Extracellular Vesicles (EVs) Lecture Notes PDF

Session #5 Department of Biological sciences BIO 613 CELL BIOLOGY Extracellular Vesicles 1 Dr. Mohamad RIMA Summary way of communication Extracellular...

Session #5 Department of Biological sciences BIO 613 CELL BIOLOGY Extracellular Vesicles 1 Dr. Mohamad RIMA Summary way of communication Extracellular Vesicles (EVs) Definition, types, biogenesis, composition, functions EVs in Health and Disease Isolation and Characterization (Workshop) 2  Extracellular vesicles (EVs) secreted by the cell EVs are small, membrane-bound vesicles released by cells into the extracellular space. Function: cell-cell communication Delivering cargo from donor to recipient cells and modulating their physiological condition. 3 types Classical EVs are exosomes, microvesicles, and apoptotic bodies. 3 Ref. Extracellular Vesicles: New Classification and Tumor Immunosuppression  Extracellular vesicles (EVs) how were they produced Category EV Class Size Markers Biogenesis Exosome Small EV 40–150 nm CD63, CD9, CD81 Multivesicular endosome Microvesicle Large EV ~150–1000 nm Annexin A1 Plasma membrane shedding Apoptotic body Large EV 1–5 µm Annexin V Apoptosis Small EVs are 200 nm in diameter. These three classical EV types are mutually exclusive based on their biogenesis mechanism 5 Ref. Extracellular Vesicles: New Classification and Tumor Immunosuppression Exosome biogenesis Exosome biogenesis starts within the endosomal system: Early endosomes mature into late endosomes (Multivesicular bodies MVBs). MVBs fuse with the plasma membrane, and release exosomes. 6 Microvesicles biogenesis Microvesicles are formed through direct outward budding and shedding from the plasma membrane. 7 Apoptotic body biogenesis Apoptotic bodies are generated in the process of apoptosis. Containing information and substances from dying cells. Previously regarded as garbage bags until they were discovered to be capable of delivering useful materials to healthy recipient cells (ex. Autoantigens). 8 Classical EVs comparison exocytosis e endocytosis eg mitochondria signature 9  so we have macromolecules inside and also within the plasma membrane EVs contain a broad spectrum of proteins, lipids, and nucleic acids. Based on the database ExoCarta (http://www.exocarta.org), there are EVs composition more than 9700 proteins, 3400 mRNA, and 2800 miRNAs that have been identified in exosomes. 11 Extracellular vesicles, from the pathogenesis to the therapy of neurodegenerative diseases  Exosomes content Exosomes are abundantly loaded with : Tetraspanins (CD63, CD81, and CD9) Active soluble proteins (Growth factors) Transcription factors Transmembrane proteins Nucleic acids including DNA, mRNAs, and non-coding RNAs like miRNAs Lipids including sphingomyelin, phosphatidylserine, cholesterol 12 Extracellular vesicles, from the pathogenesis to the therapy of neurodegenerative diseases  MVs content MVs also contain various proteins, lipids, and nucleic acids, but they differ from exosomes: signature of MV MVs express CD40, integrins, and highly likely cytoskeletal proteins due to their plasma membrane origin.actin or tubuline Small MVs also express CD63 (like exosomes), but the expression levels of CD81 and CD9 are significantly lower in MVs vs exosomes. always check the size also Membranes of MVs are highly enriched in cholesterol, phosphatidylserine, and diacylglycerol. because it is budding from the cell membrane 13 Extracellular vesicles, from the pathogenesis to the therapy of neurodegenerative diseases EV functions Initially: EVs were considered to be “garbage bags”, not affecting cells. trashing whatever is not needed in the cell Today: Cell-to-cell communication. Decide cell fate (apoptosis, growth, cell cycle, migration, invasion, and differentiation of recipient cells). function of the cell Regulate gene expression. they contain transcription factors Innate and adaptive immunity (inflammation, antigen presentation, and the development and activation of B cells and T cells). 14  alarm the immune system to act for whatever type of intervention is needed, example DNA damage in the cell and that may lead to cancer …. cells will release EV to send this alarm to the immune system as a first line of defence EVs and immunity a b a. EVs present antigen on their surface MHC molecules directly to T cells. b. EVs attach to (or are possibly recycled to) the surface bound on membrane of DC of dendritic cells (DCs), in which case the DC plasma membrane concentrates a large number of EV-associated peptide–MHC complexes for efficient immune synapse formation. 