Therapeutic Effects of Mesenchymal/Stromal Stem Cells and Their Derived Extracellular Vesicles in Rheumatoid Arthritis

Abstract

Currently available therapies for rheumatoid arthritis (RA) are inadequate to alleviate the inflammation and reduce joint damage. While the immune-regulatory effect of human mesenchymal/stromal stem cells (MSCs) extracellular vesicles (EVs) has been tested in many inflammation-related diseases, little is known regarding their effect on patients with RA. Thus, we assessed the effect of human MSCs and MSC-EVs (from naïve or IFN-β-primed MSCs) on CD4+ T cells from patients with RA. Moreover, we investigated the effect of MSC-EVs on RA patients-derived synovial fibroblasts (FLS). MSC-EVs were prepared using a PEG precipitation followed by ultracentrifugation-based protocol. Applied to RA CD4+ T cells, EVs from IFN-β-primed MSCs, suppressed the expression of more key RA-associated cytokines (IL-4, GM-CSF IFN-γ, IL-2, TNF-α), and decreased CD4+ T-cell polyfunctionality than MSCs or EVs from naïve MSCs. MSCs mediated a slight decrease in the frequency of T-regulatory cells, while MSC-EVs rescued the frequency of T-regulatory cells. MSCs significantly inhibited CD4+ T-cell proliferation (P < .05), while no inhibition was observed in response to EV preparations. EVs from IFN-β-primed MSCs inhibited (P < .01) RA FLS migration and downregulated (P < .05) RA FLS surface markers CD34 and HLA-DR. Collectively, we demonstrated the immune-modulatory function of MSCs and their derived EVs in RA CD4+ T cells, which could be further enhanced by priming MSCs with IFN-β. Moreover, EVs from IFN-β-primed MSCs more efficiently inhibit RA FLS migration, and expression of RA FLS-related surface markers, suggesting these EVs as a potent therapy for RA.

Current Methods for Analysing Mesenchymal Stem Cell-Derived Extracellular Vesicles

“…We can evaluate the morphology and size, but it is challenging to actually quantify EVs [ 18 ]. MSC-EV samples are usually loaded onto nitrocellulose [ 45 ] or formvar grids coated with carbon [ 23 , 28 ] or copper [ 24 ]. Precisely due to this film on the grid, we are only left with EVs larger than the openings on the film, causing inaccurate counting [ 57 ].…”

“…Since they are typically found on EVs from a range of cell types, the transmembrane proteins CD9, CD63 and CD81—collectively known as tetraspanins—are determined most of the time [ 21 ]. In addition to these proteins, the expression of CD90, CD73 [ 22 ], CD105 [ 23 ], programmed death-ligand 1 protein (PD-L1) [ 24 ], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [ 15 ], syntenin-1, calnexin [ 15 , 23 , 25 , 26 ], glial fibrillary acidic protein (GFAP) [ 25 ], apoptosis-linked-gene-2-interacting protein X (ALIX) [ 26 , 27 ], tumour susceptibility gene 101 (TSG101) [ 25 , 26 , 27 ], amyloid-beta (Aβ), cis-Golgi matrix protein (GM130) [ 28 ] and heat shock protein 70 (HSP70) [ 29 ] is measured in research on MSC-derived EVs. The above analysis used purchased cultures of human BM [ 25 , 28 ] and human placenta-derived MSCs [ 27 ], MSCs isolated directly from human BM tissue [ 22 , 23 ], mouse BM [ 24 ], chorionic villus tissue from a human placenta [ 26 ] and human tonsil tissue [ 29 ].…”

“…In addition to these proteins, the expression of CD90, CD73 [ 22 ], CD105 [ 23 ], programmed death-ligand 1 protein (PD-L1) [ 24 ], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [ 15 ], syntenin-1, calnexin [ 15 , 23 , 25 , 26 ], glial fibrillary acidic protein (GFAP) [ 25 ], apoptosis-linked-gene-2-interacting protein X (ALIX) [ 26 , 27 ], tumour susceptibility gene 101 (TSG101) [ 25 , 26 , 27 ], amyloid-beta (Aβ), cis-Golgi matrix protein (GM130) [ 28 ] and heat shock protein 70 (HSP70) [ 29 ] is measured in research on MSC-derived EVs. The above analysis used purchased cultures of human BM [ 25 , 28 ] and human placenta-derived MSCs [ 27 ], MSCs isolated directly from human BM tissue [ 22 , 23 ], mouse BM [ 24 ], chorionic villus tissue from a human placenta [ 26 ] and human tonsil tissue [ 29 ]. Samples are usually run on a 4–20% polyacrylamide gel before being deposited onto a nitrocellulose membrane and treated with the primary and secondary antibodies conjugated with horseradish peroxidase (HRP) [ 15 , 25 , 26 ].…”

“…One of flow cytometry’s newest and growing applications is imaging flow cytometry, which allows single-EV analyses with enhanced fluorescence sensitivity, a minimal background and the capacity for image confirmation. The labelling of CD9, CD63 [ 23 , 37 ] and CD81 [ 23 ] was used in a study of BM-MSC-EVs as drug delivery systems [ 37 ] and as a potential therapy for rheumatoid arthritis [ 23 ]. Fluorescent polystyrene beads are a very helpful tool in accurately acquiring EVs and identifying the area on the dot plot where the EVs should be.…”

Mesenchymal stem cell‐derived extracellular vesicles: Recent therapeutics and targeted drug delivery advances

“…In this context, there is compelling evidence that these membrane‐based nanoparticles, more importantly, mesenchymal stem cell‐derived EVs (MSC‐EVs), have undergone tremendous advancements in their utility as biomarkers and therapeutics of various organ conditions such as lung injury/disease (Sharma et al., 2022, Zhao et al., 2022), liver regeneration/injury (Lu et al., 2022, Zhang et al., 2023), bone repair/bone tissue regeneration (Cha et al., 2023, Huang et al., 2023, Warmink et al., 2023), graft‐versus‐host disease (Madel et al., 2023), wound healing (Kim et al., 2023, Las Heras et al., 2022), COVID‐19 induced infection (Chutipongtanate et al., 2022), blood‐brain‐barrier disruption (BBB) (Qiu et al., 2022), and various neurodegenerative disorders (Xu et al., 2022). Studies indicate that these EVs are the primary and fundamental paracrine effectors of MSCs and can effectively replicate the therapeutic properties of MSCs (Kou et al., 2022, Tsiapalis et al., 2023). Herein, we describe the characteristics of MSC‐EVs and factors determining their secretion profile in different cells, emphasizing their therapeutic application and drug delivery potential across different disease models.…”