Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Extracellular vesicles (EVs), such as exosomes and microvesicles, have been identified as mediators of a newly-discovered intercellular communication system. They are essential signaling mediators in various physiological and pathophysiological processes. Depending on their origin, they fulfill different functions. EVs of mesenchymal stem/stromal cells (MSCs) have been found to promote comparable therapeutic activities as MSCs themselves. In a variety of in vivo models, it has been observed that they suppress pro-inflammatory processes and reduce oxidative stress and fibrosis. By switching pro-inflammatory into tolerogenic immune responses, MSC-EVs very likely promote tissue regeneration by creating a pro-regenerative environment allowing endogenous stem and progenitor cells to successfully repair affected tissues. Accordingly, MSC-EVs provide a novel, very promising therapeutic agent, which has already been successfully applied to humans. However, the MSC-EV production process has not been standardized, yet. Indeed, a collection of different protocols has been used for the MSC-EV production, characterization and application. By focusing on kidney, heart, liver and brain injuries, we have reviewed the major outcomes of published MSC-EV in vivo studies.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

Background and Purpose— Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods— Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV–treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results— sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O 2 ) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O 2 ) but also reduced serum IgG extravasation—a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions— Leukocytes and specifically PMNs contribute to MSC-sEV–induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview— An online visual overview is available for this article.

Within the last two decades mesenchymal stem/stromal cells (MSCs) emerged after hematopoietic stem cells as the second most investigated and applied somatic stem cell entity so far. MSCs mediate immunosuppressive as well as pro-regenerative activities. Against the initial assumption, MSCs may not primarily exert their therapeutic functions in a cellular but rather in a paracrine manner. Here, extracellular vesicles (EVs), such as exosomes and microvesicles, have been identified as major mediators of these paracrine effects. Meanwhile, MSC-EVs have been applied to an increasing amount of different animal models and were tested in a patient suffering from steroid-refractory acute graft-versus-host disease (acute GvHD) as well as in a patient cohort with chronic kidney disease. So far, the MSC-EV administration appears to be safe in humans and all tested animal models. Improvements were reported in all settings. Thus, MSC-EVs appear as promising novel therapeutic agents which might help to improve disease associated symptoms in millions of patients. Here, we review some of the milestones in the field, briefly discuss challenges and highlight clinical aspects of acute GvHD and its treatment with MSCs and MSC-EVs.

Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not ‘normoxic’ (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS–MS revealed 52 proteins differentially abundant in sEVs from hypoxic and ‘normoxic’ MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix–receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix–receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

Background: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of childhood mortality and disability. Stem cell-based regenerative therapies seem promising to prevent long-term neurological deficits. Our previous work in neonatal HI revealed an unexpected interaction between mesenchymal stem/stromal cells (MSCs) and the brains' microenvironment leading to an altered therapeutic efficiency. MSCs are supposed to mediate most of their therapeutic effects in a paracrine mode via extracellular vesicles (EVs), which might be an alternative to cell therapy. In the present study, we investigated the impact of MSC-EVs on neonatal HI-induced brain injury.Methods: Nine-day-old C57BL/6 mice were exposed to HI through ligation of the right common carotid artery followed by 1 h hypoxia (10% oxygen). MSC-EVs were injected intraperitoneally 1, 3, and 5 days after HI. One week after HI, brain injury was evaluated by regional neuropathological scoring, atrophy measurements and immunohistochemistry to assess effects on neuronal, oligodendrocyte and vessel densities, proliferation, oligodendrocyte maturation, myelination, astro-, and microglia activation. Immunohistochemistry analyses were complemented by mRNA expression analyses for a broad set of M1/M2- and A1/A2-associated molecules and neural growth factors.Results: While total neuropathological scores and tissue atrophy were not changed, MSC-EVs significantly protected from HI-induced striatal tissue loss and decreased micro- and astroglia activation. MSC-EVs lead to a significant downregulation of the pro-inflammatory cytokine TNFa, accompanied by a significant upregulation of the M2 marker YM-1 and the anti-inflammatory cytokine TGFb. MSC-EVs significantly decreased astrocytic expression of the A1 marker C3, concomitant with an increased expression of neural growth factors (i.e., BDNF, VEGF, and EGF). These alterations were associated with an increased neuronal and vessel density, coinciding with a significant increase of proliferating cells in the neurogenic sub-ventricular zone juxtaposed to the striatum. MSC-EV-mediated neuroprotection went along with a significant improvement of oligodendrocyte maturation and myelination.Conclusion: The present study demonstrates that MSC-EVs mediate anti-inflammatory effects, promote regenerative responses and improve key developmental processes in the injured neonatal brain. The present results suggest different cellular target mechanisms of MSC-EVs, preventing secondary HI-induced brain injury. MSC-EV treatment may be a promising alternative to risk-associated cell therapies in neonatal brain injury.