PerSPecTIveS S t e m C e l l S — e S S ay
The promise of human induced pluripotent stem cells for research and therapy Shin-ichi Nishikawa, Robert A. Goldstein and Concepcion R. Nierras
Abstract | Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer. Induced pluripotent stem (iPS) cells are the product of somatic cell reprogramming to an embryonic-like state. This occurs by the introduction of a defined and limited set of transcription factors and by culturing these cells under embryonic stem (ES)-cell conditions1 (FIG. 1). The method was first described by Shinya Yamanaka and colleagues using mouse fibroblasts, in which it was demonstrated that the retroviral-mediated introduction of four transcription factors — octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group (HMG)-box protein-2 (SOX2), MYC and Kruppel-like factor-4 (KLF4) — could induce pluripotency. The protocol has since been applied to other types of mouse cells2,3 and to human somatic cells4–7. Although the original gene set for reprogramming1 comprised Oct3/4, Sox2, Myc and Klf4, recent studies5,8 have shown that other combinations of factors can substitute for MYC and KLF4 and produce iPS cells. The reprogramming procedure is straightforward9, robust and has been independently replicated by multiple groups. However, the detailed mechanism for reprogramming is unknown and is a topic of intense scrutiny in several laboratories. iPS-cell technology is a novel method for generating pluripotent stem cells10–12. The method is striking in that it can convert NATuRE REvIEwS | molecular cell biology
somatic cells directly into pluripotent cells, in a manner that is totally independent of the availability of embryonic cells. This is in contrast to other methods for generating pluripotent stem cells, such as the derivation of ES cells from the inner cell mass of the pre-implantation embryo at the blastocyst stage13, the fusion of somatic cells with pre-existing ES cells14,15 and derivation of ES cells from embryos generated by nuclear transfer16 (TABLE 1). Opponents of stem-cell research have welcomed iPS-cell technology as a method for achieving an embryonic-like state without the ethical dilemma of destroying human embryos. Therefore, iPS-cell technology is especially attractive for researchers in countries in which the use of embryonic cells is restricted. Both iPS cells and ES cells can be used as the pluripotent starting material for differentiated cells or tissues in regenerative medicine17,18. As sources of pluripotent cells, iPS cells are inevitably compared with ES cells, but the pressing scientific question to ask is: how closely do iPS cells resemble conventional ES cells? Mouse iPS cells have been shown to be functionally equivalent to mouse ES cells3,19,20, as they express mouse ES-cell markers, have similar geneexpression profiles, form teratomas when
injected into nude hosts and contribute to the cell types of chimeric animals, including the germ line. Mouse iPS cells have been differentiated into haematopoietic precursor cells and have been shown to rescue lethally irradiated mice21. In addition, iPS cells were successfully derived from a mouse model of sickle cell anaemia and the defective gene was replaced by homologous recombination. The treated iPS cells were then differentiated into haematopoietic precursors, and these cells were used successfully to treat the sickle cell mouse. The systematic characterization of human ES cell lines is ongoing22. Nonetheless, comparative gene-expression profiles of human ES cells and human iPS cells are now starting to emerge5,7. Although geneexpression profiles are closely correlated, there are some differences between human ES cells and iPS cells, as well as differences among different iPS-cell clones7 and among human ES cell lines. The detailed implications or consequences of these differences are unknown. In this article, we discuss the promise of iPS cells and the challenges of using these cells for therapy. Along with others, we strongly advocate continued support of research using both ES and iPS cells23,24. The use of both of these cell types allows the comparison of developmental processes, with the promise of accelerating our understanding of human development. Comparative analyses might also provide useful information as cell therapies are developed using human ES cell and iPS cell sources. iPS cells for patient-specific therapy As mentioned above, iPS-cell technology has been successfully applied to human somatic cells, but the efficiency of derivation remains low4,5,25. Human iPS cells might be an ideal cell source for cell therapy10, given that iPS cells can be derived from the patient to be treated and thus are genetically identical cells that would avoid immune rejection. However, human iPS cells have not yet been directed to differentiate into a specific functional tissue or organ, although gene-expression profiles of in vitro differentiated human iPS cells vOLuME 9 | SEPTEMBER 2008 | 725
PersPectives
Di
?
