Immortalization eliminates a roadblock during cellular reprogramming into iPS cells

Abstract

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells1–3. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that additional 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 fibroblasts into iPS cells decreases upon serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, we here demonstrate that cells with low endogenous ARF levels and immortal fibroblasts deficient for components of the p16INK4a/ARF/p53 pathway yield iPS colonies with a 3-fold faster kinetics and at a significantly higher efficiency compared with wild-type (WT) cells, endowing almost every somatic cell with the potential to form iPS cells. Importantly, acute genetic ablation of 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 pluripotent cell lines and tumor cells.

Mechanism and methods to induce pluripotency

“…Only a few cells can overcome this barrier and become iPSCs. A number of studies demonstrated that when key components (such as p53 and p21) of the DNA damage pathway were deleted, the percentage of iPS cell generation significantly increased (Zhao et al, 2008a;Banito et al, 2009;Hong et al, 2009;Kawamura et al, 2009;Li et al, 2009;Marión et al, 2009;Utikal et al, 2009b). Suppression of the p53 pathway may compromise a cell's genome stability.…”

“…As the DNA damage pathway poses a major barrier towards regaining pluripotency (Zhao et al, 2008b;Banito et al, 2009;Hong et al, 2009;Kawamura et al, 2009;Li et al, 2009;Marión et al, 2009;Utikal et al, 2009b), chemical compounds that alleviate the stress of DNA damage response may help cells to overcome this barrier. To this end, Vitamin C has been shown to significantly increase the formation of both mouse and human iPSC by suppressing p53 induced cell senescence (Esteban et al, 2010).…”

Mutant p53 subverts p63 control over KLF4 expression in keratinocytes

“…Thus, overexpression of this TF has profound and lasting consequences on the expression of growth-promoting genes. This process is influenced by the p53 status Kawamura et al, 2009;Mario´n et al, 2009;Utikal et al, 2009).…”

Pluripotency of Male Germline Stem Cells

“…Established GSCs from p53 knockout mice generate ES-like cells at a higher rate compared to wild-type, even after long periods in culture, indicating that p53 negatively controls the reprogramming process (Kanatsu-Shinohara et al, 2004). The importance of p53 activity has been confirmed in a series of publications describing enhanced iPS formation by p53 knockout or knockdown, although SSC reprogramming may not always take the same route before pluripotency is achieved (Hong et al, 2009;Kawamura et al, 2009;Li et al, 2009;Marión et al, 2009;Utikal et al, 2009). Genetic and epigenetic alterations of other tumor suppressors and cell cycle regulators are known to affect cell reprogramming and ES cell maintenance (Boominathan, 2009;Ruiz et al, 2011).…”