Mesenchymal stromal/stem cell-derived extracellular vesicles in tissue repair: challenges and opportunities

Mesenchymal stem/stromal cells (MSCs) are important players in tissue homeostasis and regeneration owing to their immunomodulatory potential and release of trophic factors that promote healing. They have been increasingly used in clinical trials to treat multiple conditions associated with inflammation and tissue damage such as graft versus host disease, orthopedic injuries and cardiac and liver diseases. Recent evidence demonstrates that their beneficial effects are derived, at least in part, from their secretome. In particular, data from animal models and first-in-man studies indicate that MSC-derived extracellular vesicles (MSC-EVs) can exert similar therapeutic potential as their cells of origin. MSC-EVs are membranous structures loaded with proteins, lipids, carbohydrates and nucleic acids, which play an important role in cell-cell communication and may represent an attractive alternative for cell-based therapy. In this article we summarize recent advances in the use of MSC-EVs for tissue repair. We highlight several isolation and characterization approaches used to enrich MSC-derived EVs. We discuss our current understanding of the relative contribution of the MSC-EVs to the immunomodulatory and regenerative effects mediated by MSCs and MSC secretome. Finally we highlight the challenges and opportunities, which come with the potential use of MSC-EVs as cell free therapy for conditions that require tissue repair

A kaleidoscopic view of extracellular vesicles in lysosomal storage disorders

Extracellular vesicles (EVs) are a heterogeneous population of stable lipid membrane particles that play a critical role in the regulation of numerous physiological and pathological processes. EV cargo, which includes lipids, proteins, and RNAs including miRNAs, is affected by the metabolic status of the parental cell. Concordantly, abnormalities in the autophagic-endolysosomal pathway, as seen in lysosomal storage disorders (LSDs), can affect EV release as well as EV cargo. LSDs are a group of over 70 inheritable diseases, characterized by lysosomal dysfunction and gradual accumulation of undigested molecules. LSDs are caused by single gene mutations that lead to a deficiency of a lysosomal protein or lipid. Lysosomal dysfunction sets off a cascade of alterations in the endolysosomal pathway that can affect autophagy and alter calcium homeostasis, leading to energy imbalance, oxidative stress, and apoptosis. The pathophysiology of these diseases is very heterogenous, complex, and currently incompletely understood. LSDs lead to progressive multisystemic symptoms that often include neurological deficits. In this review, a kaleidoscopic overview will be given on the roles of EVs in LSDs, from their contribution to pathology and diagnostics to their role as drug delivery vehicles. Furthermore, EV cargo and surface engineering strategies will be discussed to show the potential of EVs in future LSD treatment, both in the context of enzyme replacement therapy, as well as future gene editing strategies like CRISPR/Cas. The use of engineered EVs as drug delivery vehicles may mask therapeutic cargo from the immune system and protect it from degradation, improving circulation time and targeted delivery

Injectable hydrogels for sustained delivery of extracellular vesicles in cartilage regeneration

Extracellular vesicles (EVs) are a population of small vesicles secreted by essentially all cell types, containing a wide variety of biological macromolecules. Due to their intrinsic capabilities for efficient intercellular communication, they are involved in various aspects of cellular functioning. In the past decade, EVs derived from stem cells attracted interest in the field of regenerative medicine. Owing to their regenerative properties, they have great potential for use in tissue repair, in particular for tissues with limited regenerative capabilities such as cartilage. The maintenance of articular cartilage is dependent on a precarious balance of many different components that can be disrupted by the onset of prevalent rheumatic diseases. However, while cartilage is a tissue with strong mechanical properties that can withstand movement and heavy loads for years, it is virtually incapable of repairing itself after damage has occurred. Stem cell-derived EVs (SC-EVs) transport regenerative components such as proteins and nucleic acids from their parental cells to recipient cells, thereby promoting cartilage healing. Many possible pathways through which SC-EVs execute their regenerative function have been reported, but likely there are still numerous other pathways that are still unknown. This review discusses various preclinical studies investigating intra-articular injections of free SC-EVs, which, while often promoting chondrogenesis and cartilage repair in vivo, showed a recurring limitation of the need for multiple administrations to achieve sufficient tissue regeneration. Potentially, this drawback can be overcome by making use of an EV delivery platform that is capable of sustainably releasing EVs over time. With their remarkable versatility and favourable chemical, biological and mechanical properties, hydrogels can facilitate this release profile by encapsulating EVs in their porous structure. Ideally, the optimal delivery platform can be formed in-situ, by means of an injectable hydrogel that can be administered directly into the affected joint. Relevant research fulfilling these criteria is discussed in detail, including the steps that still need to be taken before injectable hydrogels for sustained delivery of EVs can be applied in the context of cartilage regeneration in the clinic

Injectable hydrogels for sustained delivery of extracellular vesicles in cartilage regeneration