ffe re nt
iat io
n
ES cell
Reprogramming Differentiation Somatic cell
iPS cell
Mouse gene combinations for iPS induction Oct3/4, Sox2, c-Myc, Klf4
Oct3/4, Sox2, Klf4
Oct3/4, Sox2, N-Myc, Klf4
Oct3/4, Sox2, Lin28, Nanog
Figure 1 | reprogramming between differentiNature Reviews | Molecular Biology ated and default epigenetic states.Cell The pluripotent cells of the early embryo give rise to all types of somatic cells, which are characterized by distinct epigenetic states. Differentiated somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by the introduction of a defined and limited set of transcription factors. Four combinations have been used successfully: octamer-binding transcription factor-3/4 (Oct3/4), SrY-related high-mobility-group (HMG)-box protein-2 (Sox2), c-Myc and Kruppel-like factor-4 (Klf4) (REF. 1); Oct3/4, Sox2, N-Myc and Klf4 (REF. 8); Oct3/4, Sox2 and Klf4 (REF. 25); and Oct3/4, Sox2, Lin28 and Nanog5. The epigenetic state of iPS cells is similar (but might not be identical) to that of embryonic stem (eS) cells7; the relationship between eS cells and iPS cells is unclear, as indicated by the question mark. The detailed implications or consequences of these differences are not yet known. iPS cells can potentially be directed back to differentiated cells, although this has not yet been demonstrated.
have shown that they express different lineage markers7. Furthermore, there are numerous challenges that remain before iPS cells might be considered for patient-specific therapy. The technical challenges that we need to overcome to be able to use iPS cells for therapy include: the use of retroviral vectors to introduce reprogramming factors into cells; the need to use a selection marker (either inserted into the starting cell by homologous recombination or included as part of the vector) to identify reprogrammed cells; the use of the oncogene MYC to achieve reprogramming; and the integration of retroviral vectors into the genome. These manipulations genetically modify the starting cells, and genetically 726 | SEPTEMBER 2008 | vOLuME 9
modified cells face significant regulatory hurdles for therapeutic applications. Researchers have begun to address these challenges. Two groups have developed morphological criteria for selecting mouse iPS cells without the use of a selection marker8,21. For human iPS cells, geneexpression patterns are used as surrogate markers for pluripotency7. Several groups have now reported that MYC is not essential for producing human iPS cells5,25. As a result of this and other research, we believe that it is reasonable to expect that these technical challenges will be solved in the near future. The most desirable scenario would be to achieve high-efficiency reprogramming using only transient expression of the relevant factors and without using any oncogenes. Human ES-cell lines have shown significant differences in differentiation potential for specific lineages26. Acknowledgement of these lineage-specific differences is a reason for the establishment of stem-cell banks22. By contrast, iPS cells might offer the tantalizing possibility that their differentiation can be completely directed to any developmental lineage. However this concept will have to be proven experimentally. Given the promise of patient-specific therapy, it might be prudent to consider the establishment of cell banks of iPS cells. These repositories would increase the number of patients who might be helped by iPS-cell-derived therapies. It will be necessary to characterize the developmental potential of human iPS cell lines, and to compare the developmental potential of human ES and iPS cells. Human ES cells that have been grown in long-term culture have shown genetic instability22, but the stability of iPS cells is yet to be investigated. In particular, the identical genetic and epigenetic profile of the iPS-derived cell type and the patient will need to be confirmed as cell production is scaled up. After the expansion and differentiation of iPS cells has been achieved, the resulting cells or tissues can be evaluated using the same regulatory criteria as those that have been developed from ES cells27. These criteria include extensive documentation requirements to ensure, for instance, that no contamination by animal products has occurred during derivation and culture, and to prevent the introduction of potentially carcinogenic undifferentiated cells. Safety will be paramount for Phase I studies, and it will be necessary to show that differentiated cells that have developed from iPS cells will not develop into teratomas.
