Abstract
BackgroundInduced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Methodology/Principal FindingsHere, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G2 phase of the cell cycle, displaying a lack of the G1/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.Conclusions/SignificanceHigh degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.
Highlights
Induced pluripotent stem cells are produced by reprogramming somatic cells with a defined set of transcriptional factors
Pluripotency and radiosensitivity in human induced pluripotent stem cells Induced pluripotent stem cells share numerous similarities with embryonic stem (ES) cells, including self-renewal, differentiation into all three germ layers, and expression of markers found in ES cells, such as OCT4, NANOG, SOX2, SSEA-3 and SSEA-4 [2]
In order to confirm that we are investigating DNA damage response of pluripotent cells we examined expression of the pluripotency markers NANOG, SSEA-4 and OCT4 in both untreated cells and cells irradiated with one Gray (Gy) of cirradiation (Figure 1)
Summary
Induced pluripotent stem (iPS) cells are produced by reprogramming somatic cells with a defined set of transcriptional factors They share numerous characteristics with embryonic stem (ES) cells, such as the ability to undergo self-renewal and differentiation, as well as expression of the same pluripotency markers NANOG, OCT4, SOX2 and SSEA-4 [1]. OCT4, SOX2 and NANOG are master transcriptional regulators of the pluripotent state in embryonic stem (ES) cells [4,5,6,7] These three transcription factors bind to and activate expression of genes that are involved in maintaining pluripotency, while repressing genes involved in differentiation [8]. Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. Before these cells can be used in therapeutic designs, it is essential to understand their genetic stability
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