Development and characterization of a humanized mouse model of osteoarthritis.

OBJECTIVE: In light of the role of immune cells in OA pathogenesis, the development of sophisticated animal models closely mimicking the immune dysregulation during the disease development and progression could be instrumental for the preclinical evaluation of novel treatments. Among these models, immunologically humanized mice may represent a relevant system, particularly for testing immune-interacting DMOADs or cell therapies before their transfer to the clinic. Our objective, therefore, was to develop an experimental model of OA by destabilization of the medial meniscus (DMM) in humanized mice. METHOD: Irradiated 5-week-old NOD/LtSz-scid IL2Rγnull (NSG) mice were humanized by intravenous injection of CD34+ human hematopoietic stem cells. The engraftment efficiency was evaluated by flow cytometry 17 weeks after the humanization procedure. Humanized and non-humanized NSG mice underwent DMM or sham surgery and OA development was assessed 1, 6, and 12 weeks after the surgery. RESULTS: 120 days after the humanization, human T and B lymphocytes, macrophages and NK cells, were present in the blood and spleen of the humanized NSG mice. The DMM surgery induced articular cartilage and meniscal alterations associated with an increase in OA and the meniscal score. Moreover, the surgery triggered an inflammatory response that was sustained at a low grade in the DMM group. CONCLUSIONS: Our study shows for the first time the feasibility of inducing OA by DMM in humanized mice. This novel OA model could constitute a useful tool to bridge the gap between the preclinical and clinical evaluation of immune interacting DMOADs and cell-based therapies.

PPARß/δ directs the therapeutic potential of mesenchymal stem cells in arthritis.

OBJECTIVES: To define how peroxisome proliferator-activated receptor (PPAR) ß/δ expression level in mesenchymal stem cells (MSCs) could predict and direct both their immunosuppressive and therapeutic properties. PPARß/δ interacts with factors such as nuclear factor-kappa B (NF-κB) and regulates the expression of molecules including vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1. Since these molecules are critical for MSC function, we investigated the role of PPARß/δ on MSC immunosuppressive properties. METHODS: We either treated human MSCs (hMSCs) with the irreversible PPARß/δ antagonist (GSK3787) or derived MSCs from mice deficient for PPARß/δ (PPARß/δ-/- MSCs). We used the collagen-induced arthritis (CIA) as model of immune-mediated disorder and the MSC-immune cell coculture assays. RESULTS: Modulation of PPARß/δ expression in hMSCs either using GSK3787 or hMSCs from different origin reveals that MSC immunosuppressive potential is inversely correlated with Ppard expression. This was consistent with the higher capacity of PPARß/δ-/- MSCs to inhibit both the proliferation of T lymphocytes, in vitro, and arthritic development and progression in CIA compared with PPARß/δ+/+ MSCs. When primed with proinflammatory cytokines to exhibit an immunoregulatory phenotype, PPARß/δ-/- MSCs expressed a higher level of mediators of MSC immunosuppression including VCAM-1, ICAM-1 and nitric oxide (NO) than PPARß/δ+/+ MSCs. The enhanced NO2 production by PPARß/δ-/- MSCs was due to the increased retention of NF-κB p65 subunit on the κB elements of the inducible nitric oxide synthase promoter resulting from PPARß/δ silencing. CONCLUSIONS: Our study is the first to show that the inhibition or knockdown of PPARß/δ in MSCs primes their immunoregulatory functions. Thus, the regulation of PPARß/δ expression provides a new strategy to generate therapeutic MSCs with a stable regulatory phenotype.

Functional cartilage repair capacity of de-differentiated, chondrocyte- and mesenchymal stem cell-laden hydrogels in vitro.

