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Source of ESC Genomic Instability Uncovered

Original article from STEM CELLS

"Decrease in Abundance of Apurinic-Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells"

Unfortunately, one of the characteristics of long term cultivation of human embryonic stem cells (hESCs) in vitro is the accumulation of chromosomal abnormalities (Spits et al and Lefort et al) which may be selected for to allow continued proliferation of hESCs under sub-optimal in vitro growth conditions (Spits et al, Baker et al and Harrison et al) .   Failure of base excision repair (BER), one mode of DNA damage repair, is associated with the increase in mutant frequency in germ cells with increasing parental age and is associated with the downregulation of apurinic/apyrimidinic endonuclease 1 (APE1) (Intano et al), which led researchers from the laboratory of Vladimír Rotrekl at Masaryk University, Brno, Czech Republic to study if such a mechanism may lead to the accumulation of mutations in hESCs.   They now report in Stem Cells that BER is less efficient during prolonged in vitro growth of hESCs and is correlated to a decrease in APE1 expression; thereby suggesting that this may be an important source of genomic instability and may allow for culture adaptation (Krutá et al).

The hESC lines utilised in this study began to show karyotypic instability between passage 13 and 64 (as measured by a mix of normal and aberrant mitoses) with stable karyotypic change observed between passages 42 and 76. These alterations included extra copies of chromosomes 1, 7, 10, and 12, and translocations of chromosome 12 and 17 and were all associated with accelerated growth rate. Throughout the culture period, telomerase activity was found to be high at every stage analysed, and non-homologous end joining (NHEJ) activity was similar throughout passages 33 to 290. However, BER activity (measured as the efficiency of short patch BER (Intano et al and Singhal et al) on G:U lesions studies (Walter et al)) was found to decrease in later passages (from 150). This decrease was correlated to a decrease in APE1 protein level between passages 59 and 168, although APE1 nuclear localisation was not altered. However, other proteins involved in BER (POL-β and OGG1) were not altered during long term passaging and, additionally, OCT4, NANOG and SSEA3 protein levels stayed at a similar levels suggesting that the loss in BER activity was not linked to the loss of pluripotency. Confirming these findings was the increase in BER activity in late passage hESCs after the addition of recombinant APE1, with levels returning to those similar to early passage hESCs.

DNA damage induction by ionizing radiation (IR) caused higher numbers of double strand breaks (DSBs) immediately after IR in early passage hESCs compared with late passage hESCs, although there was no difference in DSB levels at 15 minutes, while background levels of DNA damage in all hESCs was similar. By 45 and 60 minutes, DNA damage levels had returned to background level in early passage hESCs, but this took 90 minutes in late passage hESCs. Finally, downregulation of APE1 by siRNA prior to IR treatment in early passage hESCs caused a delay in repair, to a level similar to that observed in late passage hESCs.

Overall, this points to a failure in BER, caused specifically by a reduction in APE1, as a possible major source of genomic instability in hESCs, which can lead to culture adaptation. Moving forward with this knowledge, understanding the regulation of APE1 may be important to delineate why specifically APE1 is targeted and may perhaps allow the targeted suppression of culture adaptation and the acquisition of deleterious changes in hESCs which may cause their progeny to be functionally lacking and potentially tumourigenic.


References

  • Baker DE et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 2007; 25: 207–215.
  • Harrison NJ et al. Culture adaptation of embryonic stem cells echoes germ cell malignancy. Int J Androl 2007; 30: 275–281, discussion 281.
  • Intano GW et al. Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice. Mol Cell Biol 2002; 22: 2410–2418.
  • Intano GW et al. Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity. Nucleic Acids Res 2001; 29: 1366–1372.
  • Lefort N et al. Human embryonic stem cells reveal recurrent genomic instability at 20q11.21. Nat Biotechnol 2008; 26: 1364–1366.
  • Singhal RK et al. DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. J Biol Chem 1995; 270: 949–957.
  • Spits C et al. Recurrent chromosomal abnormalities in human embryonic stem cells. Nat Biotechnol 2008; 26: 1361–1363.
  • Walter CA et al. Mutation frequency declines during spermatogenesis in young mice but increases in old mice. Proc Natl Acad Sci USA 1998; 95: 10015–10019.

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.