# Extracellular Vesicles From Mesenchymal Stromal Cells Drive Muscle and Neuronal Regeneration Through TNFα Modulation

## TL;DR

This study shows that extracellular vesicles from mesenchymal stromal cells help regenerate muscle and nerve tissue by reducing inflammation through TNFα modulation.

## Contribution

The study identifies TNFα modulation as a key mechanism by which extracellular vesicles promote muscle and neuronal regeneration.

## Key findings

- EVs protect muscle cells from death and stimulate proliferation by downregulating TNFα.
- EVs reduce neuroinflammation and promote axon sprouting in spinal cord models by inhibiting TNFα.
- GMP-grade EVs show potential for clinical applications in tissue regeneration.

## Abstract

Muscle defects caused by accidents, tumour resection and congenital malformations affect paediatric and adult patients. In this context, the neural‐muscle regeneration potential of mesenchymal stromal/stem cells derived from extracellular vesicles (EV) has been demonstrated by our group and others, but the mechanism by which EVs act remains unknown. This work aimed to investigate the neural‐muscle regeneration mechanism shown by EVs in vivo using three‐dimensional (3D) multicellular in vitro models. We used (1) human muscle decellularised tissue (ECM) engineered with human muscle precursor cells (hMPC) together with macrophages THP‐1 (M0) and (2) organotypic spinal cord from rat foetuses. We also studied neuroinflammation in 2D with primary microglia cells stimulated with lipopolysaccharide (LPS). Samples treated with good manufacturing practices (GMP)‐grade EVs were assessed, combining functional analyses, protein and gene expression. In the functional muscle model, EVs protect the cells from death after damage, decreasing cCAS3 and stimulating cell proliferation. The protein array and gene results highlighted that EVs act through the downregulation of the TNFα factor. In parallel, in both neuroinflammation‐induced microglia and organotypic spinal cord‐damaged models, EVs regulated the neuroinflammation by inhibiting TNFα and promoting neural axon sprouting. In summary, EVs guard great potential for tissue regeneration by TNFα modulation, promoting muscle‐neural regeneration.

Workflow of the work. (1) Human muscle biopsies were collected, and the human muscle precursor cells (hMPC) were isolated, followed by co‐culture with human monocytes (THP‐1). In parallel, human muscles were decellularised to obtain an extracellular matrix (ECM) free of cells. ECM were recellularised by injecting hMPC and THP1 into the matrix. The recellularised ECM was induced to skeletal muscle loss by damaging the sample with cardiotoxin (CTX). (2) Good Manufacturing Practice (GMP) grade mesenchymal stromal cells were expanded and the GMP‐grade extracellular vesicles (EVs) were isolated. (3) Recellularised damaged ECM was treated with EVs, and a reduction/downregulation of TNFα was observed. (4) Spinal cord was isolated from a rat foetus to create an organotypic spinal cord culture to analyse the axonal psprouting. (5) EVs were used to treat organotypic spinal cord culture supported sprouting of the neural projections and induced a reduction /downregulation of TNFα expression was observed (Image created using Biorender.com).