Equine Bone Marrow-Derived MSCs and Their EVs Exhibit Different Immunomodulatory Effects on Cartilage Explants in an In Vitro Osteoarthritis Model.

Joint injury often leads to cartilage damage and posttraumatic inflammation, which drives continued extracellular matrix degradation culminating in osteoarthritis. Mesenchymal stem cells (MSCs) have been proposed as a biotherapeutic to modulate inflammation within the joint. However, concerns have been raised regarding the immunogenicity of MSCs cultured in traditional fetal bovine serum (FBS) containing media, and the potential of xenogenic antigens to activate the immune system causing rejection and destruction of the MSCs. Xenogen-free alternatives to FBS have been proposed to decrease MSC immunogenicity, including platelet lysate (PL) and equine serum. The objective of this study was to compare the immunomodulatory properties of BM-MSCs culture-expanded in media supplemented with autologous PL (APL), pooled PL (PPL), equine serum (ES) or FBS. We hypothesized that BM-MSCs culture expanded in media with xenogen-free supplements would exhibit superior immunomodulatory properties to those cultured in FBS containing media. Bone marrow-derived MSCs (BM-MSCs) were isolated from six horses and culture expanded in each media type. Blood was collected from each horse to isolate platelet lysate. The immunomodulatory function of the BM-MSCs was assessed via a T cell proliferation assay and through multiplex immunoassay quantification of cytokines, including IL-1β, IL-6, IL-8, IL-10, and TNFα, following preconditioning of BM-MSCs with IL-1β. The concentration of platelet-derived growth factor BB (PDGF-BB), IL-10, and transforming growth factor-β (TGF-β) in each media was measured via immunoassay. BM-MSCs cultured in ES resulted in significant suppression of T cell proliferation (p = 0.02). Cell culture supernatant from preconditioned BM-MSCs cultured in ES had significantly higher levels of IL-6. PDGF-BB was significantly higher in APL media compared to FBS media (p = 0.016), while IL-10 was significantly higher in PPL media than ES and FBS (p = 0.04). TGF-β was highest in APL media, with a significant difference in comparison to ES media (p = 0.03). In conclusion, expansion of equine BM-MSCs in ES may enhance their immunomodulatory abilities, while PL containing media may have some inherent therapeutic potential associated with higher concentrations of growth factors. Further studies are needed to elucidate which xenogen-free supplement optimizes BM-MSC performance.

ACE2-enriched extracellular vesicles enhance infectivity of live SARS-CoV-2 virus.

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

Aim: Mesenchymal stem cells (MSCs) are a promising therapeutic strategy for osteoarthritis (OA), largely due to their regenerative potential, which is attributed in part to their secretome. The secretome includes soluble factors and extracellular vesicles (EVs). Given that MSCs are sensitive to various culture conditions, this study aims to investigate the effects of different media supplemented with either fetal bovine serum (FBS) (F), platelet lysate (P), or serum/xeno-free (S/X) on the composition and therapeutic potential of the secretome from bone marrow-derived MSCs (BMSCs).Methods: BMSCs were cultured in F, P, or S/X media, with secretomes collected after starvation. The secretomes were analyzed for soluble factors, EVs, and miRNAs. Inflammatory responses were assessed in an in vitro OA model using inflamed chondrocytes and gene expression was evaluated by qRT-PCR.Results: The secretomes from all conditions exhibited a similar molecular fingerprint. Proteomic analysis identified 98 common proteins encompassing growth factors and inflammatory mediators. EVs showed similar size and phenotype, with a slight difference in CD44 expression in EVs derived from P-expanded MSCs. Despite the high overall similarity, miRNA profiling identified 13 key players, with subtle differences in the miRNA composition of EVs from FBS-expanded BMSCs. All secretomes exhibited anti-inflammatory effects, with the FBS-expanded secretome showing the most pronounced therapeutic potential.Conclusion: The secretomes derived from different culture conditions share key molecular components. EVs may contribute to variations in therapeutic outcomes through their cargo. Optimizing MSC expansion conditions is crucial for enhancing the therapeutic potential of MSC-derived secretomes in OA treatment. Further research is needed to clarify the specific role of factors, miRNAs, and EVs in modulating OA pathology.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC-EVs) have been proposed as a novel therapeutic tool with numerous clinically related advantages. However, their characteristics and functionality are dependent on the source of MSCs and their cell culture conditions. Fetal bovine serum (FBS) provides a source of nutrients and growth factors to the cultured cells. However, certain pitfalls are associated with its supplementation to the culture media, including introduction of exogenous FBS-derived EVs to the cultured cells. Thus, recent practices recommend utilization of serum-free (SF) media or EV-depleted FBS. On the contrary, evidence suggests that the immunomodulatory ability of MSC-EVs can be improved by exposing MSCs to an inflammatory (IF) environment. The objective of this study was to (1) compare EVs isolated from two tissue sources of MSCs that were exposed to various cell culture conditions and (2) to evaluate their anti-inflammatory effects. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were exposed to either a SF media environment, an IF environment, or media supplemented with 5% EV-depleted FBS. Following isolation of MSC-EVs, the isolates were quantified and evaluated for particle size, phenotypic changes, and their immunomodulatory potential. A statistically significant difference was not identified on the yield and protein concentration of different isolates of EVs from BM-MSCs and UC-MSCs, and all isolates had a circular appearance as evaluated via electron microscopy. A significant difference was identified on the phenotype of different EVs isolates; however, all isolates expressed classical markers such as CD9, CD63, and CD81. The addition of BM-derived MSC-EVs from FBS environment or UC-derived MSC-EVs from IF environment resulted in statistically significant downregulation of IL-6 messenger RNA (mRNA) in stimulated leukocytes. This study confirms that EVs produced by different MSC sources and cell culture conditions affect their phenotype and their immunomodulatory capacities.

