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

Nature advance online publication 9 August 2009 | doi:10.1038/nature08287;
Received 15 May 2009; Accepted 17 July 2009; Published online 9 August 2009
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature08287.html

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

Rosa M. Marión^{1, 5}, Katerina Strati^{1, 5}, Han Li², Matilde Murga³, Raquel Blanco¹, Sagrario Ortega⁴, Oscar Fernandez-Capetillo³, Manuel Serrano², and Maria A. Blasco¹

1. Telomeres and Telomerase Group,
2. Tumor Suppression Group,
3. Genetic Instability Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid E-28029, Spain
4. Transgenic mice Unit, Biotechnology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid E-28029, Spain
5. These authors contributed equally to this work.

Correspondence to: Maria A. Blasco¹ M.A.B. (Email: mblasco@cnio.es).

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 (1, 2), 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.

http://www.nature.com/nature/journal/vaop/ncurrent/fig_tab/nature08287_F1.html#figure-title

Fig. 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.

http://www.nature.com/nature/journal/vaop/ncurrent/fig_tab/nature08287_F4.html#figure-title

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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