Encapsulation of metabolically standardized MSC as a novel osteoarthritis therapy – METAB-OA

The METAB-OA project combines cell biology, biomaterials engineering, and preclinical models to develop improved cell-based therapies for osteoarthritis.

Mesenchymal stromal cells (MSCs) derived from adipose tissue were first isolated and metabolically reprogrammed to modulate their functional state. Their biological properties were comprehensively assessed using molecular and cellular approaches, including gene expression analyses, metabolic profiling, and functional assays evaluating immune regulation and interaction with key inflammatory cells.

To enhance their delivery and persistence in the joint, MSCs were encapsulated within injectable biomaterials. Microgels were produced using micromolding and extrusion-based bioprinting, enabling control over size, reproducibility, and scalability. The physical and biological properties of these systems were characterized to optimize cell protection, viability, and functional performance.

The therapeutic potential of MSCs, with or without metabolic preconditioning, was then evaluated in vitro and in preclinical models of osteoarthritis. The project evolved toward the use of a murine inflammatory osteoarthritis model, better suited to study immune mechanisms and treatment responses. These in vivo approaches allowed assessment of the impact of MSCs on joint inflammation and tissue remodeling in a physiologically relevant context.

In parallel, high-resolution analyses were performed on synovial tissues using single-cell RNA sequencing (scRNA-seq). This state-of-the-art technology enabled detailed characterization of the cellular composition of the joint and the identification of treatment-induced changes at the single-cell level. In particular, it provides unique insights into immune cell dynamics, including macrophage populations, and their modulation by MSC-based therapies.

Overall, this integrated methodological framework makes it possible to link metabolic reprogramming, delivery strategies, and in vivo responses, and to better understand the mechanisms underlying the therapeutic effects of MSCs in osteoarthritis.

The METAB-OA project demonstrated that mesenchymal stromal cells (MSCs) can be functionally enhanced by modifying their metabolic state. By reprogramming these cells toward a glycolytic profile, their ability to regulate immune responses was significantly improved in laboratory settings. In particular, reprogrammed MSCs more effectively reduced the proliferation of pro-inflammatory immune cells, such as T lymphocytes, and promoted a shift toward less inflammatory immune profiles. They also helped reduce the presence of pro-inflammatory macrophages, which play a key role in joint inflammation.

A major finding of the project is that this metabolic reprogramming is not permanent. The enhanced functional state of MSCs gradually fades, with cells returning to their original state within about one week. This result identifies a key biological limitation of current approaches and highlights an important challenge for developing long-lasting therapies. At the same time, it provides a clear direction for future research, emphasizing the need for more stable strategies.

The project also showed that MSCs can be successfully delivered using injectable biomaterials designed to protect the cells and support their activity within the joint. Microgels were developed using micromolding and extrusion-based bioprinting, demonstrating good reproducibility and scalability. However, the beneficial effects of metabolically reprogrammed MSCs were not maintained after encapsulation, mainly due to the transient nature of the preconditioning.

In preclinical models of osteoarthritis, MSCs with or without preconditioning were administered into the joint. The results indicate that their therapeutic effects depend strongly on both the type of preparation and the inflammatory environment. Rather than inducing uniform responses, MSCs influence specific immune cell populations in a context-dependent manner.

A major advance of the project is the use of single-cell RNA sequencing (scRNA-seq) to analyze synovial tissue from treated animals. This approach provided an unprecedented level of detail on the cellular composition of the joint and revealed how different immune cells respond to MSC-based therapies. In particular, macrophages were identified as central players in both disease progression and therapeutic response.

Overall, METAB-OA improved understanding of how the metabolic state of MSCs influences their function, identified critical limitations of current strategies, and provided new insights into immune regulation within the joint. These findings represent an important step toward the development of more effective and durable cell-based therapies for osteoarthritis.

A key strength of the METAB-OA project is the demonstration that the therapeutic potential of mesenchymal stromal cells (MSCs) can be significantly enhanced by modulating their metabolic state. This finding establishes a direct and actionable link between cellular metabolism and immunomodulatory activity, providing a novel strategy to improve cell-based therapies for osteoarthritis.

Another major contribution of the project is the identification of a critical limitation: the enhancement induced by pharmacological metabolic reprogramming is transient. While this may appear as a constraint, it represents a highly informative result, as it clarifies why MSC therapeutic effects are often short-lived in vivo. This insight has reoriented the project toward more mechanistic approaches and highlights the need for strategies capable of stabilizing MSC functions over time.

