"Linking the p53 tumour suppressor pathway to somatic cell reprogramming".

Teruhisa Kawamura ^{1., 2,  7}, Jotaro Suzuki ^{1, 3, 7}, Yunyuan V. Wang ¹, Sergio Menendez ⁴, Laura Batlle Morera ⁴, Angel Raya ^{4,  5,  6}, Geoffrey M. Wahl ¹, and  Juan Carlos Izpisúa Belmonte ^{1, 4}

¹ Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA
² Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Kyoto 606-8501, Japan
³ Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki 305-8585, Japan
⁴ Center of Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
⁵ Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluis Companys 23, 08010 Barcelona, Spain
⁶ Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Dr. Aiguader 88, 08003 Barcelona, Spain
⁷ These authors contributed equally to this work.

Correspondence to: Geoffrey M. Wahl (Email: wahl@salk.edu).
Correspondence and requests for materials to J.C.I.B. (Email: belmonte@salk.edu)

Abstract:

Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes 1, 2, 3, 4, 5, 6, 7, 8, but the low frequency and tendency to induce malignant transformation 9 compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

Supplementary Information:
http://www.nature.com/nature/journal/v460/n7259/suppinfo/nature08311.html

1. Krizhanovsky V, and Lowe SW,
"The promises and perils of p53".

2. Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega S, Fernandez-Capetillo O, Serrano M, Blasco MA.
"A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity".

3. Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, Khalil A, Rheinwald JG, and Hochedlinger K.
"Immortalization eliminates a roadblock during cellular reprogramming into iPS cells".

Supplementary Information:
http://www.nature.com/nature/journal/v460/n7259/suppinfo/nature08311.html

FIGURE 4. Downregulation of p53 activity increases reprogramming efficiency of human somatic cells.

FIGURE 4. Downregulation of p53 activity increases reprogramming efficiency of human somatic cells.

a, HEFs were infected with retroviruses encoding Oct4, Sox2 and Klf4 (three factors, or 3F) or Oct4, Sox2, Klf4 and c-Myc (four factors, or 4F) in combination with lentiviruses expressing control or p53 shRNA. Emerging colonies of iPS cells were immunostained with anti-Nanog antibody (top). p53-knockdown efficiency was examined by western blot (bottom).

b, Human primary keratinocytes were co-infected with four factors and retroviruses expressing GFP or p53DD. After 2 weeks, cells were stained for alkaline phosphatase activity (top). Expression of p53DD resulted in stabilization of wild-type p53 (bottom). Actin was used as a loading control.

c, The average number of iPS-cell-like colonies obtained from 10⁴ keratinocytes reprogrammed with three or four factors and retroviruses encoding GFP or p53DD, in the absence or presence of Nutlin-3a (n = 3). iPS-cell-like colonies were scored as having human ES-cell-like morphology and positive alkaline phosphatase staining. Owing to the numerous colonies generated in four-factor p53DD keratinocytes, quantification was done using 10⁴ cells. Error bars, s.d.

d, e, Colonies of human keratinocyte-derived iPS cells generated by three factors and p53DD show strong immunoreactivity for pluripotency-associated transcription factors (Oct4, Sox2, and Nanog) and surface markers (TRA-1-60 and TRA-1-81) (d) and differentiate in vitro into cell types that express markers of endoderm (alpha-fetoprotein, Foxa2), mesoderm (Gata4, sarcomeric alpha-actinin), and ectoderm (Tuj1, tyrosine hydroxylase (TH)) (e).

Additional References:

1. Frenster JH, and Hovsepian JA,  (October, 2007c)
 “Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms”.

37. Berx G, Raspe E, Christfori G, Thiery JP, and Sleeman JP, (November, 2007)
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Clin Exp Metastasis, 2007;24(8):587-97, Epub 2007 Nov3.

38. Sarrió D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, and Palacios J,  (Feb. 2008)
"Epithelial-Mesenchymal Transition in Breast Cancer Relates to the Basal-like Phenotype",
Cancer Research 68, 989-997, February 15, 2008.

39. Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Sharp PA, and  Jacks T., (March, 2008)
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40. Haigis KM, Kendall KR, Wang Y, Cheung A, Haigis MC, Glickman JN, Niwa-Kawakita M,  Sweet-Cordero A, Sebolt-Leopold J, Shannon KM, Settleman J, Giovannini M,  and  Jacks T.  (March 30, 2008)
"Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon".
Nature Genetics 40, 600 - 608 (2008). Published online: 30 March 2008 | doi:10.1038/ng.115)

41. Boyerinas B, Park S-M, Shomron N, Hedegaard MM, Vinther J, Andersen JS, Feig C, Xu J,  Burge CB, and Peter ME,  (April, 15, 2008)
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Cancer Research vol. 68, no. 8, pp. 2587-2591  (April 15, 2008).

42. Marcucci G, Radmacher MD, Maharry K, Mrózek K, Ruppert AS, Paschka P, Vukosavljevic T, Whitman SP, Baldus CD, Langer C, Liu C-G, Carroll AJ, Powell BL, Garzon R, Croce CM, Kolitz JE, Caligiuri MA, Larson RA, and Bloomfield CD,  (May 1, 2008)
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New England Journal of Medicine vol. 358: no. 18, pp. 1919-1928 May 1, 2008.)

43. Mani SA, Guo W, Liao M-J, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F,  Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, and Weinberg RA,  (May 16, 2008)
"The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells".
Cell, vol: 133, pp. 704-715 (May 16,  2008).

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