Abstract
Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay, we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives, but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response, revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK, a key NHEJ component, by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast, NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus, DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells.
Highlights
Pluripotent human embryonic stem cells are derived from the inner cell mass (ICM) of spare blastocysts and are able to differentiate into various cell types
It is possible that the chromosomal aberrations observed in human embryonic stem cells (hESCs) might reflect events similar to those that occur in a developing embryo at the blastocyst stage
We show that DNA-PK-dependent non-homologous end joining (NHEJ) suppresses the formation of chromatid breaks after irradiation during late G2, and most of the radiation-induced chromatid exchanges observed in hESCs result from DNA-PK activity
Summary
Pluripotent human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of spare blastocysts and are able to differentiate into various cell types. These cells are often used as an in vitro model of the ICM. The pluripotent cell state is maintained for a very limited time; hESCs can be grown indefinitely in culture and their capacity to self renew and to differentiate into any cell type can be preserved for prolonged periods of time. Cells with normal karyotypes are selected by an unknown mechanism, but hESCs accumulate chromosomal www.impactaging.com
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