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. 2009 Aug 27;460(7259):1149-53.
doi: 10.1038/nature08287. Epub 2009 Aug 9.

A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity

Rosa M Marión  1 , Katerina Strati, Han Li, Matilde Murga, Raquel Blanco, Sagrario Ortega, Oscar Fernandez-Capetillo, Manuel Serrano, Maria A Blasco
Affiliations

A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity

Rosa M Marión et al. Nature. .

Abstract

The reprogramming of differentiated cells to pluripotent cells (induced pluripotent stem (iPS) cells) is known to be an inefficient process. We recently reported that cells with short telomeres cannot be reprogrammed to iPS cells despite their normal proliferation rates, probably reflecting the existence of 'reprogramming barriers' that abort the reprogramming of cells with uncapped telomeres. Here we show that p53 (also known as Trp53 in mice and TP53 in humans) is critically involved in preventing the reprogramming of cells carrying various types of DNA damage, including short telomeres, DNA repair deficiencies, or exogenously inflicted DNA damage. Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells.

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Figures

Figure 1
Figure 1. p53-deficiency allows reprogramming of MEFs with short telomeres
a, b, Relative reprogramming efficiencies are shown, with the fold changes indicated. Student’s t-test (two-tailed) is used for statistics. Error bars, standard error. n = experiments with independent MEFs. 3F, three factors; WT, wild type. c, Reprogramming plates stained with alkaline phosphatase. The number of parental cells used is indicated. d, Reprogramming of BJ human fibroblasts with four factors (4F) together with a human shRNA against the human p53 gene. Fold changes relative to BJ + 4F (BJ) are indicated. e, Reprogramming plates stained with alkaline phosphatase. f, Relative reprogramming efficiencies of wild-type MEFs exposed to UVC (UV) or ionizing radiation (IR), and expressing three factors together with a retrovirus expressing mouse p53 shRNA or Bcl2. Error bars, standard deviation. Ctrl, control.
Figure 2
Figure 2. p53-deficiency abrogates apoptosis at the onset of iPS cell formation
a, b, Apoptosis and necrosis are determined on days 9 and 10 (a) and 11-13 (b) post-infection. Data are mean and s.e.m. A Student’s t-test is used for statistics. c, Representative FACS profiles at day 9 of reprogramming. PI, propidium iodide. 7-AAD, 7-amino-actinomycin D. d, Western blots of p53 and p21 protein levels at day 9 post-infection. As a control, wild-type MEFs were γ-irradiated (10 Gy). Two reprogramming experiments per genotype are shown. e, Quantification of p53 and p21 westerns shown in d. Values are in arbitrary units (a.u.). Error bars, standard error.
Figure 3
Figure 3. DDR activation during reprogramming
a, b, γH2ax and 53BP1 foci 9 days after infection (a) and in iPS cell clones (b). Results are the mean of two experiments. Two-hundred cells were analysed per genotype/experiment. Error bars, s.d. c, Left, telomere-induced DNA damage foci (TIF) 9 days after infection. n = cells with γH2ax foci analysed. Values correspond to two reprogramming experiments. Error bars, standard error. Student’s t-test was used for statistics. Right, representative images of γH2ax (red) and Trf1 (green) staining. Arrows indicate co-localization events (yellow). Original magnification, ×63. d, Representative images that are quantified in a. White arrows, pan-nuclear γH2ax; yellow arrows, co-localization of γH2ax and 53BP1. Scale bars, 10 μm. e, Western blot (right) showing Atm phosphorylation 9 days after infection. Atm1987P denotes Atm protein phosphorylated at Ser 1987. Numbers above each lane represent the relative quantification of Atm levels. Two western blots were used for quantification (left). Error bars, standard deviation. f, Relative reprogramming efficiencies of 53BP1−/− and Atm−/− MEFs compared to wild-type MEFs. Fold changes are indicated. Student’s t-test is used for statistics. Error bars, standard error. n = experiments performed with independent MEF cultures. g, Reprogramming plates stained with alkaline phosphatase.
Figure 4
Figure 4. p53-null iPS cells show chromosomal instability
a, b, Frequency of end-to-end fusions (a) and breaks/fragments (b) in the indicated cells. n = metaphase number. The number of aberrations out of the chromosomes scored is indicated. Student’s t-test was used for statistics. Error bars, s.e.m. c, Representative metaphases. Red arrows, end-to-end fusions. Original magnification, ×100. d, Percentage of signal-free ends. n = telomeres used for the analysis. Chi-square test is used for statistics. e, Average telomere elongation (kilobase (kb)) in iPS cell clones compared to parental MEFs. n = telomeres used for the analysis. At least two independent iPS cell clones were used per genotype. MEF passage number = 3; iPS cell passage number = 4-6. Error bars, s.e.m. f, Summary illustrating that p53 constitutes a main barrier to reprogramming of cells with increased DNA damage by preventing that they become iPS cells.
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