Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

Abstract

To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to "dedifferentiate" into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels, cell cycle profiles, repair protein expression and NHEJ repair efficacy, indicating reprogramming of the DNA repair pathways. Human iPSC however show a partial apoptotic response to irradiation, compared to hESC. We suggest that DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming.

A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors

“…39 However, recent studies in human ESC and iPSC suggest that this may be different in humans and both HR and NHEJ have a key role in repair of DSBs with specific roles at different stages of cell cycle progression. 27,28 Our results discussed in this manuscript suggest that this is indeed the case for impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs, enhanced apoptosis and increased genomic instability, despite the existence of a functional HR in these cells. On a more practical level, our study raises interesting questions on the ability of iPSC and more broadly embryonic and adult stem cells to preserve their genome integrity during ex vivo expansion.…”

“…These findings point to NHEJ as the main repair pathway used by human pluripotent stem cells to repair DNA damage caused by IR, corroborating previous findings. 27,28 Overexpression of DNA LIGASE IV restores NHEJ activity ( Figure 3a) and the ability to respond to IR, albeit not to the same extent as the wild-type control cells (Figures 3b-d).…”

“…32,33 Literature precedence suggests that both HR and NHEJ are utilized in human ESC and iPSC to repair DSBs; however, the main pathway used to repair DSBs caused by IR is highfidelity NHEJ. 27,28 To investigate the impacts of deficient NHEJ-mediated-DSB repair, we subjected all iPSC lines to IR and analysed the impacts by immunofluorescent microscopy analysis of DSB site specific phosphorylated H2A.X (gH2A.X) at different time points (Figures 3b-d). This analysis shows that all LIG4-iPSC lines are deficient in their DSB repair activity and are unable to repair the IR-caused damage even after 24 hours, despite the higher HR capacity (Figure 3a).…”

“…Previous work done by our group has shown that both human and mouse ESC possess efficient DNA repair and reactive oxygen species scavenging ability; however, this stress defence capability is downregulated during their differentiation. 25,26 Both HR and NHEJ (but not MMEJ) have been suggested to function in human ESC and the more recently generated human induced pluripotent stem cells (iPSCs) to maintain this genomic stability; 27,28 however, the impacts of dysfunctional NHEJ or HR have not been assessed either at the pluripotent stage or during their differentiation. In this manuscript, we have taken advantage of iPSC technology and have created a human iPSC model of LIG4 deficiency to investigate the impacts of deficient NHEJ on pluripotent stem cell renewal and their differentiation to haematopoietic lineages.…”

Poly(ADP-ribose) polymerase 1 regulates nuclear reprogramming and promotes iPSC generation without c-Myc

“…A recent study demonstrated that iPSCs (Armstrong et al, 2010), which are similar to ESCs, maintain genomic stability by elevated non homologous end joining (NHEJ) activity and DNA repair efficacy (Fan et al, 2011). Notably, Parp1 and PARylation have been linked to the regulation of chromatin remodeling and genome stability (Deng, 2009).…”