So welcome to the Basics of Extracellular Vesicles. I am Suresh Mathivanan from La Trobe University Melbourne Australia. I'm going to talk about biogenesis, release and proteomic cargo of extracellular vesicle subtypes. In the first part I'll give and introduction on biogenesis and release of extracellular vesicles. Before that a bit of history on extracellular vesicles. In extracellular vesicles where initially discovered they were considered as garbage bags. It is shown to remove transferring receptor from reticular sites. There are quite a few reports which supports this hypothesis that extracellular vesicles can function as garbage bags and can be important In the maturation of certain cell types. But we have moved beyond that observation and now we are able to show that can transfer signaling molecules between cells both in and fashion. When it comes to the naming of the nomenclature of the extracellular vesicles, researchers initially chose to name them based on the cell of the sample source from which they derived. For instance prostate cancer derived [INAUDIBLE] called prostasomes, while dendritic cell derived [INAUDIBLE] were called dexosomes. This was merely because when researchers initially saw that Exosomes in the culture media. They were not sure whether these exosomes are specific to the cell type that they are working with. Or it is conserved across various cell forms. So this naming on the nomenclature which were used for extracellular vesicles led to a lot of confusion among researchers. Currently we know more about extracellular vesicles. And based on the biogenesis. Again this is based on the current knowledge in the field. We can classify these extracellular vesicles by three subtypes. This is exosomes, the ectosomes and the apoptotic bodies Exosomes are relatively well-studied and they're coming from an endocytic part. So when there's a receptor internalization which is led to endocytosis in a cell, that results in early endosomes and the early endosomes can mature into a late endosomes. Inward budding of the MVB results in the progressive accumulation of intraluminal vesicles inside this multivascular bodies. And this multivascular bodies can per take two functions. This two pathways can either be pathway where multivascular body can fuse with Lysomes and degrade it content. Or it can take the exocytic pathway, where it can fuse with the plasma membrane and release its contents into the extracellular space. Once the ILVs are released into the extracellular space, th ey are called as exosomes. So what governments does transition off the multivascular bodies to exocytic pathway or the degradative pathway reminds the the uncharacterise. So when it come to Ectosomes, these are again the sickles larger than the exosomes which are secreted from the cells, and they are coming from the plasma membrane, mainly through outward budding. So then there's the interaction between the cytoskeletal proteins and the plasma membrane is gradually lost. This is mainly because of a, that the cytosolic increase of calcium. And the protein degrading enzymes which can induce the deassembly of the cytoskeleton. Some of the enzymes which can do this are, one of the examples, calpain, which again, is dependent on calcium itself. So in this way, the plasma membrane can now get detached or it loses the contact from the cytoskeleton and then it forms the budding. So then, the lipid translocases get activated. And then these induce changes in the lipid bilayer favoring the budding and membrane this results in the release of ectosomes, or shedding microvesicles from the cells. Lastly, apoptotic bodies. Again, this has been studied for quite some time, and people don't pay too much attention on these bodies because There are mere debris of the cell and they are getting cleared out in the system. So there are quite a few ways by which apoptotic bodies are generated from a cell. In this one example you can see when there is apoptosis is induced. Again you can see a preclusive structures which are formed from the cell. Their membrane, the vesticles are formed and this is called as apoptopodia. Where apoptopodic are released from this membrane protrusions in to the excess cellular space. Again, the formation of the apoptotic bodies is cell type dependent. And there's also cases where the cell structures or even form small bodies through blooming. Again it is based on the stimuli as well and this is a work in progress, where a lot of people are trying to characterize, what are the way by which apoptotic bodies are formed? So this study, which I've showed here, is largely by one of my colleague in called Ivan Poon, who was able to show in this protrusion. And this is a null observation which was especially found in monocytes. So when we look at the known features of these EV subtypes based on the introduction that I spoke about, you can see when it comes to size of these vesicles Exosomes are normally 30-150 nano-metre in diameter. When it comes to ectosomes they're a bit larger, they're 100-1000 nano-metre in diameter while apoptotic bodies are thought to be from 50-5000 nano-metre. Well this is not completely unproven by multiple studies. When it comes to the as I described before, Exosomes are endocytic where they derive from the multicellular bodies. While ectosomes are the shedding microvesicles are coming from the plasma membrane directly. While apoptotic bodies is aided to cellular disassembly and the fragmentation of the cell components. So, again, these vesicles have a lot of proteins, RNA, and the lipid as well. And nucleic acids are also found in these vesicles. And based on the content, and also the lipid bilayer, and the size of them, they have a density and they Kind of float in a particular density when we try to purify then by either sucrose or by optical density. And based on literature and many other studies which have been carried out throughout the world, we can see exozomes settle in the density between 1.10 to 1.14 grams per mil. But this is varied based on the cell type as well. Again, ectosomes in that case, because they are bigger in size, but the cargo may not be always correlating with the size. But the density may lies between 1.12 to 1.20 and as you can see here there is an overlap between the density in terms of the exosomes and ectosomes and even in terms fo the size as well. In terms of apoptotic bodies the density of these vesicles are not unlisted. We have tried to characterize them, and similar to other groups we saw listability of these extracted vesicles when you're trying to purify them, but vesicles are best centrifugation approaches. So then it comes to the protein markers, which are present in the particular vesicle subtypes, that people have been thinking of quite a few markers. You can see Alics, TSG101, CD63, CD81, CD9, which are the type which are more often found in ectosomes. Are considered to be exosomal markers. When it comes to ectosomes of the shedding microvesicles, there're not too much studies which have been done on these vesicle