Molecular Obstacles to Clinical Translation of iPSCs
2016; Elsevier BV; Volume: 19; Issue: 3 Linguagem: Inglês
10.1016/j.stem.2016.06.017
ISSN1934-5909
AutoresNatàlia Tàpia, Hans R. Schöler,
Tópico(s)Animal Genetics and Reproduction
ResumoThe ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) using defined factors provides new tools for biomedical research. However, some iPSC clones display tumorigenic and immunogenic potential, thus raising concerns about their utility and safety in the clinical setting. Furthermore, variability in iPSC differentiation potential has also been described. Here we discuss whether these therapeutic obstacles are specific to transcription-factor-mediated reprogramming or inherent to every cellular reprogramming method. Finally, we address whether a better understanding of the mechanism underlying the reprogramming process might improve the fidelity of reprogramming and, therefore, the iPSC quality. The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) using defined factors provides new tools for biomedical research. However, some iPSC clones display tumorigenic and immunogenic potential, thus raising concerns about their utility and safety in the clinical setting. Furthermore, variability in iPSC differentiation potential has also been described. Here we discuss whether these therapeutic obstacles are specific to transcription-factor-mediated reprogramming or inherent to every cellular reprogramming method. Finally, we address whether a better understanding of the mechanism underlying the reprogramming process might improve the fidelity of reprogramming and, therefore, the iPSC quality. In 2005, we were in a lab retreat discussing how many genes would be required to reprogram somatic cells to pluripotency. No one suggested just four proteins. The following year, 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 reported the generation of induced pluripotent stem cells (iPSCs) using a reprogramming cocktail of Oct4, Sox2, Klf4, and Myc (hereafter referred to as OSKM), a discovery that has undoubtedly changed the field of regenerative medicine and our understanding of cellular identity. iPSC technology is based on the assumption that a set of transcription factors expressed in embryonic stem cells (ESCs) is responsible for maintaining a pluripotent fate and is sufficient for establishing a de novo pluripotency program. Finding this specific combination of factors is an extraordinary accomplishment considering that ESCs express thousands of proteins. Nicely, the same cocktail of proteins was later shown to also reprogram human somatic cells to pluripotency (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). The beauty of this finding is that a simple experiment with a very low probability for success answered a complex question. We wonder whether Yamanaka received funding for this specific experiment, considering that many grants are awarded based on the probability of generating the expected results. In the last 10 years, iPSCs have been thoroughly scrutinized, and their value as a disease model and a source of cells has been intensively debated. Indeed, genetic mutations and chromosomal aberrations detected in iPSCs have raised concerns about their tumorigenic potential (Yamanaka, 2012Yamanaka S. Induced pluripotent stem cells: past, present, and future.Cell Stem Cell. 2012; 10: 678-684Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). Likewise, epigenetic aberrations have questioned iPSC differentiation potential and immune tolerance after autologous transplantation (Okita et al., 2011Okita K. Nagata N. Yamanaka S. Immunogenicity of induced pluripotent stem cells.Circ. Res. 2011; 109: 720-721Crossref PubMed Scopus (93) Google Scholar). However, further analyses have demonstrated that these abnormalities are mostly due to technical limitations, thus excluding these reprogramming errors as an intrinsic characteristic of transcription factor-mediated reprogramming. Here we review the immunogenic and tumorigenic features attributed to some iPSC clones, the relationship between these undesirable traits and incomplete reprogramming, and the mechanisms underlying different reprogramming methods as a strategy for improving iPSC reprogramming fidelity and thus the utility of iPSCs in molecular and biomedical applications. One of the main expectations of iPSC technology is to supply cells for autologous transplantation. Indeed, patient-specific iPSC