Extracellular vesicles (EVs) are a population of small vesicles secreted by essentially all cell types, containing a wide variety of biological macromolecules. Due to their intrinsic capabilities for efficient intercellular communication, they are involved in various aspects of cellular functioning. In the past decade, EVs derived from stem cells attracted interest in the field of regenerative medicine. Owing to their regenerative properties, they have great potential for use in tissue repair, in particular for tissues with limited regenerative capabilities such as cartilage. The maintenance of articular cartilage is dependent on a precarious balance of many different components that can be disrupted by the onset of prevalent rheumatic diseases. However, while cartilage is a tissue with strong mechanical properties that can withstand movement and heavy loads for years, it is virtually incapable of repairing itself after damage has occurred. Stem cell-derived EVs (SC-EVs) transport regenerative components such as proteins and nucleic acids from their parental cells to recipient cells, thereby promoting cartilage healing. Many possible pathways through which SC-EVs execute their regenerative function have been reported, but likely there are still numerous other pathways that are still unknown. This review discusses various preclinical studies investigating intra-articular injections of free SC-EVs, which, while often promoting chondrogenesis and cartilage repair in vivo, showed a recurring limitation of the need for multiple administrations to achieve sufficient tissue regeneration. Potentially, this drawback can be overcome by making use of an EV delivery platform that is capable of sustainably releasing EVs over time. With their remarkable versatility and favourable chemical, biological and mechanical properties, hydrogels can facilitate this release profile by encapsulating EVs in their porous structure. Ideally, the optimal delivery platform can be formed in-situ, by means of an injectable hydrogel that can be administered directly into the affected joint. Relevant research fulfilling these criteria is discussed in detail, including the steps that still need to be taken before injectable hydrogels for sustained delivery of EVs can be applied in the context of cartilage regeneration in the clinic

Mesenchymal stem/stromal cells-derived extracellular vesicles as a potentially more beneficial therapeutic strategy than MSC-based treatment in a mild metabolic osteoarthritis model

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) hold promise as a disease modifying treatment in osteoarthritis (OA). Obesity, and its associated inflammation, contribute to OA development and metabolic OA represents a specific and significant group of the OA patient population. Given their immunomodulatory properties, MSC and MSC-EVs are especially interesting for this group of patients as a therapeutic option. Here, we were the first to compare the therapeutic efficacy of MSCs and MSC-EVs in a mild OA model taking these metabolic aspects into consideration. METHODS: Male Wistar-Han rats (Crl:WI(Han) (n = 36) were fed a high fat diet for 24 weeks, with unilateral induction of OA by groove surgery after 12 weeks. Eight days after surgery rats were randomized in three treatment groups receiving MSCs, MSC-EVs or vehicle injection. Pain-associated behavior, joint degeneration, and local and systemic inflammation were measured. RESULTS: We demonstrated that despite not having a significant therapeutic effect, MSC-EV treatment results in lower cartilage degeneration, less pain behaviour, osteophytosis and joint inflammation, than MSC treatment. Suggesting that MSC-EVs could be a more promising therapeutic strategy than MSCs in this mild metabolic OA model. CONCLUSION: In summary, we find that MSC treatment has negative effects on the joint in metabolic mild OA. This is an essential finding for the significant group of patients with metabolic OA phenotype, and might help to understand why clinical translation of MSC treatment shows varying therapeutic efficacy thus far. Our results also suggest that MSC-EV-based treatment might be a promising option for these patients, however MSC-EV therapeutic efficacy will need improvement

Mesenchymal stem/stromal cells-derived extracellular vesicles as a potentially more beneficial therapeutic strategy than MSC-based treatment in a mild metabolic osteoarthritis model

Background: Mesenchymal stromal/stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) hold promise as a disease modifying treatment in osteoarthritis (OA). Obesity, and its associated inflammation, contribute to OA development and metabolic OA represents a specific and significant group of the OA patient population. Given their immunomodulatory properties, MSC and MSC-EVs are especially interesting for this group of patients as a therapeutic option. Here, we were the first to compare the therapeutic efficacy of MSCs and MSC-EVs in a mild OA model taking these metabolic aspects into consideration. Methods: Male Wistar-Han rats (Crl:WI(Han) (n = 36) were fed a high fat diet for 24 weeks, with unilateral induction of OA by groove surgery after 12 weeks. Eight days after surgery rats were randomized in three treatment groups receiving MSCs, MSC-EVs or vehicle injection. Pain-associated behavior, joint degeneration, and local and systemic inflammation were measured. Results: We demonstrated that despite not having a significant therapeutic effect, MSC-EV treatment results in lower cartilage degeneration, less pain behaviour, osteophytosis and joint inflammation, than MSC treatment. Suggesting that MSC-EVs could be a more promising therapeutic strategy than MSCs in this mild metabolic OA model. Conclusion: In summary, we find that MSC treatment has negative effects on the joint in metabolic mild OA. This is an essential finding for the significant group of patients with metabolic OA phenotype, and might help to understand why clinical translation of MSC treatment shows varying therapeutic efficacy thus far. Our results also suggest that MSC-EV-based treatment might be a promising option for these patients, however MSC-EV therapeutic efficacy will need improvement

Efficacy of MSC for steroid-refractory acute GVHD associates with MSC donor age and a defined molecular profile

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Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - An ISEV position paper

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNAencoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolationmethods, optimisation of methodologies to isolate and characteriseminute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly