טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNaama Koifman
SubjectNanostructural Study of Exteracellular Vesicles
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisors Professor Emeritus Talmon Yeshayahu
Assistant Professor Aharon Anat
Full Thesis textFull thesis text - English Version


Abstract

Extracellular vesicles are nanometric vesicles, ranging from several tens of nanometers to more than 1 µm in size. They may originate from the cell membrane (“microparticles”), or from the endosomal system (“exosomes”). If they originate from the disintegration of cells undergoing apoptosis, they are known as “apoptotic bodies”. Although they are found in all body fluids under healthy physiological conditions, their concentration increases significantly during certain pathologies and during pregnancy, and thus they have excellent diagnostic potential as disease biomarkers. The current available techniques used for the characterization of EVs provide information on EV populations, but they lack in morphological characterization on the single vesicle level.


We have applied advanced direct imaging techniques, namely, cryogenic electron microscopy (cryo-EM) for the nanostructural characterization of EVs. Stimulated and unstimulated cells were imaged while undergoing shedding by cryogenic scanning electron microscopy (cryo-SEM), and the isolated extracellular vesicles were imaged by cryogenic transmission electron microscopy (cryo-TEM).


Cryo-SEM of stimulated and unstimulated leukemic monocytes (THP1) indicated distinct differences in membrane morphology of cells under starvation conditions, as compared to cells stimulated by the lipopolysaccharide (LPS) endotoxin. EVs were isolated from the shedding cells and characterized by cryo-TEM complemented by nanoparticle tracking analysis (NTA). Although we could not identify distinct morphology differences by cryo-TEM, NTA indicated differences in size and concentration following different stimulations. LPS was found to have an intensifying effect on cellular shedding, while starvation had reduced effect on shedding, and may lead to cellular arrest.


EVs are heterogeneous in nanostructure and size, they bear granular vs. smooth membranes, and may contain cellular material, smaller vesicle, protein and genetic material, and cellular fibers. Apart from nanostructural characterization, we have optimized a protocol of immunogold labeling in the liquid state, to identify EVs rich in negatively-charged phospholipids, mostly phosphatidylserine (PS), thus adding an important single-vesicle compositional characterization aspect to our work, advancing the ability to link morphology and biology of this complex multicomponent system. The optimized methodology is simple, readily available to researchers, and relatively inexpensive. The labeling protocol was initially tested and optimized on a model system of liposomes, composed from PS only, or of a mixture of phosphatidylcholine (PC) and PS. We demonstrated the specificity of the labeling for PS, using biotinylated annexin-V and gold conjugated streptavidin. Labeling was then applied to EVs from leukemic monocytes (THP1) breast cancer cells (MDA-MB-468), and platelets. 


Cryo-SEM imaging of platelets isolated from healthy donors provided a detailed view of these small blood components that are crucial for hemostasis. Non-activated platelets appeared oblate, and the pores of the OCS were observed on their membrane. In some of the pores we identified vesicle-like structures, suggesting a mechanism for vesicle release different than the membrane flipping model. A platelet plug composed of platelets activated by thrombin, was imaged, showing the aggregation and nanostructural variations of activated platelets and shed EVs composing the clot. This is the first time, as far as we know, that platelets were imaged by cryo-SEM, which combines excellent preservation with high-resolution information unavailable by other imaging techniques.