# Deconstructing heterogeneity of replicative senescence in human mesenchymal stem cells at single cell resolution

## TL;DR

This study uses single-cell analysis to uncover distinct pre-senescent and senescent states in human mesenchymal stem cells during replicative senescence.

## Contribution

The study identifies new pre-senescent and senescent cell states in mesenchymal stem cells and their regulatory gene networks.

## Key findings

- esMSCs transition through newly identified pre-senescent states before entering three distinct senescent states.
- Regulatory gene networks show reduced connectivity and altered gene expression as cells enter senescence.
- The findings reconcile different senescence programs within a single cell type and suggest potential senotherapeutic strategies.

## Abstract

Following prolonged cell division, mesenchymal stem cells enter replicative senescence, a state of permanent cell cycle arrest that constrains the use of this cell type in regenerative medicine applications and that in vivo substantially contributes to organismal ageing. Multiple cellular processes such as telomere dysfunction, DNA damage and oncogene activation are implicated in promoting replicative senescence, but whether mesenchymal stem cells enter different pre-senescent and senescent states has remained unclear. To address this knowledge gap, we subjected serially passaged human ESC-derived mesenchymal stem cells (esMSCs) to single cell profiling and single cell RNA-sequencing during their progressive entry into replicative senescence. We found that esMSC transitioned through newly identified pre-senescent cell states before entering into three different senescent cell states. By deconstructing this heterogeneity and temporally ordering these pre-senescent and senescent esMSC subpopulations into developmental trajectories, we identified markers and predicted drivers of these cell states. Regulatory networks that capture connections between genes at each timepoint demonstrated a loss of connectivity, and specific genes altered their gene expression distributions as cells entered senescence. Collectively, this data reconciles previous observations that identified different senescence programs within an individual cell type and should enable the design of novel senotherapeutic regimes that can overcome in vitro MSC expansion constraints or that can perhaps slow organismal ageing.

The online version contains supplementary material available at 10.1007/s11357-023-00829-y.