Induced pluripotent stem cells: origins, applications, and future perspectives

Induced pluripotent stem cells: origins, applications, and future perspectives first-author corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:yxm411@sdu.edu.cnjggao@sdu.edu.cn †E-mail:yxm411@sdu.edu.cnjggao@sdu.edu.cn yxm411@sdu.edu.cn corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:yxm411@sdu.edu.cnjggao@sdu.edu.cn †E-mail:yxm411@sdu.edu.cnjggao@sdu.edu.cn yxm411@sdu.edu.cn School of Life Sciences, Shandong University, Jinan 250100, China School of Life Sciences, Shandong University, Jinan 250100, China Embryonic stem (ES) cells are widely used for different purposes, including gene targeting, cell therapy, tissue repair, organ regeneration, and so on. However, studies and applications of ES cells are hindered by ethical issues regarding cell sources. To circumvent ethical disputes, great efforts have been taken to generate ES cell-like cells, which are not derived from the inner cell mass of blastocyst-stage embryos. In 2006, Yamanaka et al. first reprogrammed mouse embryonic fibroblasts into ES cell-like cells called induced pluripotent stem (iPS) cells. About one year later, Yamanaka et al. and Thomson et al. independently reprogrammed human somatic cells into iPS cells. Since the first generation of iPS cells, they have now been derived from quite a few different kinds of cell types. In particular, the use of peripheral blood facilitates research on iPS cells because of safety, easy availability, and plenty of cell sources. Now iPS cells have been used for cell therapy, disease modeling, and drug discovery. In this review, we describe the generations, applications, potential issues, and future perspectives of iPS cells. Induced pluripotent stem cells Origin Peripheral blood cells Application Potential issues Introduction 1 Embryonic stem (ES) cells are pluripotent cells which were derived from the inner cell mass of blastocyst-stage embryos. Pluripotency is the potential that one type of cells can differentiate into various kinds of cells, such as muscle cells, neural cells, and even germ cells. Based on this property, ES cells can generate any type of cell to meet the requirements of different applications. ES cells are also capable of self-renewal; they can be semi-permanently cultured on feeder cells, which supply necessary growth factors for ES cells. Mouse ES cells were established by Evans and Kaufman ( 1981 1981 ). At present, most of gene-targeted and transgenic mice are generated via mouse ES cells. Basic research on gene functions can be carried out on these animal models. However, research and clinical applications on human ES cells, which were established by Thomson et al. ( 1998 1998 ), have been restricted by ethical issues regarding cell sources and immunological rejection in cell therapy. Most debates on ethics are concentrated on the morality of destroying human embryos for the benefit of other people. Obtaining stem cells from oocytes and embryos is full of disputes regarding the onset of personhood and reproduction. Furthermore, immunizing antigens from different ES cells are obviously different. After transplanted into a recipient, somatic cells, which were derived from human ES cells, may be rejected by the recipient’s immune system. Therefore, it is necessary to make patient-specific pluripotent stem cells. Scientists have attempted to reprogram somatic cells to develop a new kind of stem cell with self-renewal properties and pluripotency through many methods, such as nuclear transfer (Wilmut et al., 1997 1997 ; Chesné et al., 2002 2002 ) and cell fusion (Ying et al., 2002 2002 ). However, all methods cannot do without the pluripotency of the cell nucleus in animals. As early as 1962, in fact, Gurdon ( 1962 1962 ) found that the differentiation of animal cells is reversible. In the classic experiment, he replaced the nuclei in unfertilized frogs’ eggs with the nuclei from intestinal cells. The modified eggs finally still developed into normal tadpoles. This means that the DNA of adult cells in frogs still contains all the genetic information as would be found in the nucleus of the zygote. In 1996, the birth of Dolly indicated that a mammal could be successfully cloned from adult cells (Wilmut et al., 1997 1997 ). In 2006, a Japanese group made a remarkable breakthrough. Takahashi and Yamanaka ( 2006 2006 ) generated induced pluripotent stem (iPS) cells by over-expressing a few types of transcription factors. iPS cells without ethical arguments have self-renewal properties and pluripotency just like ES cells. In 2012, Gurdon and Yamanaka received the Nobel Prize in physiology or medicine for their researches. Here we review the initial development of iPS cells and their applications in pharmacology and medicine, especially the usage of peripheral blood as an easily accessible source for deriving iPS cells. In addition, we also provide discussions on potential issues and future perspectives for iPS cells. Generations of iPS cells 1 When mouse somatic cells are hybridized with mouse ES cells, their nuclei can be reprogrammed. The hybridized somatic cells have the ability of differentiating into endoderm, mesoderm, and ectoderm cells. These findings demonstrated that reprogramming factors expressed in ES cells could induce pluripotency in somatic cells. The most difficult part in reprogramming somatic cells is how to find these reprogramming factors which can convert somatic cells to pluripotent stem cells. Now this problem has been solved by the Fbx15-Neo reporter system. Fbx15 is a gene which was expressed specifically in ES cells. Normal fibroblasts cannot survive in the presence of Geneticin (G418), an analog of Neomycin (Neo) used for screening ES cells. Therefore, candidate reprogramming factors can be screened via fibroblasts with a Neo resistance gene in their Fbx15 locus. Fibroblast reprogrammed by the candidate reprogramming factors can activate the Fbx15 locus, which leads to the expression of the Neo resistance gene. Thereby, the fibroblasts can survive in the presence of G418. Takahashi and Yamanaka ( 2006 2006 ) selected 24 genes, which were important transcripts of ES cells and oncogenes as candidate reprogramming factors. Different combinations of these candidates were introduced into mouse embryonic fibroblasts in order to screen proper reprogramming factors via the Fbx15-Neo reporter system. If these candidate genes could reprogram the fibroblasts, G418-resistant stem cell-like colonies would appear about two weeks later. Finally, the 24 candidates were narrowed down to four transcription factor genes. After introduction of the retroviral mediated factors Oct3/4 , Sox2 , Klf4 , and c-Myc , mouse embryonic fibroblasts were reprogrammed into ES cell-like cells called iPS cells. This was a revolutionary breakthrough that immediately sparked immense interest so that many scientists subsequently focused on this research. However, some key pluripotent genes were not fully activated by the four transcription factor genes. Therefore, the fibroblasts were only partially reprogrammed. When the iPS cells were injected into mouse blastocysts, they could not generate postnatal chimeras or contribute to the germline. In fact, pluripotent iPS cell lines were not established until 2007. Live chimeras and germline transmitted mice were derived from these iPS cells through blastocyst injection (Maherali et al., 2007 2007 ; Okita et al., 2007 2007 ; Wernig et al., 2007 2007 ). Zhao et al. ( 2009 2009 ) and Kang et al. ( 2009 2009 ) used iPS cells to generate germline transmitted mice through tetraploid complementation. As mentioned above, research in different laboratories indicates that iPS cells, which are similar to ES cells, have the potential to differentiate into any cell type. Takahashi et al. ( 2007 2007 ) and Yu et al. ( 2007 2007 ) independently reprogrammed human somatic cells to iPS cells. The former used Oct3/4 , Sox2 , Klf4 , and c-Myc on human dermal fibroblasts when the latter used Oct3/4 , Sox2 , Nanog , and Lin28 on human somatic cells. Both researches demonstrated that human iPS cells resemble human ES cells