Regulators, companies and scientists could use the knowledge gained from tissues developed from human ES cells in clinical trials for regeneration as a near-term paradigm for ES-cell-based treatments28. A major consideration for ensuring the therapeutic effectiveness of cell therapy, regardless of the cell source, will be the environment in which the cells are placed. Cell therapy might not be effective in the context of an autoimmune disease such as type 1 diabetes (which would involve the use of replacement pancreatic β-cells), because the transplanted cells may be destroyed by the host immune response. Therefore, additional treatments (such as immunosuppressive treatments) might be required. Even a ‘simple’ scenario, such as providing replacement nerve cells in spinal cord injury or providing replacement cardiomyocytes in heart disease, must account for either an inflammatory (in the injured spinal cord) or fibrotic (in the diseased heart) cellular milieu. The development and implementation of iPS-cell-based or ES-cell-based therapies will be far more complex than simple differentiation to replacement cells. Regulatory requirements will elongate the research timeline and increase the cost of such therapies. Patient-specific therapy is therefore unlikely to be widespread in the near future. The high costs for this therapy might mean it will never be applicable to large numbers of people. iPS cells as a research tool iPS cells demonstrate that it is possible to reprogramme the epigenetic state of somatic cells to a state that is comparable to that of ES cells. This state has, in theory, full developmental potential. In contrast to ES cells, however, iPS-cell technology also provides the opportunity to reiterate or to redirect the developmental process: that is, fully differentiated somatic cells can be de-differentiated to iPS cells and then redifferentiated to their original state, or even redirected to another differentiated state. Epigenetic changes in the DNA methylation state and chromatin organization are thought to maintain the differentiated state. Human ES cell lines have shown variation in their epigenetic status22, and this might explain differences in their differentiation potential in vitro. Although the exact epigenetic state generated by the iPS cell procedure might be variable, it is thought to be comparable to ES cells. Previous studies have shown that ES cells can self-renew and maintain the expression of OCT3/4, www.nature.com/reviews/molcellbio
PersPectives Table 1 | Comparison of pluripotent stem cells derived by different methods iPS cells
eS cells
eS cells from nuclear transfer
Stem cells from fusion of somatic cells with eS
reported in humans
Yes
Yes
No
Yes
embryos or donor oocytes required
No
Yes
Yes
existing eS cell required
Stemness markers* expressed
Yes
Yes
Yes
Yes
Teratomas produced
Yes
Yes
Yes
Yes
utility as a research tool
Allows repeated development
Allows the study of development
cell line with predetermined characteristics
Allows the study of development
can be used as models for human diseases
Yes
Some
Yes
Some
can be used in a screen to identify drugs
Yes
Yes
Likely
Yes
Variable fates
Likely
Yes
Unknown
Unknown
Develop into specific human To be shown tissues
Yes
Not shown in humans
No
genetically match the patient
Unknown
No
Unknown
No
additional information
cells are genetically modified in current methods
cells are allogeneic and might cause immune rejection
Human oocytes are in limited supply
Tetraploid
references
4–7
12
16
14,15
* ‘Stemness markers’ refers to genes that are consistently expressed in human embryonic stem-cell lines (see REFS 7, 22). eS cell, embryonic stem cell; iPS cell, induced pluripotent stem cell.
SOX2 and NANOG in the absence of DNA methyltransferase-1 (DNMT1), which is essential for the maintenance of the DNA methylation pattern29, or in the absence of polycomb repressive complex-2 (PRC2), which is involved in the maintenance of suppressive chromatin organization through the methylation of histone H3 (REF. 30). Hence, the maintenance of a distinct epigenetic state is apparently not necessary for the two important features of ES cells — pluripotency and self-renewal. whether or not the maintenance of iPS cells requires specific epigenetic mechanisms is an important question that is still being tested. we predict that the epigenetic status of an iPS cell can be attained from any somatic cell, and that the epigenetic differentiated state of any somatic cell can be acquired from the iPS-cell state. This epigenetic reprogramming provides an important basis for the promise of iPS-cell technology, as it implies the possibility of converting a given somatic cell lineage to other lineages through an iPS cell (FIG. 1). Such reprogramming was not possible in human cells before iPS-cell technology. The potential to make any somatic cell from another somatic cell creates exciting research questions and clinical applications. NATuRE REvIEwS | molecular cell biology
iPS cells can provide models of human disease. The first use of iPS-cell technology might be the production of cell lines from patients with various diseases31 (FIG. 2). For genetic diseases, iPS cells provide a new opportunity to analyse the pathways that lead to disease pathogenesis based on a particular genetic trait at the cellular level. A good example would be Huntington’s disease32, a neurodegenerative disease that is caused by CAG repeats in the gene that encodes huntingtin protein. The process that leads to cell death by expression of polyglutamine-containing molecules remains unclear, despite the presence of various cellular and animal models. Part of the difficulty is the absence of an appropriate human experimental system. If iPS cells from patients could be induced to develop into the relevant types of neuron, it would be the optimal study material for investigating the process of neural degeneration induced by these extra-long CAG repeats. The only other way to produce such cells would be by somatic cell nuclear transfer, a procedure that has not been successful in human cells. Neural cells from patients with Huntington’s disease would be important tools for searching for drugs that reduce the toxicity of polyglutamine.
