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

In recent years, mesenchymal stem cells (MSCs) have become one of the most widely utilized stem cell types in regenerative medicine, owing to their ability to modulate the inflammatory process^1,2. MSCs have shown promising results also in veterinary medicine, particularly in the treatment of equine osteoarthritis (OA), a prevalent joint disorder in horses characterized by a chronic inflammation that leads to the progressive damage of the articular cartilage and surrounding synovial structures, involving both cellular and humoral mediators^3,4.
It is well established that MSCs exert their effect via paracrine mechanisms, especially through extracellular vesicles (EVs)⁵. EVs, classified as exosomes or microvesicles, are described as particles delimited by a lipid bilayer containing proteins, lipids, mRNA and regulatory RNAs, such as microRNAs (miRNAs)⁶. The primary advantage associated with their use over cell-based therapies is represented by the absence of immunogenicity and tumorigenicity².
It has been demonstrated that the molecular content of EVs can be modulated by the surrounding extracellular microenvironment⁷. Further research is needed to explore the role of miRNAs in EVs and how MSC preconditioning (e.g. pro-inflammatory cytokines treatments) 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.

 

Aims

The aims of this study can be developed into three main issues:
The first is to characterize the miRNA content within EVs secreted by MSCs (MSC-EVs), aiming to identify a panel of miRNAs linked to the inflammatory response in OA, that will be considered for further analyses.
The second issue is to evaluate whether a significant modification in the miRNA expression occurs across different MSCs preconditioning treatments.
The third one is to better understand whether modifications in the molecular cargo of MSC-EVs are able to differently influence target cells that play a role in the chronic inflammatory response associated with equine osteoarthritis.

 

Materials and methods

1st year

MSCs from bone marrow and fat will be isolated and their identity confirmed through immunophenotyping (flow cytometry) and tri-lineage differentiation (in adipocytes, chondrocytes, osteoblasts)⁸. To minimize individual variability, pools of MSCs from 3 horses will be produced. Sample size will be defined according to an expected biologically relevant difference in miRNA expression.
In parallel, starting from previously obtained MSCs (3 horses), EVs will be isolated via ultracentrifugation and characterized for size and concentration using Nanosight LS300, which will perform an automated Nanoparticle Tracking Analysis (NTA). MiRNAs will be extracted and analysed through RT-qPCR: 23 miRNAs linked to the inflammatory response of OA (from literature) will be tested. The detectable ones will be used for further analyses.

2nd year

The effect of different medium conditionings will be tested on MSCs. Such conditionings (e.g. pro-inflammatory cytokines IL-1β and TNF-α; conditioned medium from cultured leukocytes subpopulations) will reflect the inflammatory environment of OA.
To set up these assays, leukocytes subpopulations will be isolated and cultured.
After treating MSCs, EVs will be isolated and characterized as previously described, and changes in miRNA expression will be identified through RT-qPCR.

3rd year

EV effect will be assessed on target cells involved in OA chronic inflammation (e.g. primary chondrocytes exposed to pro-inflammatory cytokines): 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.

 

Expected results

The expected outcomes aim to identify specific molecular mediators within MSC-EVs and determine which in vitro culture conditions may elicit the most significant effects on target cells involved in the chronic inflammatory response of equine osteoarthritis.
The lack of standardization in cell culture and EV isolation protocols presents a challenge in studying chronic joint diseases in horses using in vitro models. In this context, the harmonization of EV harvesting and isolation procedures, combined with the development of a cell-free product derived from MSCs represents a crucial step in advancing osteoarthritis research.
These findings could pave the way for a cell-free treatment to improve the management of diseases such as osteoarthritis, ultimately reducing drug usage and enhancing the overall health and well-being of horses.

 

References

1- 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.

2- 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.

3- Arévalo-Turrobiarte M., Baratta M., Ponti G., Chiaradia E., Martignani E. Extracellular vesicles from equine mesenchymal stem cells decrease inflammation markers in chondrocytes in vitro. Equine Vet J. 2022;54:1133–1143.

4- 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.

5- 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.

6- 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.

7- 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.

8- Dominici M. Le Blanc K., Mueller I., Slaper-Cortenbach I., Marini F., Krause D., Deans R., Keating A., Prockop D., Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8(4): 315-317.

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