in many aspects, such as morphology, proliferation, pluripotency markers, gene expression profiles, epigenetic status, and differentiation potential. These findings revealed that human iPS cells have the capability of replacing human ES cells. Human iPS cells provide the correct direction of addressing the ethical disputes over stem cell sources and immunological rejection in cell therapy. Since the first iPS cell line was established by Yamanaka in 2006, scientists have made efforts to improve the safety and efficiency of the reprogramming process, including single (Si-Tayeb et al., 2010 2010 ) and multiple transient transfections (Okita et al., 2008 2008 ), non-integrating vectors (Stadtfeld et al., 2008a 2008a ; Yu et al., 2009 2009 ; Okita et al., 2011 2011 ), excisable vectors (Kaji et al., 2009 2009 ; Lacoste et al., 2009 2009 ; Woltjen et al., 2009 2009 ), direct protein transduction (Kim D. et al., 2009 2009 ; Zhou et al., 2009 2009 ; Cho et al., 2010 2010 ), RNA-based Sendai viruses (SeVs) (Fusaki et al., 2009 2009 ; Nishimura et al., 2010 2010 ; Seki et al., 2010 2010 ), mRNA-based transcription factor delivery (Warren et al., 2010 2010 ; Yakubov et al., 2010 2010 ), microRNA transfections (Maehr et al., 2009 2009 ), and the use of chemical compounds (Desponts and Ding, 2010 2010 ; Li and Ding, 2010 2010 ). Recently, small-molecule compounds have been used to generate mouse iPS cells from somatic cells (Hou et al., 2013 2013 ). Small-molecule compounds have advantages over other inducers because they can be cell-permeable, nonimmunogenic, easily synthesized, and cost-effective. Moreover, their effects on inhibiting and activating the function of specific proteins are often reversible and can be reversed by varying the concentrations. It is a milestone in the field of iPS cells. In the future, this chemical reprogramming strategy will be hotspots for reprogramming different somatic cells. Cell sources for deriving iPS cells 1 Moreover, many other cell sources are also used in research on iPS cells. Up to now, iPS cells have been derived from many different species, such as mice, humans, rats, marmosets, rhesus monkeys, pigs, and rabbits (Table 1 1 ). However, most iPS cell lines cannot generate live chimeras. Because of the successful reprogramming of the fibroblasts, many different cell types have been analyzed for their capacity to be reprogrammed. The cell types successfully reprogrammed contain hepatocytes, gastric epithelial cells, keratinocytes, stomach cells, mesenchymal cells, neural stem cells, pancreatic cells, B and T lymphocytes, blood progenitor cells, cord blood cells, peripheral blood cells, and so on (Table 1 1 ). HUVEC: human umbilical vein endothelial cell; O: Oct3/4 ; S: Sox2 ; K: Klf4 ; M: c-Myc ; E: Esrrb ; L: Lin28 ; N: Nanog ; Nr: Nr52a Species Cell type Factor or chemical Vector Reference 17 Mouse Fibroblast OKSM or OKS Retrovirus Takahashi and Yamanaka, 2006 2006 ; Nakagawa et al., 2008 2008 ; Wernig et al., 2008 2008 Fibroblast OSE or KSNr Retrovirus Feng et al., 2009 2009 ; Heng et al., 2010 2010 Fibroblast mir302/367 cluster Lentivirus Anokye-Danso et al., 2011 2011 Fibroblast OKSM PB transposon and 2A peptides Kaji et al., 2009 2009 Fibroblast Proteins (OKSM) Poly-arginine Zhou et al., 2009 2009 Fibroblast OKSM Plasmid or adenovirus Okita et al., 2008 2008 ; Stadtfeld et al., 2008a 2008a Dermal papilla OKM or OK Retrovirus Tsai et al., 2010 2010 Melanocyte OKM Drug-inducible lentivirus Utikal et al., 2009 2009 Mature B and T cell OKSM Retrovirus Eminli et al., 2009 2009 Myeloid progenitor OKSM Retrovirus Eminli et al., 2009 2009 Hematopoietic stem cell OKSM Retrovirus Eminli et al., 2009 2009 Pancreatic β cell OKSM Drug-inducible lentivirus Stadtfeld et al., 2008b 2008b Intestinal epithelial cell OKSM Drug-inducible lentivirus Wernig et al., 2008 2008 Hepatocyte OKS Retrovirus Aoi et al., 2008 2008 Gastric epithelial cell OKSM Retrovirus Aoi et al., 2008 2008 Adipose stem cell OKSM Retrovirus Sugii et al., 2010 2010 Neural stem cell OK or O Retrovirus Kim et al., 2008 2008 ; 2009b 2009b 5 20 Human Fibroblast OKSM or OKS Retrovirus Takahashi et al., 2007 2007 ; Nakagawa et al., 2008 2008 Fibroblast OSLN Lentivirus Yu et al., 2007 2007 Fibroblast OKSM or OKS Floxed lentivirus Soldner et al., 2009 2009 Fibroblast OS and valproic acid Retrovirus Huangfu et al., 2008 2008 Fibroblast Proteins (OKSM) Poly-arginine Kim D. et al., 2009 2009 Fibroblast OKSM Adenovirus Zhou and Freed, 2009 2009 HUVEC OKSM Retrovirus Lagarkova et al., 2010 2010 Peripheral blood cell OKSM Drug-inducible lentivirus Loh et al., 2010 2010 ; Staerk et al., 2010 2010 Cord blood endothelial cell OSLN Lentivirus Haase et al., 2009 2009 Cord blood stem cell OKSM or OS Retrovirus Eminli et al., 2009 2009 ; Giorgetti et al., 2009 2009 Adipose stem cell OKSM Lentivirus Sun et al., 2009 2009 Adipose stem cell OKS Retrovirus Aoki et al., 2010 2010 Amniotic cell OKSM Retrovirus Li C. et al., 2009 2009 Amniotic cell OSN Lentivirus Zhao et al., 2010 2010 Neural stem cell O Retrovirus Kim J.B. et al., 2009a 2009a Marrow mesenchymal cell OKSM or OK Retrovirus Park et al., 2008 2008 Adipose stem cell OSLN Nonviral minicircle DNA Park et al., 2008 2008 Hepatocyte OKSM Retrovirus Liu et al., 2010 2010 Astrocyte OKSM Retrovirus Ruiz et al., 2010 2010 Keratinocyte OKSM or OKS Retrovirus Aasen et al., 2008 2008 5 Pig Fibroblast OKSM Drug-inducible lentivirus Wu et al., 2009 2009 5 Rabbit Hepatocyte and stomach cell OKSM Lentivirus Honda et al., 2010 2010 5 4 Rat Fibroblast OKS Retrovirus Chang et al., 2010 2010 Fibroblast OKSM Lentivirus Liao et al., 2009 2009 Neural progenitor cell OKS Retrovirus Chang et al., 2010 2010 Liver progenitor cell OKS Retrovirus Li W. et al., 2009 2009 5 Marmoset Fibroblast OKSM Retrovirus Wu et al., 2010 2010 5 Rhesus monkey Fibroblast OKSM Retrovirus Liu et al., 2008 2008 Advantages of peripheral blood over other cell sources for iPS cells 1 The generation of patient-specific iPS cells is a critical step in cell therapy and other clinical applications. As shown in Table 1 1 , human iPS cells are normally derived from dermal fibroblasts because of their accessibility and relatively high reprogramming efficiency. However, skin biopsy and a prolonged period of expansion in cell culture are required prior to using dermal fibroblasts. During skin biopsy, the exposure of the dermis to ultraviolet light might increase the risk for chromosomal aberrations. In addition, it cannot be ignored that patients would experience the pain and the risk of infection when obtaining dermal fibroblasts. These issues limit the wide application of iPS cells. Blood cells are the most easily accessible source of patients’ tissues for reprogramming because it is not need to maintain cell cultures extensively prior to reprogramming experiments. Furthermore, the venipuncture is safer than skin biopsy. And numerous peripheral blood samples have already been frozen and stored in blood banks, so we can directly generate human iPS cells via these samples. The reprogramming of peripheral blood cells started with research on mice in 2008. Hanna et al. ( 2008 2008 ) utilized retroviral-mediated factors ( Oct3/4 , Klf4 , Sox2 , and c-Myc ) to reprogram mouse B lymphocytes. In the experiment, they improved the reprogramming efficiency by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α or knockdown of the B cell transcription factor Pax5. Hong et al. ( 2009 2009 ) generated iPS cells from mouse T lymphocytes by the introduction of Oct3/4 , Sox2 , Klf4 , and c-Myc in a p53-null background. After mouse peripheral blood cells were reprogrammed, Haase et al. ( 2009 2009 ) generated human iPS cells from cord blood (CB). It is an advantage that CB can be obtained from public and commercial CB banks without any risk to donors. Ye Z. et al. ( 2009 2009 ) derived human iPS cells from previously frozen CB and CD34 + cells of healthy adult donors. However, the use of CB is still limited because it can only be obtained from neonates. Loh et al. ( 2010 2010 ), Seki et al. ( 2010 2010 ), and Staerk et al. ( 2010 2010 ) independently derived iPS cells from human peripheral blood