RESUMO
We present a protocol to evaluate the impact of senescence secretome on reprogramming to pluripotency using both cellular and mouse models. First, we describe the in vitro reprogramming procedure using conditioned medium derived from senescent cells. Next, to explore the impact of senescence on in vivo reprogramming, we detail the steps to identify senescent and reprogrammed cells in mouse skeletal muscle, followed by semi-automatic quantification. This protocol can be used to study the effect of paracrine senescence on cellular plasticity. For complete details on the use and execution of this protocol, please refer to von Joest et al. (2022).1.
Assuntos
Reprogramação Celular , Senescência Celular , Camundongos , Animais , Reprogramação Celular/genética , Senescência Celular/genética , Células CultivadasRESUMO
Cellular senescence is an irreversible growth arrest with a dynamic secretome, termed the senescence-associated secretory phenotype (SASP). Senescence is a cell-intrinsic barrier for reprogramming, whereas the SASP facilitates cell fate conversion in non-senescent cells. However, the mechanisms by which reprogramming-induced senescence regulates cell plasticity are not well understood. Here, we investigate how the heterogeneity of paracrine senescence impacts reprogramming. We show that senescence promotes in vitro reprogramming in a stress-dependent manner. Unbiased proteomics identifies a catalog of SASP factors involved in the cell fate conversion. Amphiregulin (AREG), frequently secreted by senescent cells, promotes in vitro reprogramming by accelerating proliferation and the mesenchymal-epithelial transition via EGFR signaling. AREG treatment diminishes the negative effect of donor age on reprogramming. Finally, AREG enhances in vivo reprogramming in skeletal muscle. Hence, various SASP factors can facilitate cellular plasticity to promote reprogramming and tissue repair.