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Inducing iPSCs to Escape the Dish

2011; Elsevier BV; Volume: 9; Issue: 2 Linguagem: Inglês

10.1016/j.stem.2011.07.006

1934-5909

Bonnie L. Barrilleaux, Paul S. Knoepfler,

Tissue Engineering and Regenerative Medicine

Induced pluripotent stem cells (iPSCs) hold great promise for autologous cell therapies, but significant roadblocks remain to translating iPSCs to the bedside. For example, concerns about the presumed autologous transplantation potential of iPSCs have been raised by a recent paper demonstrating that iPSC-derived teratomas were rejected by syngeneic hosts. Additionally, the reprogramming process can alter genomic and epigenomic states, so a key goal at this point is to determine the clinical relevance of these changes and minimize those that prove to be deleterious. Finally, thus far few studies have examined the efficacy and tumorigenicity of iPSCs in clinically relevant transplantation scenarios, an essential requirement for the FDA. We discuss potential solutions to these hurdles to provide a roadmap for iPSCs to "jump the dish" and become useful therapies. Induced pluripotent stem cells (iPSCs) hold great promise for autologous cell therapies, but significant roadblocks remain to translating iPSCs to the bedside. For example, concerns about the presumed autologous transplantation potential of iPSCs have been raised by a recent paper demonstrating that iPSC-derived teratomas were rejected by syngeneic hosts. Additionally, the reprogramming process can alter genomic and epigenomic states, so a key goal at this point is to determine the clinical relevance of these changes and minimize those that prove to be deleterious. Finally, thus far few studies have examined the efficacy and tumorigenicity of iPSCs in clinically relevant transplantation scenarios, an essential requirement for the FDA. We discuss potential solutions to these hurdles to provide a roadmap for iPSCs to "jump the dish" and become useful therapies. The goal of stem cell-based regenerative medicine is to treat disease states using cells, including the differentiated progeny of pluripotent stem cells (PSCs), as the therapeutic modality. In this way, regenerative medicine has the potential to transform conventional medicine, which has been dominated by surgery and drugs for centuries. The pluripotent nature of human embryonic stem cells (hESCs), which allows their potential use to repair almost any tissue, is only beginning to be harnessed for human therapies. Goldring et al., 2011Goldring C.E.P. Duffy P.A. Benvenisty N. Andrews P.W. Ben-David U. Eakins R. French N. Hanley N.A. Kelly L. Kitteringham N.R. et al.Assessing the safety of stem cell therapeutics.Cell Stem Cell. 2011; 8: 618-628Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar have recently reviewed safety issues pertaining to a range of promising stem cell-based therapeutics, including three clinical trials using ESCs to repair nerve cells and retinal pigment cells, which are not amenable to replacement by adult stem cells. However, three key issues have slowed the potential clinical use of hESCs: ethical issues, because a human blastocyst must be used to create the lines; immunological issues, because hESCs would be used for allotransplants; and safety issues, because hESCs can form teratomas and sometimes other, more malignant tumors. When human induced pluripotent stem cells (hiPSCs) were first reported (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14964) Google Scholar), part of the tremendous excitement surrounding them was their high level of similarity to hESCs, but at the same time, iPSCs had key potential advantages over hESCs. They seemed poised to avoid two out of the three central challenges facing the clinical use of hESCs: ethical and immune rejection issues. By using iPSCs for potential future regenerative medicine therapies, patients could, at least in theory, be given autologous transplants of iPSC-derived cells without using a human blastocyst and without immunosuppressive therapy. Not surprisingly, in the almost 5 years since the initial publication on murine iPSCs (miPSCs) (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar), as we have learned a great deal more about iPSCs, clinical expectations have become more realistic. While iPSCs are undoubtedly remarkably similar to hESCs, some laboratories report a number of differences that cast doubt upon the complete equivalence of the two cell types. In addition, iPSCs have their own unique issues that present different kinds of roadblocks to their future use in regenerative medicine therapies. These include the use of oncogenes for reprogramming and the time required to produce and characterize a new iPSC line, which may render autologous hiPSCs inherently unsuitable to treat acute conditions such as myocardial infarction and spinal cord injury. Even the immune tolerance of autologous iPSCs has recently been called into question (Zhao et al., 2011Zhao T. Zhang Z.