will provide a new therapeutic concept of those intractable diseases in the future.

As described above, disease-specific iPSCs are remarkable powerful tools and resources for both research and therapy. The inner ear tissues of a living human are difficult to obtain because the inner ear is encapsulated in the hard bone, and the puncture to that capsule for biopsy results in the serious deterioration of the inner ear function. Furthermore, the amount of the inner ear tissue obtained from the biopsy would be limited and it would be difficult to maintain the obtained tissues in a long-term culture. Disease-specific iPSC technologies will be powerful tools to establish inner ear disease models for the investigation of the pathogenic mechanisms, drug discovery, and invention of new treatment strategies. A wide variety of diseases related to inner ear dysfunction of monogenic, chromosomal, or mitochondrial etiology will be the target of entities that are modeled by patient-specific iPSCs.

There are several advantages of iPSCs over ESCs for research and future clinical applications [1]. The first point is the ethics. The generation of ESCs is ethically problematic because it usually accompanies with the destruction of embryos, which are continuous to the living individual. Some researchers justify the generation of human ESCs because they are generated from the fertilized eggs that are destined to be discarded after in vitro fertilization. For those who do not accept this justification, the generation of ESCs from blastomeres [67] or from preimplanted morula [67, 68] might be acceptable because it does not involve the activity of destroying the whole embryos. However, there is no consensus on propriety of intervening embryos to generate ESCs. On the other hand, iPSCs do not raise this ethical problem because they are derived from somatic cells. Secondly, disease-specific iPSCs can be easily obtained from patients as mentioned above. Theoretically, ESCs generated by nuclear transfer (ntESCs) [69, 70] enable the generation of pluripotent stem cells that code genomic information of transferred nucleus. However, it is costly to prepare for every ntESC, where the nucleus originates from each patient. Thirdly, the premise that iPSCs and their progeny would not cause any immune response in the host due to self-tolerance was supported by two reports that syngeneic iPS-derived cells did not elicit immune responses [71, 72]. But there is an opposing report that teratomas from syngeneic murine iPSCs were immunorejected by a host, whereas those from mouse ESCs were immunotolerant [73]. Kaneko and Yamanaka rebutted this report maintaining that immunorejection of syngeneic iPSCs may have been caused by gene integration [74].

However, human ESC research is still important because there is a serious concern about the stem cell-related malignancy. The generation of iPSCs originally accompanied with gene integration. Such genomic modification is a risk factor for insertional mutagenesis, which may cause malignant cell transformation [75]. To reduce this risk, non-integrating gene delivery methods for the expression of defined pluripotent factors have been reported (Fig. 31.1): iPSCs generated with adenoviruses [76], sendai viruses [77], plasmid vectors [78], removable transposon systems [79], direct delivery of recombinant reprogramming proteins [80], and synthetic modified messenger RNA [81]. The use of a proto-oncogene c-Myc is also a concern, which now can be replaced with L-Myc [82] or eliminated [29]. Genetic defects in iPSCs result in not only tumorigenesis but also differentiation defectiveness. Koyanagi-Aoi et al. [83] identified low-quality iPSC lines out of a relatively large number of human iPSC and ESC lines. iPSC lines of low quality were marked by higher expression levels of several genes expressed from long terminal repeats of specific human endogenous retroviruses and molecular signatures such as RNA expression and DNA methylation patterns. Those iPSCs were prone to form teratoma when neurally induced and transplanted into mouse brains. Thus, human iPSCs have considerable variation in terms of complete differentiation and tumorigenesis.

In terms of quality control, ESCs are still superior, because clinical grade human ESC lines have already been established [84]. Therefore, the use of iPSCs in the clinic will require much more research and understanding, and human ESCs will provide an accurate reference for iPSCs, which will allow them to be standardized for their use in clinical applications.

Since Ito et al. first demonstrated the regeneration of the auditory pathway by transplants of fetal brain tissue [85], several stem cell-based approaches to cure deafness using mesenchymal stem cells, neural stem cells, ESCs, and iPSCs have been adopted. Current major targets are cochlear hair cells and SGNs, which are primarily compromised and unregenerable cells in sensorineural hearing loss.