Senescence did not alter the chondroprotective effect of extracellular vesicles from mesenchymal stromal cells

Background:Age is the most important risk factor in degenerative osteoarthritis (OA) and is associated with the accumulation of senescent cells that contribute to functional decline of joint. We previously demonstrated that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) largely mediate the therapeutic effect of parental cells in OA.Objectives:Here, we assessed the impact of senescence on the characteristics of EVs from adipose tissue-derived MSCs (ASC-EVs) and their properties in an in vitro model of OAMethods:ASCs were induced to senescence using 25µM etoposide for 24 hours. Senescence was assessed by quantifying proliferation rate, SA-βGal activity, nuclear γH2AX foci number, phalloidin staining and expression of cyclin dependent kinase inhibitors (CDKI) (RT-qPCR). ASC-EVs were isolated by differential ultracentrifugation and characterized by size, concentration, total protein content, structure (cryo-TEM) and immunophenotype. In vitro OA model used chondrocytes isolated from OA patients, which were stimulated with IL1β for 48h before culture with ASCs or ASC-EVs for 7 days. Expression of chondrocytic and inflammatory markers was quantified by RT-qPCR and SASP factors were quantified by ELISA in supernatants.Results:Senescence-induced ASCs experienced growth arrest and increase of SA-βGal staining, of p21 CDKI expression, of nuclear γH2AX foci, of stress fibers and of several SASP factors (IL6, IL8, MMP3) confirming the expression of main senescence features. Senescent ASCs produced 4-fold more EVs than healthy ASCs and senescent ASC-EVs were larger. In vitro, both healthy and senescent ASCs decreased fibrotic markers (type III COLLAGEN), catabolic and hypertrophic markers (MMP3, MMP13, AP) and increased COX2 expression in OA chondrocytes. By contrast, healthy ASCs decreased the expression of IL6 while senescent ASCs highly increased IL6. Looking at the role of ASC-EVs on OA chondrocytes, we found out that both healthy and senescent ASC-EVs were able to increase the expression of AGG and type II COLLAGEN while they decreased the expression of MMP13, AP, type X COLLAGEN, HMOX1 and IL6. Finally, healthy and senescent ASC-EVs decreased the number of SA-βGal positive chondrocytes but did not impact the expression of p21 in IL1β-induced chondrocytes.Conclusion:Our results indicated a chondroprotective effect of ASC-EVs, independently of the senescent state of parental cells and suggested that EVs might act through different mechanisms than ASCs, which warrants further investigationDisclosure of Interests:Jérémy Boulestreau: None declared, Marie Maumus Employee of: Bauerfeind France, Pauline Rozier: None declared, Christian Jorgensen Shareholder of: Medxcell sciences, Consultant of: Medxcell sciences, Daniele Noel Shareholder of: Medxcell sciences, Consultant of: Medxcell sciences

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Extracellular Vesicles Are More Potent Than Adipose Mesenchymal Stromal Cells to Exert an Anti-Fibrotic Effect in an In Vitro Model of Systemic Sclerosis

International Journal of Molecular Sciences ◽

10.3390/ijms22136837 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6837

Author(s):

Pauline Rozier ◽

Marie Maumus ◽

Claire Bony ◽

Alexandre Thibault Jacques Maria ◽

Florence Sabatier ◽

...

Keyword(s):