derivatives have been assumed to be tolerated by the immune system, thereby evading life-long immunosuppressive treatment for the prevention of allograft rejection. Somatic cell nuclear transfer (SCNT) can also generate autologous pluripotent cells. However, SCNT ESCs retain the mitochondria from the recipient oocyte, which induce alloimmunity after transplantation in mice genetically matched to the reprogrammed nucleus (Deuse et al., 2015Deuse T. Wang D. Stubbendorff M. Itagaki R. Grabosch A. Greaves L.C. Alawi M. Grünewald A. Hu X. Hua X. et al.SCNT-derived ESCs with mismatched mitochondria trigger an immune response in allogeneic hosts.Cell Stem Cell. 2015; 16: 33-38Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The high expectations regarding the immune tolerance of patient-specific iPSCs started to be questioned when 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 reported that the transplantation of iPSCs into syngeneic murine recipients led to the formation of immunogenic teratomas (Figure 1A). Those authors showed that iPSC-derived teratomas expressed a subset of antigens that were not detected in the teratomas generated after ESC transplantation and speculated that the expression of these aberrant antigens was due to the incomplete reprogramming of iPSCs (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). These findings raised doubts about the practical applications of iPSC technology in cell replacement therapies. The main criticism of the report by 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 was that it assessed the immunogenicity of iPSC-derived teratomas rather than pure populations of iPSC-differentiated cells, which are the cells to be used for transplantation in medical treatments (Okita et al., 2011Okita K. Nagata N. Yamanaka S. Immunogenicity of induced pluripotent stem cells.Circ. Res. 2011; 109: 720-721Crossref PubMed Scopus (93) Google Scholar). To address this issue, Araki et al., 2013Araki R. Uda M. Hoki Y. Sunayama M. Nakamura M. Ando S. Sugiura M. Ideno H. Shimada A. Nifuji A. Abe M. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.Nature. 2013; 494: 100-104Crossref PubMed Scopus (384) Google Scholar generated iPSC-chimeric mice from which terminally differentiated tissues were isolated and subsequently transplanted into genetically matched recipients (Figure 1B). These in vivo-differentiated tissues showed limited immunogenicity; thus the authors conclude that iPSC derivatives do not elicit an immune response (Araki et al., 2013Araki R. Uda M. Hoki Y. Sunayama M. Nakamura M. Ando S. Sugiura M. Ideno H. Shimada A. Nifuji A. Abe M. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.Nature. 2013; 494: 100-104Crossref PubMed Scopus (384) Google Scholar). Surprisingly, those authors briefly stated in their discussion that cardiomyocytes obtained through in vitro iPSC differentiation generated a significant T immune response after transplantation into syngeneic mice. These apparently contradictory results suggest that the immune reaction mounted against in vitro iPSC-differentiated cardiomyocytes results from an incomplete or abnormal differentiation process that is not observed when the iPSCs are terminally differentiated and matured through in vivo chimera formation. Similar findings have been described in ESCs. Indeed, in vitro ESC differentiation has been shown to induce aberrant antigen expression in ESC derivatives, which, in turn, elicits immunogenicity (Tang and Drukker, 2011Tang C. Drukker M. Potential barriers to therapeutics utilizing pluripotent cell derivatives: intrinsic immunogenicity of in vitro maintained and matured populations.Semin. Immunopathol. 2011; 33: 563-572Crossref PubMed Scopus (31) Google Scholar). Because in vitro iPSC-differentiated cells will likely serve as the main source of cells for therapeutic applications, Guha et al., 2013Guha P. Morgan J.W. Mostoslavsky G. Rodrigues N.P. Boyd A.S. Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells.Cell Stem Cell. 2013; 12: 407-412Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar specifically evaluated the immunogenicity of in vitro iPSC-derived cells after transplantation into syngeneic mouse recipients. To this end, iPSCs were first differentiated in vitro into one representative cell type of each embryonic germ layer and then transplanted into the kidney capsule of isogenic mice (Figure 1C). An immune response was not observed, thus leading to the conclusion that autologous iPSC-differentiated cells are not immunogenic in autologous recipients. However, an important caveat when interpreting these results is that the ectopic transplantation site does not reflect the actual clinical scenario. Furthermore, the findings of Guha et al., 2013Guha P. Morgan J.W. Mostoslavsky G. Rodrigues N.P. Boyd A.S. Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells.Cell Stem Cell. 2013; 12: 407-412Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar do not correlate with the immune response observed with the in vitro iPSC-derived cardiomyocytes described by Araki et al., 2013Araki R. Uda M. Hoki Y. Sunayama M. Nakamura M. Ando S. Sugiura M. Ideno H. Shimada A. Nifuji A. Abe M. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.Nature. 2013; 494: 100-104Crossref PubMed Scopus (384) Google Scholar, which were also ectopically transplanted. These opposite results suggest that the immunogenicity of iPSC-derived cells might depend on the final cell type, the similarity of the in vitro-differentiated cells to their in vivo counterparts, including maturation status, or the reprogramming quality of the initial iPSCs. Interestingly, one study compared the immune response to endothelial cells obtained from in vitro-differentiated iPSCs with endothelial cells isolated from in vivo murine aortas (de Almeida et al., 2014de Almeida P.E. Meyer E.H. Kooreman N.G. Diecke S. Dey D. Sanchez-Freire V. Hu S. Ebert A. Odegaard J. Mordwinkin N.M. et al.Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance.Nat. Commun. 2014; 5: 3903PubMed Google Scholar). The authors' results pointed to in vitro iPSC-derived endothelial cells and in vivo-isolated endothelial cells as being similarly tolerated by isogenic hosts. The authors concluded that the differences in antigen expression between the iPSC progeny and their in vivo equivalent cells were not sufficient to trigger an immune response after transplantation. However, a gene expression comparison between iPSC-derived and in vivo-isolated endothelial cells was not shown. Thus, the degree of transcriptional divergence the immune system can tolerate because of reprogramming infidelity, genomic instability, or suboptimal differentiation remains unknown. Recently, the immunogenicity of human iPSC-derived cells was investigated using a humanized mouse model with a reconstituted human immune system. Zhao et al., 2015Zhao T. Zhang Z.N. Westenskow P.D. Todorova D. Hu Z. Lin T. Rong Z. Kim J. He J. Wang M. et al.Humanized Mice Reveal Differential Immunogenicity of Cells Derived from Autologous Induced Pluripotent Stem Cells.Cell Stem Cell. 2015; 17: 353-359Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar showed that human iPSC-derived smooth muscle cells were immunogenic, but retinal pigment epithelial cells were not, after transplantation into the skeletal muscle and the eye, respectively (Figure 1D). The authors claimed that the expression of some immunogenic antigens detected in the iPSC-derived smooth muscle cells, but not in the retinal pigment epithelial cells, was responsible for the immune response. Again, the aberrant antigen expression in the smooth muscle cells may result from a suboptimal differentiation protocol or incomplete iPSC reprogramming, which induces abnormal gene expression upon cellular differentiation into smooth muscle cells. Overall, the immune response to iPSC progeny still requires more thorough investigation, specifically regarding the type and amount of gene expression differences between iPSC-derived somatic cells and their in vivo counterparts that can be tolerated by the immune system after transplantation into clinically relevant sites. Finally, future studies should evaluate the immune tolerance to the progeny of genetically corrected iPSCs because the immune system might not show tolerance to the wild-type gene to which it had never been exposed (Wood et al., 2016Wood K.J. Issa F. Hester J. Understanding Stem Cell Immunogenicity in Therapeutic Applications.Trends Immunol. 2016; 37: 5-16Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The probability that genomic mutations occur during the reprogramming process has raised concerns about the tumorigenic potential of iPSCs, bringing into question the safety of iPSCs for clinical applications. For this reason, the genomic integrity of human iPSCs (hiPSCs) has been thoroughly studied (reviewed by Liang and Zhang, 2013Liang G. Zhang Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application.Cell Stem Cell. 2013; 13: 149-159Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). A karyotype analysis on more than 1,700 hiPSC and human ESC (hESC) samples showed that the incidence of abnormal chromosome number was similar in both cell types (Taapken et al., 2011Taapken S.M. Nisler B.S. Newton M.A. Sampsell-Barron T.L. Leonhard K.A. McIntire E.M. Montgomery K.D. Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells.Nat. Biotechnol. 2011; 29: 313-314Crossref PubMed Scopus (220) Google Scholar). Some chromosomal aberrations, such as partial gain of chromosome 12, were commonly found in hiPSCs and hESCs. Interestingly, duplication of the chromosome 12 short arm is a recurrent chromosomal abnormality also detected in testicular germ cell tumors (van Echten et al., 1995van Echten J. Oosterhuis J.W. Looijenga L.H. van de Pol M. Wiersema J. te Meerman G.J. Schaffordt Koops H. Sleijfer D.T. de Jong B. No recurrent structural abnormalities apart from i(12p) in primary germ cell tumors of the adult testis.Genes Chromosomes Cancer. 1995; 14: 133-144Crossref PubMed Scopus (115) Google Scholar). In contrast, trisomy 8 and trisomy 17 were preferentially detected in hiPSCs and hESCs, respectively. Interestingly, the chromosomal abnormalities described in hiPSCs and hESCs differ from those reported in human embryos, suggesting that the specific type of chromosomal aberrations detected in hESCs and hiPSCs might be involved in cell culture adaptation (Taapken et al., 2011Taapken S.M. Nisler B.S. Newton M.A. Sampsell-Barron T.L. Leonhard K.A. McIntire E.M. Montgomery K.D. Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells.Nat. Biotechnol. 2011; 29: 313-314Crossref PubMed Scopus (220) Google Scholar). Indeed, hiPSCs exhibiting a partial gain of chromosome 12 overexpress NANOG and GDF3, which might provide a selective growth advantage (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). Furthermore, chromosomes 12 and 17 encode the neurotrophin p75NGFR receptor and the ligand neurotrophin 3, which are known to increase hESC survival at clonal density. However, neurotrophins are usually not included in hESC medium formulations, suggesting that chromosomal aberrations found in hESCs might be selected to compensate for deficient hESC culture conditions (Pyle et al., 2006Pyle A.D. Lock L.F. Donovan P.J. Neurotrophins mediate human embryonic stem cell survival.Nat. Biotechnol. 2006; 24: 344-350Crossref PubMed Scopus (218) Google Scholar). Techniques of higher resolution than karyotyping have provided closer insight into the subchromosomal stability of hiPSCs. One study showed a higher frequency of copy number variations (CNVs) in hiPSCs than in the donor tissue, suggesting that these genomic aberrations were introduced during the reprogramming process (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). However, 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 used SNP arrays in which low-frequency donor cell variations could not be detected, a limitation that increased the rate of false-positive CNVs in hiPSCs. Subsequent studies using a more sensitive method, such as next-generation sequencing, showed fewer or no detectable de novo CNVs in hiPSCs (Abyzov et al., 2012Abyzov A. Mariani J. Palejev D. Zhang Y. Haney M.S. Tomasini L. Ferrandino A.F. Rosenberg Belmaker L.A. Szekely A. Wilson M. et al.Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.Nature. 2012; 492: 438-442Crossref PubMed Scopus (294) Google Scholar, Cheng et al., 2012Cheng L. Hansen N.F. Zhao L. Du Y. Zou C. Donovan F.X. Chou B.K. Zhou G. Li S. Dowey S.N. et al.NISC Comparative Sequencing ProgramLow incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression.Cell Stem Cell. 2012; 10: 337-344Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, Young et al., 2012Young M.A. Larson D.E. Sun C.W. George D.R. Ding L. Miller C.A. Lin L. Pawlik K.M. Chen K. Fan X. et al.Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells.Cell Stem Cell. 2012; 10: 570-582Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Interestingly, Cheng et al., 2012Cheng L. Hansen N.F. Zhao L. Du Y. Zou C. Donovan F.X. Chou B.K. Zhou G. Li S. Dowey S.N. et al.NISC Comparative Sequencing ProgramLow incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression.Cell Stem Cell. 2012; 10: 337-344Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar demonstrated that most of the sequence variations found in hiPSCs are within the range expected from normal mitotic mutations in human adult somatic cells, with half of these mutations being also detected in the parental somatic cells. Indeed, preexisting somatic mosaicism in the donor cell tissue is currently considered to be the major source of CNVs and single-nucleotide variations observed among hiPSCs (Cheng et al., 2012Cheng L. Hansen N.F. Zhao L. Du Y. Zou C. Donovan F.X. Chou B.K. Zhou G. Li S. Dowey S.N. et al.NISC Comparative Sequencing ProgramLow incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression.Cell Stem Cell. 2012; 10: 337-344Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, Young et al., 2012Young M.A. Larson D.E. Sun C.W. George D.R. Ding L. Miller C.A. Lin L. Pawlik K.M. Chen K. Fan X. et al.Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells.Cell Stem Cell. 2012; 10: 570-582Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Similarly, mtDNA mutations, which stochastically arise in individual somatic cells and accumulate in somatic tissues during aging, can be captured during the reprogramming process and clonally expanded in individual iPSC lines (Kang et al., 2016Kang E. Wang X. Tippner-Hedges R. Ma H. Folmes C.D.L. Marti Gutierrez N. Lee Y. Van Dyken C. Ahmed R. Li Y. et al.Age-related accumulation of somatic mitochondrial DNA mutations in adult-derived human iPSCs.Cell Stem Cell. 2016; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Nevertheless, it cannot be excluded that some of these DNA and mtDNA mutations are introduced de novo during the reprogramming process. Although the frequency and type of genomic changes have not been correlated with any of the methods used for reprogramming (Bhutani et al., 2016Bhutani K. Nazor K.L. Williams R. Tran H. Dai H. Džakula Ž. Cho E.H. Pang A.W. Rao M. Cao H. et al.Whole-genome mutational burden analysis of three pluripotency induction methods.Nat. Commun. 2016; 7: 10536Crossref PubMed Scopus (88) Google Scholar, Taapken et al., 2011Taapken S.M. Nisler B.S. Newton M.A. Sampsell-Barron T.L. Leonhard K.A. McIntire E.M. Montgomery K.D. Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells.Nat. Biotechnol. 2011; 29: 313-314Crossref PubMed Scopus (220) Google Scholar), the use of integrative methods is highly discouraged for preventing genome mutagenesis at the insertion site and for minimizing the likelihood of disrupting active genes and tumor suppressor genes as well as activating oncogenes. Collectively, all of these studies rule out that the reprogramming of iPSCs is intrinsically a mutagenic process and point to the donor cell type and in vitro cell culture as the main causes for the observed genomic variability. It is worth noting that most reprogramming experiments have been conducted on fibroblasts because these cells can be obtained routinely in the clinic without using any invasive procedures. However, fibroblasts can tolerate high levels of genomic insults, and thus somatic cell types with lower mutation tolerance rates should be preferentially employed. The human genome contains about 100 copies of long interspersed nuclear elements (LINE)-1 retrotransposons. These elements retain retrotransposition competence and pose a risk to genomic integrity because of their potential for introducing insertional mutations after transposition. For this reason, epigenetic mechanisms such as histone modifications and DNA methylation prevent the mobility of LINE-1. However, epigenetic remodeling during hiPSC reprogramming induces a LINE-1 peak of expression that is attenuated in late-passage hiPSCs (Klawitter et al., 2016Klawitter S. Fuchs N.V. Upton K.R. Muñoz-Lopez M. Shukla R. Wang J. Garcia-Cañadas M. Lopez-Ruiz C. Gerhardt D.J. Sebe A. et al.Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.Nat. Commun. 2016; 7: 10286Crossref PubMed Scopus (92) Google Scholar). One study has detected retroviral transposition events in hiPSCs (Klawitter et al., 2016Klawitter S. Fuchs N.V. Upton K.R. Muñoz-Lopez M. Shukla R. Wang J. Garcia-Cañadas M. Lopez-Ruiz C. Gerhardt D.J. Sebe A. et al.Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.Nat. Commun. 2016; 7: 10286Crossref PubMed Scopus (92) Google Scholar), whereas another has not detected any in mouse iPSCs (Quinlan et al., 2011Quinlan A.R. Boland M.J. Leibowitz M.L. Shumilina S. Pehrson S.M. Baldwin K.K. Hall I.M. Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangement during reprogramming.Cell Stem Cell. 2011; 9: 366-373Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These contradictory results might be due to the different iPSC reprogramming approaches used in each study—Sleeping Beauty transposon-based plasmids (Klawitter et al., 2016Klawitter S. Fuchs N.V. Upton K.R. Muñoz-Lopez M. Shukla R. Wang J. Garcia-Cañadas M. Lopez-Ruiz C. Gerhardt D.J. Sebe A. et al.Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.Nat. Commun. 2016; 7: 10286Crossref PubMed Scopus (92) Google Scholar) versus lentiviral vectors (Quinlan et al., 2011Quinlan A.R. Boland M.J. Leibowitz M.L. Shumilina S. Pehrson S.M. Baldwin K.K. Hall I.M. Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangement during reprogramming.Cell Stem Cell. 2011; 9: 366-373Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar); the distinctively different properties of human primed (Klawitter et al., 2016Klawitter S. Fuchs N.V. Upton K.R. Muñoz-Lopez M. Shukla R. Wang J. Garcia-Cañadas M. Lopez-Ruiz C. Gerhardt D.J. Sebe A. et al.Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.Nat. Commun. 2016; 7: 10286Crossref PubMed Scopus (92) Google Scholar) and mouse naive (Quinlan et al., 2011Quinlan A.R. Boland M.J. Leibowitz M.L. Shumilina S. Pehrson S.M. Baldwin K.K. Hall I.M. Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangement during reprogramming.Cell Stem Cell. 2011; 9: 366-373Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) pluripotency states; or the method employed for detecting de novo retrotransposition events—genome-wide DNA sequencing with (Klawitter et al., 2016Klawitter S. Fuchs N.V. Upton K.R. Muñoz-Lopez M. Shukla R. Wang J. Garcia-Cañadas M. Lopez-Ruiz C. Gerhardt D.J. Sebe A. et al.Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.Nat. Commun. 2016; 7: 10286Crossref PubMed Scopus (92) Google Scholar) or without (Quinlan et al., 2011Quinlan A.R. Boland M.J. Leibowitz M.L. Shumilina S. Pehrson S.M. Baldwin K.K. Hall I.M. Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangement during reprogramming.Cell Stem Cell. 2011; 9: 366-373Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) a previous retrotransposon enrichment step. Therefore, more analyses are required to clarify this discrepancy. Because genomic instability is associated with cancer, genomic abnormalities have been postulated to compromise the safety of hiPSCs in cell replacement therapies. However, there is little evidence linking genomic aberrations in hiPSCs with tumorigenesis (Peterson and Loring, 2014Peterson S.E. Loring J.F. Genomic instability in pluripotent stem cells: implications for clinical applications.J. Biol. Chem. 2014; 289: 4578-4584Crossref PubMed Scopus (91) Google Scholar). Future studies need to establish safety standards for genomic integrity that distinguish genomic insults with an oncogenic potential from harmless genomic variations amplified from the donor tissue. More importantly, recent studies have described genomic aberrations arising from the direct differentiation of hiPSCs (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, Varela et al., 2012Varela C. Denis J.A. Polentes J. Feyeux M. Aubert S. Champon B. Piétu G. Peschanski M. Lefort N. Recurrent genomic instability of chromosome 1q in neural derivatives of human embryonic stem cells.J. Clin. Invest. 2012; 122: 569-574Crossref PubMed Scopus (39) Google Scholar), pointing to hiPSC expansion and differentiation as a major source of genetic abnormalities, as has also been described previously in hESCs (Tang and Drukker, 2011Tang C. Drukker M. Potential barriers to therapeutics utilizing pluripotent cell derivatives: intrinsic immunogenicity of in vitro maintained and matured populations.Semin. Immunopathol. 2011; 33: 563-572Crossref PubMed Scopus (31) Google Scholar). In conclusion, most of the genomic variations in iPSCs have either been inherited from the cell of origin or induced during long-term culture. However, one cannot rule out that some mutations are introduced during the reprogramming process. It is worth to highlight that not all hiPSC lines acquire genetic abnormalities during extended in vitro culture. Therefore, prior to their use in a therapeutic context, exhaustive genome-wide sequencing screenings of hiPSCs and their derivatives need to be implemented to select cells without genomic aberrations (Figure 2). Several studies had initially reported differences in the transcriptional and epigenetic p
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