-N. Rong Z. Xu Y. Immunogenicity of induced pluripotent stem cells.Nature. 2011; 474: 212-215Crossref PubMed Scopus (1116) Google Scholar). At the same time, the tremendous potential of iPSCs for disease modeling has generated a great deal of excitement about iPSC-based "disease models in a dish" (Saha and Jaenisch, 2009Saha K. Jaenisch R. Technical challenges in using human induced pluripotent stem cells to model disease.Cell Stem Cell. 2009; 5: 584-595Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). The crucial question facing the iPSC field at this time is whether iPSCs can escape the confines of the dish and go beyond disease modeling to get to the clinic to more directly help patients, as was originally hoped. Here we outline the main hurdles facing translation of iPSCs to the bedside and discuss the most promising solutions. One of the most exciting aspects of the development of iPSCs was their potential use for patient-specific autologous transplants. While this remains an important potential attribute of iPSCs and their derivatives, enthusiasm was tempered a bit recently by the report of Zhao et al., 2011Zhao T. Zhang Z.-N. Rong Z. Xu Y. Immunogenicity of induced pluripotent stem cells.Nature. 2011; 474: 212-215Crossref PubMed Scopus (1116) Google Scholar who found that while murine ESC (mESC)-derived teratomas were accepted by syngeneic recipients, teratomas derived from miPSCs were rejected with massive CD4+ T cell infiltration. What might be the cause of this rejection in what should be a syngeneic context? It was not a result of MYC-based reprogramming or transgene integration, as miPSCs generated without MYC and with nonintegrating episomal vectors also encountered a significant immunologic response. Rather, the immunogenicity was apparently caused by overexpression of a few specific genes in miPSC-derived teratomas, suggesting that subtle epigenetic changes could have important therapeutic consequences. However, for many reasons the jury is still out on the immunity issue. We would argue that the focus of the Zhao study only on teratomas might very well have greatly overestimated the likelihood of autologous iPSCs to elicit an immune response. Because some tumors can be highly immunogenic, the teratoma context may confer an enhanced immunogenicity upon iPSC derivatives that does not manifest in iPSC-derived normal tissues. At least one of the overexpressed genes, HORMAD1, is expressed in developing germ cells and has been characterized as a tumor-specific antigen (Chen et al., 2005Chen Y.-T. Venditti C.A. Theiler G. Stevenson B.J. Iseli C. Gure A.O. Jongeneel C.V. Old L.J. Simpson A.J.G. Identification of CT46/HORMAD1, an immunogenic cancer/testis antigen encoding a putative meiosis-related protein.Cancer Immun. 2005; 5: 9PubMed Google Scholar). Its expression could therefore be a result of germ cell differentiation within the teratoma, or a result of the tumor formation process itself, rather than an inherent characteristic of the iPSC lines studied. Teratoma assays require injecting large numbers of undifferentiated cells, which is very different from the way the cells will be used clinically. Indeed, there are hints that iPSCs that have been predifferentiated in vitro do not share the immune-activating properties of teratomas. A study from the Jaenisch group in which iPSCs were used successfully to treat sickle cell anemia without immune rejection seems to suggest that in some circumstances, iPSC derivatives are not immunogenic (Hanna et al., 2007Hanna J. Wernig M. Markoulaki S. Sun C.-W. Meissner A. Cassady J.P. Beard C. Brambrink T. Wu L.-C. Townes T.M. Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.Science. 2007; 318: 1920-1923Crossref PubMed Scopus (1234) Google Scholar). However, in this study the recipient mice were subjected to both radiation and immunosuppression, making it more difficult to draw conclusions. iPSC immunogenicity is a new, critical open question, but one that can be readily addressed by transplantation of normal cells or tissues derived from miPSCs into nonimmunodeficient, nonimmunosuppressed mice. Because the Zhao study was only conducted in mice, another important open question is whether similar findings would be observed in a human context with hiPSCs. We predict that different iPSC lines will exhibit a range of immunotolerance in autologous hosts, so it may be fruitful to generate a panel of hiPSC lines from each patient and test them for autologous T cell reactivity in vitro. One potential way to begin addressing the immune tolerance of hiPSCs and their derivatives in vivo would be to study transplantation into mice with humanized immune systems capable of rejecting human allografts. Human peripheral blood mononuclear cells (PBMCs) can be used to reconstitute the immune system of immunodeficient mice, resulting in effective rejection of allogeneic human pancreatic islets (Vlad et al., 2008Vlad G. D'Agati V.D. Zhang Q.Y. Liu Z. Ho E.K. Mohanakumar T. Hardy M.A. Cortesini R. Suciu-Foca N. Immunoglobulin-like transcript 3-Fc suppresses T-cell responses to allogeneic human islet transplants in hu-NOD/SCID mice.Diabetes. 