|Source of ESC mutations Uncovered?|
Original article from STEM CELLS
The long term culture of human embryonic stem cells (hESCs) is known to have some detrimental effects, such as the acquirement of an abnormal karyotype, increased copy number variations, loss of heterozygosity, increased rate of proliferation and resistance to apoptosis (Draper et al, Baker et al and Narva et al). These changes are all indicative of the gain of a tumourigenetic phenotype, so it is not surprising that culture-adapted hESCs form teratocarcinomas when transferred into severe combined immunodeficiency (SCID) mice, while non-adapted hESCs lead to teratoma formation (Solter, Blum and Benvinisty and Andrews). How long term culture leads to this effect is relatively unknown, although it is thought that uncontrolled cell cycle checkpoints and abnormal DNA damage response and repair are among the mechanisms contributing to hESC adaptation and major karyotypic changes (Rodriguez-Jiminez et al, Mantel et al and Momcilovic et al). In a study described in Stem Cells, researchers from the laboratories of Peter W. Andrews and Thierry Nouspikel at theUniversity of Sheffield, United Kingdom have now analyzed one mode of DNA damage repair; Nucleotide Excision Repair (NER) with respect to the DNA damage response and cell cycle checkpoints in hESCs and have found that point mutations result from a combination of defects in the DNA damage signaling pathway which leads to incomplete arrest at cell cycle checkpoints, the rapid proliferation rate of hESCs and insufficient NER activity (Hyka-Nouspikel et al).
Although mild UV irradiation (5 J/m2 254 nm) causes little apoptosis in fibroblasts and lymphoblasts, the same treatment of low passage hESCs caused massive apoptosis (70% by 6 hours), yet those that survived irradiation rapidly resumed proliferation and increased the number of viable cells within 24 hours. Further analysis found that irradiated hESCs in S-phase did not accumulate in G2 and indeed some hESCs re-entered S-phase, suggesting that the G1/S phase checkpoint was not operational. UV irradiation mainly activates the ATR-Chk1 pathway in response to DNA damage and in irradiated hESCs Chk1 was phosphorylated and activated, alongside some ATM activation. The G1/S checkpoint also involves p53 phosphorylation and activation with subsequent induction of p21, which can inhibit Cdk2 and Cdk4, stopping the cell cycle. In irradiated hESCs, phospho-p53 increased leading to increases in p21 mRNA but not protein, again suggesting that the G1/S checkpoint was not operational, as has been previously shown in another recent Stem Cells paper (Dolezalova and Mraz et al).
Interestingly, cells which survived irradiation showed no alteration in pluripotency-associated cell-surface markers (SSEA3, SSEA4, TRA-1-60, or TRA-1-81) or clonogenic potential, ability to form embryoid bodies (EBs) or differentiate into the three germ layers suggesting that the UV-irradiated surviving hESCs are still pluripotent. Interestingly, re-irradiation of surviving cells demonstrated that these have an increased tolerance as denoted by a lower level of apoptosis, suggesting that DNA repair is more efficient in UV-resistant hESCs. The two main UV-induced lesions are cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine pyrimidone photoproducts [(6-4) PPs], with (6-4) PPs being good NER substrates. In line with this, (6-4) PPs were rapidly removed, while CPDs, which require a specialized damaged DNA binding (DDB) complex, were not. Indeed, at 24 hours when hESCs began to resume proliferation, CPDs lesions still remained, which constitute strong blocks for replication forks. This can be overcome by switching of polymerases (replicative polymerases to specialized TLS polymerases) orchestrated by proliferating cell nuclear antigen (PCNA) monoubiquitination, which was observed after irradiation in this study. However, the TLS lacks proof reading capabilities and its use constitutes an error-prone process (McCulloch and Kunkel) and through the use of specialized PCR analysis to pick up a pattern that is considered a signature UV-induced mutation, increased mutagenesis levels were observed.
In conclusion, this study demonstrates that apoptosis seems to be the main response of hESCs to DNA damage, however due to the lack of p21 protein synthesis and therefore a deficient G1/S checkpoint, this strategy is not efficient. This is further combined with the relatively low efficiency of DNA damage repair through NER in surviving cells leading to the propagation of point mutations. Additionally, these findings represent an important consideration in ESC-based cell replacement therapy; ESCs used for such purposes are likely to be cultured long-term and, while cultured hESCs are unlikely to be exposed to UV, other stressors are likely to cause DNA damage events. If indeed surviving cells with DNA damage are less prone to apoptosis, these might be positively selected for, resulting in cultures enriched with ESCs carrying mutations.
Baker DE et al.
Blum B, Benvenisty N.
Dolezalova D, Mraz M et al.
Draper JS et al.
Hyka-Nouspikel N et al.
Mantel C et al.
McCulloch SD, Kunkel TA.
Momcilovic O et al.
Narva E et al.
Rodriguez-Jimenez FJ et al.
STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.