Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis.

BACKGROUND AIMS: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. METHODS: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. RESULTS: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. CONCLUSIONS: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS.

A Cellular Model of Amyotrophic Lateral Sclerosis to Study the Therapeutic Effects of Extracellular Vesicles from Adipose Mesenchymal Stem Cells on Microglial Activation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to progressive paralysis and death. Increasing evidence indicates that neuroinflammation plays an important role in ALS's pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate/accelerate MN death. In particular, the role of the microglia in ALS remains unclear, partly due to the lack of experimental models that can fully recapitulate the complexity of ALS's pathology. In this study, we developed and characterized a microglial cell line, SIM-A9-expressing human mutant protein Cu+/Zn+ superoxide dismutase_1 (SIM-A9hSOD1(G93A)), as a suitable model in vitro mimicking the microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells induced a change in their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which is known to activate cell death processes and apoptosis. Afterward, we used our microglial model as an experimental set-up to investigate the therapeutic action of extracellular vesicles isolated from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represent a promising therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. Here, we demonstrated that treatment with ASC-EVs is able to modulate activated ALS microglia, reducing their metabolic activity and polarizing their phenotype toward an anti-inflammatory one through a mechanism of reduction of reactive oxygen species.

Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy.

BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. METHODS: In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 µg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10. RESULTS: The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. CONCLUSIONS: Our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.

Adipose mesenchymal stem cells-derived extracellular vesicles exert their preferential action in damaged central sites of SOD1 mice rather than peripherally.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.

CSF levels of Chitinase3like1 correlate with early response to cladribine in multiple sclerosis.

Background: Cladribine has been introduced as a high-efficacy drug for treating relapsing-remitting multiple sclerosis (RRMS). Initial cohort studies showed early disease activity in the first year after drug initiation. Biomarkers that can predict early disease activity are needed. Aim: To estimate cerebrospinal fluid (CSF) markers of clinical and radiological responses after initiation of cladribine. Methods: Forty-two RRMS patients (30F/12M) treated with cladribine were included in a longitudinal prospective study. All patients underwent a CSF examination at treatment initiation, clinical follow-up including Expanded Disability Status Scale (EDSS) assessment, and a 3T MRI scan after 6,12 and 24 months, including the evaluation of white matter (WM) and cortical lesions (CLs). CSF levels of 67 inflammatory markers were assessed with immune-assay multiplex techniques. The 'no evidence of disease activity' (NEDA-3) status was assessed after two years and defined by no relapses, no disability worsening measured by EDSS and no MRI activity, including CLs. Results: Three patients were lost at follow-up. At the end of follow-up, 19 (48%) patients remained free from disease activity. IFNgamma, Chitinase3like1, IL32, Osteopontin, IL12(p40), IL34, IL28A, sTNFR2, IL20 and CCL2 showed the best association with disease activity. When added in a multivariate regression model including age, sex, and baseline EDSS, Chitinase 3 like1 (p = 0.049) significantly increased in those patients with disease activity. Finally, ROC analysis with Chitinase3like1 added to a model with EDSS, sex, age previous relapses, WM lesion number, CLs, number of Gad enhancing lesions and spinal cord lesions provided an AUC of 0.76 (95%CI 0.60-0.91). Conclusions: CSF Chitinase 3 like1 might provide prognostic information for predicting disease activity in the first years after initiation of cladribine. The drug's effect on chronic macrophage and microglia activation deserves further evaluation.

Association of Levels of CSF Osteopontin With Cortical Atrophy and Disability in Early Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: To evaluate CSF inflammatory markers with accumulation of cortical damage as well as disease activity in patients with early relapsing-remitting MS (RRMS). METHODS: CSF levels of osteopontin (OPN) and 66 inflammatory markers were assessed using an immune-assay multiplex technique in 107 patients with RRMS (82 F/25 M, mean age 35.7 ± 11.8 years). All patients underwent regular clinical assessment and yearly 3T MRI scans for 2 years while 39 patients had a 4-year follow-up. White matter lesion number and volume, cortical lesions (CLs) and volume, and global cortical thickness (CTh) were evaluated together with the 'no evidence of disease activity' (NEDA-3) status, defined by no relapses, no disability worsening, and no MRI activity, including CLs. RESULTS: The random forest algorithm selected OPN, CXCL13, TWEAK, TNF, IL19, sCD30, sTNFR1, IL35, IL16, and sCD163 as significantly associated with changes in global CTh. OPN and CXCL13 were most related to accumulation of atrophy after 2 and 4 years. In a multivariate linear regression model on CSF markers, OPN (p < 0.001), CXCL13 (p = 0.001), and sTNFR1 (p = 0.024) were increased in those patients with accumulating atrophy (adjusted R-squared 0.615). The 10 markers were added in a model that included all clinical, demographic, and MRI variables: OPN (p = 0.002) and IL19 (p = 0.022) levels were confirmed to be significantly increased in patients developing more CTh change over the follow-up (adjusted R-squared 0.619). CXCL13 and OPN also revealed the best association with NEDA-3 after 2 years, with OPN significantly linked to disability accumulation (OR 2.468 [1.46-5.034], p = 0.004) at the multivariate logistic regression model. DISCUSSION: These data confirm and expand our knowledge on the prognostic role of the CSF inflammatory profile in predicting changes in cortical pathology and disease activity in early MS. The data emphasize a crucial role of OPN.