The Ink4/Arf locus is a barrier for iPS cell reprogramming

Abstract

The mechanisms involved in the reprogramming of differentiated cells into induced Pluripotent Stem (iPS) cells by Oct4, Klf4 and Sox2 (3F) remain poorly understood 1 . The Ink4/Arf tumour suppressor locus encodes three potent inhibitors of proliferation, namely p16 Ink4a , p15 Ink4b and Arf, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals 2-4 . We show here that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated upon differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the 3F together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of stemness. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus implying that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive impact on the efficiency of iPS generation, increasing both the kinetics of reprogramming and the number of emerging iPS colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming through activation of p53 and p21; whereas, in human fibroblasts, INK4a is more important than ARF. Finally, organismal aging upregulates the Ink4/Arf locus 2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with an shRNA. All together, we conclude that the silencing of Ink4/Arf locus is rate limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS.The Ink4/Arf tumour suppressor locus encodes three important tumour suppressors that activate two critical anti-proliferative pathways, namely, the Rb and p53 pathways, whose activation prevents the propagation of aberrant cells, either by apoptosis or senescence (see scheme in Supplementary Fig. S1) 4 . Briefly, the paralogs p16 Ink4a and p15 Ink4b bind and inhibit the cyclin D-dependent kinases Cdk4 and Cdk6, which in turn are important to relieve the cell-cycle inhibitory activity of the Rb tumour suppressor. On the other hand, Arf

Blockade of senescence‐associated micro RNA ‐195 in aged skeletal muscle cells facilitates reprogramming to produce induced pluripotent stem cells.

“…3A) as reported in earlier findings (Li et al ., 2009; Kim et al ., 2010; Cheng et al ., 2011; Wang et al ., 2011; Rohani et al ., 2014). We reprogrammed SkMs from both young and old mice using classic Yamanaka factors, and colonies were stained with alkaline phosphatase (ALP) on day 16–18 and counted to assess reprogramming efficiency.…”

“…Previous studies have shown that older and senescent cells are more resistant to reprogramming (Li et al ., 2009; Kim et al ., 2010; Cheng et al ., 2011; Wang et al ., 2011; Rohani et al ., 2014). These observations have established the importance of current efforts underway to understand the biology of cell reprogramming in the context of chronological and replicative age.…”

“…Previous reports demonstrated that age‐dependent decline in reprogramming efficiency is correlated with activated intrinsic senescence pathways in cells from old donors (Zhao et al ., 2008; Kawamura et al ., 2009; Li et al ., 2009). Reprogramming SkMs into iPSCs reverses several cellular and molecular characteristics associated with aging, including senescence, telomere truncation, mitochondrial dysfunction, and global changes in gene expression.…”

“…This could be of significant interest to elderly people because many age‐associated deficits that were once thought to be permanent may be reversible (Lapasset et al ., 2011). Interestingly, gene expression profiles of iPSCs derived from old mice and human subjects can also be reset to an embryonic‐like state (Li et al ., 2009; Lapasset et al ., 2011; Wang et al ., 2011). For example, reprogramming can trigger an increase in telomere length in cells of both old mice and humans, reversing the erosion characteristics of aged and senescent cells.…”

“…Unlike ESCs, iPSCs are not subject to immune rejection because they are derived from a patient's own cells (Isobe et al ., 2014), and present no ethical harvesting concerns related to the obtaining human ESCs for derivation. Although murine studies reveal that cell reprogramming efficiency declines with age, the potential to generate iPSCs from a patient's own cells has emerged as a promising strategy for autologous cell‐based therapy (Li et al ., 2009; Kim et al ., 2010; Cheng et al ., 2011; Wang et al ., 2011; Rohani et al ., 2014). …”