Extracellular vesicles (EVs) are lipid bilayer-delimited pieces of cells that are released from the plasma membrane as "ectosomes" and from the endosomal system as "exosomes." EVs serve both to dispose of toxic materials and to facilitate intercellular communication, so they have many roles to play in disease, including neurodegenerative diseases such as Alzheimer's. Although the long-distance, inter-organ actions of EVs are an exciting topic of research, EVs in solid tissues are most likely to act locally, near the site of release. Our group has adapted a rigorous method for EV separation from tissues. Here, we discuss this method and some of the challenges and opportunities it presents. We then examine molecular profiling of EVs from brain of Alzheimer's disease patients and matched controls and assess possible contributions of APOE genotype. We review the implications of our findings for peripheral biomarkers of disease, using single-particle characterization technologies. All results are shared in the context of the "Minimal Information for Studies of EVs" (MISEV), the consensus document on best practices in the field that was issued by the International Society for Extracellular Vesicles and most recently updated as MISEV2018 (Théry and Witwer, et al, J Extracell Vesicles, 2018). We expect that rigorous and reproducible EV science will continue to develop and contribute to personalized medicine in the coming years.

Learning Objectives:

1. Explain the challenges and opportunities in studying extracellular vesicles in tissue

2. Identify associations between Alzheimer's disease and EV cargo

3. Introduce rigorous approaches to EV separation and characterization

Extracellular vesicles (EVs) are nanosized vesicles containing active proteins, lipids, and different types of genetic material such as non coding RNA species, related to the characteristics of the originating cell. They possess a distinctive capacity to communicate over long distances. EVs have been involved in the modulation of several pathophysiological conditions and, more importantly, stem cell-derived EVs appear as a new promising therapeutic option. Convincing evidence of the regenerative potential of EVs released by stem cells and, in particular, mesenchymal stromal cells has been shown in different injury models. My presentation will focus on the potential of stem cells-EVs in acute and chronic renal injury, describing mechanisms of action and active cargo. Besides, the clinical application of stem cell-EVs will be discussed.

Learning Objectives:

1. Rationale of the application of Stem Cell-EVs for regeneration

2. Role of EV cargo for Regenerative medicine

3. EV in vivo Biodistribution

4. Limits and promises for clinical application

MicroRNAs (miRs) are small non-coding RNAs whose expression is altered in several types of human cancers. Recent evidence supports their inter-cellular transfer through extracellular vesicles (EVs) secreted both by the cancer cells and by the surrounding cells of the Tumor Microenvironment (TME). These findings have suggested that the behavior of cancer cells can be significantly modified by the cross-talk with the surrounding TME as mediated by the exchange of EV-miRs. This lecture will focus on some of the most relevant implications of vesicular miRs in the biology of cancer cells and its surrounding TME.

Learning Objectives:

1. Explain how to classify different types of Extracellular Vesicles

2. Identify different possible outcomes of miR transfer to and from cancer cells

3. Learn how to exploit this knowledge to identify new therapeutic targets

Extracellular vesicles (EV) from many cell types have demonstrated therapeutic potential against many different diseases. Inhibiting progress in this area is the capability to produce EVs in sufficient quantity to deploy them in in vivo research applications and ultimately the clinic.  Current methods can utilize large numbers of flasks and serum starvation in a batch mode process, or large volume conventional bioreactor systems. It is clear that culture conditions affect the concentration, composition and therapeutic activity of secreted extracellular vesicles. 3-D capillary bioreactors represent the most in vivo like way to culture cells. They can support the culture of large numbers of cells at high densities, 1-2X10e8 cells/ml, 2X10e10 total cells.  Cells are bound to a porous support with a 20kDa molecular weight cut off (MWCO) so cell passaging is not required and EVs cannot cross the fiber in either direction. Secreted EVs are concentrated, free from contaminating serum EVs, show reduced membrane fragment contamination and in some cases have demonstrated enhanced bioactivity. The functional principles of 3-D capillary bioreactors will be shown, and examples of the current and potential future of clinical EV scale-up will be discussed.

