Equine osteoarthritis: Strategies to enhance mesenchymal stromal cell-based acellular therapies

International audienceOsteoarthritis (OA) is a degenerative disease that eventually leads to the complete degradation of articular cartilage. Articular cartilage has limited intrinsic capacity for self-repair and, to date, there is no curative treatment for OA. Humans and horses have a similar articular cartilage and OA etiology. Thus, in the context of a One Health approach, progress in the treatment of equine OA can help improve horse health and can also constitute preclinical studies for human medicine. Furthermore, equine OA affects horse welfare and leads to significant financial losses in the equine industry. In the last few years, the immunomodulatory and cartilage regenerative potentials of mesenchymal stromal cells (MSCs) have been demonstrated, but have also raised several concerns. However, most of MSC therapeutic properties are contained in their secretome, particularly in their extracellular vesicles (EVs), a promising avenue for acellular therapy. From tissue origin to in vitro culture methods, various aspects must be taken into consideration to optimize MSC secretome potential for OA treatment. Immunomodulatory and regenerative properties of MSCs can also be enhanced by recreating a pro-inflammatory environment to mimic an in vivo pathological setting, but more unusual methods also deserve to be investigated. Altogether, these strategies hold substantial potential for the development of MSC secretome-based therapies suitable for OA management. The aim of this mini review is to survey the most recent advances on MSC secretome research with regard to equine OA

Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

International audienceContext: Osteoarthritis (OA) is an invalidating articular disease characterized by cartilage degradation and inflammatory events. In horses, OA is associated with up to 60% of lameness and leads to reduced animal welfare along with extensive economic losses; currently, there are no curative therapies to treat OA. The mesenchymal stromal cell (MSC) secretome exhibits anti-inflammatory properties, making it an attractive candidate for improving the management of OA. In this study, we determined the best storage conditions for conditioned media (CMs) and tested whether priming MSCs with cytokines can enhance the properties of the MSC secretome. Methods: First, properties of CMs collected from bone-marrow MSC cultures and stored at −80°C, −20°C, 4°C, 20°C or 37°C were assessed on 3D cultures of equine articular chondrocytes (eACs). Second, we primed MSCs with IL-1β, TNF-α or IFN-γ, and evaluated the MSC transcript levels of immunomodulatory effectors and growth factors. The primed CMs were also harvested for subsequent treatment of eACs, either cultured in monolayers or as 3D cell cultures. Finally, we evaluated the effect of CMs on the proliferation and the phenotype of eACs and the quality of the extracellular matrix of the neosynthesized cartilage. Results: CM storage at −80°C, −20°C, and 4°C improved collagen protein accumulation, cell proliferation and the downregulation of inflammation. The three cytokines chosen for the MSC priming influenced MSC immunomodulator gene expression, although each cytokine led to a different pattern of MSC immunomodulation. The cytokine-primed CM had no major effect on eAC proliferation, with IL-1β and TNF-α slightly increasing collagen (types IIB and I) accumulation in eAC 3D cultures (particularly with the CM derived from MSCs primed with IL-1β), and IFN-γ leading to a marked decrease. IL-1β-primed CMs resulted in increased eAC transcript levels of MMP1, MMP13 and HTRA1 , whereas IFNγ-primed CMs decreased the levels of HTRA1 and MMP13 . Conclusion: Although the three cytokines differentially affected the expression of immunomodulatory molecules, primed CMs induced a distinct effect on eACs according to the cytokine used for MSC priming. Different mechanisms seemed to be triggered by each priming cytokine, highlighting the need for further investigation. Nevertheless, this study demonstrates the potential of MSC-CMs for improving equine OA management

Mesenchymal stromal cells-derived exosomes: a new treatment for horse osteoarthritis?

