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.

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, p16^INK4a, 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.

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 p16^INK4a/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 p16^INK4a) 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 …