2 ways: 1- EV directly engulfed by the dendritic cells => the dendritic cell will now => to activate the immune system express what was expressed by EV to immune system cells to activate them 2- EV will stick of dendritic cells in a way to activate immune cells => present antigen 2 immune cells 15 The roles of extracellular vesicles in the immune system  important to keep in mind even if EV can play a positive role by inducing inflammation and triggering immune system to tissue where defect detected BUT at the same time it can have detrimental effect on immune system by inhibiting their activity or inducing their cell death => what will choose if EV have positive or negative effects => EVs and immunity the key element is the content and the markers n the surface of the membrane Immunoregulatory molecules on the surface of extracellular vesicles (EVs) Ex. The immune-checkpoint molecules programmed death ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) and the apoptosis- inducing ligands FASL and TNF- related apoptosis inducing ligand (TRAIL), interact with cognate ligands and receptors expressed by T cells and natural killer (NK) cells to inhibit their activity or induce apoptosis. based on what is present on membrane of EV => output can be detrimental. 16 The roles of extracellular vesicles in the immune system  how cells will be responding to EV: when EV secreted from cell1 will reach cell 2 they can be engulfed through endocytosis -> enter -> become part of the early endosome -> sorted: some will be secreted as new exosomes, other parts (if TF present) may go to the nucleus to regulate gene expression EV functions 17 The biology, function, and biomedical applications of exosomes EV may have advantageous effects (positive), at the same time just like they can spread cancer they may also spread several types of diseases one of the is AGING AND SENESCENCE: if we take EV from an old individual that has plenty of senescent cells and we inject them in young individual this will be promoting the premature aging of this individual !!!! when doing blood transfusion we cannot control the age of the person that donated !!!!!! what am i taking in this blood (EXOSOMES!!!! good or bad) that may spread disease. Isolation and Characterization Workshop Techniques for EV isolation. cell line, blood, biological fluid Methods for EV characterization. Students will be divided in 4 groups. Each group will be responsible of finding, preparing and presenting one technique/method for EV isolation and characterization 18 Isolation and Characterization Techniques for EV isolation (ultracentrifugation, size exclusion chromatography, etc.). Methods for EV characterization (electron microscopy, flow exosomes or MV or APbodies cytometry, etc.). smaller than exosomes: exomeres 19 Papers for Evs isolation 20 based on the molecular weight and the volume of macromolecule, based on the speed by which we centrifuge this macromolecule we may have it pelleted => separate tiny molecules if speed is really high cell in culture producing EVs spin down => cell Ultracentrifugation pelleted supernatant containing EVs (tiny structure cannot be take speed and run it at very pelleted yet high XG, we may pellet large EVs like apoptotic bodies and microvesicles exosome pellet the small EVs like exomeres and exosome 21  another technique: more advances and specific: ultracentrifugation using density gradient EV isolation techniques size exclusion chromatography 22 Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography  DIFF IN THE SIZE shu ma aaml i will have some impurities(10% hsebo ) EV characterization techniques Transmission electron microscopy (TEM) has nanometre resolution and can be used to distinguish single extracellular vesicles (EVs) from non-EV particles. Transmission electron microscopy of EVs in conditioned media. Serum-free, cell-depleted 24 h conditioned medium of THP-1 cells was ultracentrifuged at 100 000g for 60 min and subjected for electron microscopic imaging. Extracellular vesicles from all size-based subpopulations were fluorescent microscope: AKID NO, le2anno ma b farjine shozi hl2d zghiri present simultaneously. APO: apoptotic body, MV: microvesicle , EXO: exosome. 