International audienceOsteoarthritis (OA) is a degenerative osteoarticular pathology that can affect all the synovial joints, and is marked by the gradual deterioration of hyaline cartilage. This disorder compromises the well-being of horses, and leads to early career retirement, resulting in significant financial burdens for the equine industry. Over the last few years, the use of the immunomodulatory and cartilage regenerative potentials of mesenchymal stromal cells (MSCs) have emerged, but has also raised several concerns. As MSCs therapeutic properties are mainly driven by their secretome, exosomes derived from MSCs appear to be a promising therapeutic strategy for OA management. However, to our knowledge, the purification and characterization of equine exosomes were not well documented and needed more in-depth investigation.Here, we first purified exosomes from MSCs by membrane affinity capture (MAC) and size-exclusion chromatography (SEC), and validated a panel of equine exosomes markers (CD9, CD63, CD81, CD82, Alix and Tsg101) for their characterization. In addition, we showed that purified exosomes were intact and could be internalized by equine articular chondrocytes (eACs). Through the comparison of the two isolation methods, exosomes from MAC exhibited superior results in enhancing the neo-synthesis of a hyaline-like matrix by eACs. This enhancement was highlighted by the modulation of collagen levels, an increase in Pcna expression, and a decrease in Htra1 synthesis. However, due to unexpected effects induced by the MAC elution buffer on eACs, an additional ultrafiltration step was incorporated into the isolation protocol.Furthermore, our investigation revealed that exosomes derived from MSCs primed with equine pro-inflammatory cytokines, such as IL-1β, TNF-α, or IFN-γ, improved the hyaline-like phenotype of eACs, particularly IL-1β and TNF-α. Collectively, these findings underscore the significance of the exosome purification method and the potential benefits of pro-inflammatory cytokine priming to enhance the therapeutic efficacy of exosomes for the treatment of equine OA

Tumor Suppressive Role of miR-342-5p and miR-491-5p in Human Osteosarcoma Cells

International audienceOsteosarcomas are the most common type of malignant bone tumor. These tumors are characterized by the synthesis of an osteoid matrix. Current treatments are based on surgery and combination chemotherapy. However, for metastatic or recurrent tumors, chemotherapy is generally ineffective, and osteosarcomas are sometimes unresectable. Thus, the use of microRNAs (miRNAs) may represent an attractive alternative for the development of new therapies. Using high-throughput functional screening based on impedancemetry, we previously selected five miRNAs with potential chemosensitizing or antiproliferative effects on chondrosarcoma cells. We validated the tumor-suppressive activity of miR-491-5p and miR-342-5p in three chondrosarcoma cell lines. Here, we carried out individual functional validation of these five miRNAs in three osteosarcoma cell lines used as controls to evaluate their specificity of action on another type of bone sarcoma. The cytotoxic effects of miR-491-5p and miR-342-5p were also confirmed in osteosarcoma cells. Both miRNAs induced apoptosis. They increased Bcl-2 homologous antagonist killer (Bak) protein expression and directly targeted Bcl-2 lymphoma-extra large (Bcl-xL). MiR-342-5p also decreased B-cell lymphoma-2 (Bcl-2) protein expression, and miR-491-5p decreased that of Epidermal Growth Factor Receptor (EGFR). MiR-342-5p and miR-491-5p show tumor-suppressive activity in osteosarcomas. This study also confirms the potential of Bcl-xL as a therapeutic target in osteosarcomas

Pro-Inflammatory Cytokine Priming and Purification Method Modulate the Impact of Exosomes Derived from Equine Bone Marrow Mesenchymal Stromal Cells on Equine Articular Chondrocytes

Osteoarthritis (OA) is a widespread osteoarticular pathology characterized by progressive hyaline cartilage degradation, exposing horses to impaired well-being, premature career termination, alongside substantial financial losses for horse owners. Among the new therapeutic strategies for OA, using mesenchymal stromal cell (MSC)-derived exosomes (MSC-exos) appears to be a promising option for conveying MSC therapeutic potential, yet avoiding the limitations inherent to cell therapy. Here, we first purified and characterized exosomes from MSCs by membrane affinity capture (MAC) and size-exclusion chromatography (SEC). We showed that intact MSC-exos are indeed internalized by equine articular chondrocytes (eACs), and then evaluated their functionality on cartilaginous organoids. Compared to SEC, mRNA and protein expression profiles revealed that MAC-exos induced a greater improvement of eAC-neosynthesized hyaline-like matrix by modulating collagen levels, increasing PCNA, and decreasing Htra1 synthesis. However, because the MAC elution buffer induced unexpected effects on eACs, an ultrafiltration step was included to the isolation protocol. Finally, exosomes from MSCs primed with equine pro-inflammatory cytokines (IL-1β, TNF-α, or IFN-γ) further improved the eAC hyaline-like phenotype, particularly IL-1β and TNF-α. Altogether, these findings indicate the importance of the exosome purification method and further demonstrate the potential of pro-inflammatory priming in the enhancement of the therapeutic value of MSC-exos for equine OA treatment