IntroductionIn the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a “tri-culture” model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants.MethodsGene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures).ResultsIn general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young’s and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only.ConclusionsOur results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs’ chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies.

Background Synovial mesenchymal stem cells (SMSCs) have been discussed as promising tools for protecting chondrocytes from loss and inhibiting osteoarthritis (OA). This work infocuses on the function of SMSC-derived extracellular vesicles (EVs) in chondrocytes during OA and the molecular mechanism. Methods EVs were extracted from SMSCs and identified. Chondrocytes were treated with interleukin (IL)-1β to induce an OA-like condition in vitro and then treated with EVs. The proliferation, apoptosis, migration, extracellular matrix (ECM) degradation and inflammation in chondrocytes were examined. Key microRNAs (miRNAs) carried by EVs were screened using a microarray analysis, and the downstream molecules involved were explored using bioinformatic analysis. Rescue experiments were performed to validate the involvements of these molecules in EV-mediated events. Results EVs restored proliferation and migration while reduced apoptosis, ECM degradation and the secretion of pro-inflammatory cytokines in chondrocytes induced by IL-1β. miR-130b-3p was significantly elevated in chondrocytes after EVs treatment. Knockdown of miR-130b-3p blocked the protective roles of EVs against IL-1β-induced damage to chondrocytes. miR-130b-3p was found to target LDL receptor related protein 12 (LRP12) mRNA in chondrocytes. Overexpression of LRP12 counteracted the effects of EVs as well and activated the AKT/β-catenin signaling pathway. Conclusion This study provided evidence that EVs alleviate chondrocyte damage during OA through miR-130b-3p-mediated inhibition of the LRP12/AKT/β-catenin axis. This study may offer novel thoughts into the protection of chondrocytes and the management of OA.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors.Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function.Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines.Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Producing vesicle-free cell culture additive for human cells extracellular vesicles manufacturing.

Extracellular vesicles (EVs) have been recently identified as vital components of cell-based therapies based on the observation that conditioned media from cultured stromal cells reproduce some of the beneficial effects of intact cells. In order to obtain clinically active EVs derived from Mesenchymal Stromal Cells (MSCs) different procedures have been reported in the literature. Usually, non-confluent cells are incubated with culture medium for 48 h either with EV-depleted Fetal Bovine Serum (FBS) or without FBS. Our aim was to compare the effects of EVs isolated by ultracentrifugation from human umbilical cord MSC conditioned media obtained using these two conditions: with EV-depleted FBS (UC) or without FBS (UCw/o) on the mRNA expression levels of extracellular matrix related genes using the mouse chondrogenic cell line ATDC-5. We observed a deleterious effect on chondrogenic cells treated with UCw/o, showing higher mRNA expression levels of different metalloproteinases and decorin (Dcn) and lower collagen (Col1a1 and Col2a1) and aggrecan (Acan) mRNA levels. To elucidate whether this deleterious effect was induced by the EVs or by any proteins co-purified in the EV pellet, we used size exclusion chromatography (SEC) to further purify the EV pellet, obtaining an EV enriched fraction (EV or EVw/o) and a protein enriched fraction (Prot or Protw/o). Our results pointed that the negative effect on the chondrogenic cell line was due to the contaminant proteins coisolated with the EVs by ultracentrifugation and not from the EVs themselves. Thus, these results highlight the importance of working with well purified EV preparations to specifically achieve their therapeutic effect.

Stem cell derived extracellular vesicles for vascular elastic matrix regenerative repair

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-drivenOA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 µm sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.