Extracellular vesicles (EVs) are becoming promising tools for clinical application, either as sources of disease-specific molecular signatures for the unraveling of disease pathophysiology and ...establishment of novel biomarkers, or as platforms for cell-free nanotherapy. Yet, an unsolved issue is to define standardized techniques for EV isolation allowing data comparison across laboratories worldwide. Considering the difficulties to find this necessary consensus, it has to be stressed out that the outcome of the downstream analysis might be deeply biased by the isolation method, among other variables. Thus, it is crucial that the researcher is aware of the strengths and weaknesses of each method keeping their intended use in mind, and to sufficiently report the methodology details for the results to be comparable and solid. This review aims to present the most widely used EV isolation methods, from the initial differential ultracentrifugation (dUC) to newest approaches.
•A pan-method for EVs’ isolation does not exist.•EVs’ isolation method biases the outcome of downstream analysis.•EV final use will determine the strategy of isolation.•The best EV isolation strategy might be a combination of different methods to reach the ideal purity and recovery.•Correct reporting of the EV isolation method in papers will lead to comparable and solid results.
Primary ventricular fibrillation (PVF) is a life-threatening complication of ST-segment elevation myocardial infarction (STEMI). It is unclear what roles viral infection and/or systemic inflammation ...may play as underlying triggers of PVF, as a second hit in the context of acute ischaemia. Here we aimed to evaluate whether the circulating virome and inflammatory proteome were associated with PVF development in patients with STEMI. Blood samples were obtained from non-PVF and PVF STEMI patients at the time of primary PCI, and from non-STEMI healthy controls. The virome profile was analysed using VirCapSeq-VERT (Virome Capture Sequencing Platform for Vertebrate Viruses), a sequencing platform targeting viral taxa of 342,438 representative sequences, spanning all virus sequence records. The inflammatory proteome was explored with the Olink inflammation panel, using the Proximity Extension Assay technology. After analysing all viral taxa known to infect vertebrates, including humans, we found that non-PVF and PVF patients only significantly differed in the frequencies of viruses in the Gamma-herpesvirinae and Anelloviridae families. In particular, most showed a significantly higher relative frequency in non-PVF STEMI controls. Analysis of systemic inflammation revealed no significant differences between the inflammatory profiles of non-PVF and PVF STEMI patients. Inflammatory proteins associated with cell adhesion, chemotaxis, cellular response to cytokine stimulus, and cell activation proteins involved in immune response (IL6, IL8 CXCL-11, CCL-11, MCP3, MCP4, and ENRAGE) were significantly higher in STEMI patients than non-STEMI controls. CDCP1 and IL18-R1 were significantly higher in PVF patients compared to healthy subjects, but not compared to non-PVF patients. The circulating virome and systemic inflammation were not associated with increased risk of PVF development in acute STEMI. Accordingly, novel strategies are needed to elucidate putative triggers of PVF in the setting of acute ischaemia, in order to reduce STEMI-driven sudden death burden.
In light of pioneering findings in the 1980s and an estimation of more than 130 million global annual births, umbilical cord blood (UCB) is considered to be the most plentiful reservoir of cells and ...to have regenerative potential for many clinical applications. Although UCB is used mainly against blood disorders, the spectrum of diseases for which it provides effective therapy has been expanded to include non-hematopoietic conditions; UCB has also been used as source for regenerative cell therapy and immune modulation. Thus, collection and banking of UCB-derived cells have become a popular option. However, there are questions regarding the cost versus the benefits of UCB banking, and it also raises complex ethical and legal issues. This review discusses many issues surrounding the conservation of UCB-derived cells and the great potential and current clinical applications of UCB in an era of new therapies. In particular, we describe the practical issues inherent in UCB collection, processing, and long-term storage as well as the different types of 'stem' or progenitor cells circulating in UCB and their uses in multiple clinical settings. Given these considerations, the trend toward UCB will continue to provide growing assistance to health care worldwide.
Compelling evidence supports the therapeutic benefit of extracellular vesicles (EVs). EVs are nanostructures with a lipid bilayer membrane that are secreted by multiple cells, including mesenchymal ...stromal cells (MSCs), as means of cellular communication. MSC-EVs, resembling their MSC origin, carry protected immunomodulatory and pro-regenerative cargoes to targeted neighboring or distant cells and tissues. Though treatments focused on MSC-EVs have emerged as greatly versatile approaches to modulate multiple inflammatory-related conditions, crucial concerns, including the possibility of increasing therapeutic outcomes by pre-conditioning parental MSCs or engineering derived EVs and clarification of the most relevant mechanisms of action, remain. Here, we summarize the large amount of preclinical research surrounding the modulation of beneficial effects by MSC-EVs.
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•MSC-EV are a potential therapy through immunomodulation in multiple disease models.•Common MSC-EV mechanisms of action affect the NFκB and Stat3 pathways.•A variety of miRNA and EV-associated proteins account for foremost MSC-EV effects.•The bioengineering of MSC-EVs is a feasible option to enhance MSC-EV function.