iPS cells might prove to be even more useful in understanding the development of complex genetic disorders, such as type 1 diabetes and Parkinson’s disease. In these diseases, it is difficult to analyse the pathogenic process in a prospective manner, as our current genetic knowledge is insufficient to predict who will develop the disease. However, if iPS cells could be derived from the somatic cells of such patients, and if immune cells or dopamineproducing neurons, respectively, could then be induced, it would represent a new way to prospectively investigate pathogenesis. iPS cells can be used to examine the effects of known genotypes on the cellular phenotype. An important possible application comes from the fact that iPS cells can be generated from any human who is taking a medicine. Thus, any effect or lack of effect of a particular drug that is detected during clinical treatment can be re-analysed using iPS cells from patients. This would greatly facilitate studies such as those to monitor drug safety once drugs are on the market. iPS cells can model normal development. iPS cells could be used to model normal development in humans, which could help vOLuME 9 | SEPTEMBER 2008 | 727
PersPectives
Maturation and disease development
Genetic analysis Statistics, Epidemiology, Animal model studies
Cell therapy Normal embryogenesis Mechanism of disease Effect of drugs Teratology
Differentiation of various cell lineages from iPS cells
Generation of patient-specific iPS cells
Figure 2 | iPS cells as models for human disease. For manyNature non-infectious complex diseases, it is Reviews | Molecular Cell Biology difficult to predict who will develop a particular disease. Strategies to understand disease begin with the patient and include genetic analyses, epidemiology, statistics and studies in animal models. Induced pluripotent stem (iPS)-cell technology expands the opportunities for research. Patient-derived iPS cells can be used to examine the disease process at a cellular level. iPS cells can be used to test responses to possible drugs and might be used to develop patient-specific cell therapy.
to understand disease pathogenesis and to develop suitable treatments in some cases. In type 1 diabetes, where it is necessary to replace β-cells that are destroyed by disease, knowledge of the steps that are necessary to produce progenitor, precursor and functional β-cells would be crucial for developing replacement therapy. It might also be possible to analyse what happens when a differentiated somatic cell type, such as a nerve cell, is reprogrammed by iPS-cell technology. Proof of concept for this application of iPS-cell technology is provided in a recent report that shows that PAX5, a key molecule for maintaining the terminally differentiated B lymphocyte state, inhibits the induction of iPS cells in mice33. Not only would this concept provide information about developmental pathways, but it might also lead to a better understanding of the mechanisms that are used in tissue repair or regeneration. A long-term dividend of such knowledge would be the ability to repair or regenerate tissues in situ. It has often been difficult to investigate developmental failure. iPS cells from patients with birth defects provide a powerful tool for analysing the effect of various teratogens or of specific genetic traits on cellular processes during embryogenesis. Moreover, in vitro differentiation culture of iPS cells will be a useful tool for screening potential teratogens. 728 | SEPTEMBER 2008 | vOLuME 9
iPS cells for cancer research. Another area that might be ripe for iPS-cell technology is cancer research. By illuminating the genetic and epigenetic changes that occur during normal development, human ES cells have provided opportunities to study the oncogenic process in a prospective manner. iPS-cell technology further expands what can be done: iPS cells can be derived from cancerous somatic cells to investigate the significance of the genetic background of the patient in the development of cancer. These data would contribute to the existing large datasets of genetic profiles of various cancers34, and could also be used in comparative analyses. Cancer cells themselves would also be a useful starting material from which to derive iPS cells. Previous attempts to reprogramme cancer cells by nuclear transfer into unfertilized eggs showed that the generation of ES cells from a nuclear transfer embryo is more difficult using cancer cell nuclei35 than normal somatic cell nuclei. Because of technical problems with nuclear transfer, the molecular basis underlying this difficulty remains uninvestigated. we now know that cancer cells inhibit iPS-cell induction, whereas normal cells do not. Here again the underlying mechanism is unknown. Given that cancer development involves the accumulation of mutations, we could evaluate which genetic alterations in cancer cells inhibit iPS-cell induction.
