Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization.

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macrophage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo ). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. Stem Cells Translational Medicine 2017 Stem Cells Translational Medicine 2017;6:1018-1028.

Lo Sicco C ，Reverberi D ，Balbi C ，Ulivi V ，Principi E ，Pascucci L ，Becherini P ，Bosco MC ，Varesio L ，Franzin C ，Pozzobon M ，Cancedda R ，Tasso R ... -  《-》

A Method for Isolating and Characterizing Mesenchymal Stromal Cell-derived Extracellular Vesicles.

The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages. The topics comprise description of the MSC-conditioned medium containing vesicles preparation, EV isolation, and characterization mainly by specifically set up flow cytometry and electron microscopy approaches, and in vitro methodologies involved in testing the EV anti-inflammatory capacity. The procedures described here can be easily reproduced and can be employed regardless of the type of progenitor cells used to secrete EVs. © 2018 by John Wiley & Sons, Inc.

Lo Sicco C ，Reverberi D ，Pascucci L ，Tasso R ... -  《-》

Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery.

Ren W ，Hou J ，Yang C ，Wang H ，Wu S ，Wu Y ，Zhao X ，Lu C ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

Atorvastatin-pretreated mesenchymal stem cell-derived extracellular vesicles promote cardiac repair after myocardial infarction via shifting macrophage polarization by targeting microRNA-139-3p/Stat1 pathway.

Extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (MSCs) pretreated with atorvastatin (ATV) (MSCATV-EV) have a superior cardiac repair effect on acute myocardial infarction (AMI). The mechanisms, however, have not been fully elucidated. This study aims to explore whether inflammation alleviation of infarct region via macrophage polarization plays a key role in the efficacy of MSCATV-EV. MSCATV-EV or MSC-EV were intramyocardially injected 30 min after coronary ligation in AMI rats. Macrophage infiltration and polarization (day 3), cardiac function (days 0, 3, 7, 28), and infarct size (day 28) were measured. EV small RNA sequencing and bioinformatics analysis were conducted for differentially expressed miRNAs between MSCATV-EV and MSC-EV. Macrophages were isolated from rat bone marrow for molecular mechanism analysis. miRNA mimics or inhibitors were transfected into EVs or macrophages to analyze its effects on macrophage polarization and cardiac repair in vitro and in vivo. MSCATV-EV significantly reduced the amount of CD68+ total macrophages and increased CD206+ M2 macrophages of infarct zone on day 3 after AMI compared with MSC-EV group (P < 0.01-0.0001). On day 28, MSCATV-EV much more significantly improved the cardiac function than MSC-EV with the infarct size markedly reduced (P < 0.05-0.0001). In vitro, MSCATV-EV also significantly reduced the protein and mRNA expressions of M1 markers but increased those of M2 markers in lipopolysaccharide-treated macrophages (P < 0.05-0.0001). EV miR-139-3p was identified as a potential cardiac repair factor mediating macrophage polarization. Knockdown of miR-139-3p in MSCATV-EV significantly attenuated while overexpression of it in MSC-EV enhanced the effect on promoting M2 polarization by suppressing downstream signal transducer and activator of transcription 1 (Stat1). Furthermore, MSCATV-EV loaded with miR-139-3p inhibitors decreased while MSC-EV loaded with miR-139-3p mimics increased the expressions of M2 markers and cardioprotective efficacy. We uncovered a novel mechanism that MSCATV-EV remarkably facilitate cardiac repair in AMI by promoting macrophage polarization via miR-139-3p/Stat1 pathway, which has the great potential for clinical translation.

Ning Y ，Huang P ，Chen G ，Xiong Y ，Gong Z ，Wu C ，Xu J ，Jiang W ，Li X ，Tang R ，Zhang L ，Hu M ，Xu J ，Xu J ，Qian H ，Jin C ，Yang Y ... -  《BMC Medicine》

Mesenchymal stem cell-secreted extracellular vesicles carrying TGF-β1 up-regulate miR-132 and promote mouse M2 macrophage polarization.

The effects of mesenchymal stem cells (MSCs) on different types of diseases are controversial, and the inner mechanisms remain unknown, which retards the utilization of MSCs in disease therapy. In this study, we aimed to elucidate the mechanisms of MSCs-extracellular vesicles (EVs) carrying transforming growth factor-beta 1 (TGF-β1) in M2 polarization in mouse macrophages via the microRNA-132 (miR-132)/E3 ubiquitin ligase myc binding protein 2 (Mycbp2)/tuberous sclerosis complex 2 (TSC2) axis. Mouse MSCs were isolated for adipogenic and osteogenic induction, followed by co-culture with mouse macrophages RAW264.7. Besides, mouse macrophages RAW264.7 were co-cultured with MSCs-EVs in vitro, where the proportion of macrophages and inflammation were detected by flow cytometry and ELISA. The experimental data revealed that MSCs-EVs promoted M2 polarization of macrophages, and elevated interleukin (IL)-10 expression and inhibited levels of IL-1β, tumour necrosis factor (TNF)-α and IL-6. MSC-EV-treated macrophages RAW264.7 increased TGF-β1 expression, thus elevating miR-132 expression. MiR-132 directly bound to Mycbp2, as confirmed by luciferase activity assay. Meanwhile, E3 ubiquitin ligase Mycbp2 could ubiquitinate TSC2 protein. Furthermore, silencing TGF-β1 inhibited M2 polarization of MSC-EV-treated macrophages. Taken conjointly, this study provides evidence reporting that MSC-secreted EVs carry TGF-β1 to promote M2 polarization of macrophages via modulation of the miR-132/Mycbp2/TSC2 axis.

Wang Y ，Han B ，Wang Y ，Wang C ，Zhang H ，Xue J ，Wang X ，Niu T ，Niu Z ，Chen Y ... -  《-》