OBJECTIVE: The long-term performance of cell-seeded matrix-based cartilage constructs depends on (1) the development of sufficient biomechanical properties, and (2) lateral integration with host tissues, both of which require cartilage-specific matrix deposition within the scaffold. In this study, we have examined the potential of tissue-engineered cartilage analogs developed using different cell types, i.e., mesenchymal stem cells (MSCs) vs chondrocytes and de-differentiated chondrocytes, in an established "construct in cartilage ring" model. DESIGN: Cell-laden constructs of differentiated chondrocytes, de-differentiated chondrocytes after two, five or eight population doublings, and MSCs were either implanted into a native cartilage ring immediately after fabrication (immature group) or pre-treated for 21 days in a transforming growth factor-ß3 (TGF-ß3) containing medium prior to implantation. After additional culture for 28 days in a serum-free, chemically defined medium, the extent of lateral integration, and biochemical and biomechanical characteristics of the implants as hybrid constructs were assessed. RESULTS: The quality of integration, the amount of accumulated cartilage-specific matrix components and associated biomechanical properties were found to be highest when using differentiated chondrocytes. De-differentiation of chondrocytes negatively impacted the properties of the implants, as even two population doublings of the chondrocytes in culture significantly lowered cartilage repair capacity. In contrast, MSCs showed chondrogenic differentiation with TGF-ß3 pre-treatment and superior integrational behavior. CONCLUSIONS: Chondrocyte expansion and de-differentiation impaired the cell response, resulting in inferior cartilage repair in vitro. With TGF-ß3 pre-treatment, MSCs were able to undergo sustained chondrogenic differentiation and exhibited superior matrix deposition and integration compared to de-differentiated chondrocytes.

PPARß/δ-dependent MSC metabolism determines their immunoregulatory properties.

Mesenchymal stem cell (MSC)-based therapy is being increasingly considered a powerful opportunity for several disorders based on MSC immunoregulatory properties. Nonetheless, MSC are versatile and plastic cells that require an efficient control of their features and functions for their optimal use in clinic. Recently, we have shown that PPARß/δ is pivotal for MSC immunoregulatory and therapeutic functions. However, the role of PPARß/δ on MSC metabolic activity and the relevance of PPARß/δ metabolic control on MSC immunosuppressive properties have never been addressed. Here, we demonstrate that PPARß/δ deficiency forces MSC metabolic adaptation increasing their glycolytic activity required for their immunoregulatory functions on Th1 and Th17 cells. Additionally, we show that the inhibition of the mitochondrial production of ATP in MSC expressing PPARß/δ, promotes their metabolic switch towards aerobic glycolysis to stably enhance their immunosuppressive capacities significantly. Altogether, these data demonstrate that PPARß/δ governs the immunoregulatory potential of MSC by dictating their metabolic reprogramming and pave the way for enhancing MSC immunoregulatory properties and counteracting their versatility.

Mesenchymal Stem Cells Regulate the Innate and Adaptive Immune Responses Dampening Arthritis Progression.

Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to immunomodulate cells from both the innate and the adaptive immune systems promoting an anti-inflammatory environment. During the last decade, MSCs have been intensively studied in vitro and in vivo in experimental animal model of autoimmune and inflammatory disorders. Based on these studies, MSCs are currently widely used for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) characterized by complex deregulation of the immune systems. However, the therapeutic properties of MSCs in arthritis are still controverted. These controversies might be due to the diversity of MSC sources and isolation protocols used, the time, the route and dose of MSC administration, the variety of the mechanisms involved in the MSCs suppressive effects, and the complexity of arthritis pathogenesis. In this review, we discuss the role of the interactions between MSCs and the different immune cells associated with arthritis pathogenesis and the possible means described in the literature that could enhance MSCs therapeutic potential counteracting arthritis development and progression.

Glucocorticoid-induced leucine zipper governs the therapeutic potential of mesenchymal stem cells by inducing a switch from pathogenic to regulatory Th17 cells in a mouse model of collagen-induced arthritis.

