# Uncovering the bequeathing potential of apoptotic mesenchymal stem cells via small extracellular vesicles for its enhanced immunomodulatory and regenerative ability

## TL;DR

This study shows that inducing apoptosis in mesenchymal stem cells improves the immunomodulatory and regenerative abilities of their extracellular vesicles.

## Contribution

The novel approach of inducing apoptosis in MSCs to enhance sEVs' immunomodulatory and regenerative potential is introduced.

## Key findings

- sEVs from apoptotic MSCs (sEVsApo) showed enhanced immunomodulation by suppressing T-cell proliferation and promoting T-regulatory cells.
- sEVsApo reduced mitochondrial ROS and improved oxidative phosphorylation in vitro and ameliorated fibrosis and inflammation in a CLD model in vivo.
- WJ-sEVs demonstrated superior performance compared to other tissue sources in terms of immunomodulatory effects.

## Abstract

Mesenchymal Stem Cells-derived Small Extracellular Vesicles endowed with regenerative cargo from their parent cells, have emerged as a promising avenue for cell-free therapeutics in regenerative medicine. Notably, deliberate induction of apoptosis in MSCs before sEV isolation has been identified as a strategy to augment the regenerative capabilities of MSCs-sEVs. This study explores a novel approach to enhance the immunomodulatory potential of MSC-sEVs through apoptosis induction and optimal tissue source to ensure consistent and improved clinical outcomes.

Apoptosis was induced in tissue-specific MSCs using Staurosporine. sEVsV and sEVsApo were isolated via ultracentrifugation. Invitro immune response was assessed via T-cell proliferation, T-regulatory cell induction & macrophage polarization assay. Mitochondrial bioenergetics was studied using MitoSOX staining and Seahorse assay in H2O2-treated HuH7 cells. These findings were validated invivo in the CCL4-induced Chronic Liver Disease model via Histopathological staining, biochemical parameters, and fibrotic, pro-inflammatory, and anti-inflammatory markers and assessed the mechanism by targeting TGF-β/SMAD pathway.

Our results demonstrate that sEVsApo exhibited significantly higher concentrations and superior immunomodulatory effects by suppressing CD3 + T-cell proliferation, promoting T-regulatory cell differentiation, and polarized macrophages towards M2-phenotype. In terms of tissue specificity, it was observed that WJ-sEVs were faring better. sEVsApo effectively reduced mitochondrial ROS & significantly improved oxidative phosphorylation. Invitro findings were corroborated in an invivo CLD model, wherein sEVsApo ameliorated fibrosis and inflammation, by inhibiting TGF-β/ SMAD2/3 pathway.

This study concludes that apoptosis induction can be considered as minimum manipulation strategy to enhance the immunoregulatory and regenerative potential of MSCs-sEVs, thereby expanding their implication in immune disorder.

The online version contains supplementary material available at 10.1186/s13287-025-04370-x.