Systemic Sclerosis ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

In Vitro Model ◽

Specific Treatment ◽

Complex Disorder ◽

Molecular Features ◽

Vitro Model

Systemic sclerosis (SSc) is a complex disorder resulting from dysregulated interactions between the three main pathophysiological axes: fibrosis, immune dysfunction, and vasculopathy, with no specific treatment available to date. Adipose tissue-derived mesenchymal stromal cells (ASCs) and their extracellular vesicles (EVs) have proved efficacy in pre-clinical murine models of SSc. However, their precise action mechanism is still not fully understood. Because of the lack of availability of fibroblasts isolated from SSc patients (SSc-Fb), our aim was to determine whether a TGFβ1-induced model of human myofibroblasts (Tβ-Fb) could reproduce the characteristics of SSc-Fb and be used to evaluate the anti-fibrotic function of ASCs and their EVs. We found out that Tβ-Fb displayed the main morphological and molecular features of SSc-Fb, including the enlarged hypertrophic morphology and expression of several markers associated with the myofibroblastic phenotype. Using this model, we showed that ASCs were able to regulate the expression of most myofibroblastic markers on Tβ-Fb and SSc-Fb, but only when pre-stimulated with TGFβ1. Of interest, ASC-derived EVs were more effective than parental cells for improving the myofibroblastic phenotype. In conclusion, we provided evidence that Tβ-Fb are a relevant model to mimic the main characteristics of SSc fibroblasts and investigate the mechanism of action of ASCs. We further reported that ASC-EVs are more effective than parental cells suggesting that the TGFβ1-induced pro-fibrotic environment may alter the function of ASCs.

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Role of extracellular vesicles from adipose tissue‐ and bone marrow‐mesenchymal stromal cells in endothelial proliferation and chondrogenesis

Stem Cells Translational Medicine ◽

10.1002/sctm.21-0107 ◽

2021 ◽

Author(s):

Cansu Gorgun ◽

Maria Elisabetta Federica Palamà ◽

Daniele Reverberi ◽

Maria Cristina Gagliani ◽

Katia Cortese ◽

...

Keyword(s):

Bone Marrow ◽

Adipose Tissue ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Endothelial Proliferation

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Mesenchymal Stromal Cells Derived Extracellular Vesicles Ameliorate Acute Renal Ischemia Reperfusion Injury by Inhibition of Mitochondrial Fission through miR-30

Stem Cells International ◽

10.1155/2016/2093940 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 52

Author(s):

Di Gu ◽

Xiangyu Zou ◽

Guanqun Ju ◽

Guangyuan Zhang ◽

Erdun Bao ◽

...

Keyword(s):

Reperfusion Injury ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Apoptosis Pathway ◽

Acute Renal Ischemia

Background. The immoderation of mitochondrial fission is one of the main contributors in ischemia reperfusion injury (IRI) and mesenchymal stromal cells (MSCs) derived extracellular vesicles have been regarded as a potential therapy method. Here, we hypothesized that extracellular vesicles (EVs) derived from human Wharton Jelly mesenchymal stromal cells (hWJMSCs) ameliorate acute renal IRI by inhibiting mitochondrial fission through miR-30b/c/d.Methods. EVs isolated from the condition medium of MCS were injected intravenously in rats immediately after monolateral nephrectomy and renal pedicle occlusion for 45 minutes. Animals were sacrificed at 24 h after reperfusion and samples were collected. MitoTracker Red staining was used to see the morphology of the mitochondria. The expression of DRP1 was measured by western blot. miR-30 in EVs and rat tubular epithelial cells was assessed by qRT-PCR. Apoptosis pathway was identified by immunostaining.Results. We found that the expression of miR-30 in injured kidney tissues was declined and mitochondrial dynamics turned to fission. But they were both restored in EVs group in parallel with reduced cell apoptosis. What is more, when the miR-30 antagomirs were used to reduce the miRNA levels, all the related effects of EVs reduced remarkably.Conclusion. A single administration of hWJMSC-EVs could protect the kidney from IRI by inhibition of mitochondrial fission via miR-30.

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The extracellular vesicles-derived from mesenchymal stromal cells: A new therapeutic option in regenerative medicine

Journal of Cellular Biochemistry ◽

10.1002/jcb.26726 ◽

2018 ◽

Vol 119 (10) ◽

pp. 8048-8073 ◽

Cited By ~ 29

Author(s):

Vajihe Taghdiri Nooshabadi ◽

Soura Mardpour ◽

Aliakbar Yousefi-Ahmadipour ◽

Amir Allahverdi ◽

Mehrnaz Izadpanah ◽

...

Keyword(s):

Regenerative Medicine ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Option

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Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis

Frontiers in Immunology ◽

10.3389/fimmu.2021.627605 ◽

2021 ◽

Vol 12 ◽

Author(s):

Anna Maria Tolomeo ◽

Ignazio Castagliuolo ◽

Martina Piccoli ◽

Michele Grassi ◽

Fabio Magarotto ◽

...

