PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Eugenio Martignani

Curriculum vitae

Curriculum Vitae

Phd thesis

Title: An in vitro investigation of extracellular vesicles as mediators of the crosstalk between horse mesenchymal stem cells and the inflammatory environment in equine osteoarthritis

 

Scientific background/state of the art

Osteoarthritis (OA), a degenerative chronic joint disease characterized by the progressive damage to the articular cartilage, subchondral bone and surrounding synovial structures, represents the most common cause of retirement from athletic activities in horses^1,2. Although current pharmacological treatments can be used to alleviate symptoms, the restoration of damaged cartilage is not achieved^3,4.

As a result, mesenchymal stem cells (MSCs) have emerged as a promising alternative in regenerative medicine owing to their paracrine effects, largely mediated by extracellular vesicles (EVs)^3,5,6. EVs are particles released into the microenvironment used as means of communication between cells, transporting proteins, lipids, mRNA and regulatory RNAs, such as microRNAs (miRNAs)⁷. It is well established that EV molecular content can be modulated by the surrounding extracellular microenvironment⁸; however, further research is still needed to explore the role of miRNAs in EVs and how MSC preconditioning influences their molecular content. Such insights could be pivotal in enhancing our understanding of EVs as mediators in the crosstalk between MSCs and target cells, offering potential advancements in the treatment of equine OA.

 

Specific aims of the project, Methods, Results

The overall objective of the project will be achieved through three successive milestones:

 

1. Characterization of the miRNA content within EVs secreted by MSCs (MSC-EVs) in order to detect a subset of miRNAs linked to the inflammatory response in OA.
   
   Following already standardized protocols, bone marrow and fat were sampled from 3 horses at a slaughterhouse. MSCs were isolated and their identity confirmed through immunophenotyping (flow cytometry) and tri-lineage differentiation (in adipocytes, chondrocytes, osteoblasts).
   Moreover, previously obtained MSCs (3 horses, from previous projects of the Physiology Lab) were used to isolate EVs via ultracentrifugation and characterize them (using an external service) for size distribution and concentration using Nanosight LS300, which performed an automated Nanoparticle Tracking Analysis (NTA).
   A literature analysis was carried out to identify a panel of miRNAs (23) linked to the inflammatory response of OA. Subsequently miRNAs were extracted from isolated EVs and RT-qPCR was performed to assess the presence of the selected miRNA panel.
   
   Currently, MSCs were successfully isolated and their identity confirmed through flow cytometry and tri-lineage differentiation.
   MSC-EVs were isolated and NTA showed a concentration mean of 2,57 x10¹⁰ particles/mL, and a size mean of 139,40 nm. Moreover, 9 out of the 23 miRNAs were detected in EVs.

 

2. Evaluation of significant modifications in the miRNA expression across different MSC preconditioning treatments that mime an inflammatory development or status
   
   To mimic the effect of an inflammatory environment on EVs released by MSCs, the effect of different supplements (e.g. pro-inflammatory cytokines IL-1β and TNF-α) will be tested on 6 MSC pools of 3 horses (produced to reduce individual variability). EV isolation and characterization will be performed via NTA and RT-qPCR, where the 23 miRNA preliminary panel will be tested to identify changes in their expression.

 

3. Assessment of the influence of different molecular cargos of MSC-EVs on target cells playing a role in the chronic inflammatory response associated with equine OA.
   
   An inflammation model will be used to assess EV effects on target cells involved in OA chronic inflammation. Primary chondrocytes exposed to pro-inflammatory cytokines will receive supplementation of EVs collected from MSCs conditioned with different factors: functional performances of EVs will be evaluated through in vitro assays (proliferation, wound healing), and changes in gene expression will be analysed through RT-qPCR.

 

Future developments

During the first part of the 2^nd year, sample collection and characterization will continue. The remainder of the year will focus on assessing significant modifications in the miRNA expression across different MSC preconditioning treatments and the in vitro model will be set up partially in collaboration with Ghent University.
The 3^rd year will be focused on the assessment of the effect of different MSC-EV molecular cargos on target cells involved in the chronic inflammatory response of equine OA.

 

References

1. Giorgino R., Albano D., Fusco S., Peretti G. M., Mangiavini L., Messina C. Knee Osteoarthritis: Epidemiology, Pathogenesis, and Mesenchymal Stem Cells: What Else Is New? An Update. Int. J. Mol. Sci. 2023, 24, 6405.
   
   
2. O’brien T., Hollinshead F., Goodrich L. Extracellular vesicles in the treatment and prevention of of osteoarthritis: can horses help us translate this therapy to humans? Extracell Vesicles Circ Nucleic Acids 2023;4:151-69.
   
   
3. Hotham W., Thompson C., Szu-Ting L., Henson F. The anti-inflammatory effects of equine bone marrow stem cell-derived extracellular vesicles on autologous chondrocytes. Vet Rec Open. 2021;8:e22.
   
   
4. Webster A., Pezzanite L., Hendrickson D., Griffenhagen G. Review of intra-articular local anaesthetic administration in horses: Clinical indications, cytotoxicity, and outcomes. Equine Vet J. 2024;56:870–883.
   
   
5. Liu Y., Sun L., Li Y., Holmes C. Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities. J Extracell Vesicles. 2024;13:e12472.
   
   
6. Martinez-Arroyo O., Ortega A., Forner M., Cortes R. Mesenchymal Stem Cell-Derived Extracellular Vesicles as Non-Coding RNA Therapeutic Vehicles in Autoimmune Diseases. Pharmaceutics 2022, 14, 733.
   
   
7. Tang J., Wang X., Lin X., Wu C. Mesenchymal stem cell-derived extracellular vesicles: a regulator and carrier for targeting bone-related diseases. Cell Death Discov. 2024;10:212.
8. Kang M., Huang C., Gajendrareddy P., Lu Y., Shirazi S., Ravindran S., Cooper L. Extracellular Vesicles From TNFa Preconditioned MSCs: Effects on Immunomodulation and Bone Regeneration. Front. Immunol. 2022;13:878194.

Training:

• Webinar “Ultracentrifugation Meets Nano-Flow Cytometry: A Practical Workflow for Extracellular Vesicle Enumeration and Integrity Assessment” (Beckman Coulter), 25/09/2025

• Webinar “Extracellular Vesicle Essentials 1: Methods for Isolating and Characterizing Extracellular Vesicles” (Promega, INOVIQ), 09/07/2025

• Cycle of Seminars on Scientific Writing (Springer Nature), 7/05/2025 – 25/06/2025

• Course “Light Field and Fluorescence Microscopy” (Nikon), 19/06/2025

• Workshop “Biology and Applications of Extracellular Vesicles” (University of Tuscia), 12/02/2025

ORCID: orcid.org/0009-0009-5755-1886