OBJECTIVE: Mesenchymal stem cells (MSCs) are potent immunosuppressive cells that have shown promise in the treatment of rheumatoid arthritis (RA). Deciphering the intrinsic characteristics of MSCs that correlate with their biologic activity will facilitate their clinical use. Recently, the role of glucocorticoid-induced leucine zipper (GILZ) in the development of RA has been documented. The aim of this study was to evaluate whether GILZ expression by MSCs may contribute to their therapeutic effect. METHODS: MSCs were isolated from GILZ-deficient (GILZ(-/-) ) mice and wild-type mice. MSCs (1 × 10(6) cells) were injected twice via the tail vein into mice with collagen-induced arthritis (CIA). RESULTS: In vitro, we showed that GILZ is a key factor involved in the immunosuppressive potential of MSCs. MSCs derived from GILZ(-/-) mice did not suppress the proliferation of CD4+ T cells and were less efficient than MSCs derived from WT mice in altering Th17 cell polarization. Thus, we investigated the role of GILZ in an experimental model of arthritis and demonstrated that although WT MSCs significantly reduced paw swelling in arthritic mice, GILZ(-/-) MSCs did not. Moreover, the magnitude of the effects of GILZ(-/-) MSCs on Th17 cell frequency was significantly lower than that of WT MSCs. The therapeutic effect of MSCs correlated with the generation of Treg cells bearing the CD4 + RORγt+IL-17(low) IL-10+ signature, and Th17 cell polarization was GILZ dependent. CONCLUSION: This study demonstrates that GILZ has an essential role in the therapeutic effectiveness of MSCs in arthritis by favoring Th17 cell polarization toward a regulatory phenotype. Therefore, potentiation of GILZ expression in MSCs could represent a means to enhance their therapeutic effect in autoimmune diseases.

Mesenchymal progenitor cells derived from traumatized human muscle.

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate cell type for cell-based tissue engineering and regenerative medicine. Currently, clinical applications for MSCs require additional surgical procedures to harvest the autologous MSCs (i.e. from bone marrow) or commercial allogeneic alternatives. We have recently identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue that has been surgically debrided from traumatic orthopaedic extremity wounds. The purpose of this study was to evaluate whether MPCs derived from traumatized muscle may provide a clinical alternative to bone-marrow MSCs, by comparing their morphology, proliferation capacity, cell surface epitope profile and differentiation capacity. After digesting the muscle tissue with collagenase, the MPCs were enriched by a direct plating technique. The morphology and proliferation rate of the muscle-derived MPCs was similar to bone-marrow derived MSCs. Both populations expressed cell surface markers characteristic for MSCs (CD 73, CD 90 and CD105), and did not express markers typically absent on MSCs (CD14, CD34 and CD45). After 21 days in specific differentiation media, the histological staining and gene expression of the MPCs and MSCs was characteristic for differentiation into osteoblasts, chondrocytes and adipocytes, but not into myoblasts. Our findings demonstrate that traumatized muscle-derived MPCs exhibit a similar phenotype and resemble MSCs derived from the bone marrow. MPCs harvested from traumatized muscle tissue may be considered for applications in tissue engineering and regenerative medicine following orthopaedic trauma requiring circumferential debridement.

Engineering mesenchymal stem cells for immunotherapy.

Allogeneic hematopoietic stem cell transplantation, after sublethal irradiation of recipient animals, is capable of inducing donor-specific tolerance facilitating subsequent organ transplantation. This approach could reintroduce tolerance in autoimmune diseases and it has been applied to treat autoimmune diseases with, however, a great susceptibility of recurrence. Mesenchymal stem cells (MSCs) present within the bone marrow could be critical to the immunosuppressive effect of the treatment. This tolerance induction may be useful in allogeneic transplantations, where low incidence of graft-versus-host disease was observed when the hematopoietic graft was coinjected with MSCs. In this paper, we discuss the use of MSCs in different therapeutic strategies either as immunosuppressive agents or genetically engineered to express molecules acting against the autoimmune process.

Surface modification of extracellular vesicles with polyoxazolines to enhance their plasma stability and tumor accumulation.

Extracellular vesicles (EVs) are future promising therapeutics, but their instability in vivo after administration remains an important barrier to their further development. Many groups evaluated EV surface modification strategies to add a targeting group with the aim of controlling EV biodistribution. Conversely, fewer groups focused on their stabilization to obtain "stealth" allogenic EVs. Modulating their stabilization and biodistribution is an essential prerequisite for their development as nano-therapeutics. Here, we explored polyoxazolines with lipid anchors association to the EV membrane (POxylation as an alternative to PEGylation) to stabilize EVs in plasma and control their biodistribution, while preserving their native properties. We found that this modification maintained and seemed to potentiate the immunomodulatory properties of EVs derived from mesenchymal stem/stromal cells (MSC). Using a radiolabeling protocol to track EVs at a therapeutically relevant concentration in vivo, we demonstrated that POxylation is a promising option to stabilize EVs in plasma because it increased EV half-life by 6 fold at 6 h post-injection. Moreover, EV accumulation in tumors was higher after POxylation than after PEGylation.