23 Differential detergent sensitivity of extracellular Q1 vesicle subpopulations Light vs electron microscope Electron microscope (EM) provides much greater resolution than the light microscope. EM uses electrons instead of light to produce a magnified image of cells. The interaction of the electrons with the sample forms the image as the beam is transmitted through the specimen. EM has a higher resolution of ~ 0.1 nm 24  EV characterization techniques Flow cytometry (FC) Conventional FC can be used to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples. used to assess the proteins on the surface of the cell (use florescent antibodies that bind to protein on surface) detect fluorescence through FC or intracellular inside the cell EV very small even the largest EV cant be detected through flow cytometry => i use magnetic beads 25 High sensitivity detection of extracellular vesicles immunecaptured from urine by conventional fow cytometry  EV characterization techniques Flow cytometry (FC) EV size ranging from 20 nanometer to to 1 micrometer (apoptotic body) we use beads with large diameter => yaane bghayer size mn size l EV (small yellow balls la size l beads (purple balls) with the changes diameter i will be able to detect it through cytometry eza my antigen b aleb l cell, i will use something to i have a shift permeabilize (create pores) entry for antibodies in the signal => antibody background did bind ma behsbo (yaane DPP4 cannot bind did bind) 26 kl ma rouh aal yamin my intensity is increasing  EV characterization techniques (FC) peak=> i have the binding EVs immobilised on 6 μm APC-beads were stained using biotinylated antibody followed by PE-conjugated streptavidin and analysed by fow cytometry. A gate containing only single beads was created in the Forward Scatter (FSC)/Side Scatter (SSC) plot. A second gate, within single beads, confrmed the APC fuorescence of microbeads 27 High sensitivity detection of extracellular vesicles immunecaptured from urine by conventional fow cytometry  EV characterization techniques Nanoparticle Tracking Analysis (NTA): first method making EVs visible in their natural dispersed state. A technique used for measuring and analyzing single particle size and concentration. When a laser beam is passed through the sample chamber, the particles in suspension that fall in the laser beam path disperse light in such a way that they can be observed through a 20x magnification microscope. 28 EV characterization techniques (NTA) nanoparticle + florescence Adding fluorescent dye Analysis of only the extracellular vesicles (EVs) present in a heterogeneous sample. Exclude protein aggregates, membrane fractions, and other background particles EV-specific particle size distribution and concentration. 29 EVs in diseases Being a key intercellular communicator, EVs have emerged as a key contributor to the pathogenesis of various diseases: and metastasis Tumor cells release EVs that promote tumor growth and invasion in vivo. EVs that carry tumor suppressors, could inhibited tumor growth. In the NS, EVs are key contributor to the pathogenesis of various neurodegenerative diseases. promoting the appearance Senescent cells release EVs that promote secondary senescence. hallmark of senscent cells 30  EVs therapeutic potential EVs have shown innate immunosuppressive and anti- inflammatory properties when isolated from stem/progenitor cells and have also been considered vehicles to be edited for drug delivery. if we take EVs from stem cells => EV will activate the immune system and prevent inflammation and also be immunosuppressant also skin, hair growth collagen 31 Extracellular Vesicles as Biomarkers and Therapeutic Tools: From Pre-Clinical to Clinical Applications  EV produced by CAR T cells engineered in the lab EVs therapeutic potential Example of antitumour effects of extracellular vesicles released by genetically engineered cells. Chimeric antigen receptor (CAR) T cells release extracellular vesicles (EVs) carrying surface CARs. These EVs also contain perforin and granzyme B and can cause tumour cell death upon recognition of the CAR-specific tumour antigen. 32 The roles of extracellular vesicles in the immune system  -communication - therapeutic EVs therapeutic potential - check for disease Study of EVs and their biomarkers in healthy and disease conditions helped in developing EV- based therapeutic strategies. Also, EVs are used for diagnosis. biomarkers to check the state of ind, healthy diseases… if a specific treatment is good, how the body is responding to the treatment le2anno available b diff samples (blood). take blood sample i extract the EV and i see if they have DPP4 -> diagnosis tool 33  EV secretion and isolation … collection samples The possibility of isolating EVs from different biofluids makes EVs valuable biomarkers to be analyzed for the diagnosis of several conditions. 34 Extracellular Vesicles as Biomarkers and Therapeutic Tools: From Pre-Clinical to Clinical Applications Summary 35