cells. Loh et al. ( 2010 2010 ) separated mononuclear cells (PBMCs) and CD34 + cells (PBCD34 + ) from peripheral blood samples, which were then collected through venipuncture and Ficoll density centrifugation. After infection with lentiviruses expressing Klf4 , Sox2 , Oct3/4 , and c-Myc , PBCD34 + cells showed a reprogramming efficiency of 0.002%, whereas PBMCs showed relatively low values of 0.0008% to 0.001%. Staerk et al. ( 2010 2010 ) utilized a doxycycline-inducible lentivirus construct to derive iPS cells from T lymphocytes and myeloid cells which were cultured in IL-7 or G-CSF, GM-CSF, IL-3, and IL-6; this lentivirus construct could encode four reprogramming factors ( Oct3/4 , Sox2 , Klf4 , and c-Myc ) into a polycistronic expression cassette (pHAGE2-TetOminiCMV-hSTEMCCA). Their results showed that the reprogramming efficiency of T lymphocytes was higher than that of myeloid cells. Because T lymphocytes exhibited a higher proliferation rate and had a better long-term growth potential in vitro than myeloid cells, Seki et al. ( 2010 2010 ) induced T lymphocytes into iPS cells by a temperature-sensitive mutant SeV vector encoding human Oct3/4 , Sox2 , Klf4 , and c-Myc with an efficiency of 0.1%. This SeV vector is a non-integrating type, and it could not proliferate at standard culture temperatures. So these characteristics significantly increase the safety for the generation of iPS cells. Chou et al. ( 2011 2011 ) generated iPS cells from newborn CB and adult peripheral blood mononuclear cells with an improved EBNA1/OriP plasmid. By this new reprogramming vector, iPS cells were derived from peripheral blood cells within 14 d, instead of 28 to 30 d as in a previous work on fibroblasts. The research and findings provide the evidence that human iPS cells from peripheral blood cells are comparable to human ES cells in terms of morphology, surface antigens, pluripotency gene expression, DNA methylation, and differentiation potential. iPS cells from mononuclear cells of peripheral blood can be considered reliable and safe. Therefore, methods of generating iPS cells from human peripheral blood cells will accelerate research on and promote clinical applications of iPS cells in the future. Interestingly, human iPS cells can also differentiate into peripheral blood cells because of their pluripotency. Lei et al. ( 2012 2012 ) made human iPS cells differentiate into both conventional and antigen-specific T lymphocytes for T cell-based immunotherapy by utilizing either in vitro or in vivo induction systems. The recently established human iPS cells by Ebihara et al. ( 2012 2012 ) represent potentially unlimited safe sources of donor-free red blood cells for blood transfusion, as they can proliferate indefinitely in vitro without the potential for infectious disease via blood transfusion. Applications of iPS cells 1 Similar to ES cells, mouse iPS cells are able to differentiate into any type of cell, and even have the capability of germline transmission (Maherali et al., 2007 2007 ; Okita et al., 2007 2007 ; Wernig et al., 2007 2007 ; Kang et al., 2009 2009 ; Zhao et al., 2009 2009 ). This means that we can obtain various differentiated cells in large quantities for research and therapy. For example, Zhu et al. ( 2012 2012 ) and Easley et al. ( 2012 2012 ) independently investigated the differentiation potential of human and mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. The derivation of male germ cells from iPS cells represents potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development. Up to now, iPS cells have had main applications in three major areas: human disease modeling (Zhang et al., 2012 2012 ), regenerative medicine, and drug discovery (Fig. 1 1 ). Modified from Stadtfeld and Hochedlinger ( 2010 2010 ) Disease modeling 2 For many human genetic diseases, therapeutic research is hindered by problems regarding the source of experimental materials. iPS cells can overcome these issues by establishing disease-specific models. Disease-specific iPS cells can form cell lineages reflecting the defects caused by