Therapeutic effects of extracellular vesicles derived from genetically modified mesenchymal stem cells on a mice model of neuropathic pain.

Introduction: Neuropathic pain, a prevalent type of chronic pain, lacks effective therapy. Mesenchymal stem cells (MSC) have shown promise in treating such conditions, primarily through their paracrine action. This action involves the secretion of biologically active compounds, including extracellular vesicles (EV). Pre-clinical research has demonstrated the potential of MSC and MSC-derived extracellular vesicles in neuropathic syndromes. Notably, genetic modifications of MSC have been found to enhance their therapeutic effect. In this context, the present study investigates the therapeutic effects of human umbilical cord MSC overexpressing leukemia inhibitory factor (MSC-LIF) and EV isolated from them (EV-LIF) in a model of painful neuropathy.

Methods: MSC-LIF were serum starved for 48h before the supernatant collection for EV isolation. EV-LIF were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Dotblot. The neuropathic pain model induced by partial sciatic nerve ligation (PSL) in C57Bl/6 mice was used (Animal Ethics Committee IGM/FIOCRUZ 020/2022). Seven days after model induction, each mouse received a single intravenous injection of 1x10⁶ MSC-LIF, EV-LIF (2.47x10⁹ ± 7.13x10⁷ particles/mL), or vehicle (100µL saline). The antinociceptive effect was assessed using Hargreaves and von Frey filaments tests. Effects on physical capacity were assessed using a treadmill test.

Results: Each 1x10⁶ MSC produced 2.47x10⁹ ±7.13x10⁸ EV particles/mL. The EV were of an average size of 167.1 ± 1.8 nm, with the 10th and 90th percentiles at 121.5 ± 1.2 nm and 233.0 ± 4.2 nm, respectively. The EV showed typical spherical morphology and expressed CD9, CD81, and TSG101 markers. A single injection of MSC-LIF or EV-LIF provided consistent and long-lasting relief of PSL-associated thermal hypernociception throughout the experimental period, with no significant differences between the effects of MSC-LIF or EV-LIF. However, the mechanical antinociceptive effects of both MSC-LIF and EV-LIF were transitory, with EV-LIF showing a greater magnitude of effect than MSC-LIF. Furthermore, each of the treatments restored the physical capacity of neuropathic mice, whose run time and walked distance was similar to non-neuropathic mice on the treadmill test.

Conclusion: MSC-LIF and EV-LIF similarly improve nociceptive and functional parameters in the PSL neuropathic pain model. Plus, these data indicate that EV preserves the therapeutic properties of genetically modified MSC and has the potential to be used as a cell-free therapy for neuropathic pain.