Keyword(s):

Beneficial Effect ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Dextran Sulfate ◽

Experimental Colitis ◽

Culture Conditions ◽

Experimental Conditions ◽

Intestinal Fibrosis ◽

Cells Of Origin

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1β, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.

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Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells Protect Cardiac Cells Against Hypoxia/Reoxygenation Injury by Inhibiting Endoplasmic Reticulum Stress via Activation of the PI3K/Akt Pathway

Cell Transplantation ◽

10.1177/0963689720945677 ◽

2020 ◽

Vol 29 ◽

pp. 096368972094567

Author(s):

Changyi Zhang ◽

Hongwu Wang ◽

Godfrey C.F. Chan ◽

Yu Zhou ◽

Xiulan Lai ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Umbilical Cord ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cardiac Cells ◽

Akt Pathway ◽

Human Umbilical Cord ◽

Hypoxia Reoxygenation

Endoplasmic reticulum (ER) stress is implicated in the pathogenesis of many diseases, including myocardial ischemia/reperfusion injury. We hypothesized that human umbilical cord mesenchymal stromal cells derived extracellular vesicles (HuMSC-EVs) could protect cardiac cells against hyperactive ER stress induced by hypoxia/reoxygenation (H/R) injury. The H/R model was generated using the H9c2 cultured cardiac cell line. HuMSC-EVs were extracted using a commercially available exosome isolation reagent. Levels of apoptosis-related signaling molecules and the degree of ER stress were assessed by western blot. The role of the PI3K/Akt pathway was investigated using signaling inhibitors. Lactate dehydrogenase leakage and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) analysis were used for evaluating the therapeutic effects of HuMSC-EVs in vitro. The results showed that ER stress and the rate of apoptosis were increased in the context of H/R injury. Treatment with HuMSC-EVs inhibited ER stress and increased survival in H9c2 cells exposed to H/R. Mechanistically, the PI3K/Akt pathway was activated by treatment with HuMSC-EVs after H/R. Inhibition of the PI3K/Akt pathway by a specific inhibitor, LY294002, partially reduced the protective effect of HuMSC-EVs. Our findings suggest that HuMSC-EVs could alleviate ER stress–induced apoptosis during H/R via activation of the PI3K/Akt pathway.

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Equine Adipose-Derived Mesenchymal Stromal Cells Release Extracellular Vesicles Enclosing Different Subsets of Small RNAs

Stem Cells International ◽

10.1155/2019/4957806 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 8

Author(s):

Stefano Capomaccio ◽

Katia Cappelli ◽

Cinzia Bazzucchi ◽

Mauro Coletti ◽

Rodolfo Gialletti ◽

...

Keyword(s):

Electron Microscopy ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Small Rnas ◽

Rna Binding ◽

Bioinformatic Analysis ◽

Protein Coding ◽

Significant Enrichment ◽

Pathway Analyses

Background. Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role. Methods. EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was isolated, enriched for small RNAs (<200 nt), and sequenced by Illumina technology. After bioinformatic analysis with state-of-the-art pipelines for short sequences, mapped reads were used to describe EV RNA cargo, reporting classes, and abundances. Enrichment analyses were performed to infer involved pathways and functional categories. Results. Electron microscopy showed the presence of vesicles ranging in size from 30 to 300 nm and expressing typical markers. RNA analysis revealed that ribosomal RNA was the most abundant fraction, followed by small nucleolar RNAs (snoRNAs, 13.67%). Miscellaneous RNA (misc_RNA) reached 4.57% of the total where Y RNA, RNaseP, and vault RNA represented the main categories. miRNAs were sequenced at a lower level (3.51%) as well as protein-coding genes (1.33%). Pathway analyses on the protein-coding fraction revealed a significant enrichment for the “ribosome” pathway followed by “oxidative phosphorylation.” Gene Ontology analysis showed enrichment for terms like “extracellular exosome,” “organelle envelope,” “RNA binding,” and “small molecule metabolic process.” The miRNA target pathway analysis revealed the presence of “signaling pathways regulating pluripotency of stem cells” coherent with the source of the samples. Conclusion. We herein demonstrated that e-AdMSC release EVs enclosing different subsets of small RNAs that potentially regulate a number of biological processes. These findings shed light on the role of EVs in the context of MSC biology.

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