In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinsons disease and Alzheimers disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.

The 2012 Nobel Prize in Physiology or Medicine was awarded for The discovery that mature cells can be reprogrammed to become pluripotent. First, we would like to consider the significance of this research. The lives of mammals, including humans, begin with the fertilization of an egg by a sperm cell. In humans, a blastocyst composed of 70-100 cells forms by approximately 5.5 days after fertilization. The blastocyst is composed of the inner cell mass, the cell population that has the ability to differentiate into the various cells that constitute the body (pluripotency), and the trophoblast, the cells that develop into the placenta and extra-embryonic tissues and do not contribute cells to the body. In the embryonic stage, the pluripotent cells of the inner cell mass differentiate into the three germ layers, endoderm, mesoderm, and ectoderm, which will form specific organs and tissues containing somatic stem cells with limited differentiation potencies. These somatic stem cells continue to divide and differentiate, and, by adulthood, an individual composed of 60 trillion cells is produced. Somatic stem cells born in the fetal period actively divide, and are involved in the formation and growth of various organs. However, even in the adult, somatic stem cells persist in niches in every organ and tissue, and play an important role in maintaining organ and tissue homeostasis. When cells in the inner cell mass are removed at the blastocyst stage and cultured in vitro, pluripotent embryonic stem (ES) cells are obtained. Thus, in the normal process of development, cell differentiation of the three germ layers proceeds from the simple stages of the fertilized egg and blastocyst, and ultimately produces an individual consisting of a complex cellular society.

In 1893, August Weismann argued that only germ cells (eggs and spermatozoa) maintain a determinant, which was described as heritable information essential to decide on the functions and features of all somatic cells in the body [1]. In his germ plasm theory, the determinants are lost or irreversibly inactivated in differentiated somatic cells.

It took more than 50 years for researchers to rewrite this dogma. In 1962, Sir John Gurdon demonstrated the acquisition of pluripotency by reprogramming cells to their initial stage using a novel research technique, i.e., producing cloned individuals by transferring somatic cell nuclei into eggs [2]. However, for many years, that result was regarded as a special case limited to frogs alone. The production of Dolly the sheep by transferring the nucleus of a somatic cell (mammary gland epithelial cell) by Sir Ian Wilmut in the late 1990s [3] showed that cloning could also be applied to mammals.

These brilliant previous works led to our studies that culminated in the induction of pluripotency in mouse somatic cells in 2006, using retroviral vectors to introduce four genes that encode transcription factors i.e., Oct4, Sox2, Klf4, and c-Myc. We designated these cells as iPS cells [4]. In 2007, we succeeded in generating human iPS cells using genes encoding the same four transcription factors [1]. The results of this research showed that although the developmental process was thought to be irreversible, by introducing key genes into differentiated adult cells the cells could be reset to a state in the extremely early stage of development in which they possessed pluripotency. That is, the results demonstrated that the differentiation process was reversible. This startling discovery made it necessary to rewrite the embryology textbooks.

Three major lines of research led us to the production of iPS cells [

] (Figure

). The first, as described above, was nuclear reprogramming initiated by Sir John Gurdon in his research of cloning frogs by nuclear transfer in 1962 [

] and by Sir Ian Wilmut, who cloned a mammal for the first time in 1997 [

]. In addition, Takashi Tada showed that mouse ES cells contain factors that induce reprogramming in 2001 [

]. The second line of research was factor-mediated cell fate conversion, initiated by Harold Weintraub, who showed that fibroblasts can be converted into the muscle lineage by transduction with the

gene, which encodes a muscle lineage-specific basic helix-loop-helix transcription factor in 1987 [

]. The third line of research was the development of mouse ES cells, initiated by Sir Martin Evans and Gail Martin in 1981 [

,

]. Austin Smith established culture conditions for mouse ES cells and identified many factors essential for pluripotency including leukemia inhibitory factor (LIF) in 1988 [

]. Later, he developed the method to induce the ground state of mouse ES cell self-renewal using inhibitors for mitogen-activated protein kinase and glycogen synthase kinase 3 [

], which supports the establishment of fully reprogrammed mouse iPS cells. Furthermore, James Thomson generated human ES cells [

] and established their optimal culture conditions using fibroblast growth factor-2 (FGF-2). Without these previous studies, we could never have generated iPS cells. Interest rapidly escalated, and, in tandem with the birth of iPS cell technology, pluripotency leapt into the mainstream of life sciences research in the form of reprogramming technology [

]. However, there remain many unanswered questions regarding reprogramming technology. What are the reprogramming factors in the egg cytoplasm that are active in cloning technology? What do they have in common with the factors required to establish iPS cells and what are the differences? What kind of epigenetic changes occur in association with the reprogramming?

The history of investigations of cellular reprogramming that led to the development of iPS cells. Our generation of iPS cells in 2006 [4] became possible due to three scientific lines of investigation: 1) nuclear reprogramming, 2) factor-mediated cell fate conversion, and 3) ES cells. See the text for details (modified from Reference [5] with permission).

