Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering

International audienceOsteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering

New therapeutic strategies for the treatment of horse joint disorders

Le cartilage articulaire est un tissu possédant une faible capacité de réparation intrinsèque. Dès lors, la répétition de traumatismes articulaires induit un microenvironnement propice à la dégradation du cartilage et, in fine, l’émergence de l’arthrose. Les traitements utilisés à l’heure actuelle visent uniquement à soulager la douleur, réduire l’inflammation et la progression de l’arthrose. Ainsi, le traitement des lésions chondrales équines revêt une importance majeure puisque les affections locomotrices constituent la première cause de baisse de performances et d’arrêt prématuré de la carrière du cheval sportif. De plus, le cheval est le modèle animal qui possède le cartilage articulaire le plus semblable à celui de l’Homme et, en conséquence, représente un modèle d’étude pertinent pour les lésions chondrales humaines. Dans ce contexte, notre étude s’est attachée à développer de nouvelles stratégies pour le traitement des lésions chondrales basées sur la différenciation chondrogénique de CSM en vue de produire in vitro un substitut cartilagineux implantable en site articulaire. Ainsi, nous avons d’abord isolé et caractérisé des CSM équines à partir de prélèvements de moelle osseuse (MO) et de sang de cordon ombilical (SCO), puis, nous avons réalisé la différenciation en cultivant les CSM durant 14 jours en hypoxie ou normoxie au sein d’un biomatériau (éponges de collagène de types I/III), en présence de BMP-2 et TGF-β1 et de siRNA ciblant le collagène de type I et HtrA1, molécules atypiques du cartilage hyalin. Bien que ce protocole nous ait permis d’obtenir un substitut cartilagineux riche en marqueurs du cartilage hyalin comme le collagène de type II et l’agrécane, la présence du collagène de type I persistait. Nous avons donc tenté d’optimiser le protocole en allongeant le temps de culture, en utilisant le TGF-β3, et en modifiant la stratégie d’interférence par l’ARN. Cette étape nous a permis de conclure sur l’effet bénéfique de l’allongement de la culture à 28 jours et l’efficacité des facteurs chondrogéniques initialement utilisés. Néanmoins, la stratégie d’interférence par l’ARN demeure encore perfectible. Finalement, nous avons comparé la qualité du substitut cartilagineux obtenu après différenciation en fonction de la source de CSM utilisée. Les CSM de MO semblent les plus adaptées mais le protocole que nous avons utilisé n’est probablement pas le plus efficace pour induire la différenciation des CSM de SCO. Dans une partie complémentaire, bien que ces résultats soient préliminaires, nous avons montré que le sécrétome des CSM pourrait être un formidable outil afin d’améliorer le traitement des lésions chondrales. Dans leur ensemble, les résultats obtenus permettent d’avoir un regard optimiste concernant la mise en place de thérapies cellulaire et tissulaire du cartilage, aussi bien en médecine équine qu’humaine.Articular cartilage is a tissue with low intrinsic repair abilities. Therefore, repeated traumas lead to cartilage degradation and ultimately, to the emergence of osteoarthritis (OA). Current therapies aim to reduce pain, inflammation and to prevent the progression of OA. Thus, treatment of equine chondral lesions is of major importance since locomotor disorders are the main causes of poor performance and early retirement of the athlete horses. In addition, the horse is an animal model with the most human-like articular cartilage and, therefore, represents the best relevant model to study human chondral lesions and arthropathies. In this context, our study focused on developing new strategies for the treatment of chondral lesions based on the chondrogenic differentiation of equine mesenchymal stem cells (MSC) in order to produce an in vitro neo-synthetized cartilaginous substitute, which could be implantable in the chondral lesion site. Thus, we first isolated and characterized equine MSC derived from bone marrow (BM) and umbilical cord blood (UCB). Then, we have differentiated MSC by culturing them for 14 days in hypoxia or normoxia, in a biomaterial (types I / III collagen sponges), in the presence of BMP-2 and TGF-β1 and siRNA targeting type I collagen and HtrA1, two atypical hyaline cartilage molecules overexpressed in OA. Although this protocol allowed us to obtain a cartilaginous substitute composed of large amounts of hyaline cartilage markers such as type II collagen and aggrecan, the presence of type I collagen persisted. We therefore tried to optimize the protocol by extending the culture time, using TGF-β3, and modifying the RNA-interference strategy. We have concluded on the beneficial effect of the lengthening of the culture to 28 days and the effectiveness of the chondrogenic factors initially used. Nevertheless, the RNA-interference strategy still remains perfectible. Finally, we compared the quality of the neo-synthetized cartilaginous substitute according to the source of MSC used. BM-MSC seem to be the most suitable, but the protocol we used is probably not the most effective for inducing UCB-MSC differentiation. In a complementary part, although these results are very preliminary, we have shown that the MSC secretome could be a tremendous tool to improve current therapies of chondral lesions. Overall, the results obtained make it possible to look ahead with optimism, in order to obtain future efficient cartilage tissue engineering therapies, both in equine and human medicines

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

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

Caractérisation phénotypique des chondrocytes fœtaux humains

International audienceL’arthrose est une pathologie dégénérative irréversible du cartilage articulaire. Différentes stratégies thérapeutiques ont été développées sans que l’on puisse actuellement la guérir. De nouvelles approches ont émergé comme la transplantation de chondrocytes autologues (TCA) initiée par Brittberg et ses collaborateurs en 1994. La TCA a permis le développement de plusieurs techniques d’ingénierie tissulaire permettant d’obtenir des organoïdes cartilagineux comme modèles d’étude du cartilage ou comme substitut biologique. Ces procédés restent perfectibles, entre autres du fait d’un phénomène de dédifférenciation chondrocytaire lors de l’indispensable phase d’amplification cellulaire. Il a été démontré l’intérêt de l’utilisation des chondrocytes fœtaux humains (CFH) de tête fémorale (TF) ou de genou comme source cellulaire. Du fait du processus d’ossification endochondrale, les foyers de CFH sont multiples. L’objectif de ce travail est de caractériser le phénotype de CFH issus de TF, de côtes (C) et de disques intervertébraux (DIV) en culture monocouche, ainsi que leur capacité de prolifération. Le potentiel de redifférenciation chondrocytaire des CFH est évalué en culture 3D, en hypoxie, en présence ou en absence du facteur chondrogénique, la « Bone Morphogenetic Protein-2 » (BMP-2) et ils sont comparés aux chondrocytes articulaires humains (CAH) adultes arthrosiques. Les résultats montrent une capacité de prolifération et de dédifférenciation différentes selon l’origine anatomique des CFH. Nous avons confirmé la capacité de redifférenciation des CFH. Par ailleurs, l’utilisation des CFH de DIV en présence de la BMP-2 semble la plus avantageuse. En accord avec les données de la littérature, nous avons également montré la supériorité des CFH vis-à-vis des CAH dans l’obtention d’organoïdes cartilagineux

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