The project also demonstrated the feasibility of combining cell engineering with biomaterial-based delivery systems. Encapsulation of MSCs in injectable hydrogels was successfully achieved using controllable technologies such as micromolding and bioprinting. Although these approaches are promising, further optimization is required to fully exploit their potential and ensure scalability for future applications.

A major breakthrough lies in the use of single-cell RNA sequencing (scRNA-seq) to analyze synovial tissue in an inflammatory osteoarthritis model. This approach provides unprecedented resolution of the cellular landscape within the joint and represents, to our knowledge, the first such analysis in this context following MSC treatment with different preconditioning strategies. These data have identified macrophages and other immune cell subsets as central players in both disease progression and therapeutic response.

Looking ahead, the project opens strong perspectives for the development of next-generation therapies. A key priority will be to achieve long-term control of MSC function, particularly through approaches such as genetic engineering to maintain a stable therapeutic phenotype. In parallel, improving delivery strategies and targeting specific immune pathways within the joint will be essential to enhance treatment efficacy.

Overall, METAB-OA combines conceptual innovation, technological advances, and high-resolution mechanistic insights. It lays the groundwork for more effective, durable, and precisely controlled cell-based therapies for osteoarthritis.

Osteoarthritis (OA), the most prevalent joint disease, is characterized by structural and functional alterations of all joint components. The most promising cell-based therapy approach for OA relies on the use of pleiotropic mesenchymal stromal/stem cells (MSC) that display tissue-protective and immunoregulatory properties. The intra-articular (IA) injection of MSC in pre-clinical studies as well as in phase I and II clinical trials has been investigated and reported as a safe and well-tolerated treatment for OA. Moreover, Partner 1 (INSERM U1183-IRMB/Montpellier) has demonstrated the capacity of MSC to reduce the clinical severity of OA in experimental animal models by decreasing inflammation and protecting cartilage and bone from degradation. However and despite significant clinical improvement, the benefit of IA injection of MSC in OA patients still remains limited to the short-term. In that respect, it is believed that the poor in vivo survival rate of IA MSC, that strongly limits their residence time in the injured joint, associated to their biological heterogeneity, could collectively reduce their long-term beneficial effects in OA.
Phenotypic and metabolic heterogeneity of MSC subtypes is dynamic and can be exacerbated by manufacturing process at clinical scale. To overcome this first limitation, Partner 1 has recently demonstrated that MSC metabolism governs their immunosuppressive and anti-inflammatory properties and developed an approach of cell expansion able to standardize MSC metabolism. Additionally, Partner 2 (INSERM U1229-RMeS, Nantes) has developed cytoprotective hydrogels and micromolding procedures for cell encapsulation that could contribute to enhance MSC survival and persistence in the injured joint.
Considering the safety and promising therapeutic effect of MSC in OA and the potential role of the MSC metabolic switch in their immunomodulatory properties, METAB-OA aims to investigate whether the metabolic stability of adipose tissue-derived MSC can improve MSC anti-OA potential and whether MSC encapsulation in injectable micro-sized hydrogels can maintain MSC metabolism and survival upon injection and provide a suitable microenvironment supporting MSC functions. Thus the specific objectives of the METABOA, are to: (i) generate pharmacologically-induced glycolytic AT-MSC (iAT-MSC) with enhanced anti-OA properties, (ii) encapsulate iAT-MSC in micromolded hydrogels to protect them from cell death upon injection and limit their dispersion outside the joint space and (iii) investigate whether iAT-MSC encapsulated in alginate microgels and injected in the knee joints of OA rabbits exhibit enhanced and prolonged therapeutic effects.
To address these issues, METAB-OA will be organized in 4 work packages (WP): • WP0: Project management, communication and valorization, • WP1: Generation of a homogeneous population of AT-MSC (iAT-MSC) with enhanced immunosuppressive and chondro-protective properties, • WP2: Generation of micromolded hydrogels to encapsulate and protect iAT-MSC and • WP3: Proof of concept of the efficacy of encapsulated iAT-MSC in a medium-sized animal model of OA. In case of success, METAB-OA innovative project will undoubtedly allow us to consider clinical trials and thus pave the way of new therapeutic avenues in the medical care of OA patients.