A mitochondrial strategy for safeguarding the reprogrammed genome
© Prigione and Adjaye; licensee BioMed Central Ltd. 2014
Received: 28 January 2014
Accepted: 11 March 2014
Published: 29 March 2014
Genomic aberrations induced by somatic cell reprogramming are a major drawback for future applications of this technology in regenerative medicine. A new study by Ji et al. published in Stem Cell Reports suggests a counteracting strategy based on balancing the mitochondrial/oxidative stress pathway through antioxidant supplementation.
Reactive oxygen species (ROS) are common by-products of cellular respiration. They can act as second messengers exerting physiological roles . However, if ROS levels increase beyond a certain threshold, functional oxidative damage to macromolecules can occur, leading to protein, lipid or genomic aberrations and eventually cell death . To preserve genome integrity, cells have developed a fine-tuned machinery to counteract ROS by keeping them in equilibrium with reducing equivalents [1, 2]. The maintenance of redox balance is thus critical for cells both in steady states and during adaptations to different conditions. Now, a new study by Ji et al.  demonstrates that supporting redox homeostasis is important also during the induction of pluripotency.
The authors detected increased levels of ROS and oxidative DNA damage during the early stages of human retroviral-based reprogramming using four factors (4F: OCT4, SOX2, KLF4, c-MYC), in agreement with previous reports [4, 5]. Notably, the concurrent supply of antioxidants (vitamin C or N-acetyl-cysteine, NAC) appeared capable of reducing both ROS and genomic double-strand breaks, resulting in lower apoptotic rates. These effects were not a consequence of altered transgene activity, since antioxidants did not modify the 4F expression or their silencing. Remarkably, induced pluripotent stem cells (iPSCs) lines generated with antioxidant supplementation displayed significantly fewer de novo copy number variations (CNVs), i.e. genomic variants that were not already present in the parental fibroblast population. To rule out that the reduction in the number of CNVs was not due to additional non-antioxidant related mechanisms influencing reprogramming, which have been found associated with vitamin C supplementation [6, 7], the authors demonstrated that CNVs were similarly lowered by vitamin C and NAC treatment. It must also be noted that culture media typically employed for human reprogramming (e.g. KSR and mTeSR) contains vitamin C, suggesting that in its absence the levels of ROS would be higher. Hence, supporting the redox balance through the addition of reducing molecules may protect the somatic genome, leading to iPSCs with fewer genomic alterations.
Their findings also raise a series of important questions. For example, how is it that somatic-coding mutations are not affected by the introduction of antioxidants? The authors suggest that oxidative DNA lesions might be less error-prone and therefore more easily corrected. Moreover, is it possible to employ additional conditions that potentiate the effects of the antioxidant cocktail? In this regard, hypoxia or the addition of a hypoxia mimetic might be beneficial, given that hypoxia enhances iPSC derivation , by inducing a faster glycolytic transition . Likewise, do antioxidants protect against mitochondrial mutations acquired during reprogramming ?
Another central issue that remains to be addressed is the relationship between antioxidant supplementation, reprogramming methods and genomic aberrations. Although mutations have been found to occur also using non-integrating strategies , the levels of nuclear and mtDNA alterations may be diminished under these conditions . Indeed, non-integrating episomal plasmids elicit a lower ROS response than viral-based reprogramming . A systematic comparison using various iPSC techniques with and without antioxidant treatment would help to clarify this matter.
Finally, the data by Ji et al.  underscores the unique features of c-MYC within the 4F cocktail. c-MYC is a key inducer of glycolytic reconfiguration  but also appears as the major contributor of reprogramming-mediated oxidative stress. In fact, the use of the other three factors did not generate a drastic elevation of ROS nor was their basal level affected by antioxidant supplementation . Nonetheless, genomic aberrations and metabolic conversion can occur also in the absence of c-MYC [19, 20]. Hence, reprogramming strategies should ideally avoid the inclusion of c-MYC, and it remains unclear whether such strategies would also benefit from the addition of antioxidants.
Overall, the work by Ji et al.  has relevant implications, as the occurrence of reprogramming-mediated genomic alterations is currently a major obstacle hindering the use of iPSCs in medical applications . Further manipulation of the mitochondrial/oxidative stress pathway may thus pave the way for the development of safer reprogramming approaches.
The authors declare no competing financial or commercial interests and acknowledge support from the Fritz Thyssen Foundation (grant AZ. 10.11.2.160 to A.P.) and the European Union (funding/FP7 (FP7/2007-2013)/Grant Agreement n° 305299 /AgedBrainSYSBIO to J.A.).
- Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J: Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007,39(1):44–84. 10.1016/j.biocel.2006.07.001PubMedView ArticleGoogle Scholar
- Finkel T, Holbrook NJ: Oxidants, oxidative stress and the biology of ageing. Nature 2000,408(6809):239–247. 10.1038/35041687PubMedView ArticleGoogle Scholar
- Ji J, Sharma V, Qi S, Guarch ME, Zhao P, Luo Z, Fan W, Wang Y, Mbabaali F, Neculai D, Esteban MA, McPherson JD, Batada NN: Antioxidant supplementation reduces genomic aberrations in human induced pluripotent stem cells. Stem Cell Reports 2014,2(1):44–51. 10.1016/j.stemcr.2013.11.004PubMed CentralPubMedView ArticleGoogle Scholar
- Esteban MA, Wang T, Qin B, Yang J, Qin D, Cai J, Li W, Weng Z, Chen J, Ni S, Chen K, Li Y, Liu X, Xu J, Zhang S, Li F, He W, Labuda K, Song Y, Peterbauer A, Wolbank S, Redl H, Zhong M, Cai D, Zeng L, Pei D: Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 2010,6(1):71–79. 10.1016/j.stem.2009.12.001PubMedView ArticleGoogle Scholar
- Mah N, Wang Y, Liao MC, Prigione A, Jozefczuk J, Lichtner B, Wolfrum K, Haltmeier M, Flottmann M, Schaefer M, Hahn A, Mrowka R, Klipp E, Andrade-Navarro MA, Adjaye J: Molecular insights into reprogramming-initiation events mediated by the OSKM gene regulatory network. PLoS One 2011,6(8):e24351. 10.1371/journal.pone.0024351PubMed CentralPubMedView ArticleGoogle Scholar
- Wang T, Chen K, Zeng X, Yang J, Wu Y, Shi X, Qin B, Zeng L, Esteban MA, Pan G, Pei D: The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell 2011,9(6):575–587. 10.1016/j.stem.2011.10.005PubMedView ArticleGoogle Scholar
- Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, Kono T, Shioda T, Hochedlinger K: Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 2010,465(7295):175–181. 10.1038/nature09017PubMed CentralPubMedView ArticleGoogle Scholar
- Prigione A, Fauler B, Lurz R, Lehrach H, Adjaye J: The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells 2010,28(4):721–733. 10.1002/stem.404PubMedView ArticleGoogle Scholar
- Folmes CD, Nelson TJ, Martinez-Fernandez A, Arrell DK, Lindor JZ, Dzeja PP, Ikeda Y, Perez-Terzic C, Terzic A: Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metab 2011,14(2):264–271. 10.1016/j.cmet.2011.06.011PubMed CentralPubMedView ArticleGoogle Scholar
- Gruning NM, Ralser M: Cancer: sacrifice for survival. Nature 2011,480(7376):190–191. 10.1038/480190aPubMedView ArticleGoogle Scholar
- Prigione A, Lichtner B, Kuhl H, Struys EA, Wamelink M, Lehrach H, Ralser M, Timmermann B, Adjaye J: Human induced pluripotent stem cells harbor homoplasmic and heteroplasmic mitochondrial DNA mutations while maintaining human embryonic stem cell-like metabolic reprogramming. Stem Cells 2011,29(9):1338–1348.PubMedGoogle Scholar
- Prigione A, Rohwer N, Hoffmann S, Mlody B, Drews K, Bukowiecki R, Blumlein K, Wanker EE, Ralser M, Cramer T, Adjaye J: HIF1alpha modulates cell fate reprogramming through early glycolytic shift and upregulation of PDK1–3 and PKM2. Stem Cells 2014,32(2):364–376. 10.1002/stem.1552PubMedView ArticleGoogle Scholar
- Yanes O, Clark J, Wong DM, Patti GJ, Sanchez-Ruiz A, Benton HP, Trauger SA, Desponts C, Ding S, Siuzdak G: Metabolic oxidation regulates embryonic stem cell differentiation. Nat Chem Biol 2010,6(6):411–417. 10.1038/nchembio.364PubMed CentralPubMedView ArticleGoogle Scholar
- Armstrong L, Tilgner K, Saretzki G, Atkinson SP, 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(4):661–673. 10.1002/stem.307PubMedView ArticleGoogle Scholar
- Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S: Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 2009,5(3):237–241. 10.1016/j.stem.2009.08.001PubMedView ArticleGoogle Scholar
- Young MA, Larson DE, Sun CW, George DR, Ding L, Miller CA, Lin L, Pawlik KM, Chen K, Fan X, Schmidt H, Kalicki-Veizer J, Cook LL, Swift GW, Demeter RT, Wendl MC, Sands MS, Mardis ER, Wilson RK, Townes TM, Ley TJ: Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell 2012,10(5):570–582. 10.1016/j.stem.2012.03.002PubMed CentralPubMedView ArticleGoogle Scholar
- Cheng L, Hansen NF, Zhao L, Du Y, Zou C, Donovan FX, Chou BK, Zhou G, Li S, Dowey SN, Ye Z, Chandrasekharappa SC, Yang H, Mullikin JC, Liu PP: Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell 2012,10(3):337–344. 10.1016/j.stem.2012.01.005PubMed CentralPubMedView ArticleGoogle Scholar
- Miller DM, Thomas SD, Islam A, Muench D, Sedoris K: c-Myc and cancer metabolism. Clin Cancer Res 2012,18(20):5546–5553. 10.1158/1078-0432.CCR-12-0977PubMed CentralPubMedView ArticleGoogle Scholar
- Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K: Somatic coding mutations in human induced pluripotent stem cells. Nature 2011,471(7336):63–67. 10.1038/nature09805PubMed CentralPubMedView ArticleGoogle Scholar
- Folmes CD, Martinez-Fernandez A, Faustino RS, Yamada S, Perez-Terzic C, Nelson TJ, Terzic A: Nuclear reprogramming with c-Myc potentiates glycolytic capacity of derived induced pluripotent stem cells. J Cardiovasc Transl Res 2013,6(1):10–21. 10.1007/s12265-012-9431-2PubMed CentralPubMedView ArticleGoogle Scholar
- Pera MF: Stem cells: The dark side of induced pluripotency. Nature 2011,471(7336):46–47. 10.1038/471046aPubMedView ArticleGoogle Scholar
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