Learning Objectives:

1. What are the three fundamental differences between a 3-D hollow fiber bioreactor and a T flask?

2. What is the definition of a Dalton?

3. In what ways is a 3-d hollow fiber bioreactor more in vivo like?

Studying exosomes is extremely challenging, due to enormous technical difficulties in defining and isolating such small and molecularly diverse nanovesicles. The progress in the biomedical field has been rather slow because of the lack of effective isolation approach. Extracellular vesicles (EVs) shed by cells are a highly complex population with a size range of 10-1000 nm, which is mainly composed of exosomes and microvesicles that differ in cell origin and biogenesis. Due to the substantial size overlap among these membrane vesicles, confusion on the origin and nomenclature of EVs has spread through the field. This talk reviews both historic and cutting edge exosome isolation technologies and platforms along with exosome methods of production, isolation and purification which have been shown to have significant effects on yield, viability and function. Further, exosome subtypes are critical to future research, biomarker identification and therapeutic development. Currently, the most-recognized isolation approach is ultracentrifugation, which has been shown to exhibit marked heterogeneity in isolated exosomes with variable sizes and protein compositions, indicating the inability to differentiate exosome subpopulations and other vesicle types (e.g., microvesicles and apoptotic blebs). Moreover, the isolation efficiency of ultracentrifugation is rather low (~5-25%) and involves multiple time-consuming steps for repetitive centrifugation and filtration (>10 hrs). Size-exclusion methods are prone to dilution, contaminations, and pressure-caused damage. To date, there are no well-defined methods for exosome isolation in high-efficiency and high-throughput, which severely hindered the broad applications of exosomes in the biomedical field. We close by introducing Clara ExoRelease Beads, a new purification method called “immuno-isolation and release” that offers standardization, reproducibility, and subtyping while preserving exosome functionality.

Learning Objectives:

1. Understand methods of isolating and enriching exosomes along with their pros/cons of the different techniques

2. What do we mean when we talk about subtyping, and what is its importance?

Chronic kidney disease (CKD) is a major public health threat, affecting 11-15% of the U.S. population alone. Currently, there are no effective therapies to cure CKD. Drug therapies are not potent, and directed only at slowing deterioration of renal function and treating metabolic consequences of CKD. With end-stage renal disease (ESRD) the only options are dialysis or transplantation.  To address the large unmet need for CKD therapies we identified potent renal protective effects of amniotic fluid stem cell derived extracellular vesicles (AFSC-EVs) in preclinical studies. AFSC-EVs are uniquely suited and represent an innovative therapeutic strategy for treatment of CKD. Using Alport syndrome (AS) as a model of CKD, we have previously shown that administration of amniotic fluid stem cells (AFSC) provides therapeutic value, improves renal function and prolongs animal survival. We established that the beneficial effect of AFSC is mainly attributed to secretion of extacellular vesicles (EVs) that contribute to the paracrine activity of AFSC in the kidney, and that administration of EVs alone recapitulate the protective effects of AFSC providing functional and survival benefits. AFSC-EVs are characterized by an array of exosomal and mesenchymal surface markers and miRNA cargo typical to the cell of their origin. In light of potential translation to treat CKD, in addition to mouse lines we characterized EVs derived from human AFSC. We have established that hAFSC are genotypically stable, can be easily scaled up and are high producers of EV. We have reduced EV heterogeneity by use of human clonal cell lines of AFSC, which can produce EVs with comparable expression profile and efficacy in vivo in our animal model of CKD, suggesting high reproducibility. We developed specific identity and potency assays thus guaranteeing the generation of lots of EVs with similar characteristics for therapeutic application. We performed extensive characterization of clonally derived EVs by proteomic and miRNA-seq analysis and using hollow fiber bioreactor system we are also able to increase EV production to clinical scale without losing characteristic purity, potency and efficacy. In order to advance the translation of hEVs and to explore their safety, targeting and effectiveness, we also developed a state of the art in vivo monitoring capabilities using novel magnetic nanoparticle agent for labeling EVs to study EV biodistribution by means of MRI technology. Our biodistribution studies showed that when injected in vivo into AS mice, hAFSC-EVs localized in the kidney, corrected proteinuria and prolonged the life-span of treated mice. In conclusion, we strongly believe that our findings will significantly enhance the development of EVs for paracrine regulation of glomerular diseases, with potential implications for patients affected by CKD.

Learning Objectives:

1. To understand the structure and function of the glomerulus, development of chronic kidney disease  and the pathophysiology of Alport syndrome

2. To understand the characterization of functional extracellular vesicles using in vitro and in vivo models, and potential imaging strategies for live tracking of EVs in vivo