2008; 57: 1878-1886Crossref PubMed Scopus (45) Google Scholar) and skin grafts (Issa et al., 2010Issa F. Hester J. Goto R. Nadig S.N. Goodacre T.E. Wood K. Ex vivo-expanded human regulatory T cells prevent the rejection of skin allografts in a humanized mouse model.Transplantation. 2010; 90: 1321-1327Crossref PubMed Scopus (154) Google Scholar). A similar experiment could be performed using hiPSCs autologous to the PBMCs in order to detect rejection of immune-matched iPSC grafts. It is currently unclear whether the immune capacity of these chimeric mice is sufficiently complex to mediate rejection of autologous iPSC derivatives that may differ only slightly from native human tissue, but if so, the results would begin to bridge the gap between immunologic experiments involving miPSCs in the murine immune context and clinical trials in human patients. We also predict that the specific tissue into which the stem cells are transplanted may greatly influence the extent of immune response in the recipient. Ultimately, if necessary, iPSC derivatives could be given as a transplant to patients with some degree of immunosuppression, such as the short-term leukocyte costimulatory blockade reported by Pearl et al., 2011Pearl J.I. Lee A.S. Leveson-Gower D.B. Sun N. Ghosh Z. Lan F. Ransohoff J. Negrin R.S. Davis M.M. Wu J.C. Short-term immunosuppression promotes engraftment of embryonic and induced pluripotent stem cells.Cell Stem Cell. 2011; 8: 309-317Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar to enhance stem cell engraftment, but that would in some ways defeat the purpose of using iPSCs versus ESCs. If iPSCs are to be used for therapies as we hope, we must understand the functional meaning, if any, of the different kinds of mutations that occur in iPSC lines to define a clinically acceptable level of genomic integrity. While some changes may be an inevitable result of extensively cultivating imperfect somatic cells, it is critical to determine their functional impact on the iPSCs, including any effect of mutational load on tumorigenicity, and how any risk of deleterious mutations can be minimized. Multiple kinds of genomic changes have been observed in hiPSCs, which may ultimately affect the therapeutic readiness of the cells (Figure 1). Chromosomal aneuploidy and translocations, megabase-scale duplications/deletions, and point mutations have all been described (Gore et al., 2011Gore A. Li Z. Fung H.-L. Young J.E. Agarwal S. Antosiewicz-Bourget J. Canto I. Giorgetti A. Israel M.A. Kiskinis E. et al.Somatic coding mutations in human induced pluripotent stem cells.Nature. 2011; 471: 63-67Crossref PubMed Scopus (998) Google Scholar, Hussein et al., 2011Hussein S.M. Batada N.N. Vuoristo S. Ching R.W. Autio R. Närvä E. Ng S. Sourour M. Hämäläinen R. Olsson C. et al.Copy number variation and selection during reprogramming to pluripotency.Nature. 2011; 471: 58-62Crossref PubMed Scopus (757) Google Scholar, Laurent et al., 2011Laurent L.C. Ulitsky I. Slavin I. Tran H. Schork A. Morey R. Lynch C. Harness J.V. Lee S. Barrero M.J. et al.Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.Cell Stem Cell. 2011; 8: 106-118Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, Mayshar et al., 2010Mayshar Y. Ben-David U. Lavon N. Biancotti J.-C. Yakir B. Clark A.T. Plath K. Lowry W.E. Benvenisty N. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). As much as these mutations are cause for concern, nonetheless at this point there is no evidence proving or disproving that these mutations actually matter in a functional sense. At the level of gross chromosomal abnormalities, karyotyping is routinely used to characterize genomic problems in hiPSCs as well as hESCs. Alternately, when gene expression profile data are available, these can also be used to identify chromosomal regions of overexpression or underexpression (Mayshar et al., 2010Mayshar Y. Ben-David U. Lavon N. Biancotti J.-C. Yakir B. Clark A.T. Plath K. Lowry W.E. Benvenisty N. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). These analyses point to chromosome 12 as a common duplication in both hiPSCs and hESCs after extended culturing (Baker et al., 2007Baker D.E.C. Harrison N.J. Maltby E. Smith K. Moore H.D. Shaw P.J. Heath P.R. Holden H. Andrews P.W. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo.Nat. Biotechnol. 2007; 25: 207-215Crossref PubMed Scopus (507) Google Scholar, Mayshar et al., 2010Mayshar Y. Ben-David U. Lavon N. Biancotti J.-C. Yakir B. Clark A.T. Plath K. Lowry W.E. Benvenisty N. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). This chromosome contains the pluripotency genes Nanog and GDF3 as well as many cell cycle-related genes that may contribute to the selection of cells with these changes during culture. Duplication of chromosome 17 was previously reported to be an aberration specific to hESCs (Mayshar et al., 2010Mayshar Y. Ben-David U. Lavon N. Biancotti J.-C. Yakir B. Clark A.T. Plath K. Lowry W.E. Benvenisty N. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 521-531Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar), but this duplication has also recently been observed in hiPSCs (Ben-David et al., 2011Ben-David U. Mayshar Y. Benvenisty N. Large-scale analysis reveals acquisition of lineage-specific chromosomal aberrations in human adult stem cells.Cell Stem Cell. 2011; 9 (this issue): 97-102Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These chromosomal anomalies are not a result of the reprogramming method used, because gains of whole or partial chromosomes have been identified in hiPSCs produced using a variety of techniques including nonintegrating methods such as synthetic mRNAs (Ben-David et al., 2011Ben-David U. Mayshar Y. Benvenisty N. Large-scale analysis reveals acquisition of lineage-specific chromosomal aberrations in human adult stem cells.Cell Stem Cell. 2011; 9 (this issue): 97-102Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Karyotypes produced by G-banding can be used to detect large-scale chromosomal abnormalities such as aneuploidy and translocations. However, hiPSCs can also contain genomic changes at a smaller scale, undetectable by standard karyotyping, which nonetheless could have outsized consequences for cell biology. These smaller genetic alterations can be more labor-intensive to identify, requiring array- or sequencing-based high-throughput techniques. Extensive copy number variation (CNV) has been detected in early-passage hiPSCs using a high-resolution single nucleotide polymorphism (SNP) array. These CNVs tend to occur in common fragile sites, indicating that they are likely a result of replication stress (Hussein et al., 2011Hussein S.M. Batada N.N. Vuoristo S. Ching R.W. Autio R. Närvä E. Ng S. Sourour M. Hämäläinen R. Olsson C. et al.Copy number variation and selection during reprogramming to pluripotency.Nature. 2011; 471: 58-62Crossref PubMed Scopus (757) Google Scholar). It has been reported that in some cases, high-passage hiPSCs contain fewer CNVs than their early-passage precursors (Hussein et al., 2011Hussein S.M. Batada N.N. Vuoristo S. Ching R.W. Autio R. Närvä E. Ng S. Sourour M. Hämäläinen R. Olsson C. et al.Copy number variation and selection during reprogramming to pluripotency.Nature. 2011; 471: 58-62Crossref PubMed Scopus (757) Google Scholar). This suggests that most reprogramming-associated CNVs are detrimental to survival of the cells and are selected against during culture, but further study is required. The remaining CNVs that survive this selective pressure tend toward deletion of tumor suppressor genes and amplification of oncogenes (Laurent et al., 2011Laurent L.C. Ulitsky I. Slavin I. Tran H. Schork A. Morey R. Lynch C. Harness J.V. Lee S. Barrero M.J. et al.Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.Cell Stem Cell. 2011; 8: 106-118Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar), highlighting the importance of monitoring these changes in cells that are intended for therapeutic use. Still smaller genomic lesions have also been identified, including cancer-related point mutations in karyotypically normal hiPSCs (Gore et al., 2011Gore A. Li Z. Fung H.-L. Young J.E. Agarwal S. Antosiewicz-Bourget J. Canto I. Giorgetti A. Israel M.A. Kiskinis E. et al.Somatic coding mutations in human induced pluripotent stem cells.Nature. 