a certain disease in patients. Some human diseases for which models have been established by patient-specific iPS cells include amyotrophic lateral sclerosis (Dimos et al., 2008 2008 ), spinal muscular atrophy (Ebert et al., 2009 2009 ), Parkinson’s disease (Soldner et al., 2009 2009 ), β-thalassemia (Ye L. et al., 2009 2009 ), Rett syndrome (Hotta et al., 2009 2009 ), adenosine deaminase deficiency-related severe combined immunodeficiency, Shwachman-Bodian-Diamond syndrome, Gaucher disease, Duchenne muscular dystrophy, Becker muscular dystrophy, Huntington’s disease, type 1 diabetes mellitus, Down syndrome, and Lesch-Nyhan syndrome (carriers) (Park et al., 2008 2008 ). Disease models based on iPS cells can result in both insights into the mechanisms of pathogenesis and the development of new drugs. Patient- and disease-specific therapies represent the most valuable outcome of the whole area of iPS cells. Considering the generation of a wide variety of cell types from iPS cells, these provide the potential to treat disorders of virtually all tissue systems in the body. Regenerative medicine 2 Immunological rejection is a major issue in organ transplantation and cell therapy, because long-term treatment with immunosuppressive drugs would produce serious side effects. Patient-specific iPS cells have the immune markers of the patient, so they solve the problem of immunological rejection. In addition, disease-causing mutations can also be restored by gene targeting in patient-specific iPS cells. Repaired cells can differentiate into targeted cells. After transplanted into the diseased area, they can alleviate disease symptoms. To illustrate this, using a mouse model, Hanna et al. ( 2007 2007 ) proved that iPS cells can be used to cure sickle cell anemia, a genetic blood disorder that renders red blood cells nonfunctional. The disease-causing mutation was repaired in iPS cells generated from the mouse model via gene targeting. The repaired iPS cells then differentiated into blood-forming progenitor cells. These healthy progenitors were transplanted into an anemic mouse where they can proliferate and generate normal red blood cells, thereby curing the disease. In fact, the application of patient-specific iPS cells for tissue replacement and cell therapy indicates what the ultimate goal of regenerative medicine is. At present, however, many limitations still affect the possibility to apply this personalized medicine. The main limitations are related to technical issues, including the development of safe and efficient methods for iPS generation as well as the choice of the most appropriate cell type for reprogramming. Drug discovery 2 Before using novel drugs for treatment, we need to obtain reliable data on their potential toxic effects on humans. In drug discovery, the effects and side effects of novel drugs are usually tested in laboratory animals, such as mice, dogs, and pigs. However, these tests are costly and humans and animals have relatively significant differences. Moreover, animal tests are not effectively standardized. Now we can efficiently test novel drugs on disease models generated from patient-specific iPS cells. This approach will greatly facilitate research on pharmacology and toxicology. Some drugs have already been tested on iPS cells derived from patients suffering from various diseases, such as spinal muscular atrophy (Ebert et al., 2009 2009 ), familial dysautonomia (Lee et al., 2009 2009 ), and LEOPARD syndrome (Carvajal-Vergara et al., 2010 2010 ). The fact that novel drugs alleviate “symptoms” in patient-specific iPS cells demonstrates their therapeutic potential. This method now has been applied to many other diseases and will benefit many patients. Potential issues regarding iPS cells 1 Crucial experiments based on iPS cell technology have shed light on human diseases at the cellular and molecular levels. The application of iPS cells in drug discovery can reduce costs and increase the chances of success. Above all, iPS cells circumvent ethical disputes on ES cells and patient-specific iPS cells may resolve