Apart from basic research in embryology, broad interest has been drawn to the following possible applications of iPS cell research: (1) regenerative medicine, including elucidating disease pathologies and drug discovery research using iPS cell disease models, and (2) medical treatments (Figure

). In this review, we describe these potential applications in the context of the results of our own research.

The application of iPS cell technologies to medical science. iPS cell technologies can be used for medical science including 1) cell therapies and 2) disease modeling or drug development. See the text for details.

iPS cells can be prepared from patients themselves and therefore great expectations have been placed on iPS cell technology because regenerative medicine can be implemented in the form of autografts presumably without any graft rejection reactions. Although there have been some controversies [

], the immunogenicity of terminally differentiated cells derived from iPS cells can be negligible [

]. Moreover, there has been substantial interest in the possibility of regenerative medicine without using the patients own cells; that is, using iPS cell stocks that have been established from donor somatic cells that are homozygous at the three major human leukocyte antigen (HLA) gene loci and match the patients HLA type [

]. The development of regenerative medicine using iPS cells is being pursued in Japan and the USA for the treatment of patients with retinal diseases, including age-related macular degeneration [

], spinal cord injuries [

], Parkinsons disease (PD) [

,

], corneal diseases [

], myocardial infarction [

,

], diseases that cause thrombocytopenia, including aplastic anemia and leukemia [

,

], as well as diseases such as multiple sclerosis (MS) and recessive dystrophic epidermolysis bullosa [

] (Table

).

Planned clinical trials of iPS cell-based therapies

Masayo Takahashi, (RIKEN)

Retinal Pigment Epithelium (sheet)

Age-related macular degeneration (wet type)

Alfred Lane, Anthony Oro, Marius Wernig (Stanford University)

Keratinocytes

Recessive dystrophic epidermolysis bullosa (RDEB)

Mahendra Rao (NIH)

DA neurons

Parkinsons disease

Koji Eto (Kyoto University)

Megakaryocyte

Thrombocytopenia

Jun Takahashi (Kyoto University)

DA neurons

Parkinsons disease

Steve Goldman, (University of Rochester)

Oligodendrocyte precursor cell

Multiple Sclerosis

Hideyuki Okano, Masaya Nakamura (Keio University)

Neural stem/progenitor cells

Spinal Cord Injury

Shigeto Shimmura (Keio University)

Corneal endothelial cells

Corneal endothelial dysfunction

Koji Nishida (Osaka University)

Corneal epithelial cells (sheet)

Corneal epithelial dysfunction and trauma (e.g. StevensJohnson syndrome)

Yoshiki Sawa (Osaka University)

Cardiomyocytes (sheet)

Heart Failure

Keiichi Fukuda (Keio University)

Cardiomyocytes (sphere)

Heart Failure

Yoshiki Sasai and Masayo Takahashi (RIKEN)

Neuroretinal sheet including photoreceptor cells

Retinitis pigmentosa

Advanced Cell Technology

Megakaryocytes

Refractory thrombocytopenia

In 1998, Hideyuki Okano, in collaboration with Steven Goldman, demonstrated for the first time the presence of neural stem/progenitor cells (NS/PCs) in the adult human brain using a neural stem cell marker, the ribonucleic acid (RNA)-binding protein Musashi1 [30, 31]. Research on nerve regeneration then commenced in earnest. That same year, we began regenerative medicine research on neural stem cell transplantation in a rat model of SCI, and have since made progress in developing NS/PC transplantation therapies in experiments on animal models of SCI. First, motor function was restored by transplanting rat fetal central nervous system (CNS)-derived NS/PCs into a rat SCI model [32]. The same study also showed that the sub-acute phase is the optimal time for NS/PC transplantation after SCI. In this study, at least part of the putative mechanism by which NS/PC transplantation restored function was identified in animal models of SCI. Both the cell autonomous effect (such as synaptogenesis between graft-derived neurons and host-derived neurons) and non-cell autonomous (trophic) effects mediated cytokines released from the graft-derived cells are likely contributing to tissue repair and functional recovery. Subsequently, a non-human primate SCI model was developed using the common marmoset, and motor function in that model was restored by transplanting human fetal CNS-derived stem cells [33]. In the same study, a behavioral assay for motor function associated with SCI was developed. Based on these studies, a preclinical research system for cell transplantation therapy was established in a non-human primate SCI model.

