Research Article

Tissue damage and senescence provide critical signals for cellular reprogramming in vivo

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Science  25 Nov 2016:
Vol. 354, Issue 6315, aaf4445
DOI: 10.1126/science.aaf4445

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For cell reprogramming, context matters

Differentiated cells in a culture dish can assume a new identity when manipulated to express four transcription factors. This “reprogramming” process has sparked interest because conceivably it could be harnessed as a therapeutic strategy for tissue regeneration. Mosteiro et al. used a mouse model to study the signals that promote cell reprogramming in vivo. They found that the factors that trigger reprogramming in vitro do the same in vivo; however, they also inflict cell damage. The damaged cells enter a state of senescence and begin secreting certain factors that promote reprogramming, including an inflammatory cytokine called interleukin-6. Thus, in the physiological setting, cell senescence may create a tissue context that favors reprogramming of neighboring cells.

Science, this issue p. 10.1126/science.aaf4445

Structured Abstract

INTRODUCTION

The ectopic expression of transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) enables reprogramming of adult differentiated cells into pluripotent cells, known as induced pluripotent stem cells (iPSCs), that are functionally equivalent to embryonic stem cells. Expression of OSKM in vivo leads to widespread cell dedifferentiation and reprogramming within tissues and eventually to the formation of teratomas (tumors arising from iPSCs). The molecular mechanisms operating during in vitro OSKM-driven reprogramming have been extensively characterized; however, little is known about in vivo reprogramming.

RATIONALE

The process of OSKM reprogramming is inefficient both in vitro and in vivo. A number of cell-intrinsic barriers have been identified in vitro, most of which are activated by cellular damage and are particularly prominent in aged cells. Mechanistically, these cell-intrinsic barriers for reprogramming are primarily mediated by the tumor suppressors p53, p16INK4a, and ARF (the latter two are encoded by the Ink4a/Arf gene locus). In this work, we have investigated the effect of these tumor suppressors, cellular damage, and aging on in vivo reprogramming.

RESULTS

We found that the expression of OSKM in vivo not only triggers reprogramming of some cells but also inflicts extensive damage on many other cells, driving them into a state known as cellular senescence. Senescent cells are characterized by their inability to proliferate and by their secretion of inflammatory cytokines. We have observed a positive correlation between senescence and OSKM-driven reprogramming. For example, tissues lacking p16INK4a/ARF do not undergo senescence, and their ability to reprogram is severely compromised. By contrast, in tissues lacking p53, damage is rampant; this leads to maximal levels of senescence, exacerbated cytokine production, and increased in vivo reprogramming.

To explore the connection between senescence and reprogramming, we manipulated these processes in vivo through pharmacological interventions. In particular, an increase in senescence produced by palbociclib (a drug that functionally mimics p16INK4a) results in higher levels of reprogramming. Conversely, a reduction in senescence achieved by navitoclax (a proapoptotic drug with selectivity against senescent cells) leads to decreased in vivo reprogramming. We found that the cross-talk between senescence and reprogramming is mediated by the cytokine-rich microenvironment associated with senescent cells. This is based, among other evidence, on the observation that pharmacological inhibition of NFκB, a major driver of cytokine production, reduces in vivo reprogramming. Analysis of the inflammatory cytokines produced by senescent cells, both in vivo and in vitro, led us to identify interleukin-6 (IL-6) as a critical secreted factor responsible for the ability of senescent cells to promote reprogramming. In support of this, blockade of IL-6 or its downstream kinase effector PIM potently reduced in vivo reprogramming. These observations can be recapitulated in vitro, where reprogramming efficiency is strongly enhanced by the presence of damaged cells or by the conditioned medium derived from damaged cells. Moreover, immunodepletion of IL-6 from the conditioned medium abolished reprogramming.

Having established that senescence promotes reprogramming, we studied whether tissue injury leading to senescence has a positive effect on OSKM-driven reprogramming. In particular, we show that bleomycin-induced tissue damage strongly promotes reprogramming in the lung. Finally, aging, which is associated with higher levels of cellular senescence, also favors OSKM-driven reprogramming both in progeric and in physiologically aged mice.

CONCLUSION

The expression of OSKM in vivo triggers two different cellular outcomes: reprogramming in a small fraction of cells, and damage and senescence in many other cells. There is a strong positive association between these two processes, due to the fact that cellular senescence creates a tissue context that favors OSKM-driven reprogramming in neighboring cells. The positive effect of senescence on reprogramming is mediated by secreted factors, of which IL-6 is a key player. This also applies to tissue injury and aging, where there is an accumulation of senescent cells that send signals to surrounding cells to promote OSKM-driven dedifferentiation and reprogramming. A similar conceptual interplay may occur in physiological conditions, where damage-triggered senescence could induce cell dedifferentiation to promote tissue repair.

Interplay between cellular senescence and OSKM-driven reprogramming.

Expression of OSKM in vivo, apart from inducing the reprogramming of a small population of cells, also induces damage and senescence in many other cells. Senescent cells release factors that promote the reprogramming of neighboring cells, with IL-6 being a critical mediator. Tissue injury and aging, through the accumulation of senescent cells, favor in vivo reprogramming.

Abstract

Reprogramming of differentiated cells into pluripotent cells can occur in vivo, but the mechanisms involved remain to be elucidated. Senescence is a cellular response to damage, characterized by abundant production of cytokines and other secreted factors that, together with the recruitment of inflammatory cells, result in tissue remodeling. Here, we show that in vivo expression of the reprogramming factors OCT4, SOX2, KLF4, and cMYC (OSKM) in mice leads to senescence and reprogramming, both coexisting in close proximity. Genetic and pharmacological analyses indicate that OSKM-induced senescence requires the Ink4a/Arf locus and, through the production of the cytokine interleukin-6, creates a permissive tissue environment for in vivo reprogramming. Biological conditions linked to senescence, such as tissue injury or aging, favor in vivo reprogramming by OSKM. These observations may be relevant for tissue repair.

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