iPS-cell technology has the potential to lead to new definitions of the cancer state, and it might be interesting to classify cancers according to their ability to generate iPS cells. It is widely recognized that, besides genetic alterations, changes to the epigenetic status also have a role in the development of cancer36. If a given cancer can be reprogrammed to an induced pluripotent state, then the epigenetic alterations responsible for the cancer phenotype would be erased during the reprogramming process without altering the genetic modifications of the cancer. Cancer-derived iPS cells could then be induced to differentiate back to the cell lineage from which the original cancer arose. By repeating the processes of induction and re-differentiation, we might be able to generate distinct epigenetic states and identify the specific alterations that occur during cancer formation. An assessment of the epigenetic effect on oncogenesis was addressed previously using nuclear transfer ES cells. The results showed that melanocytes that are differentiated from melanoma-derived nuclear transfer ES cells are not yet melanoma cells37, which demonstrates the requirement for epigenetic changes in oncogenesis. iPS cells stimulate other technologies Currently, iPS cells are generated by the transduction of a set of genes using retroviral vectors. Thus, a crucial question for the use of iPS cells as a source of cell therapy is whether it is possible to initiate epigenetic reprogramming without viral vectors or even without genetic integration events, as the integration of foreign genes could potentially initiate oncogenic processes. Efforts are in progress to develop alternative methods that avoid genetic manipulation (for example, by using peptides or chemical compounds) and deliver the required genes or proteins into the cell. High-throughput screens to identify chemical compounds that could substitute for gene transduction to achieve reprogramming are being undertaken by many laboratories. The more we understand the process of reprogramming to an iPS-cell state, the larger the potential range of methodologies. Methods for the genome-wide analysis of the epigenetic state of cells as they undergo reprogramming are also important for iPS-cell technology. Progress in this field continues at a rapid pace, from ChIPon-chip38 to ChIP-to-sequence technologies39 with high-throughput sequencers. www.nature.com/reviews/molcellbio
PersPectives we believe that the use of iPS-cell technology will influence continued developments in this field, for example, by pushing for improved analyses using fewer cells. For cell therapy, it should be noted that teratoma formation is an inherent feature of ES cells and iPS cells. As this tumorigenic activity is lost following differentiation, however, differentiated cells, including those derived from ES cells, are expected to be safe for therapeutic use, provided that undifferentiated cells are removed. Methods to detect and remove undifferentiated cells are technologies that will be required for using ES cells and iPS cells for cell therapy. The dissemination of iPS-cell technology is likely to encourage the development of in vivo models in animals, in order to most efficiently take advantage of the new scientific opportunities. For instance, given that iPS cells can be induced to develop into any cell type, it is desirable to have experimental systems that allow us to investigate the behaviour of these differentiated cells in vivo. Currently, immunodeficient mice are the most popular tools for this purpose. However, given the differences between mice and humans, humanized animals in which some of the key signalling molecules are replaced by corresponding human counterparts should be an important tool that would expand the potential of iPS-cell technology40. Animals with a humanized environment for tumour growth are also essential for validating the tumorigenicity of iPS-cell-derived populations for cell therapy. Concluding remarks iPS-cell technology brings a new appreciation for the possibility of reprogramming differentiated cells. using iPS cells, we might demonstrate that all somatic cells are interconnected by the reprogramming route, with the iPS cell as the functional equivalent of the ES cell, or its effective mimic. Any cell could then be converted to any other cell. It is still premature to predict what future new worlds will become available with iPS-cell technology, because of our limited knowledge of the molecular process that occurs during reprogramming. The most urgent issue for any study using iPS cells is basic research on the molecular basis that underlies this technique. These studies would benefit enormously from effective and continuing comparisons with ES cells. This research will help bring to reality what today seems to be an extremely promising future. NATuRE REvIEwS | molecular cell biology
Note added in proof Two groups recently reported the first iPS cell lines derived from patients with various genetic diseases (with either Mendelian or complex inheritance) and the generation of iPS-cell-derived differentiated cells41,42. Another report showed that two of the original factors identified by the Yamanaka group (OCT3/4 and KLF4 or MYC) are sufficient for generating iPS cells from adult neural stem cells43. Shin-ichi Nishikawa is at the Laboratory for Stem Cell Biology, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. Robert A. Goldstein and Concepcion R. Nierras are at the Juvenile Diabetes Research Foundation, 120 Wall Street, 19th floor, New York, New York 10005, USA. e-mails:
[email protected];
[email protected];
[email protected] doi:10.1038/nrm2466 Published online 13 August 2008 1.
2. 3. 4. 5. 6. 7.
8. 9.
10. 11. 12. 13. 14.
