Background & Aims

Mesenchymal stem cells (MSCs) are promising therapeutic agents in treating different conditions, including osteoarthritis (OA). Recent investigations have underscored the pivotal role played by MSC-derived extracellular vesicles (EVs) that enable biomolecular cargo exchange between cells. We recently demonstrated the capacity of MSC-EVs to alleviate pain in a mouse OA model, which was due to MSC-EV amelioration of OA-induced sensory neuron hyperexcitability, rather than modifying joint pathology, thereby illuminating their potential in pain management. Notably, in vitro experimentation demonstrated that MSC-EVs directly influence sensory neuron function, reversing nerve growth factor (NGF)-induced nociceptor sensitization (NGF plays a key role in OA pain). In this study, we investigated the underlying mechanisms by which MSC-EV-mediated regulation of sensory neuron activity occurs, focusing on the internalisation of EVs by sensory neurons.

Methods

MSC-EVs were obtained by ultracentrifugation of conditioned media. Using the NGF – dorsal root ganglia (DRG) neuron sensitization model, neurons were treated with human bone marrow-derived MSC-EVs and neuron excitability was evaluated using whole-cell patch-clamp electrophysiology. Recordings were performed on isolectin B4 (IB4) negative neurons, those most likely to express the NGF receptor, tropomyosin receptor kinase A (TrkA). To remove protein from the external EV surface, so-called shaving, EVs were treated with either trypsin or proteinase K. To determine the impact of shaving on EV internalisation and modulation of neuronal function, confocal microscopy and electrophysiology were used, respectively.

Results

MSC-EVs isolated were spherical vesicles with characteristic lipid bilayer structures, consistent with the known morphology of EVs. Neither trypsin nor proteinase K treatment (“shaving”) changed EV morphology, but internalisation was inhibited and a reduction in MSC-EV modulation of neuronal activity was observed.

Conclusions

Interactions between MSC-EV extravesicular proteins and DRG neurons are required for EV regulation of DRG neuron acitvity, perhaps involving MSC-EV internalization. Further research is required to fully elucidate the mechanisms by which MSC-EVs regulate DRG neuron function and thus determine their potential for refining pain management.

References

Hunter, D.J., and Bierma-Zeinstra, S. (2019). Osteoarthritis. The Lancet 393: 1745–1759.
Syx, D., Tran, P.B., Miller, R.E., and Malfait, A.-M. (2018). Peripheral mechanisms contributing to osteoarthritis pain. Current Rheumatology Reports 20: 9.
Fuloria, S., Subramaniyan, V., Dahiya, R., Dahiya, S., Sudhakar, K., Kumari, U., et al. (2021). Mesenchymal stem cell-derived extracellular vesicles: Regenerative Potential and Challenges. Biology 10: 172.
Ai, M., Hotham, W.E., Pattison, L.A., Ma, Q., Henson, F.M.D., and Smith, E.St.John. (2023). Human mesenchymal stem cells and derived extracellular vesicles reduce sensory neuron hyperexcitability and pain behaviours in murine osteoarthritis. Arthritis & Rheumatology 75: 352–363.
Allen, N.R., Taylor-Mew, A.R., Wilkinson, T.J., Huws, S., Phillips, H., Morphew, R.M., et al. (2021). Modulation of rumen microbes through extracellular vesicle released by the rumen fluke calicophoron daubneyi. Frontiers in Cellular and Infection Microbiology 11: 661830.
Frazer, Z., Yoo, C., Sroya, M., Bellora, C., DeWitt, B.L., Sanchez, I., et al. (2020). Effect of different proteinase k digest protocols and deparaffinization methods on yield and integrity of DNA extracted from formalin-fixed, paraffin-embedded tissue. Journal of Histochemistry & Cytochemistry 68: 171–184.
Aloe, L., Tuveri, M.A., Carcassi, U., and Levi-Montalcini, R. (1992). Nerve growth factor in the synovial fluid of patients with chronic arthritis. Arthritis & Rheumatism 35: 351–355.

Poster Authors

Lanhui Qiu

BSc(Hons)

University of Cambridge

Lead Author

E-mail

Alexander Cloake

PhD

University of Cambridge

Lead Author

E-mail

Luke Pattison

PhD

University of Cambridge

Lead Author

E-mail

Javier Aguilera-Lizarraga

PhD

University of Cambridge

Lead Author

E-mail

Brandon Lockey

PhD candidate

University of Cambridge

Lead Author

E-mail

Tim Williams

PhD

University of Cambridge

Lead Author

E-mail

Ewan St. John Smith

PhD

University of Cambridge

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology