pteryxin br Results br Discussion Genomic instability is

Results
Discussion Genomic instability is one of the biggest concerns in the potential clinical use of iPSCs (Rocha et al., 2013). The challenging field of regenerative medicine requires an in-depth understanding of the causes and consequences of genetic abnormalities that arise during the reprogramming process (Studer et al., 2015; Tabar and Studer, 2014; Buganim et al., 2013). Here, we study mouse and human reprogramming to iPSCs, to elucidate the role of DNA end resection as a relevant mechanism for avoiding genomic instability in this process. We demonstrate that expression of the CtIP protein, a key protein in DNA end resection (Sartori et al., 2007), is upregulated during the formation of iPSCs and is required for efficient reprogramming. A CtIP deficiency during reprogramming not only drastically impairs the reprogramming process but also endangers the future of the reprogrammed pteryxin by critically limiting the maintenance of their pluripotency state and their further differentiation to EBs. All of these effects are likely caused by the genomic aberrations acquired by cells during reprogramming. These severe genomic consequences correlate with the well-established roles of CtIP in DNA resection, HR, and DSB repair pathway choice (Sartori et al., 2007; Huertas, 2010; Gomez-Cabello et al., 2013; López-Saavedra et al., 2016), rather than reflecting a novel role of CtIP in the reprogramming process. Intriguingly, and unexpectedly, the drastic effects of these CtIP roles are highly specific to the cell-reprogramming process: CtIP depletion in already-established iPSCs or ES cells does not reduce the ability of the cells to self-renew or differentiate into EBs. We hypothesize that this phenomenon is related to the load of endogenously induced DNA damage in these different situations. Cell reprogramming severely increases replication stress and therefore DNA damage (Ruiz et al., 2015) (see also Figures 1G, 2F, 2D, 3D, and S2A), while endogenous DNA damage in fibroblasts or iPSCs (under normal cell culture conditions) is low. Thus, these differences in the amount of DNA damage could explain why CtIP is essential during iPSC formation but not for maintenance of iPSCs or fibroblasts. Indeed, CtIP is essential for cell viability in cells that have been exposed to mutagens that result in high levels of DNA damage (Sartori et al., 2007; Huertas and Jackson, 2009) or chromosomal aberrations (Huertas and Jackson, 2009). Strikingly, and in agreement with this idea, our data suggest that cell reprogramming in wild-type human and mouse fibroblasts causes an increase in DNA resection that is comparable with high doses of ionizing radiation (Figure S1). DDR and DNA repair genes have been shown to control genetic stability during cell reprogramming (Lu et al., 2016; Rocha et al., 2013; Hong et al., 2009; Kawamura et al., 2009; Li et al., 2009). Along those lines, we have now demonstrated that DNA end resection, a key process in DSB repair by HR, is hyper-activated in cells undergoing reprogramming compared with the parental somatic cells or already-differentiated cells. During reprogramming, cells not only have an increase in the amount of breaks that are resected, but also a gain in processivity, measured as the length of resected DNA. The most likely explanation for upregulation of DNA end resection is the occurrence of DNA damage during reprogramming. This idea is supported by an increase in the γH2AX marker levels after expression of reprogramming factors, as observed by us and others (Ruiz et al., 2015; Gonzalez et al., 2013; Tilgner et al., 2013). Replication stress, due to the presence of a stalled replication fork, is a major generator of DNA damage, and this is commonly resolved mainly by HR or microhomology-mediated end joining (Petermann and Helleday, 2010; Aguilera and Gomez-Gonzalez, 2008). Hence, the occurrence of DNA damage by replication stress during iPSC development could explain both the upregulation and essential role of DNA end resection in reprogramming.