Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.

https://tandfonline.com/doi/pdf/10.1080/20013078.2019.1609206

Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications

Cancer-derived extracellular vesicles (EVs) are regarded as having promising potential to be used as therapeutics and disease biomarkers. Mechanistically, EVs have been shown to function in most, if not all, steps of cancer progression. Cancer EVs, including small EVs (sEVs), contain unique biomolecular cargo, consisting of protein, nucleic acid and lipids. Through progress in the identification of this specific cargo, cancer biomarkers have been identified and developed, opening up novel and interesting opportunities for cancer diagnosis and prognosis. Intriguingly, we still lack a comprehensive understanding of the cancer-specific pathways that govern EV biogenesis in cancer cells. Filling this knowledge gap will rapidly improve cancer EV biomarkers, as it will also allow discrimination of the procancer and anticancer actions of those EVs. Even more promising is uncovering therapeutically targetable, tumour-specific EV pathways and content, which will generate novel classes of cancer therapies. This Review highlights the progress the cancer sEV field has made in the areas of biomarker discovery and validation as well as sEV-based therapeutics, highlights the challenges we are facing and identifies gaps in our knowledge, which currently prevent us from developing the full potential of sEVs in cancer diagnostic and therapy.

The evolving translational potential of small extracellular vesicles in cancer.

Exosomes are nano-sized vesicles that serve as mediators for cell-to-cell communication. With their unique nucleic acids, proteins, and lipids cargo compositions that reflect the characteristics of producer cells, exosomes can be utilized as cell-free therapeutics. Among exosomes derived from various cellular origins, mesenchymal stem cell-derived exosomes (MSC-exosomes) have gained great attention due to their immunomodulatory and regenerative functions. Indeed, many studies have shown anti-inflammatory, anti-aging and wound healing effects of MSC-exosomes in various in vitro and in vivo models. In addition, recent advances in the field of exosome biology have enabled development of specific guidelines and quality control methods, which will ultimately lead to clinical application of exosomes. This review highlights recent studies that investigate therapeutic potential of MSC-exosomes and relevant mode of actions for skin diseases, as well as quality control measures required for development of exosome-derived therapeutics.

Mesenchymal Stem/Stromal Cell-Derived Exosomes for Immunomodulatory Therapeutics and Skin Regeneration.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

https://biblio.ugent.be/publication/8654828/file/8654830.pdf

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these. Extracellular vesicles (EVs) mediate cell–cell communication in physiology and pathology but many questions remain about the mechanisms governing their delivery to recipient cells. This Expert Recommendation article highlights areas of progress and challenges in establishing the importance of EV-mediated communication in vivo. 

/pdf/challenges-and-directions-in-studying-cell-cell-142hcmlr.pdf

Challenges and directions in studying cell–cell communication by extracellular vesicles

Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.

https://www.tandfonline.com/doi/pdf/10.1080/20013078.2019.1587567

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material

Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their N2O emissions because all cells express NosZ, reducing N2O to N2, while a minority express NirS + Nor, reducing NO2− to N2O. ABSTRACT Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O2 repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed “bet-hedging,” where all cells synthesize the N2O reductase (NosZ) but only a minority synthesize nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia but with the possibility of escape if supplied with O2 or N2O. To test this, cells were exposed to gradual O2 depletion or sudden anoxia and subsequent spikes of O2 and N2O. The synthesis of NirS in single cells was monitored by using an mCherry-nirS fusion replacing the native nirS, and their growth was detected as dilution of green, fluorescent fluorescein isothiocyanate (FITC) stain. We demonstrate anoxic entrapment due to e−-acceptor deprivation and show that O2 spiking leads to bet-hedging, while N2O spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N2O spike had much lower respiration rates than those rescued by the O2 spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O2. Our results bring into relief a fitness advantage of pairing restrictive nirS expression with universal NosZ synthesis in energy-limited systems. IMPORTANCE Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their N2O emissions because all cells express NosZ, reducing N2O to N2, while a minority express NirS + Nor, reducing NO2− to N2O. We hypothesized that the cells without Nir were entrapped in anoxia, without energy to synthesize Nir, and that they could be rescued by short spikes of O2 or N2O. We confirm such entrapment and the rescue of all cells by an N2O spike but only a fraction by an O2 spike. The results shed light on the role of O2 repression in bet-hedging and generated a novel hypothesis regarding the autocatalytic nirS expression via NO production. Insight into the regulation of denitrification, including bet-hedging, holds a clue to understanding, and ultimately curbing, the escalating emissions of N2O, which contribute to anthropogenic climate forcing.

Sebastian Thalmann

Papers

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material

Preparation for Denitrification and Phenotypic Diversification at the Cusp of Anoxia: a Purpose for N2O Reductase Vis-à-Vis Multiple Roles of O2