Exosomes at the age of uncertainty

Nowadays, everyday 5 to 10 papers are published in the EV field (1853 in 2017 only with the keyword “extracellular vesicles” in Pubmed), that mostly encompasses studies of the extracellular vesicle role in a particular physiological process or particular disease. Major fundamental issues among which exosome mechanism of action has still to be investigated deeper, but it probably relies on the simultaneous action of multiple effectors in a complex process. However, today the extracellular vesicles (EVs) complexity that probably makes its major interest is also its main drawback. The field lacks of a omic-like technology to describe the effect of each protein present in EVs in a fast and convenient way. The process, still ongoing, of knock-down experiments to understand the particular effect of proteins can provide some insight to investigate each protein role in exosomes. 

Moreover, most techniques used in the field are poorly quantitative, from Western Blotting of canonical exosomal markers like CD63 that are detected in nearly any sample containing cell fractions and that is commonly sufficient to claim an exosomal preparation (even with recent ISEV position papers [17], that is usually not known or respected). Cryo–TEM only give a proxy of EV concentration or markers expression. NTA, flow cytometry or TRPS evaluate the amount of exosomes and give a rather imprecise and highly variable results depending on the purity of the sample. The best example up to date of this uncertainty is the purity index used to quantify exosome purity, namely the number of particle per NTA/protein amount ratio, that show up to a 1000 fold difference  [71], meaning that at least in theory, some samples, usually derived from patients fluids are containing nearly up to 1000 fold more contaminating particles than extracellular vesicles [100]. These contaminants, namely protein aggregates are maybe also involved in the therapeutic effect of exosomes, maybe playing a role of adjuvant, or on the contrary limiting the effect of exosomes. 

It therefore means that any conclusions from patient fluid derived exosomes are to be taken with caution, and put a major doubt on any publication claiming the enrichment of any kind of biological macromolecule in exosomes, that are maybe co-purified with a major amount of contaminants. Similarly, deciphering the therapeutic effect of exosomes produced in serum or human platelet lysate containing media from its contaminating neighbors (platelet derived EVs, etc) is of a particular difficulty. 

Such kind of misunderstanding is probably rather common in the field, but hardly detected, as shown by the “siRNA encapsulation” controversy of a particularly cited paper of the field, that was later understood to be an artifact of co-purification [109] that was due to the aggregation of siRNA and exosomes during the process. In our opinion, these errors are rather due to the lack of simple characterization methods. 

Finally, the EV community suffers, from our point of view, of unproven dogmas and hypes that hampers the investigation of valuable questions. The “tetraspanin network” hypothesis, that state that tetraspanin composition of extracellular vesicles would allow specific targeting hardly explain the fact that most exosomes in the blood are cleared within minutes in the liver, rendering a targeting effect more than difficult. The miRNA/siRNA delivering capability of extracellular vesicles is rarely compared with lipofectamine, which is by far more efficient than EVs (Vader, Piffoux et Al, unpublished results), even if lipofectamine is rather difficult to use and toxic in vivo. 

Interestingly Apoptotic Bodies are now the less investigated extracellular vesicles, and most of the actual EV community is not aware of the previously published papers in the field, not very cited (3111 papers on pubmed on “apoptotic bodies” with a pic in 2001 versus 8539 for exosome with a pic in 2017). Interestingly, the recent discovery of the role of exosomes in regenerative medicine/immune-modulation is in fact probably a re-discovery of a whole bunch of experiments made on apoptotic cells and apoptotic bodies. Interestingly the so called “efferocytosis” i.e. clearance of apoptotic cells and apoptotic bodies is dissected from a molecular point of view, implying directly chemokines, phosphatidyl serine, oxidized lipids, thrombospondin 1, and CD46 among others, and indirectly TGF-Beta, IL10, IDO and LXR through phagocyte secretion after clearance of the apoptotic cell/apoptotic body [110]. Interestingly, the immunomodulation by injection of apoptotic cells is demonstrated since 2002 to treat sepsis [111], type I diabetes, arthritis [112], GvHD [113] or even acute myocardial infarction [114]. 

In fact, the Apoptotic Bodies (AB) field describe the processes of apoptotic cells production from cultured cells by putting these cells in serum starvation for 6 to 24 hours [112, 115], just like what is actually used in many papers to produce exosomes or microvesicles [116]. 

This terminology change (shifting from apoptotic bodies to exosome and microvesicles) permitted a real unexpected return of interest in the field, but it seems that these two communities are hardly learning from each other, as depicted by the recent huge review paper published by the journal of extracellular vesicles, that clearly states in introduction that even if the paper is about EVs, it does not review at all apoptotic bodies [117]. Indeed, the role of apoptotic bodies (that probably contains exosomes and microvesicles) is seen with a “clearance” point of view, meaning that AB immunomodulatory effect is rather seen as a way to limit inflammation by efferocytosis, whereas exosomes immunomodulatory effect is seen as a fantastic property to induce an immunomodulatory environment. These two effects are probably the two faces of the same coin, and a lot more molecular mechanisms are known about the efferocytosis process, that should be of interest to the exosome community [110].

Recently, a patent depicting the use of apoptotic cells and macrophages co-culture supernatant as a potent immunomodulatory agent (EP13305009.6) was published, whose effect is probably in part mediated by apoptotic bodies or extracellular vesicles derived from apoptotic cells or macrophages. 

Beautiful great and simple experiment from the apoptotic bodies field are for example completely forgotten: Pierre M Durand et al [118] described in 2011 the proliferation effect on simple unicellulars  induced by supernatant containing vesicles produced by programed cell death (apoptotic bodies ?) or by supernatant containing vesicles coming from cells destroyed by sonication. Interestingly, programed cell death induced extracellular vesicles were highly efficient at inducing cell proliferation, whereas sonication induced extracellular vesicles was stopping other cells growth. The proposed explanation is that programed cell death derived supernatants were able to be used by the cells as nutrient, whereas sonication derived ones were not. It means that the nutrients were “prepared” and “ready/easy to eat” in programed cell death induced extracellular vesicles but not in the other. The fact that this effect is conserved among various very different species is also an interesting information. Seeing the exosome microvesicle field with an apoptotic body prism considerably changes the interpretation of many experiments, and open the question of whether EVs effect can be considered as “ready/easy to eat” pieces of nutrients. In fact, proliferation assays where extracellular vesicles induce a proliferation even in the absence of FBS could also be a proxy to quantify the extracellular vesicles ability to be good nutrients for cell. Incidentally, F Verveij et al (unpublished data) recently observed the fate of yolk cell derived EVs in zebrafish embryos, and observed that extracellular vesicles derived from this reserve of nutrients organ were in present in a massive amount in zebrafish embryo blood, and maybe have a feeding function. These findings are however hardly comparable with what happens in adult human, where the amount of extracellular vesicles is the blood is much lower. Whether this mechanism, as well as apoptosis, is conserved among species and if it is comparable to what happens in human fetuses (with placenta playing the role of yolk) is a very intriguing question. Another hypothesis is that this function would be lost after birth, and could maybe be linked with the enhanced recovery/scarring ability of fetuses. 

Following these questions, another unexplored question of huge importance (for potential implications in simplifying cell culture processes in an industrial facility) is the question of whether extracellular vesicles derived from “simple” non-stem cell types would be as efficient as stem cell derived extracellular vesicles in regenerative medicine. This question is to our knowledge not investigated, even if fibroblast were already shown to behave similarly to MSC [119].

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