Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.

Nature advance online publication 9 August 2009 | doi:10.1038/nature08285; Received 25 February 2009; Accepted 15 July 2009; Published online 9 August 2009
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature08285.html

"Immortalization eliminates a roadblock during cellular reprogramming into iPS cells".

Jochen Utikal ^{1, 2, 3, 6}, Jose M. Polo^{1, 2, 6}, Matthias Stadtfeld^1.2, Nimet Maherali^{1, 2, 4}, Warakorn Kulalert1,2, Ryan M. Walsh^{1, 2}, Adam Khalil^{1, 2}, James G. Rheinwald⁵ and Konrad Hochedlinger^{1, 2}

¹Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Harvard Stem Cell Institute, 185 Cambridge Street, Boston, Massachusetts 02114, USA
²Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA
³Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl-University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany
⁴Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA
⁵Department of Dermatology, Brigham and Women's Hospital and Harvard Skin Disease Research Center, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA
⁶These authors contributed equally to this work.

Correspondence to: Konrad Hochedlinger^{1, 2} Correspondence and requests for materials should be addressed to K.H. (Email: khochedlinger@helix.mgh.harvard.edu).

Abstract:

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, here we show that cells with low endogenous p19Arf (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) protein levels and immortal fibroblasts deficient in components of the Arf–Trp53 pathway yield iPS cell colonies with up to threefold faster kinetics and at a significantly higher efficiency than wild-type cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of Trp53 (also known as p53) in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. Our results show that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscore the similarities between induced pluripotency and tumorigenesis.

Figure 4:
http://www.nature.com/nature/journal/vaop/ncurrent/fig_tab/nature08285_F4.html#figure-title

Figure 4: Trp53 deficiency rescues reprogramming potential in cells that normally fail to form iPS cells.

a, Comparison of reprogramming potentials of sorted Thy1+, Thy1- and SSEA1+ subpopulations in wild-type and Trp53-/- cells at different time points (in days) during reprogramming in the presence or absence of doxycycline (dox).

b, Acute inactivation of Trp53 by lentivirus expressing Trp53 shRNA in secondary cells increases reprogramming efficiency at all time points.

c, Knockdown of Trp53 by Trp53 shRNA rescues the potential of Thy1- and Thy1+ subpopulations to generate iPS cells.

d, Model summarizing the presented data. During factor-induced reprogramming, cells encounter different roadblocks, such as the successful silencing of somatic genes (for example, Thy1), the activation of pluripotency genes (for example, SSEA1) and eventually the acquisition of immortality (for example, silencing of Arf). The low efficiency of the process is probably due to the capacity of rare cells to overcome these roadblocks. In immortal fibroblasts, however, almost every cell is endowed with the potential to produce iPS cells. Moreover, cells that have already encountered a roadblock can be rescued by acute inactivation of Trp53 (indicated by dashed black lines). Red bar illustrates the transition point between the somatic (blue) and the pluripotent (yellow) state.

Conclusion: Short RNA species (shRNA) are important in the initiation of iPS and /or neoplastic cells.

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