15. 16. 17.
18.
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). Aoi, T. et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 14 Feb 2008 (doi:10.1126/science.1154884) Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007). Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007). Yu, J. et al. Induced pluripotent stem cells from adult human somatic cells. Science 318, 1917–1920 (2007). Park, I.-H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–147 (2008). Lowry, W. E. et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl Acad. Sci. USA 105, 2883–2888 (2008). Blelloch, R. et al. Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell 1, 245–247 (2007). Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nature Protoc. 2, 3081–3089 (2007). Yamanaka, S. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1, 39–49 (2007). Cibelli, J. Development: is therapeutic cloning dead? Science 318, 1879–1880 (2007). Jaenisch, R. & Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008). Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998). Cowan, C. A., Atienza, J., Melton, D. A. & Eggan, K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373 (2005). Do, J. T., Han, D. W. & Scholer, H. R. Reprogramming somatic gene activity by fusion with pluripotent cells. Stem Cell Rev. 2, 257–264 (2006). Hochedlinger, K. & Jaenisch, R. Nuclear reprogramming and pluripotency. Nature 441, 1061–1067 (2006). Murry, C. E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008). Lerou, P. H. & Daley, G. Q. Therapeutic potential of embryonic stem cells. Blood Rev. 19, 321–331 (2005).
19. Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007). 20. Maherali, N. et al. Directly reprogrammed fibroblasts show epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55–70 (2007). 21. Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920–1923 (2007). 22. The International Stem Cell Initiative. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature Biotech. 25, 803–816 (2007). 23. Gottweis, H. & Minger, S. iPS cells and the politics of promise. Nature Biotech. 26, 271–272 (2008). 24. Cyranoski, D. Five things to know before jumping on the iPS bandwagon. Nature 452, 406–408 (2008). 25. Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotech. 26, 101–106 (2008). 26. Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nature Biotech. 26, 313–315 (2008). 27. US Department of Health and Human Services. Guidance for human somatic cell therapy and gene therapy. Food and Drug Administration [online], http://www.fda.gov/Cber/gdlns/somgene.pdf (1998). 28. Segers, V. F. M. & Lee, R. T. Stem cell therapy for cardiac disease. Nature 451, 937–942 (2008). 29. Rajasekhar, V. K. & Begemann, M. Concise review: roles of polycomb group proteins in development and disease: a stem cell perspective. Stem Cells 25, 2498–2510 (2007). 30. Spivakov, M. & Fisher, A. G. Epigenetic signatures of stem-cell identity. Nature Rev. Genetics 8, 263–271 (2007). 31. Holden, C. & Vogel, G. A seismic shift for stem cell research. Science 319, 560–563 (2008). 32. Walker, F. O. Huntington’s disease. Lancet 369, 218–228 (2007). 33. Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008). 34. National Cancer Institute. Cancer biomedical informatics grid. National Cancer Institute [online], https://cabig.nci.nih.gov/ (2008). 35. Blelloch, R. H. et al. Nuclear cloning of embryonal carcinoma cells. Proc. Natl Acad. Sci. USA 101, 13985–13990 (2004). 36. Esteller, M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nature Rev. Genetics 8, 286–298 (2007). 37. Hochedlinger, K. et al. Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev. 18, 1875–1885 (2004). 38. Huebert, D. J. Kamal, M., O’Donovan, A. & Bernstein, B. E. Genome-wide analysis of histone modifications by ChIP-on-chip. Methods 40, 365–369 (2006). 39. Robertson, G. et al. Genome-wide profiling of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature Methods 4, 651–657 (2007). 40. Shultz, L. D., Ishikawa, F. & Greiner, D. L. Humanized mice in translational biomedical research. Nature Rev. Immunol. 7, 118–130 (2007). 41. Dimos, J. T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 31 July 2008 (doi:10.1126/science.1158799) 42. Park, I.-H. et al. Disease-specific induced pluripotent stem cells. Cell (in the press). 43. Kim, J. B. et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454, 646–650 (2008).
Acknowledgements
The authors thank A. L. Wong and A. J. Hwa for helpful comments.
DataBaSeS UniProtKB: http://www.uniprot.org DNMT1 | KLF4 | MYC | NANOG | OCT3/4 | PAX5 | SOX2
FURtHeR INFORmatION shin-ichi Nishikawa’s homepage: http://www.riken.jp/ engn/r-world/research/lab/cdb/kann/index.html all linkS are acTiVe in The online PDf
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