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.
KeywordsAntioxidants Mitochondria Reprogramming iPSCs Genomic aberrations
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.).
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