The secretome of equine bone marrow-derived mesenchymal stem cells enhanced regenerative phenotype of equine articular chondrocytes

International audienceEquine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Many hopes lie on mesenchymal stem cell (MSC)-based therapies to repair cartilage, but these techniques face limitations inherent to cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternative to overcome those limitations while preserving MSC restorative properties (Cosenza et al., 2017). The aim of this study was to assess the effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) by indirect co-culture and/or MSC-conditioned media (CM) containing EVs. The expression of healthy cartilage/OA, and proliferation markers was evaluated in eACs cultured in monolayers or as 3D organoids. In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. Our results demonstrated that MSC secretome influences eAC phenotype by increasing cartilage functionality markers such as Prg4, collagens and proliferation associated molecule in a greater way than MSCs. Cell migration was also enhanced, which could delay OA final outcomes. To confirm the presence of nanosized EVs in MSC-CM, nanoparticle tracking assay, transmission electron microscopy and exosome-specific markers detection were assessed. Lastly, fluorescence staining revealed effective uptake of exosomes by eAC. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects

The secretome of equine bone marrow-derived mesenchymal stem cells enhanced regenerative phenotype of equine articular chondrocytes

International audienceEquine osteoarthritis (OA) is a sequential disease which leads to cartilage degradation and painful bone frictions. It induces impaired animal well-being, premature cessation of sport activity, and financial losses. Fibrocartilage synthesis occurring during cartilage destruction is a physiological response, allowing bone protection but reducing tissue mechanical resistance. To date, there is a lack of curative therapies. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to cell itself which can be overcome using their secretomethrough acellular therapy approaches.To understand the effects of equine bone marrow (BM-)MSC secretome on equine articular chondrocytes (eAC) phenotype, indirect co-culture experiments were first performed. Then, we wantedto recapitulate the effects we observed in co-culture on eAC using the MSC-conditioned medium (CM) to make acellular therapy conceivable. We assessed hyaline cartilage and fibrocartilage markers at the transcription and protein levels, and evaluated eAC migratory capacities, which are of interest during OA therapy to favor the filling of the cartilage defects. To optimize immunomodulation properties of MSC secretome for future experiments, MSC priming relevance with interleukin (IL)1-β was evaluated. Suspected to be the principal vectors of the effects observed, exosomes were isolated through chemical precipitation and then characterized to confirm their nature

Bone Marrow MSC Secretome Increases Equine Articular Chondrocyte Collagen Accumulation and Their Migratory Capacities

International audienceEquine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to the cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternatives to overcome those limitations while preserving MSC restorative properties. The effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) was analyzed with indirect co-culture and/or MSC-conditioned media (CM). The expression of healthy cartilage/OA and proliferation markers was evaluated in eACs (monolayers or organoids). In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. The presence of nanosized EVs in MSC-CM was appraised with nanoparticle tracking assay and transmission electron microscopy. Our results demonstrated that the MSC secretome influences eAC phenotype by increasing cartilage functionality markers and cell migration in a greater way than MSCs, which could delay OA final outcomes. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, the MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects

Impact of environmental enrichment on the behaviour and immune cell transcriptome of pregnant sows

International audienceThe ability to assess farm animals’ mood is important to evaluate their welfare, but practical assessment tools are still lacking. Human research has demonstrated a link between psychological states and the transcriptome of blood immune cells. Therefore, this study aimed to investigate whether blood immune cell transcriptome can be used to assess the animals’ mood using environmental enrichment as a method to generate contrasted welfare states. Pregnant sows of mixed parities were housed in two contrasting conditions throughout gestation (0 to 105 days): a conventional system on a slatted floor (C, n = 36) or an enriched system on accumulated straw with additional space per sow (E, n = 35). The behavior of multiparous sows of low (2nd and 3rd gestation; n = 29) and high (4th gestation or higher; n = 31) parity was observed from G99 to G104 and 14 sows per system were selected for biological sampling. Cortisol concentrations in saliva (G35 and G98), and in the hair (G98), were lower in E sows (P 1.2). However, parity (894 DEGs) and social dominance (437 DEGs) had a greater effect, and most of the DEGs were related to innate and adaptive immunity pathways. Therefore, these results confirm that long-term environmental enrichment decreases cortisol concentration and positively influences sow behavior. The blood transcriptome did not allow discrimination between housing conditions but it was influenced by another important factor for welfare, such as social dominance in the group