Given these findings, we began preparations for clinical studies of human fetal CNS-derived NS/PC transplantation to treat SCI patients. However, with the guidelines for clinical research on human stem cells of the Japanese Ministry of Health, Labor and Welfare that came into effect in 2006, human fetus-derived cells and ES cells became ineligible for use in regenerative medicine. Thus, we had no choice but to change our strategy (human ES cells became eligible for use in the 2013 guidelines). In 2006, one of our research groups (Yamanakas group) established iPS cells from adult mouse skin cells. Hypothesizing that it might be possible to induce NS/PCs from iPS cells, we (Okanos group) turned our attention to iPS cells as a means of obtaining NS/PCs without using fetal or ES cells. Based on conditions that were developed for experiments on mouse ES cells [34, 35], NS/PCs were induced from mouse iPS cells [36]. The following year, we succeeded in restoring motor function by transplanting these mouse iPS cell-derived NS/PCs into a mouse model of SCI, and reported that when good iPS cells -derived NS/PCs, which had been pre-evaluated as non-tumorigenic by the transplantation into the brains of immunocompromised mice, were used for transplantation, motor function was restored for a long period of time without tumors developing [37]. In 2011, we succeeded in restoring motor function by transplanting human iPS cell-derived stem cells into a mouse SCI model [38]. Moreover, in 2012, motor function was restored by transplanting human iPS (line 201B7) cell-derived NS/PCs into the marmoset SCI model, and long-term motor function was recovered without observable tumor formation [39]. This finding was of great significance in terms of preclinical research, and provided a proof of concept that could potentially lead to a treatment method.

Collectively, when mouse or human iPS cells were induced to form NS/PCs and were transplanted into mouse or non-human primate SCI models, long-term restoration of motor function was induced, without tumorigenicity, by selecting a suitable iPS cell line [17, 40]. Considering the sub-acute phase (2-4 weeks after the injury) as the optimal time for iPS cells-derived NS/PCs transplantation for SCI patients, there are following major difficulties with autograft-based cell therapy. First, it takes about a few months to establish iPS cells. Second, it also takes three months to induce them into NS/PCs in vitro. Third, one more year would be required for the quality control including their tumorigenesis.

Considering these, our collaborative team (Okano and Yamanaka laboratories) are currently planning iPS-based cell therapy for SCI patients in the sub-acute phase using clinical-grade integration-free human iPS cell lines that will be generated by Kyoto Universitys Center for iPS Cell Research and Application (CiRA). We will establish a production method, as well as a storage and management system, for human iPS cell-derived NS/PCs for use in clinical research for spinal cord regeneration, build an iPS cell-derived NS/PC stock for regenerative medicine, establish safety screenings against post-transplantation neoplastic transformation, and commence clinical research (Phase IIIa) trials for the treatment of sub-acute phase SCI (Figure

). As these studies progress, the application of iPS cells to treat chronic phase SCI and stroke will be investigated. Significant therapeutic efficacy in the treatment of chronic phase SCI has not been achieved by cell transplantation alone [

]. However, clinical studies are planned using antagonists of axon growth inhibitors, such as Semaphorin3A inhibitors [

], followed by multidisciplinary rehabilitation combination therapies. We aim to perform a clinical trial based on the Pharmaceutical Affairs Act in collaboration with drug companies and to use iPS cell-derived NS/PC stocks for regenerative medicine to establish treatment methods for stroke, MS, and Huntingtons disease.

Strategies for the development of iPS cell-based cell therapy for SCI patients. Our collaborative team (Okanos group at Keio University and Yamanakas group at Kyoto University) have been developing an iPS cell-based cell therapy for SCI since 2006. Our previous preclinical studies have shown that long-term functional restoration can be obtained by transplantation of NS/PCs derived from appropriate iPS cells clones without observable tumor formation [10]. Currently, we aim to develop iPS cells-based cell therapy for SCI patients at sub-acute phase using the clinical grade iPS cell-derived NS/PCs (i.e., the role of Okanos group described in the blue box) which have been prepared from human iPS cell stock (i.e., the role of Yamanakas group described in the yellow box).

Lesion sites are difficult to access in patients with degenerative diseases of the nervous system. Therefore, in past studies, cell biological or biochemical analyses of their pathology centered on forced expression of the causative genes in non-nervous system cultured cell lines and on mice in which the causative gene was knocked out. However, in a few instances, the animal or cell models did not necessarily reflect the human pathology. Identifying cell biological or biochemical changes in the initial stages of the disease, before onset of symptoms, has been difficult given analyses conducted on postmortem brains. However, with the development of iPS cell technologies, it became possible to establish pluripotent stem cells from the somatic cells of anyone, irrespective of race, genetic background, or whether the person exhibits disease symptoms. Thus, it is no exaggeration to say that generation of disease-specific iPS cells using iPS cell technologies is the sole means of reproducing ex vivo phenomena that occur in patients in vivo, particularly for nervous system disorders. The result has been a tremendous desire by investigators who are conducting research on neurological diseases to become engaged in disease-specific iPS cell research [4345].

A variety of disease-specific iPS cells have been used to study nervous system diseases, including amyotrophic lateral sclerosis (ALS) [

], spinal muscular atrophy [

], spinobulbar muscular atrophy [

], Friedreichs ataxia [

], Alzheimers disease (AD) [

], PD [

], Huntingtons disease [

,

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