subtypes. But the matrix is MMP2 and the cytokeratin 18 are thought to be markers of this. Again when it comes to Apoptotic bodies it's log as unknown. And they are not sure that the same Apoptotic bodies have different cell types can have the same marker. So there we are here now, no? From this we can clearly see that the EV subtypes on marked categories as well. There's still so many unknowns here and even we're not sure. Then these cellular like if different cell types are taken to account. How well the categorization of the have been understood. So it's clear that the sub types are not categorized yet. So in spite of the various biogenesis pathways, again there's not definitive markers to clearly differentiate between exosomes, ectosomes and Apoptotic bodies. So you have notice, you might notice that I've given some markers here like TGS101 in Exosomes. But again quite a few studies have shown that these can not be used as markers but rather these are enriched proteins. So again I spoke about the EV subtype markers and why is it important. Is it even important, should we even bother about researching on this? So markers are very important because now when we are working with EVs, again, these all are. Or when you look at the all the subtypes of are all found in the body of so when we isolate and purify these extracellular vesicles, we need to be sure, what is the population and what is the subtype of extracellular vesicles that we are working with. So, it will be easier for us to attribute a precise functional role to a subtype of extracellular vesicles To do that you need to have an EV marker for each and every subtype. So that's why we want to have various studies and then we want it to classify and find EV markers for every subtype. But again we need to also understand that it also depends on the question, the biological question or the research question that you are asking. In some cases the EV subtypes is not as important. Let's say now we are isolating from a normal under patient Bodily fluid. And then as long as we are able to discriminate between a normal and the patient, and this test can identify or diagnose our cancer patient early. The EV subtype or the EV marker as not as important in those cases. But when you attribute a function or there is other applications, the EV subtype marker is definitely important. So why are the EV subtypes not categorized well? Now again as I said the bi-denses partly are different and there is quite a few research going around in various parts of the world. But why are we struggling? Why are we not able to characterize these EV subtypes well? But as you can see here, there are quite a few methods by which these successful other sequels can be isolated. So the traditional method, is the ultracentrifugation. Where, the conditioned media, or the volume of fluids, can be spun down at larger speeds and then the are them. Or we can try to do the density gradient centrifugation approach or do a size exclusion chromatography or to immunicaffinity where we can couple your antibody of choice. To a magnetic bead and I salute the solute the extracellular vesicles or use the commercial kits which can also isolate extracellular vesicles in addition to other non-EV Material. But what is the current problem with this can be clearly shown in this slide. So now when you're isolating extracellular vesicles from a conditioned media, this is the gentle it can change a little bit based on the investigative plus also the cell types of the handling. So normally you do some differential a series of different to the light cells as well the apoptotic bodies, the 2000 g Twenty minutes is thought to pellet down some apoptotic bodies. On the 10,000 g spin is thought to pellet out the larger vesicles, the exosomes, while the 100,000 g centrifugation will pellet out the smaller vesicles, the exosomes. And this is the general Procedure for most of the people used in most of the labs. But this is crude isolation procedure. As we need to understand. Even in, let's say, the 10,000 g spin where we try to isolate these larger [INAUDIBLE] or ectosomes. There is a possibility we are also going to isolate Exosomes which are adding denser or which can be trapped under these larger vesicles and then can sediment along with them. Or also some of this vesicle could break because of the harsh protocols that we are employing. And even in apoptotic bodies, there is possible that there could be a heterogeneity where we are isolating a mixed up population of extracellular vesicles. So from this it's clear that we don't have a methodology where we can clearly isolate a particular EV subtype to but then our multiple options which a lot of people currently use to isolate. In this case you can enrich for an EV subtype, which is option one. For example, you can do the crude preparation like what I explained in the previous slide, and then subject them to a follow up purification like density gradient centrifugation. Where you can try to enrich of the EV subtype. Again I want to code this word Enrich because you are only Enriching for a population and you are not getting rid of 100 person that the other population is also a cor isolated from your preparation. So in the same way, now with option two, what you can do is an enrichment. You can try to increase a particular, accessible subtype of your study. For instance, if you are studying apoptotic bodies, you induce the cells with UV and try to increase the generation of apoptotic bodies, and then Try ro study them. So now you expect that what extracellular vesicles are getting are largely coming from the apoptosis. Similarly you can do for exosomes and again there are some ionophores the calcium ionophores, monensin which is known to induce exosome secretion. Similarly, you can do this for ectosomes by using calcium. So I have to emphasize caution here. Even though these reagents can increase the secretion of exosomes, ectosomes that are populated bodies. Again, these are cell type dependent. Example monensin is an ionophore which increases the calcium levels. So now, as it increases the calcium levels in the cell, it can also induce apoptosis in many cell deaths. So one need to be careful when you're taking this approach. It may not be suitable for every cell type. But,it is a good approach when you are trying to image And again, you can you can subject this to a follow up purification approach. Example, a density gradient centrifugation approach. And then, you will able to isolate these to a better purity than what you could, with a crude preparation. So in summary for this section, we can see that EV's can be classified as exosomes, ectosomes and apoptoic bodies based on a current knowledge on mode of biogenesis. And secondly, we need to understand that the current EV isolation methods can ONLY ENRICH for a EV subtype and cannot purify any one EV subtype to homogenity. So with that I would like to acknowledge people in my group. Collaborators who had helped to given this project. And also the copyright licenses for this doc.