Origins to Outcomes: A Role for Extracellular Vesicles in Precision Medicine
- Savage, John “John Savage” (Master in Molecular Medicine; LBCAM)
- Maguire, Ciaran (Manus) “Ciaran Manus Maguire” (LBCAM; AMBER Centre)
- Prina-Mello, Adriele “Adriele Prina-Mello, PhD” (LBCAM; AMBER Centre)
- Master in Molecular Medicine < School of Medicine < Trinity College Dublin – Dublin, Ireland
- Laboratory for Biological Characterization of Advanced Materials (LBCAM) < Department of Clinical Medicine < Trinity Translational Medicine Institute < School of Medicine < Trinity College Dublin - Dublin, Ireland
- AMBER Centre < CRANN Institute < Trinity College Dublin - Dublin, Ireland
Submitted: March 8, 2018; Accepted: April 19, 2018
Extracellular vesicles (EVs) are of great interest in biological research, and though they are a relatively recent discovery, they have rapidly shown great potential for use in clinical applications. The various techniques used in EV isolation along with their respective strengths, weaknesses, and potential for downstream applications are outlined here. A brief description of the different approaches in exosome characterisation are subsequently described. It has been high-lighted that despite the recent developments in these processes, there is still a great deal of refinement to be made.
EVs are produced by almost all cell types, found in many biological samples, and are implicated in multiple biological processes including cargo trafficking, cell-cell communication, and signal transduction. The presence of these EVs and their varied cargo in a biological sample can be indicative in disease diagnosis, and guide precision medicine-based approaches to disease management.
EVs have been reported to act in the benefit of the cell through moderating repair and regeneration, but they can also act to the detriment of the cell through increased tumorigenesis and metastasis. This duality is intriguing as it can allow for the use of EVs in distinct therapeutic approaches and displays their versatility in potential downstream applications.
In this review, examples of the cellular roles of EVs and their applications in pathological and regenerative contexts are explored. In reviewing some of the developments made in recent times, EVs are shown to be very promising both in diagnostic and therapeutic approaches.
- extracellular vesicles
- regenerative medicine
- EV isolation
- EV characterisation
Rationale and Purpose
Increasing body of evidence suggests that healthcare, medical decisions, practices, and treatments should be tailored towards individual patients to improve their outcomes, and there is now need for precise and personalised medicine. Extracellular vesicles are produced by almost all cell types, found in many biological samples and are implicated in multiple biological processes including cargo trafficking, cell-cell communication and signal transduction. The presence of these vesicles and their varied cargo in a biological sample can be indicative in disease diagnosis, and guide precision medicine-based approaches to disease management. EVs have been reported to act in the benefit of the cell through moderating repair and regeneration, but they can also act to the detriment of the cell through increased tumorigenesis and metastasis. This duality is intriguing as it can allow for the use of EVs in distinct therapeutic approaches and displays their versatility in potential downstream applications. In this review, examples of the cellular roles of EVs and their applications in pathological and regenerative contexts are explored.
Summary of Relevant Literature
Extracellular vesicles (EVs) can have varied cellular roles and they have been highlighted for use as potential clinical biomarkers . These small nano- to sub-micron sized lipid membrane enclosed structures, comprise varying contents, and are produced by almost all cell types . EVs were initially observed as a product of reticulocytes that were released into the extracellular environment. The study was regarding transferrin receptor endocytosis and the unintended outcome of revealing the production and secretion of EVs that contained transferrin receptors .
EVs were found to form after several intracellular processing steps. Initially, intraluminal vesicles form and are contained in multivesicular endosome (MVE) bodies. These MVE bodies subsequently are trafficked to and fuse with the plasma membrane of the cell. These MVE bodies range between 0.5 and 1 µm in diameter. Their fusion with the plasma membrane resulted in the release of multiple EVs of a diameter between 40 and 100 nm . It has also been reported that certain EV subsets can occur at lipid raft regions which are associated with high levels of cholesterol and sphingomyelin .
Upon closer examination, it was found that these EVs displayed composition and function characteristic of their cell of origin. These EVs were thought to be utilised as a mechanism of aiding cell maturation, along with the removal of excess cellular materials . However, it has been demonstrated in subsequent research that they can possess diverse contents. The con-tents of the different EV subtypes can be catalogued in various online databases, including ExoCarta , EVpedia  and Vesiclepedia .
There are far more roles for these EVs than was previously thought, and this has been discerned considering the varied contents that they can possess. They have been shown to have roles in many aspects of specific pathological processes, which are deleterious to the host. These can include immune suppression,  cellular communication through protein trafficking to drive excessive proliferation,  and they can increase cancer aggressiveness through micro RNA (miRNA) trafficking .
However, it has also been documented that EVs, particularly those released by stem cells, can have regenerative roles in a variety of disease states and tissues. Examples of this can be found in liver tissue regeneration via cargo shuttling between cells,  peripheral nervous system repair, through modifying cellular growth,  skeletal muscle regeneration through induction of stem cell differentiation , cardio protection,  and cardiac regeneration post injury, through alterations to target cell signaling .
An important caveat that should be considered in the study of EVs is that they can be produced by cells and they can be divided into many categories, including exosomes, apoptotic bodies and microvesicles . It has also been shown that even among the EVs produced from a single cell type of origin, that distinct subpopulations can occur. These subpopulations can demonstrate different marker proteins, internal cargo, and distinct downstream effects on gene expression. This reveals an additional layer of complexity in EV study but may allow for more avenues of investigation to be pursued regarding downstream applications, including therapeutics . Similarly, with the study of the heterogeneous subpopulations of EVs produced, in-sight may be granted into how EVs can impact so many diverse functions and how they can aid in tumorigenesis .
With this in mind, EVs can be utilised as biomarkers for cancers of various tissues and various diagnostic methods have been designed to utilise EVs in a clinical context, enabling personalised and precision medicine-based approaches . There are currently pilot studies for EV use as diagnostic markers [20, 21], along with early phase 1 clinical trials regarding EV use for regenerative applications  and as potential post-intervention prognostic markers in disease states  being conducted. EVs are also being used as vehicles in different therapies including small interfering RNAs (siRNAs)  and drug delivery . This has been visually summarised in Figure 1.
Considering their manifold features and broad applicable potential, the study of EVs is a complex field that involves the discussion of many aspects such as those below.
Considering the range of EVs that can be produced by a single cell type and the size of EVs produced, much consideration should be taken regarding the isolation method utilised. There are many methods that can be implemented, yet the impact of the isolation procedures on the EVs should be taken into account. It has been suggested that the EVs may be affected by the acquisition steps taken, though it can be difficult to determine the exact effects and how best to alleviate them . EVs can be isolated broadly speaking, based on size and/or affinity-based methods. A summary of these methods and their characteristics have been outlined in Table 1, Table 2, and Table 3.
A typical method of EV isolation that can be implemented considering their size is differential ultracentrifugation. This is considered the gold standard of EV isolation methods, yet there are certain contentions regarding the validity of that statement . The method of differential centrifugation can involve repeated rounds of centrifugation that use increasing centrifugal force for each round, gradually removing larger unwanted materials from a sample. It has been shown that with considerations for the type of rotor, centrifuge radius, and centrifugation duration, a more optimal protocol can be developed depending on the size of the structure of interest .