Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis

Abstract

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

Cellular and Molecular Targets of Extracellular Vesicles from Mesenchymal Stem/Stromal Cells in Rheumatoid Arthritis

“…For instance, an EV suspension (corresponding to 5.0 × 10 5 of human BM-MSCs) injected into the knee joint of antigen-induced arthritis mice significantly reduced paw swelling and histopathological changes through the suppression of Th17 polarization and synovial cellular infiltration. 65 In CIA rats, a single intravenous injection of sEVs from human UC-MSCs dose-dependently reduced joint inflammation, synovial hyperplasia and cartilage destruction. Besides, these EVs decreased T-cell proliferation, promoted the apoptosis of these cells and increased the Treg/Th17 ratio.…”

“…Therefore, EVs isolated from early-passage BM-MSCs are more immunosuppressive than those from late-passage cells while BM-MSCs in 3D cultures release more effective EVs compared with conventional 2D cultures. 37 Although recent research has suggested that normal culture conditions of MSCs are sufficient to isolate EVs with significant anti-arthritic activity in animal models, 65 different priming strategies have been used to improve their immunomodulatory properties and therapeutic potential. Pro-inflammatory priming induces important changes in the cargo of EVs and promotes the release of a higher percentage of sEVs compared to lEVs.…”

Cell‐based therapies for the treatment of rheumatoid arthritis

“…EVs isolated from MSCs were cultured normoxically (21% O 2 , 5% CO 2 ), and the AIA model was applied with normoxic EVs. These are associated with the establishment of TH17/Treg balance that resulted in reduced inflammation 68 …”

Optimized operation of a controlled stirred tank reactor system for the production of mesenchymal stromal cells and their extracellular vesicles

“…Moreover, since the concentration of MSC‐EV in the conditioned medium is typically low, scale‐out to hundreds of T‐flasks or multiplate flask systems is required, involving an extensive handling for feeding/harvesting procedures, to achieve significant final mass production. Additionally, several differences were observed, in terms of the MSC‐derived EVs cargo, between EVs isolated from culture supernatants of MSC expanded under different culture conditions, stressing the importance of controlling all culture process parameters to obtain a consistent EV content (Hyland et al, 2020; Kay et al, 2021). In this context, it turns necessary to move forward to fully controlled reactors (i.e., stirred tank reactors [STR]) to establish a reproducible and scalable process to produce MSC‐EV.…”