Novel insights from a multiomics dissection of the Hayflick limit

Abstract

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick’s original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by the transcription factors YAP1/TEAD1 and TGF-𝛽2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.

Accelerating the clock: Interconnected speedup of energetic and molecular dynamics during aging in cultured human cells

“…Our longitudinal age-related trajectories demonstrating profound bioenergetic recalibrations across the cellular lifespan agree with recent work in a single cell line of WI-38 human lung fibroblasts, indicating the increased abundance of OxPhos, TCA, and carbon metabolism proteins and transcripts across cellular passages, in parallel with the upregulation of glycolysis in senescence (Chan et al, 2022). The age-related increase in MR across the lifespan also is in line with a recent report demonstrating, in mice, an age-related elevation in both resting MR and MR associated with a given workload (i.e., metabolic efficiency) (Petr et al, 2021).…”

A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence

“…As a corollary of these conclusions from expression profiling, we note that cell cycle and DNA repair genes, classically known to be repressed during senescence (Kim et al 2013b; Chan et al 2022), did not hit a floor of expression in senescent cultures: we could detect them at even lower expression levels upon Usf2 knockdown. Since our cultures comprise >99% arrested cells within several days of irradiation (see Methods), the emerging picture is that the proliferation machinery is maintained at non-zero levels even in such a population.…”

“…Complex regulatory networks likely underlie many of the quantitative behaviors of senescent cells, including kinetics and dependence on cell type and inducer (Campisi 2013; Chan et al 2022; Casella et al 2019; Basisty et al 2020; Purcell et al 2014). Exactly how these nuances are encoded remains poorly understood.…”

“…However, given the complexity of the senescence program, many more regulators likely remain to be identified. Indeed, bioinformatic approaches have identified dozens of other transcription factor candidates (Chan et al 2022; Wang et al 2016; Xie et al 2014; Han et al 2018; Martínez-Zamudio et al 2020; Brückmann et al 2019; Tyler et al 2021; Zhang et al 2021), many of which remain unvalidated (but see (Wang et al 2016; Xie et al 2014; Han et al 2018; Martínez-Zamudio et al 2020) for recent discoveries of the roles of DLX2, FOXO3 and AP-1 in senescence).…”

“…However, given the complexity of the senescence program, many more regulators likely remain to be identified. Indeed, bioinformatic approaches have identified dozens of other transcription factor candidates in senescence (17)(18)(19)(20)(21)(22)(23)(24), many of which remain unvalidated (but see (18)(19)(20)(21) for recent discoveries of the roles of DLX2, FOXO3 and AP-1 in senescence).…”