2011; 471: 63-67Crossref PubMed Scopus (998) Google Scholar). Some of these point mutations exist in the starting cell population, while the other mutations have a less clear origin and may occur during the reprogramming process and/or during expansion of the cells. Resolving when these mutations occur is an important priority because this data may shed light on not only their functional meaning, but also on potential methods to prevent their occurrence. All of the iPSC lines analyzed by Gore et al. were derived from fibroblasts, so it is quite likely that utilizing a more genomically protected cell source may minimize preexisting DNA mutations in the starting cell population. Dermal fibroblasts are predicted to be a relatively mutation-prone cell type given their high degree of exposure to mutagenic UV light. It is currently unclear whether any human somatic cell populations have significantly less mutational load than others, although there are some hints in the literature that suggest that this is likely the case. Somatic mutation rates in the mouse differ between organs, with higher rates of chromosomal aberrations in peripheral blood lymphocytes than in bone marrow (Tucker et al., 1999Tucker J.D. Spruill M.D. Ramsey M.J. Director A.D. Nath J. Frequency of spontaneous chromosome aberrations in mice: effects of age.Mutat. Res. 1999; 425: 135-141Crossref PubMed Scopus (67) Google Scholar), and higher rates of point mutations in small intestine than in heart (Dollé et al., 2000Dollé M.E. Snyder W.K. Gossen J.A. Lohman P.H. Vijg J. Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine.Proc. Natl. Acad. Sci. USA. 2000; 97: 8403-8408Crossref PubMed Scopus (201) Google Scholar). Mutation rates in accessible human tissues for reprogramming remain to be determined, but these data from the mouse suggest that cells from highly proliferative tissues such as blood and small intestine may contain a higher mutational load and therefore be less desirable as a cell source. In addition, a tissue's relative protection from external factors may also play a role in the degree to which cells accumulate genetic lesions. For example, bone marrow cells may have a lower exposure to environmental toxins than blood or the gastrointestinal tract. As with CNVs, point mutations in iPSCs tend to cluster in cancer-associated genes, possibly pointing to connections between tumorigenic and pluripotency programming (Knoepfler, 2009Knoepfler P.S. Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine.Stem Cells. 2009; 27: 1050-1056Crossref PubMed Scopus (352) Google Scholar). There have been no tumorigenicity studies comparing iPSCs with a relatively large number of mutations to less mutated iPSCs in a clinically relevant setting, so it is currently unclear what an acceptable mutation rate for a PSC line may be from a safety perspective. In addition, the potential functional importance of specific genomic alterations observed in iPSCs remains a key open question; it will be important to test whether the mutational load of iPSCs affects therapeutically relevant parameters such as tumorigenicity, immunogenicity, and impaired differentiation capacity. It also remains unknown if mutational load decreases with culture time as has been observed in at least some cases with CNVs (Hussein et al., 2011Hussein S.M. Batada N.N. Vuoristo S. Ching R.W. Autio R. Närvä E. Ng S. Sourour M. Hämäläinen R. Olsson C. et al.Copy number variation and selection during reprogramming to pluripotency.Nature. 2011; 471: 58-62Crossref PubMed Scopus (757) Google Scholar). The difficulty of characterizing mutations and their effects, if any, on cellular functions illustrates the critical importance of developing reprogramming techniques that preserve genomic integrity. Introduction of reprogramming factors leads to increased DNA damage in the form of 8-oxoguanine, which is generally caused by oxidative stress, and histone γH2AX, a marker of double-strand breaks. DNA damage response elements including TP53/p53, CDKN2A/p16INK4a, and CDKN1A/p21CIP1 are also induced (Banito et al., 2009Banito A. Rashid S.T. Acosta J.C. Li S. Pereira C.F. Geti I. Pinho S. Silva J.C. Azuara V. Walsh M. et al.Senescence impairs successful reprogramming to pluripotent stem cells.Genes Dev. 2009; 23: 2134-2139Crossref PubMed Scopus (488) Google Scholar). Cells containing preexisting DNA damage, including irradiated cells and cells with short telomeres, tend to undergo p53-mediated growth arrest and apoptosis when put into reprogramming conditions (Marión et al., 2009Marión R.M. Strati K. Li H. Murga M. Blanco R. Ortega S. Fernandez-Capetillo O. Serrano M. Blasco M.A. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.Nature. 2009; 460: 1149-1153Crossref PubMed Scopus (842) Google Scholar). This may be one natural antitumorigenic mechanism to limit plasticity of cells containing DNA damage. Overriding these mechanisms enhances reprogramming efficiency, but potentially at the cost of allowing genetically damaged cells to be reprogrammed. The result of such strategies may be a higher proportion of unacceptably mutated iPSCs; indeed, Marión et al., 2009Marión R.M. Strati K. Li H. Murga M. Blanco R. Ortega S. Fernandez-Capetillo O. Serrano M. Blasco M.A. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.Nature. 2009; 460: 1149-1153Crossref PubMed Scopus (842) Google Scholar observed that while knocking down p53 increases reprogramming efficiency, iPSCs produced from p53−/− fibroblasts contain more chromosomal breaks and fusions than iPSCs produced from wild-type fibroblasts. Conversely, reprogramming technologies that enhance innate genomic protection could conceivably produce fewer hiPSCs, but ones with fewer genomic modifications. If this speculation can be proven, it may be preferable from a clinical perspective to use less permissive reprogramming techniques that are designed to upregulate DNA repair processes and/or select for cells with intact DNA. Few studies reporting enhancement of reprogramming have examined whether their techniques allow cells with genomic changes to be reprogrammed, especially since some smaller genomic alterations have only been characterized in hiPSCs within the past year (Gore et al., 2011Gore A. Li Z. Fung H.-L. Young J.E. Agarwal S. Antosiewicz-Bourget J. Canto I. Giorgetti A. Israel M.A. Kiskinis E. et al.Somatic coding mutations in human induced pluripotent stem cells.Nature. 2011; 471: 63-67Crossref PubMed Scopus (998) Google Scholar, Laurent et al., 2011Laurent L.C. Ulitsky I. Slavin I. Tran H. Schork A. Morey R. Lynch C. Harness J.V. Lee S. Barrero M.J. et al.Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.Cell Stem Cell. 2011; 8: 106-118Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). We would argue that focusing on developing methods to boost iPSC production efficiency is not enough. Instead, the goal should be to produce iPSCs with the fewest genomic alterations even if it is at low efficiency; for clinical purposes, theoretically all that is needed is a single bona fide iPSC line from a given patient. One key way to minimize genomic damage is to exert control over oxidative stress during reprogramming and stem cell propagation. Interestingly, hiPSCs and hESCs share a similar ability to protect against genetic damage by limiting production of reactive oxygen species (ROS) and effectively clearing ROS from the cell (Armstrong et al., 2010Armstrong L. Tilgner K. Saretzki G. Atkinson S.P. Stojkovic M. Moreno R. Przyborski S. Lako M. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells.Stem Cells. 2010; 28: 661-673Crossref PubMed Scopus (237) Google Scholar). Compared to differentiated cells, partially reprogrammed cells also share genome-protective mechanisms with fully pluripotent cells, including maintenance of low intracellular superoxide levels and relatively few mitochondria (Armstrong et al., 2010Armstrong L. Tilgner K. Saretzki G. Atkinson S.P. Stojkovic M. Moreno R. Przyborski S. Lako M. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells.Stem Cells. 2010; 28: 661-673Crossref PubMed Scopus (237) Google Scholar). However, signs of oxidative damage appear even earlier than these genome-protective cellular changes, within a few days after reprogramming factor introduction (Banito et al., 2009Banito A. Rashid S.T. Acosta J.C. Li S. Pereira C.F. Geti I. Pinho S. Silva J.C. Azuara V. Walsh M. et al.Senescence impairs successful reprogramming to pluripotent stem cells.Genes Dev. 2009; 23: 2134-2139Crossref PubMed Scopus (488) Google Scholar), suggesting that genome protection may be amenable to enhancement especially during the first few days of reprogramming. Culture conditions can impact the prevalence of karyotypic abnormalities; for example, culture at physiological (5%) oxygen tension protects cardiac stem cells and hESCs from karyotypic changes (Li and Marbán, 2010Li T.-S. Marbán E. Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells.Stem Cells. 2010; 28: 1178-1185Crossref PubMed Scopus (189) Google Scholar). Physiological oxygen tension also enhances production of iPSCs compared with either normoxic (21%) or hypoxic (1%) conditions, increasing the efficiency and rate of reprogramming murine and human fibroblasts (Yoshida et al., 2009Yoshida Y. Takahashi K. Okita K. Ichisaka T. Yamanaka S. Hypoxia enhances the generation of induced pluripotent stem cells.Cell Stem Cell. 2009; 5: 237-241Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). However, iPSCs produced in 5% oxygen conditions have just as many point mutations as those produced at atmospheric oxygen (Gore et al., 2011Gore A. Li Z. Fung H.-L. Young J.E. Agarwal S. Antosiewicz-Bourget J. Canto I. Giorgetti A. Israel M.A. Kiskinis E. et al.Somatic coding mutations in human induced pluripotent stem cells.Nature. 2011; 471: 63-67Crossref PubMed Scopus (998) Google Scholar), so it is still uncle