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. 2011 Aug 1;713(1-2):8-17.
doi: 10.1016/j.mrfmmm.2011.05.018. Epub 2011 Jun 28.

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

Jinshui Fan  1 , Carine Robert, Yoon-Young Jang, Hua Liu, Saul Sharkis, Stephen Bruce Baylin, Feyruz Virgilia Rassool
Affiliations

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

Jinshui Fan et al. Mutat Res. .

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.

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Conflict of interest statement

Conflict of interest statement

The authors indicate no potential conflict of interest.

Figures

Fig. 1
Fig. 1
ROS levels, γH2AX expression and cell cycle profile in hESC, iPSC and parental control cells. (A) Endogenous ROS was measured by staining hESC (H1 and H9), iPSC (iMSC and iLC2), and parental iPSC control cells (MSC and LC2) with H2DCFDA, followed by flow cytometric analysis. Results are representative of three independent experiments ±SD. (B) Representative immunofluorescence staining for γH2AX (red) in hESC, iPSC and parental iPSC control cells. Nuclei were counterstained with DAPI (blue, ×1000). (C) The number of γH2AX foci/cell are representative of the mean of three independent experiments ±SD. (D) Representation of cell cycle profile obtained after propidium iodide staining of hESC, iPSC and parental iPSC. (E) Graphic representation of the percentage of live cells in G1 (black bars), S (grey bars) and G2 (white bars) obtained in D. Results are representative of three independent experiments ±SD. p values are in text.
Fig. 2
Fig. 2
Immunoblotting analysis of HR and NHEJ proteins in hESC, iPSC and parental control cells. Immunoblotting analysis of (A) HR protein RAD51, NHEJ proteins XLF, DNA LigIV and XRCCIV, (B) NHEJ protein Ku70, and (C) PARP1, DNA LigIIIα and XRCC1 proteins that participate in BER, SSB and Alt NHEJ pathways was performed in WCE of hESC (H9 and H1), iPSC (iMSC and iLC2), and parental control cells (MSC and LC2). β-Actin was used as loading control.
Fig. 3
Fig. 3
In vitro NHEJ assays in hESC, iPSC and parental control cells. (A) Schematic diagram showing ligation of linear plasmid monomers to form plasmid dimers. (B) Representative autoradiographs of agarose gels showing ligation of (i) linearized pUC19 digested with BamHI endonuclease (compatible ends) and (ii) linearized pAcGFP1-N2 with KpnI/SacI (non-compatible ends), following incubation in WCE from H9, iMSC and MSC. (iii) Representative immunoblot of β-actin level in the three different WCE used for in vitro NHEJ presented in (Bi). (C) Graphic representation of relative ligation activity in H9, iMSC and MSC. Results are representative of three independent experiments ±SD. p values are shown.
Fig. 4
Fig. 4
Sequencing of DSB repair junctions in hESC, iPSC and control cells. (A) DNA sequence of DSB repair junctions were obtained after ligation of non-compatible ends linearized (Kpn1/SacI) plasmids in H9, iMSC and MSC. The occurrence of a given sequence (F) is indicated in black. Microhomologies (≥2 bp, M) found at the repair junction are indicated in green. Intact DNA sequence adjacent to the single strand overhang in the substrate is indicated in blue. Junctions with deleted sequences beyond the single strand overhang are considered inaccurate and shown in red. (B) Percentages (mean values and SD) of accurate junctions in clones from each set of experiments depicted in (A). (C) Graphic representation of the mean nucleotide loss in inaccurate junctions observed in plasmids recovered from H9, iMSC and MSC in vitro NHEJ. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 5
Fig. 5
Co-immunostaining for γH2AX and DSB repair proteins in hESC and iPSC after DNA damage induction. (A and B) Representative immunofluorescence staining for (A) γH2AX and Ku70 and (B) γH2AX and RAD51 in H9 and iMSC following 5 Gy 4 h irradiation. Cells were immunostained for Ku70 or RAD51 (green, left panel) and coimmunostained for γH2AX foci (red, middle panel). Right panel shows merged fluorescence patterns with co-immunostained regions in yellow. Nuclei are counterstained with DAPI (blue, ×1000).
Fig. 6
Fig. 6
Flow cytometric analysis of Annexin IV in irradiated hESC, iPSC and control cells. Representative dot blot of apoptotic cells detected by Annexin V–PE binding and 7AAD labeling in (A) H9, (B) iMSC and (C) MSC cells after the indicated times of irradiation (IR). Apoptotic cells in the lower right quadrant, necrotic cells in the upper right quadrant, and viable cells in the lower left quadrant were detected and the percentage of each population is presented.
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References

    1. Bhattacharya B, Puri S, Puri RK. A review of gene expression profiling of human embryonic stem cell lines and their differentiated progeny. Curr Stem Cell Res Ther. 2009;4:98–106. - PubMed
    1. Rao RR, Stice SL. Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events. Biol Reprod. 2004;71:1772–1778. - PubMed
    1. Lengner CJ. iPS cell technology in regenerative medicine. Ann N Y Acad Sci. 2010;1192:38–44. - PubMed
    1. Carpenter MK, Couture LA. Regulatory considerations for the development of autologous induced pluripotent stem cell therapies. Regen Med. 2010;5:569–579. - PubMed
    1. Thomas JW, LaMantia C, Magnuson T. X-ray-induced mutations in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 1998;95:1114–1119. - PMC - PubMed

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