Multipotent mesenchymal stromal cells (MSC) represent a promising strategy for a variety of medical applications. Although only a limited number of MSC engraft and survive after in vivo cellular ...infusion, MSC have shown beneficial effects on immunomodulation and tissue repair. This indicates that the contribution of MSC exists in paracrine signaling, rather than a cell-contact effect of MSC. In this review, we focus on current knowledge about tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and mechanisms based on extracellular vesicles (EV) that govern long-lasting immunosuppressive and regenerative activity of MSC. In this context, in particular, we discuss the very robust set of findings by Jha and colleagues, and the opportunity to potentially extend their research focus on EV isolated in concentrated conditioned media (CCM) from adipose tissue derived MSC (ASC). Particularly, the authors showed that ASC-CCM mitigated visual deficits after mild traumatic brain injury in mice. TSG-6 knockdown ASC were, then, used to generate TSG-6-depleted CCM that were not able to replicate the alleviation of abnormalities in injured animals. In light of the presented results, we envision that the infusion of much distilled ASC-CCM could enhance the alleviation of visual abnormalities. In terms of EV research, the advantages of using size-exclusion chromatography are also highlighted because of the enrichment of purer and well-defined EV preparations. Taken together, this could further delineate and boost the benefit of using MSC-based regenerative therapies in the context of forthcoming clinical research testing in diseases that disrupt immune system homeostasis.
Dilated cardiomyopathy (DCM) remains a major cause of heart failure and carries a poor prognosis despite important advances in recent years. Better disease characterization using novel molecular ...techniques is needed to refine its progression. This study explored the proteomic signature of plasma-derived extracellular vesicles (EVs) obtained from DCM patients and healthy controls using size-exclusion chromatography (SEC). EV-enriched fractions were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Raw data obtained from LC-MS/MS were analyzed against the Uniprot human database using MaxQuant software. Additional analyses using Perseus software were based on the Intensity-Based Absolute Quantification (iBAQ) values from MaxQuant analyses. A total of 90.07 ± 21 proteins (227 different proteins) in the DCM group and 96.52 ± 17.91 proteins (183 different proteins) in the control group were identified. A total of 176 proteins (74.6%) were shared by controls and DCM patients, whereas 51 proteins were exclusive for the DCM group and 7 proteins were exclusive for the control group. Fibrinogen (α, β and γ chain), serotransferrin, α-1-antitrypsin, and a variety of apolipoprotein family members (C-I, C-III, D, H or β-2-glycoprotein, and J or clusterin) were clustered in SEC-EVs derived from DCM patients relative to controls (p < 0.05). Regarding Gene Ontology analysis, response to stress and protein activation-related proteins were enriched in DCM-EVs compared with controls. Thus, the present study reports the distinct proteomic signature of circulating DCM-EVs compared with control-EVs. Furthermore, we confirm that SEC obtains highly purified EV fractions from peripheral blood samples for subsequent use in determining disease-specific proteomic signatures.
Extracellular vesicles (EVs) comprise a wide range of bilayer membrane-enclosed nanovesicles secreted by nearly all cell types and present in all human body fluids. In dilated cardiomyopathy, which is a frequent cause of heart transplantation, unraveling the specific combinations of cargo proteins within the EVs that peripherally circulate in patients hold promising potential for biomarker discovery.
For years, sufficient progress has been made in treating heart failure following myocardial infarction; however, the social and economic burdens and the costs to world health systems remain high. ...Moreover, treatment advances have not resolved the underlying problem of functional heart tissue loss. In this field of research, for years we have actively explored innovative biotherapies for cardiac repair. Here, we present a general, critical overview of our experience in using mesenchymal stem cells, derived from cardiac adipose tissue and umbilical cord blood, in a variety of cell therapy and tissue engineering approaches. We also include the latest advances and future challenges, including good manufacturing practice and regulatory issues. Finally, we evaluate whether recent approaches hold potential for reliable translation to clinical trials.
Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and ...regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV–cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells’ interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton’s Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV–cell interactions and associated functions.
The ectoenzymes CD39 and CD73 regulate the purinergic signaling through the hydrolysis of adenosine triphosphate (ATP)/ADP to AMP and to adenosine (Ado), respectively. This shifts the ...pro-inflammatory milieu induced by extracellular ATP to the anti-inflammatory regulation by Ado. Mesenchymal stem cells (MSCs) have potent immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype aiding tissue repair.
, we observed that human cardiac adipose tissue-derived MSCs (cATMSCs) and umbilical cord MSCs similarly polarize monocytes toward a regulatory M2 phenotype, which maintained the expression of CD39 and induced expression of CD73 in a cell contact dependent fashion, correlating with increased functional activity. In addition, the local treatment with porcine cATMSCs using an engineered bioactive graft promoted the
CD73 expression on host monocytes in a swine model of myocardial infarction. Our results suggest the upregulation of ectonucleotidases on MSC-conditioned monocytes as an effective mechanism to amplify the long-lasting immunomodulatory and healing effects of MSCs delivery.
Fibrosis is present in an important proportion of myocardial disorders. Injury activates cardiac fibroblasts, which deposit excess extracellular matrix, increasing tissue stiffness, impairing cardiac ...function, and leading to heart failure. Clinical therapies that directly target excessive fibrosis are limited, and more effective treatments are needed. Immunotherapy based on chimeric antigen receptor (CAR) T cells is a novel technique that redirects T lymphocytes toward specific antigens to eliminate the target cells. It is currently used in haematological cancers but has demonstrated efficacy in mouse models of hypertensive cardiac fibrosis, with activated fibroblasts as the target cells. CAR T cell therapy is associated with significant toxicities, but CAR natural killer cells can overcome efficacy and safety limitations. The use of CAR immunotherapy offers a potential alternative to current therapies for fibrosis reduction and restoration of cardiac function in patients with myocardial fibrosis.
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•New therapeutic approaches are needed to treat cardiac fibrosis.•CAR T cell immunotherapy has antifibrotic effect in cardiac fibrosis mouse models.•CAR NK cells could be an “off-the-shelf” therapy that avoids CAR T cell toxicity.