issues of immunological rejection in cell therapy. Currently, in the field of iPS cells, scientists have developed more efficient methods of deriving iPS cells from various cell sources, including those from patients who suffer from different diseases. More progress and new innovations regarding iPS cells will be made in the near future. However, some problems remain to be solved in the clinical application of iPS cells. Many genomic changes, including chromosomal aneuploidy, translocations, point mutations, megabase-scale duplications and/or deletions, and so on, have been observed in human iPS cells; these problems may limit the therapeutic potential of iPS cells. As suggested recently by Gore et al. ( 2011 2011 ), in the exome sequencing of 22 human iPS cell lines obtained from seven laboratories by five different methods, they found 124 point mutations in the iPS cell lines, but no mutations in the parental cells. This study identified many missense mutations related to the protein function modification and point mutations in genes implicated in cancers. Although the reprogramming process itself might cause genomic anomalies, not all anomalies result from it, because genomic alterations have been identified in human iPS cells produced through different techniques, including non-integrating methods such as those that use synthetic mRNAs (Warren et al., 2010 2010 ). In spite of the fact that the low efficiency of reprogramming is one of the hurdles that prevents the area from moving forward, compared with improving reprogramming efficiency, solving problems in chromosomal anomalies in iPS cells is more important. Thus, in the future, obtaining iPS cells with the fewest genomic alterations should be the focus in the field of iPS cells. The causes of chromosomal abnormalities during reprogramming somatic cells need to be investigated as well. In addition, some other questions remain: What is the mechanism for iPS cell induction? What are the optimal reprogramming factors? How do we reduce risks of insertion mutagenesis in the genome of iPS cells? How do we achieve directed differentiation? How do we evaluate the safeness of iPS cells in clinical applications? Obtaining the answers to these questions requires thorough analyses of the induction process and the epigenetics of iPS cells. Furthermore, a reliable evaluation system on clinical trials needs to be established. The clarification of these questions might improve the application of iPS technology as well as the length and quality of life for people in the future. Future perspectives 1 The generation of iPS cells is regarded as a milestone for life science (Fig. 2 2 ). In spite of the problems mentioned in the last section, the advantages of iPS cells cannot be ignored. iPS cells can avoid ethical disputes as well as immunological rejection in cell therapy. Moreover, disease models generated from iPS cells can be used to study the mechanisms of human genetic disorders and test the effects of novel drugs (Fig. 1 1 ). iPS cell biology has admittedly become a new field within stem cell research that covers various important and attractive scientific areas. Modified from Yamanaka ( 2012 2012 ) The whole area of iPS cells is a hot topic in biomedical research and is rapidly approaching its clinical utilization. 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Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study Compliance with ethics guidelines Jing ZHAO, Wen-jie JIANG, Chen SUN, Cong-zhe HOU, Xiao-mei YANG, and Jian-gang GAO declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300215 Project supported by the National Natural Science Foundation of China (Nos. 30871436, 30973297, 31171194, and 31271534), the National Basic Research Program (973) of China (Nos. 2010CB945002 and 2014CB541703), the Shandong Provincial Science and Technology Key Program (No. 2009GG10003039), and the Independent Development Foundation of Shandong University (Nos. 2012JC019 and 2012ZD030), China 2013-08-14 2013-11-21 2013-11-21 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 14